Dissertations / Theses on the topic 'Innata'

El sistema immunitari innat es basa en el reconeixement no específic d'elements conservats del metabolisme dels patògens. Aquest reconeixement es fa principalment a través de receptors de reconeixement de patrons (PRRs) codificats per la línia germinal, que són presents a cèl·lules especialitzades del sistema immunitari innat, i que són capaços de reconèixer patrons moleculars conservats associats a patògens (PAMPs). Aquest reconeixement iniciarà diferents vies de senyalització que induiran la transcripció de citoquines proinflamatòries per finalment donar lloc a una inflamació local. D'aquesta manera, el sistema immunitari innat pot ser modulat, a través de l'administració d'aquests PAMPs, simulant una trobada natural entre el sistema immunitari i els patògens. La principal hipòtesi d'aquest estudi va ser que, mitjançant l'encapsulació en un mateix sistema d'administració nanomètric de diversos PAMPs, també anomenats immunoestimulants, es podria millorar la seva administració a diferents espècies de peixos. També, que aquest sistema d'administració podria interaccionar amb les cèl·lules del sistema immunitari generant la seva activació no específica, i millorant la resposta immunitària contra diferents malalties infeccioses. En aquest context, s'ha desenvolupat un nou sistema d'administració d'immunoestimulants basat en liposomes que encapsulen el lipopolisacàrid bacterià d'Escherichia coli, i un anàleg sintètic de dsRNA viral, el poli (I:C). Els nostres resultat van mostrar que aquests liposomes eren biocompatibles i capaços de ser endocitats in vitro per hepatòcits de peix zebra (Danio rerio) i per macròfags de truita irisada (Oncorhynchus mykiss). Així mateix, els liposomes van poder modular in vitro l'expressió de diversos gens relacionats amb la immunitat. També s'ha desenvolupat un mètode per a la captació d'imatges in vivo dels liposomes nanomètrics en adults de peix zebra. Això ens va permetre seguir la dinàmica i els teixits diana dels liposomes administrats tant per injecció intraperitoneal com per immersió. Els resultats dels estudis de biodistribució van demostrar que els liposomes s'acumulaven principalment a la melsa del peix zebra i en cèl·lules del sistema immunitari com ara macròfags de truita irisada. D'altra banda, hem demostrat que aquests liposomes, administrats mitjançant injecció intraperitoneal i immersió, podrien protegir de manera efectiva el peix zebra tant d'una infecció bacteriana (Pseudomonas aeruginosa PAO1) com viral (virèmia primaveral de carpa). En conclusió, els resultats suggereixen que l'estimulació del sistema immunitari innat amb liposomes que encapsulen un lipopolisacàrid bacterià i l'anàleg sintètic de dsRNA viral, poli (I:C), podria ser una bona estratègia per aconseguir la protecció contra infeccions bacterianes i virals, i que per tant, es podria utilitzar potencialment com una eina no específica per a la prevenció d'infeccions en peix.
The innate immune system is based on the non-specific recognition of conserved elements of the pathogenic metabolism. This recognition is primarily mediated by germ line encoded pattern-recognition receptors (PRRs), present in specialized cells of the innate immune system, that are able to recognize conserved molecular patterns associated to pathogens (PAMPs). This recognition will trigger different signaling pathways that will induce the transcription of pro-inflammatory cytokines and result in local inflammation. Therefore, the innate immune system can be modulated by administration of these PAMPs, simulating a natural pathogen–immune system encounter. The main hypothesis of this study was that, by encapsulation in the same nanoscaled delivery system, of several PAMPs, also called immunostimulants, we could improve their administration to different fish species. Also, that this delivery system would interact with the cells of the immune system generating its non-specific activation and improving the immune response against different infectious diseases. In this context, a novel immunostimulant delivery nanosystem based on liposomes encapsulating a bacterial lipopolysaccharide from Escherichia coli, and a synthetic analog of viral dsRNA, poly (I:C), has been developed. Our data shows that, these biocompatible liposomes were able to be endocytosed in vitro by zebrafish (Danio rerio) hepatocytes and rainbow trout (Oncorhynchus mykiss) macrophages as well as to regulate the expression of immune related genes. We have also developed a method for in vivo imaging of nano-sized liposomes in adult zebrafish, which allowed us to follow the dynamics and the target tissues of the liposomes administered either by intraperitoneal injection or immersion. The biodistribution results showed that the delivery system accumulated mainly in the spleen of zebrafish and in immune relevant cells, such as macrophages, from rainbow trout. Moreover, we showed that these liposomes, administrated by intraperitoneal injection and immersion, could effectively protect zebrafish from bacterial (Pseudomonas aeruginosa PAO1) and viral (spring viraemia of carp virus) infections. In conclusion, these findings suggest that the stimulation of the innate immune system with liposomes encapsulating a bacterial lipopolysaccharide and the synthetic analog of viral dsRNA, poly (I:C), could be a good strategy to achieve protection against bacterial and viral infections therefore potentially working as a non-specific prevention tool in fish.

La presente tesis doctoral se encuentra integrada dentro del estudio a gran escala de la estructura-función de las ribonucleasas antimicrobianas humanas. Estas proteínas catiónicas y de bajo peso molecular son secretadas por la mayoría de los organismos vertebrados agrupándose dentro la superfamilia de la ribonucleasa A, una de las enzimas mejor caracterizadas del siglo XX. De interés remarcable podríamos considerar su amplio abanico de propiedades biológicas, teniendo en cuenta su diverso historial de propiedades biológicas no catalíticas, convirtiéndolas en un buen modelo de proteínas multifunción. Junto a su principal característica como enzima catalizador de ácidos ribonucleicos, es importante destacar también otro tipo de propiedades biológicas no menos esenciales, como su actividad antimicrobiana, que comparten miembros distantes de la familia sugiriendo una función ancestral en el sistema inmune. Además, se ha visto que la expresión de algunas RNasas humanas puede ser inducida en procesos infecciosos. En particular, las RNasas estudiadas en este trabajo, las RNasas humanas 3, 6 y 7, se expresan principalmente en eosinófilos, monocitos y células epiteliales, respectivamente. Estas proteínas muestran una alta cationicidad debido a su alta proporción de residuos básicos y una notable actividad antimicrobiana frente a una amplia gama de patógenos humanos. Nuestro grupo de investigación posee una larga trayectoria en el estudio del mecanismo de acción de las ribonucleasas humanas y el trabajo teórico-experimental que se presenta en esta tesis ha contribuido a consolidar el actual proyecto de investigación. Los principales avances llevados a cabo por la presente tesis doctoral se enumeran a continuación: - La caracterización del mecanismo antimicrobiano de la ribonucleasa 6, evaluando sus propiedades microbicidas frente a patógenos y modelos de membrana. Concretamente se ha revelado su actividad aglutinadora además de demostrarse que su actividad antimicrobiana está localizada básicamente en su extremo Nterminal. - La resolución de la primera estructura tridimensional de la ribonucleasa 6, obtenida a 1.72 Å, que ha permitido asentar las bases estructurales para futuros estudios funcionales. Análisis complementarios sobre su caracterización cinética y predicción de complejos con diferentes ligandos han revelado sitios de unión y de catálisis que posteriormente han sido confirmados mediante mutagénesis dirigida. - El estudio de la efectiva actividad antipatogena a nivel intracelular que presentan las ribonucleasas 3,6 y 7 asi como sus péptidos derivados N-terminales frente a micobacterias en un modelo de macrófagos infectados. - La expansión del conocimiento sobre las bases antipatogenas de diferentes péptidos y proteínas antimicrobianas que participan en la erradicación de las infecciones por micobacterias, asi como las terapias derivadas. - La caracterización del mecanismo antimicrobiano de los 8 peptidos N-terminales derivados de las ribonucleasas frente a Candida albicans, como modelo de patógeno eucariota Como conclusión, los resultados presentados en esta tesis contribuyen a profundizar en la comprensión de las bases moleculares del papel que desempeñan algunas ribonucleasas en el sistema inmune y expandir el proyecto al diseño de agentes terapéuticos basados en péptidos antimicrobianos con el objetivo de erradicar enfermedades infecciosas causadas por patógenos resistentes.
The present doctoral thesis is integrated into the large-scale study of the structure and function of human antimicrobial ribonucleases. These cationic and low molecular weight proteins are grouped into the ribonuclease A superfamily, considered one of the best characterized enzymes of the twentieth century. The RNase A superfamily is specific for vertebrates and has attracted remarkable interest due to the diversity of displayed biological properties; and represents an excellent example of a multifunctional protein´s family. Together with the main enzymatic activity we must highlight other biological properties such as the angiogenic, immunomodulatory and antimicrobial activities. The reported antimicrobial activity of distantly related family members in early vertebrates suggests that the family arouse with an ancestral function in host defence. Besides, the expression of several human RNases has been reported to be induced by infection. In particular, the RNases studied in this work, the human RNases 3, 6 and 7, are mainly expressed in eosinophils, monocytes and epithelial cells respectively. These proteins show a high cationicity due to the high proportion of basic residues and a remarkable antimicrobial activity against a wide range of human pathogens. Our research group has a consolidated experience in the study of the mechanism of action of human ribonucleases and the experimental work presented in this thesis is contributing to this overall research project. The main results achieved by the present PhD study are outlined below: - The characterization of the antimicrobial mechanism of RNase 6, both in bacteria cell cultures and using membrane models. Results highlight that the antimicrobial and cell agglutinating activities are mainly located at the N-terminus. - The resolution of the first three-dimensional structure of ribonuclease 6, obtained at 1.72 Å, which has set the structural basis for future functional studies. The kinetic characterization of RNase 6 mutant variants and the prediction of protein- substrate complexes have identified the enzyme nucleotide binding sites. - The study of the intracellular activity of ribonucleases 3, 6 and 7 and their derived Nterminal peptides against intracellular resident mycobacteria using a macrophage infected model. - The analysis of the anti-pathogenic mechanism of action of human antimicrobial proteins and peptides in mycobacterial infections and their applied therapies. - The comparative characterization of the antimicrobial mechanism of action of human RNases and their N-terminal derived peptides towards Candida albicans, as an eukaryote pathogen model. The results presented in this thesis will contribute to the understanding of the role of human RNases in the immune system and provide the structure- function basis to expand the initial project into the design of novel peptide mimetic therapeutics agents towards the eradication of resistant infectious diseases.

La enfermedad cardiovascular sigue siendo la principal causa de morbilidad y mortalidad en todo el mundo. La inmensa mayoría de los eventos cardiovasculares incluyendo los síndromes coronarios y accidente cerebrovascular son la causa por la rotura o erosión de las placas de ateroma en la pared arterial. La aterosclerosis es una enfermedad compleja dinámica caracterizada por la infiltración de lípidos en la pared arterial y una respuesta inflamación crónica por las células del sistema inmune innato, principalmente monocitos y macrófagos. Los altos niveles circulantes de lipoproteínas de baja densidad (LDL) son uno de los principales factores de riesgo cardiovasculares en sujetos con hipercolesterolemia familiar (FH), causada principalmente por mutaciones en LDLR afectando el metabolismo de los lípidos, que están en alto riesgo de eventos cardiovasculares prematuros. Actualmente está bien establecida la relevancia de la inflamación en el desarrollo de las placas ateroscleróticas, sin embargo, los efectos de la LDL sobre el fenotipo y la función de las células de la inmunidad innata, como monocitos y macrófagos, no son completamente entendidos. Además, la respuesta inflamatoria en los macrófagos de pacientes con HF (de SAFEHEART cohorte) y su asociación con la exposición de por vida a niveles altos de LDLc no se ha estudiado. En esta tesis, nosotros investigamos el efecto de los niveles aterogénicas LDL sobre los monocitos humanos y se demostró que la LDL facilita la diferenciación de los monocitos en macrófagos a través de un proceso que implica una expresión potenciada de moléculas de adhesión celular y la regulación disminuida de efectores de apoptosis que regulan la anoikis en el estadio temprano del macrófago. Esto sugiere un papel relevante de la LDL en la relación entre los lípidos, la inmunidad innata y la aterosclerosis. En pacientes FH, hemos demostrado que LRP5, receptor activo de internalización de lípidos, está incrementado en las células de la inmunidad innata que tiene una baja expresión del LDLR pero que conservan la función de internalización de LDL. La expresión reducida de CD163 en estos macrófagos FH también sugiere menos ateroprotección. También muestran que los macrófagos derivados de monocitos de pacientes con FH tratados de acuerdo a las guías tienen un fenotipo pro-inflamatorio sostenido, que se caracteriza por un aumento de la expresión de receptores de quimioquinas CCR3, CCR4, CXCR1 y una baja regulación de miR-505-3p, moléculas relacionadas con respuesta inflamatoria. El efecto es dependiente de la edad del paciente, lo que sugiere un patrón inflamatorio crónica en las células de la inmunidad innata que está relacionado con los cambios epigenéticas inducidos por la exposición de por vida a niveles altos de LDL.
Cardiovascular disease remains a leading cause of morbidity and mortality worldwide. The vast majority of cardiovascular events including coronary syndromes and stroke are cause by the rupture or erosion of atherosclerotic plaques in the arterial wall. Atherosclerosis is a complex dynamic disease characterized by lipid infiltration in arterial wall and a chronic inflammation response by cells of the innate immune system, mainly monocytes and macrophages. High levels of circulating low-density lipoproteins (LDL) are one of the major cardiovascular risk factors and subjects with Familial Hypercholesterolemia (FH), mainly caused by LDLR mutations affecting lipid metabolism, are at high risk of premature cardiovascular events. Currently it is well established the relevance of inflammation in the development of the atherosclerotic plaques, however, the effects of LDL on the phenotype and function of cells of the innate immunity, as monocyte and macrophage, are not fully understood. Further, the inflammatory response in macrophages of FH patients (from SAFEHEART Cohort) and its association with lifetime exposure to high LDLc levels has not been studied. In this thesis, we investigated the effect of atherogenic levels LDL on human monocyte and demonstrated that LDL facilitate monocyte differentiation into macrophage through a process that involves enhanced expression of cell-adhesion molecules and downregulation of apoptosis- effectors regulating anoikis in the early stage macrophages. This suggests a relevant role of LDL in the link between lipids, innate immunity and atherosclerosis. In patients FH, we demonstrated that LRP5, active lipid-internalizing receptor, is upregulated in innate immunity cells that have downregulated LDLR but retain the LDL internalization function. The reduced CD163 expression in these FH-macrophages also suggests less atheroprotection. We also show that monocyte-derived macrophages from FH patients treated according to guidelines have a sustained pro-inflammatory phenotype, characterized by an increased expression of chemokine receptors CCR3, CCR4, CXCR1 and a down-regulation of miR-505-3p, molecules related with inflammatory response. The effect is dependent on the age of the patient, suggesting a chronic inflammatory pattern in innate immunity cells that is related to epigenetic changes induced by lifetime exposure to high levels of LDL.

Les miopaties inflamatòries són un grup de malalties neuromusculars molt heterogeni que es caracteritzen clínicament per la presència de debilitat muscular que pot arribar a ser invalidant. Aquest grup de malalties inclou la dermatomiositis (DM), la polimiositis, la miopatia per cossos d’inclusió i recentment s’hi ha inclòs la miopatia necrotitzant autoimmune. Per arribar a un diagnòstic precís és necessari l’estudi de la biòpsia muscular entre d’altres paràmetres clínics. La biòpsia muscular d’aquest grup de pacients es caracteritza per la presència d’infiltrats inflamatoris que fenotípicament i quantitativament varia a cada entitat. A més, s’observa la sobreexpressió del complexe major d’histocompatibilitat de classe I (MHC-I) a les fibres musculars havent-li atribuït un paper patogènic. Fins ara no hi ha evidències de l’etiologia de la DM, havent-hi dues hipòtesis principals: la primera considera que la malaltia comença amb un atac autoimmune contra un antigen desconegut de l’endoteli muscular provocant la destrucció dels capil•lars i una hipòxia tissular. De fet a les biòpsies de pacients amb DM és característica una disminució dels capil·lars que, com a conseqüència, es pensa que provoca la típica atròfia perifascicular. La segona atribueix un paper principal a l’ interferó (IFN) ja que múltiples gens induïts per IFN s’han trobat sobreregulats a la DM. L’origen de l’IFN s’ha atribuït als infiltrats inflamatoris. Per estudiar específicament la contribució del múscul a la patogènia d’aquestes malalties, evitant l’efecte confusor dels infiltrats inflamatoris, vam aïllar fibres musculars patològiques mitjançant microdissecció làser a partir de biòpsies musculars d’aquests pacients. L’anàlisi transcriptòmic de les fibres aïllades va demostrar una contribució específicament a la DM de la immunitat innata, de l’IFN de tipus I (IFN-I) i de la hipòxia. En particular, es va observar una sobreregulació de RIG-I a la DM, un receptor de la immunitat innata induïble per IFN i que fisiològicament reconeix àcids nucleics vírics per iniciar una resposta antivírica a través de IFN-I. Els estudis de immunofluorescència van demostrar que la sobreexpressió de RIG-I colocalitzava amb fibres MHC-I positives a la DM. Els estudis in vitro utilitzant miotubs humans va demostrar la capacitat de RIG-I d’induïr una resposta de IFNβ –un subtipus de IFN-I- i com a conseqüència es sobreexpressava MHC-I i altre vegada RIG-I suggerint un mecanisme inflamatori auto-sustentat a la DM. A partir d’aquest primer resultat l’estudi va evolucionar al paper de la hipòxia en relació a la immunitat innata. La hipòxia està modulada principalment per un factor de transcripció clau anomenat HIF1α que indueix l’expressió de gens que continguin al seu promotor unes seqüències específiques. Els estudis in silico i in vitro van demostrar que RIG-I és un gen induït per HIF1α. Els resultats experimentals conclouen que la hipòxia és un fenomen primerenc en la seqüència d’esdeveniment fisiopatogènics en la DM ja que la sobreexpressió de RIG-I era capaç d’induir la seva activació i per tant, de promoure l’expressió d’IFN-I i en canvi, l’expressió de HIF1α no es modificava per l’estimulació amb interferons. Finalment es va avaluar la utilitat de RIG-I com a biomarcador histològic per la DM. A partir de 115 biòpsies musculars incloent malalties neuromusculars que podien confondre’s clinicament o histologicament amb la DM, vam demostrar que RIG-I és un biomarcador mes sensible que la atrofia perifascicular per la DM (50% a la DM vs 11% a la resta). De fet, en els pacients amb DM on a la biòpsia l’atròfia perifascicular no era evident, un 32% dels pacients eren RIG-I positius. Analitzant la reproductibilitat de la interpretació de l’atròfia perifascicular i RIG-I, vam trobar que RIG-I era un biomarcador més fàcilment interpretable que la atrofia perifascicular. Com a conclusió, aquesta tesi unifica les dues hipòtesis patogèniques actuals sobre la DM on la hipòxia es el primer esdeveniment que a través de RIG-I explica la signatura de IFN-I.
Inflammatory myopathies are an heterogeneous group of neuromuscular diseases that are clinically characterized by the presence of muscle weakness that leads to disability. This group of diseases includes dermatomyositis (DM), polymyositis, inclusion body myositis and recently, necrotizing autoimmune myopathies have also been included. The study of the muscle biopsy, among other parameters, is necessary to obtain an accurate diagnosis. The muscle biopsy of these patients is characterized by the presence of inflammatory infiltrates that vary phenotypically and quantitatively in each entity. However, overexpression of the major histocompatibility complex class I (MHC-I) is shared by all these entities and it is believed that it has a pathogenic role. Until now, the etiology of DM is a matter of debate but there are two main hypotheses: The first hypothesis considers that the disease starts with an autoimmune attack against unknown antigens in the endothelium leading to the destruction of capillaries promoting, eventually, tissular hypoxia and muscle fiber atrophy. In fact, in the muscle biopsies of DM patients there is a characteristic reduction in the number of capillaries and perifascicular atrophy. The second hypothesis is based on the role of type I interferons (IFN-I). Experiments of gene expression analysis have demonstrated upregulation of multiple IFN-I-induced genes in DM. The origin of this IFN-I has been attributed to perimysial inflammatory infiltrates. To study the pathological mechanisms that occur specifically in the muscle fibers we isolated MHC-I positive muscle fibers using laser-microdissection from the muscle biopsies of these patients. This technology allowed us to exclude inflammatory infiltrates that could interfere with the results. The transcriptomic profile of the isolated fibers demonstrated a significant contribution of innate immunity, IFN-I and hypoxia specifically in DM. In particular, we observed the upregulation of RIG-I in DM, a receptor of the innate immunity that recognizes nucleic acids derived from virus to initiate an antiviral response and promoting IFN-I. Immunofluorescence studies demonstrated that RIG-I is overexpressed in MHC-I-expressing fibers in DM. In vitro studies using human myotubes showed the ability of RIG-I to induce the secretion of IFNβ – an IFN-I subtype – and as a consequence, induced the expression of MHC-I and RIG-I itself, suggesting a self sustained autoimmune mechanism in DM. These results prompted us to evaluate the relation between hypoxia and innate immunity. Hypoxia is mainly modulated by a key transcription factor named HIF1α that induces the expression of genes that contain specific sequences in its promoter. Our studies in silico and in vitro demonstrated that RIG-I is an HIF-inducible gene. From these studies, we concluded that hypoxia is a primary event in the pathogenic sequence of events in DM because the overexpression of RIG-Iwas able to induce its own activation promoting the expression of IFN-I-stimulated genes. In contrast, the expression of HIF1α was not modified by the stimulation of IFNs. Finally we evaluated the utility of RIG-I as a histological biomarker for DM. We studied 115 muscle biopsies using immunohistochemistry including diseases that may be confused clinically or histologically with DM. We demonstrated that RIG-I has a higher sensitivity than perifascicular atrophy for DM diagnosis (50% in DM vs 11 % in non-DM). Interestingly, 32% of patients with DM in whom perifascicular atrophy was not evident, showed expression of RIG-I at the perifascicular areas. We analyzed the reproducibility of perifascicular atrophy and RIG-I staining and we found that RIG-I has a higher reproducibility and therefore constitutes a more easily interpretable biomarker than perifascicular atrophy. We conclude that this thesis unifies both current hypotheses on DM pathogenesis and shows that hypoxia contributes to the pathology of DM by activating the RIG-I signaling pathway and consequently inducing IFN-I expression.

Introducció. L’epidermòlisi ampul·lar adquirida (EBA) és una malaltia crónica autoinmune caracteritzada per la presència d’anticossos circulants i amb afinitat per la col·làgena VII (CVII), la qual es troba a la membrana basal (MB) dermo-epidèrmica. Es manifesta clinicament per la presència d’ampul·les tenses i erosions que afecten tant a membranes mucoses com a pell. Components del sistema de la immunitat innata han demostrat estar involucrats en el dany tissular, concretament són el sistema del complement, els receptors dels anticossos i els neutròfils. La proteïna Rac2 és una GTPasa que participa en la quimotaxi dels neutròfils i en la generació d’espècies reactives d’oxígen mitjançant l’activació de l’enzim NADPH oxidasa. La insuficiència pulmonar aguda és una síndrome induïda per una resposta immune a partir del reclutament de neutròfils, afavorint el dany tissular i causant el distrés respiratori agut. Estudis realitzats in vivo sobre ratolins amb Rac2 genoanul.lat (Rac2 GA) o mitjançant el bloqueig farmacològic amb un inhibidor de Rac2 (NSC23766) han demostrat disminuir el dany tisular pulmonar. Objectius. Estudi in vivo de la funció de la proteïna Rac2 en el desenvolupament del dany tissular mitjançant un model experimental de la EBA. Materials i mètodes. La malaltia de l’EBA es va induïr mitjançant la transferència passiva d’anticossos contra un fragment murí de la CVII (mCVIICr). IgG anti-mCVIICr es va obtenir mitjançant la immunització de conills. El sérum de conills immunitzats i no immunizats es va purificar i concentrar per a èsser injectat als ratolins. Es van realitzar dos grups d’experiments amb ratolins Rac2 GA (total de n=10) i en ratolins de soca salvatge (SS) com a controls positius (n=3). En el mateix moment, ratolins Rac2 GA (n=4) van ser injectats amb IgG inespecífica de conill com a controls negatius. Es va realitzar un tercer grup d’experiments on ratolins de SS van ser pre-tractats amb dexametasona (n=3), dapsona (n=5) i NSC23766 (n=3), un inhibidor soluble experimental de Rac2. Es va realizar valoració clínica, biòpsies de pell, immunoflorescència directa (IFD) i assaig d’immunoadsorció lligat a enzim (ELISA). Resultats. El ratolins SS injectats amb IgG anti-mCVIICr van desenvolupar la malaltia de l’EBA, amb una severitat significativament augmentada amb elevades dosis d’anticossos (p=.05). En canvi, cap ratolí Rac2 GA injectat amb IgG anti-mCVIICr va desenvolupar malaltia, encara que hi havia depòsit d’IgG i C3 a la MB, i es van detectar IgG anti-mCVIICr circulants. Els ratolins SS pre-tractats amb NSC23766 dexametasona, i dapsona van desenvolupar EBA amb una tendència inicial de menor severitat de la malatia, pero sense diferències estadísticament significatives durant el període d’observació en comparació al grup de controls positius (p=0.8). Conclusions. Donat que l’EBA no es va desenvolupar en ratolins Rac2 GA després de la transferència passiva d’IgG anti-mCVIICr, suggereix una evident implicació de la proteïna Rac2 en la patogènia de l’EBA. Aleshores, proposem la proteïna de Rac2 com a diana terapéutica potencial per poder disenyar teràpies específiques per l’EBA i altres malaties mediades per IgG. No obstant, no hi ha encara disponible un inhibidor eficaç del Rac2 per èsser utilitzat com a tractament.
Background: Epidermolysis bullosa acquisita (EBA) is a chronic autoimmune disorder characterized by the presence of circulating and tissue-bound autoantibodies against collagen VII (CVII), a protein localized at the dermal-epidermal basement membrane zone (BMZ). It is clinically manifested with tense blisters and erosions involving mucocutaneous tissues. Parts of the innate immune system have been demonstrated to be involved in tissue damage, particularly components of the complement system, antibodies’ receptors and neutrophils. Specifically, Rac2 protein is a GTPase involved in chemotaxis of neutrophils and reactive oxygen species synthesis through NADPH oxidase activation. Acute lung injury (ALI) is a syndrome induced by the immune response where the recruitment of neutrophils favors tissue damage evolving into an acute respiratory distress syndrome. In vivo studies performed in Rac2 knock-out (KO) mice and the pharmacological inhibition of Rac2 (NSC23766) in ALI have demonstrated a decrease in lung tissue injury. Objectives: To investigate the role of Rac2 protein in tissue injury developed in an in vivo experimental mouse model of EBA. Materials and Methods: EBA phenotype was induced by passive transfer of antibodies against a fragment of murine CVII (mCVIICr). Anti-mCVIICr IgG was obtained after rabbit immunization. IgG from immunized and non-immunized rabbits (used as negative controls) were purified and concentrated to inject mice. Two sets of experiments were performed with Rac2 KO mice (total n=10) and wild type mice as positive controls (n=3) by injecting rabbit anti-mCVIICr IgG. At the same time, a set of Rac2 KO mice (n=4) were injected with unspecific rabbit IgG as negative controls. A third set of experiments consisted in injecting rabbit anti-mCVIICr IgG in mice pre-treated with dexamethasone (n=3), dapsone (n=5) and NSC23766 (n=3), an experimental soluble inhibitor of Rac2. Clinical assessments, histology of skin biopsies, direct immunofluorescence (DIF), and enzyme-linked immunosorbent assays (ELISA) were performed. Results: WT mice injected with anti-mCVIICr IgG successfully developed the EBA phenotype, with significantly increased disease severity with higher doses of IgG (p=.05). In contrast, none of the Rac2 KO mice injected with anti-mCVIICr IgG developed the EBA phenotype, even though IgG and C3 were deposited at the BMZ, and circulating anti-mCVIICr IgG was detected by ELISA. Pre-treated WT mice with NSC23766, dexamethasone and dapsone developed disease initially with a trend of milder disease severity, yet we found no statistically difference in body surface area involvement at the end of the observation period when compared with positive controls (p=0.08). Conclusions: The fact that EBA lesions were not developed in Rac2 KO mice after the passive transfer of anti-mCVIICr IgG clearly suggests the involvement of Rac2 protein in EBA pathogenesis. Therefore, we propose Rac2 as a potential target for designing therapies specific for EBA and other IgG-mediated disease. However, an effective Rac2 inhibitor to be used as therapy is not yet available.

Macrophages are exquisite sensors of tissue homeostasis that can rapidly switch between pro- and anti-inflammatory or regulatory modes to respond to perturbations in the microenvironment. This functional plasticity involves a precise orchestration of gene expression patterns whose transcriptional regulators have not been fully characterised. Our project aimed at characterising the contribution of the transcription factor NFAT5 to macrophage functions in different polarisation settings. For that we generated classically and alternatively-polarised WT and NFAT5-deficient macrophages and studied the expression of typical polarisation markers by analysing their mRNA levels by RT-qPCR and also at protein levels by flow cytometry, ELISA and Western Blot. We also checked macrophage functions associated with a pro-inflammatory profile, such as bactericidal capacity and the ability to promote Th1 polarisation over Th2 responses. Finally, we performed in vivo assays to determine the pro-tumoural phenotype of NFAT5-deficient macrophages against syngeneic Lewis lung carcinoma and ID8 ovarian carcinoma cells. Overall, our revealed its predominant role in promoting pro-inflammatory macrophage functions ranging from facilitating Th1 responses to restraining tumour progression.
Els macròfags són cèl·lules del sistema immunitari innat que es troben en tots els òrgans. Als teixits, els macròfags actuen per mantenir la homeòstasi canviant ràpidament entre estats inflamatoris i antiinflamatoris per respondre eficientment a les pertorbacions que succeeixen en el seu entorn. Aquesta plasticitat funcional requereix l’expressió coordinada de gens que estan regulats per factors que encara no han estat completament caracteritzats. En aquest treball es volia estudiar la contribució del factor de transcripció NFAT5 com a regulador de les funcions dels macròfags en diferents condicions de polarització. Per això, varem generar macròfags clàssicament activats (M1) i activats de manera alternativa (M2), WT i deficients en NFAT5, i varem analitzar l’expressió de marcadors M1 i M2 a nivell de mRNA i a nivell de proteïna per tècniques de citometria de fluxe, de ELISA i de Western Blot. També es van estudiar funcions inflamatòries dels macròfags, com ara la capacitat per a eliminar bacteris intracel·lulars o la inducció de respostes Th1 en limfòcits T CD4. Finalment es varen dur a terme assajos in vivo amb tumors singènics de pulmó (Lewis lung carcinoma) i el carcinoma d’ovari (ID8) per a determinar si els macròfags deficients en NFAT5 tenen una menor capacitat antitumoral. En resum, els nostres resultats indiquen que NFAT5 contribueix preferentment en la inducció de funcions inflamatòries, com ara la inducció de respostes Th1 i la restricció de la progressió tumoral.

Ebolaviruses (EBOVs) are among the most virulent and deadly pathogens ever known, causing fulminant haemorrhagic fevers in humans and non-human primates. The 2014 outbreak of Ebola virus disease (EVD) in West Africa has claimed more lives than all previous EVD outbreaks combined. The EBOV high mortality rates have been related to the virus-induced impairment of the host innate immunity reaction due to two virus-coded proteins, VP24 and VP35. EBOV VP35 is a multifunctional protein, it is essential for viral replication as a component of the viral RNA polymerase and it also participates in nucleocapsid assembly. Early during EBOV infection, alpha-beta interferon (IFN-α/β) production would be triggered upon recognition of viral dsRNA products by cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs). However, this recognition is efficiently prevented by the double-stranded RNA (dsRNA) binding activity of the EBOV VP35 protein, which hides RLRs binding sites on the dsRNA phosphate backbone as well the 5’-triphosphate (5’-ppp) dsRNA ends to RIG-I recognition. In addition to dsRNA binding and sequestration, EBOV VP35 inhibits IFN-α/β production preventing the activation of the IFN regulatory factor 3 (IRF-3) by direct interaction with cellular proteins. Previous studies demonstrated that single amino acid changes in the VP35 dsRNA binding domain reduce EBOV virulence, indicating that VP35 is an attractive target for antiviral drugs development. Within this context, here we report the establishment of a novel method to characterize the EBOV VP35 inhibitory function of the dsRNA-dependent RIG-I-mediated IFN-β signaling pathway in a BLS2 cell culture setting. In such system, a plasmid containing the promoter region of IFN-β gene linked with a luciferase reporter gene was transfected, together with a EBOV VP35 mammalian expression plasmid, into the IFN-sensitive A549 cell line, and the IFN-induction was stimulated through dsRNA transfection. Through alanine scanning mutational studies with biochemical, cellular and computational methods we highlighted the importance of some VP35 residues involved in dsRNA end-capping binding, such as R312, K282 and R322, that may serve as target for the development of small-molecule inhibitors against EBOV. Furthermore, we identified a synthetic compound that increased IFN-induction only under antiviral response stimulation and subverted VP35 inhibition, proving to be very attractive for the development of an antiviral drug. In conclusion, our results provide the establishment of a new assay as a straightforward tool for the screening of antiviral compounds that target i) dsRNA-VP35 or cellular protein-VP35 interaction and ii) dsRNA-dependent RIG-I-mediated IFN signaling pathway, in order to potentiate the IFN response against VP35 inhibition, setting the bases for further drug development.

Intradermal infections with C. albicans are eliminated by neutrophils recruited at the site of infection with an unknown mechanism. To dissect how neutrophils are recruited at the site of infection, we analyzed the involvement of PRR that recognize C. albicans (TLR4, TLR2, Dectin1 and Dectin2) as well as CARD9, activated downstream Dectins, and MyD88 that transduces the signal derived from TLRs. WT, PRR-deficient and CARD9-deficient mice did not show any defect in neutrophil recruitment after C. albicans intradermal injection. Diversely, MyD88-deficient mice do not recruit neutrophils after C. albicans skin infection. Since MyD88 is involved in IL-1 signaling, we tested the role of IL-1β and IL-1α, two vasoactive cytokines, in the initiation of inflammation and neutrophil recruitment. In vitro and in vivo studies revealed that both IL-1β and IL-1α were involved in this process. IL-1α is constitutively expressed in epithelial, endothelial and stromal cells and can be released through proteolytic cleavage or cell death, enhancing the production of CXCL1, a chemokine with neutrophil chemoattractant activity. We confirmed that CXCL1 production in vivo depends on IL-1α release. We concluded that PRRs were not involved in the initiation of the inflammatory process during primary Candida skin exposure. Diversely, the initiation of the inflammatory process during primary infection can be due to the unspecific release of alarmins (like IL-1α) by distressed cells, considered like DAMP, stimulating neutrophils recruitment at the site of infection. To investigate whether DAMPs and PAMPs could have distinct roles in neutrophil recruitment during microbial infections, we have used models of skin infection with different types of pathogens: a fungus, C. albicans, a Gram-positive bacterium Staphylococcus aureus and a Gram-negative, Pseudomonas aeruginosa.
Intradermal infections with C. albicans are eliminated by neutrophils recruited at the site of infection with an unknown mechanism. To dissect how neutrophils are recruited at the site of infection, we analyzed the involvement of PRR that recognize C. albicans (TLR4, TLR2, Dectin1 and Dectin2) as well as CARD9, activated downstream Dectins, and MyD88 that transduces the signal derived from TLRs. WT, PRR-deficient and CARD9-deficient mice did not show any defect in neutrophil recruitment after C. albicans intradermal injection. Diversely, MyD88-deficient mice do not recruit neutrophils after C. albicans skin infection. Since MyD88 is involved in IL-1 signaling, we tested the role of IL-1β and IL-1α, two vasoactive cytokines, in the initiation of inflammation and neutrophil recruitment. In vitro and in vivo studies revealed that both IL-1β and IL-1α were involved in this process. IL-1α is constitutively expressed in epithelial, endothelial and stromal cells and can be released through proteolytic cleavage or cell death, enhancing the production of CXCL1, a chemokine with neutrophil chemoattractant activity. We confirmed that CXCL1 production in vivo depends on IL-1α release. We concluded that PRRs were not involved in the initiation of the inflammatory process during primary Candida skin exposure. Diversely, the initiation of the inflammatory process during primary infection can be due to the unspecific release of alarmins (like IL-1α) by distressed cells, considered like DAMP, stimulating neutrophils recruitment at the site of infection. To investigate whether DAMPs and PAMPs could have distinct roles in neutrophil recruitment during microbial infections, we have used models of skin infection with different types of pathogens: a fungus, C. albicans, a Gram-positive bacterium Staphylococcus aureus and a Gram-negative, Pseudomonas aeruginosa.

Les infeccions bacterianes respiratòries en pacients amb Malaltia Pulmonar Obstructiva Crònica (MPOC) i amb bronquièctasis són una de les principals causes d’empitjorament del pronòstic d’aquestes malalties. Sovint aquestes infeccions causen episodis d’agudització durant les quals la simptomatologia dels pacients s’agreuja, arribant a requerir ingrés hospitalari per controlar la infecció. Es coneix que aquests pacients pateixen una inflamació pulmonar i sistèmica que apareix durant l’estabilitat clínica i s’accentua durant les aguditzacions. A més, es creu que el microambient inflamatori pulmonar d’aquestes malalties afavoreix la infecció per determinats bacteris, com la Pseudomona aeruginosa i l’Hemophilus influenzae, associats amb major severitat. Tot i així, encara es desconeixen els mecanismes immunològics pels quals hi ha pacients que aguditzen freqüentment, anomenats aguditzadors freqüents (AF) i altres pacients que no aguditzen o ho fan de manera poc freqüent (NF). Per aquestes raons, aquesta tesi pretén estudiar els diferents elements de la resposta immune innata local implicats en la defensa de les infeccions bacterianes, en dues malalties respiratòries d’elevat impacte en la salut pública com són la MPOC i les bronquièctasis. El primer objectiu ha estat associar els nivells pulmonars de Fatty-acid binding protein 4 (FABP4) en pacients amb MPOC amb la presència d’infecció respiratòria, severitat de la malaltia i poblacions cel·lulars. El segon objectiu s’ha centrat en estudiar els nivells pulmonars dels pèptids antimicrobians (PAMs) Lactoferrina, Lisozima, LL-37 i Secretory Leukocyte Protease Inhibitor (SLPI) com a possibles marcadors pronòstic de futures aguditzacions en pacients amb bronquièctasis. Per últim, el tercer objectiu ha estat caracteritzar diferents perfils immunològics d’inflamació pulmonar en pacients amb bronquièctasis i associar aquests als paràmetres clínics.
Las infecciones bacterianas respiratorias en pacientes con Enfermedad Pulmonar Obstructiva Crónica (EPOC) y con bronquiectasias son una de las principales causas de empeoramiento del pronóstico de estas enfermedades. A menudo estas infecciones causan episodios de agudización durante las cuales la sintomatología de los pacientes se agravia, llegando a requerir ingreso hospitalario para controlar la infección. Se conoce que estos pacientes sufren una inflamación pulmonar y sistémica que aparece durante la estabilidad clínica y se acentúa durante las agudizaciones. Además, se cree que el microambient inlamatorio pulmonar de estas enfermedades favorece la infección por determinados bacterias, como la Pseudomona aeruginosa y la Hemophilus influenzae, asociados con mayor severidad. Aun así, todavía se desconocen los mecanismos inmunológicos por los cuales hay pacientes que agudizan frecuentemente, denominados aguditzadors frecuentes (AF) y otros pacientes que no agudizan o lo hacen de manera poco frecuente (NF). Por estas razones, esta tesis pretende estudiar los diferentes elementos de la respuesta inmune innata local implicados en la defensa de las infecciones bacterianas, en dos enfermedades respiratorias de elevado impacto en la salud pública como son la MPOC y las bronquiectasias. El primer objetivo ha estado asociar los niveles pulmonares de Fatty-acid binding protein 4 (FABP4) en pacientes con MPOC con la presencia de infección respiratoria, severidad de la enfermedad y poblaciones celulares. El segundo objetivo se ha centrado en estudiar los niveles pulmonares de los péptidos antimicrobianos (Palmos) Lactoferrina, Lisozima, LL-37 y Secretory Leukocyte Protease Inhibitor (SLPI) como posibles marcadores pronóstico de futuras agudizaciones en pacientes con bronquiectasias. Por último, el tercer objetivo ha estado caracterizar diferentes perfiles inmunológicos de inflamación pulmonar en pacientes con bronquiectasias y asociar estos a los parámetros clínicos.
Respiratory bacterial infections in patients with Chronic Obstructive Pulmonary Disease (COPD) and patients with bronchiectasis are one of the main causes of worsening the prognosis. These infections can cause exacerbations, known as episodes of acute worsening of disease symptoms which sometimes require hospitalization to stabilize the infection. These patients suffer from systemic and pulmonary inflammation during clinical stability and are elevated during exacerbations. In addition, the pulmonary inflammatory microenvironment of these diseases is thought to promote infection by certain bacteria, such as Pseudomona aeruginosa and Hemophilus influenzae, which are associated with greater severity. However, the immunological mechanisms by which patients frequently exacerbate, called frequent exacerbators (AF), and other patients who do not exacerbate or infrequently (NF) are still unknown. For these reasons, this thesis aims to study the different elements of the local innate immune response involved in the defense of bacterial infections, in two respiratory diseases with high public health impact such as COPD and bronchiectasis. The first objective was to associate the pulmonary and systemic levels of Fatty-acid binding protein 4 (FABP4) in COPD patients with the presence of respiratory infection, disease severity, and cell populations. The second objective was to study the pulmonary and systemic levels of antimicrobial peptides (AMPs) Lactoferrin, Lysozyme, LL-37 and Secretory Leukocyte Protease Inhibitor (SLPI) as an outcome marker of future exacerbations in patients with bronchiectasis. Finally, the third objective was to characterize different profiles of pulmonary inflammation in patients with bronchiectasis.

Memoria para optar al título Bioquímico
Como parte del sistema inmune innato existe un grupo de receptores de reconocimiento de patrones (PRRs) capaces de detectar aquellos patrones moleculares asociados a patógenos (PAMPs). Entre estos PRRs, destacan los receptores de tipo Toll o TLRs. Dentro de esta familia de receptores, TLR2 reconoce PAMPs derivados de bacterias gram positivas, los que promueven la activación de la vía de señalización intracelular dependiente del adaptador molecular MyD88, que conduce a la activación de IKK y de las MAPKs, las que tienen un impacto fundamental sobre la expresión de citoquinas proinflamatorias. El estudio de los mecanismos fisiológicos de control de esta vía de señalización constituye un área de investigación relevante en inmunología. En este sentido, los glucocorticoides, anti-inflamatorios naturales, ejercen su efecto vía un receptor citoplasmático (GR) mediante procesos de trans-represión y de expresión de proteínas reguladoras. A pesar de esto, se desconoce su efecto sobre las vías de inducción de citoquinas pro-inflamatorias mediadas por TLR2, tal como la vía de p38. Basados en estos antecedentes, el objetivo general de esta memoria de título consistió en determinar la participación de p38 en la producción de TNF-α en células A549, inducida por un agonista de TLR2, Pam3Cys-Ser-Lys4, en presencia o ausencia de dexametasona. Los objetivos específicos se centraron en: 1) determinar la expresión de TNF-α, a nivel de transcrito y proteína, en células activadas con el agonista sintético Pam3Cys-Ser-Lys4, en presencia o ausencia de dexametasona 2) evaluar la fosforilación de la proteína quinasa p38 en células estimuladas con Pam3Cys-Ser-Lys4, en presencia o ausencia de dexametasona y 3) evaluar la contribución de la proteína quinasa p38 sobre la expresión de TNF-α en células estimuladas con Pam3Cys-Ser-Lys4, en presencia o ausencia de dexametasona. Los resultados obtenidos indican que la activación de TLR2 por Pam3Cys-Ser-Lys4 induce un aumento en la expresión de TNF-α, el que no es reprimido por dexametasona. Se observó una activación de la vía p38 inducida por Pam3Cys-Ser-Lys4, que está involucrada en la expresión de TNF-α. Por otro lado, constatamos que dexametasona ejerce su efecto anti-inflamatorio clásico mediante la expresión de moléculas reguladoras de la vía de p38, como la fosfatasa MKP-1. Sin embargo, no podemos descartar un efecto independiente de la expresión de genes regulados por el glucocorticoide. Además, observamos que dexametasona no actuaría en los eventos tempranos de señalización de la vía de TLR2, como lo constatamos con la fosforilación de p38. Estos datos demostrarían un mecanismo a través del cual los glucocorticoides regulan procesos inflamatorios inducidos por TLR2, con un impacto relevante en el desarrollo de la inmunidad innata y la inflamación
As part of innate immune system, it exists a group of pattern-recognition receptors (PRRs) capable of detecting those molecular pattern associated to pathogens (pathogenassociated molecular patterns; PAMPs). Among these PRRs, Toll-like receptors (TLRs) stand out. Inside this receptor family, TLR2 recognizes PAMPs derived from gram positive bacteria, thus promoting activation of MyD88-dependent intracellular pathway. It leads to IKK and MAPKs activation, which have a fundamental impact on the expression of proinflammatory cytokines. The study of the physiological control mechanisms of this pathway constitutes an area of relevant research in immunology. In this sense, glucocorticoids, natural anti-inflammatory molecules, exert its effect by a cytoplasmatic receptor (GR) by trans-repression mechanism and regulatory proteins expression. In spite of this, the effect of glucocorticoid on the TLR2-mediated pro-inflammatory cytokine pathway induction, specially on p38, is unknown. Based on these precedents, the general aim of this study was determining the role of p38 in TNF-α production in A549 cells induced with Pam3Cys-Ser-Lys4, a TLR2 agonist, in presence or absence of dexametasona. The specific objectives were centered to: 1) determine TNF-α expression, at messenger and protein level; 2) evaluate the p38 protein kinase phosphorylation and 3) evaluate the contribution of p38 protein kinase on TNF-α expression under the stimulation with Pam3Cys-Ser-Lys4, in presence or absence of dexamethasone. The results indicate that the activation of TLR2 by Pam3Cys-Ser-Lys4 induces an increase in TNF-α expression, which is not suppressed for dexamethasone. It also was observed an activation of p38 pathway induced by Pam3Cys-Ser-Lys4, that was involved in TNF-α expression. On the other hand, that dexametasona exert its classic anti-inflammatory effect by means of expression of regulatory molecules of the p38 pathway, as phosphatase MKP-1. Nevertheless, we can’t reject an independent effect from the expression of glucocorticoid regulated genes. Besides, dexamethasona wouldn’t work on early events of TLR2 signalling pathway, as we observed for p38 phosphorylation. This data would show a mechanism by which glucocorticoids regulates the TLR2- induced inflammatory processes, with a relevant impact in the processes involved in the development of the innate immunity and inflammation

Il lavoro descritto in questa tesi si focalizza sullo studio delle interazioni tra i vettori virali e le cellule ematopoietiche staminali umane nel contesto della terapia genica, studiando sia i meccanismi di immunità innata attraverso cui i vettori virali vengono riconosciuti dalla cellula ospite, sia il ruolo di fattori di restrizione antivirali che ostacolano la trasduzione nelle cellule ematopoietiche staminali.
The work described in this thesis focuses on the study of vector-host interactions in the context of human hematopoietic stem cell gene therapy. We study the innate immune sensing mechanisms of viral vectors and the role of host antiviral restriction factors in the poor permissivity of hematopoietic stem cells to viral transduction.

Las defensinas son moléculas efectoras de la inmunidad innata con un amplio espectro antimicrobiano e importantes efectos inmunomoduladores. Dentro de su amplio espectro se incluye la inactivación del VIH, actuando directamente sobre el virus y también sobre las células diana. Nosotros hipotetizamos que las células dendríticas (DC) producirían alfa defensinas 1-3, lo que les permitirá inactivar al virus antes o después de su captura y hacer una presentación más eficiente a la célula CD4 sin producir su infección. Esta hipótesis predice que existirá una variabilidad interindividual en la producción de estas defensinas, lo que podrá constituir un factor de resistencia/susceptibilidad frente a la infección por VIH y su evolución a SIDA.
En nuestros resultados encontramos que las DC inmaduras producen y secretan alfa-defensina 1-3 y que esta producción disminuye tras la maduración de las DC con distintos agentes madurativos. Además, las citocinas pro-inflamatorias y la infección viral aumentan la producción de defensinas, mientras que el tratamiento de las células con estrógeno la disminuye. Por otro lado, las alfa-defensinas 1-3 son capaces de modular la maduración y diferenciación de las DC e inducen la secreción de IL-8 de forma dosis-dependiente. Por último, el análisis de los pacientes infectados por VIH reveló que las DC de estos pacientes producen mayores niveles de alfa-defensinas 1-3 que los controles sanos. Además, dentro del grupo de infectados, aquellos que controlan espontáneamente la infección son los mayores productores. Se encontró asimismo una correlación positiva con los niveles de células T CD4 y una asociación entre mayores niveles de alfa-defensinas 1-3 secretadas por DC y una menor progresión de la enfermedad.
En conclusión, una mayor producción de alfa-defensinas 1-3 por las DC de pacientes infectados por VIH será un factor protector frente a la evolución de la enfermedad.
Defensins are natural peptides with potent anti-HIV activity. In humans two subfamilies exist,
beta- and alpha-defensins. Alpha-Defensins 1-3 are mainly secreted by neutrophils, although other leukocytes also produce them. Besides their direct antimicrobial effect, alpha-defensins1-3 also exert immunomodulory activities, chemoattracting leukocytes and inducing cytokines and chemokines production.
We hypothesis that dendritic cells (DC) would produce alpha-defensins 1-3. The greater the production of alpha-defensins 1-3 by DC the greater the capacity to damage the HIV prior to and after its internalization and to perform a more efficient presentation to CD4+ T cells without infection. This hypothesis predicts a differential inter-individual expression of ¦Á-defensins1-3 by dendritic cells, which could be another resistance/susceptibility factor to HIV infection and AIDS progression.
In our results we found that immature DC produce and secrete alpha-defensins 1-3 and this production was down-regulated after DC maturation with different maturative agents. Defensins production was up-regulated by pro-inflammatory cytokines and viral infection and strongly down-regulated by estrogen. In addition, alpha-defensins 1-3 were able to interfere with the maturation and differentiation of DC with opposite dose dependent effects. Moreover, alpha-defensins 1-3 induce a dose dependent IL-8 secretion by DC. The analysis of HIV-infected patients revealed that immature DC from HIV-infected patients secreted significantly higher levels of alpha-defensins 1-3 than healthy controls. Within the HIV-infected group, this production was statistically increased in untrated HIV-infected patients that spontaneously control the infection while in untreated viremic and treated HIV-infected patients the production was not significantly increased. The levels ofalpha-defensins 1-3 secreted by immature DC positively correlated with CD4 T cell counts in the controllers group, but not in viremic non-controllers and treated patients. HIV-infected patients with higher alpha-defensins 1-3 secretion by immature DC showed a slower disease progression, measured as no decrease in the number of CD4+ T- cells below 350 cell/mm3, lower increase in plasmatic viral load and no initiation of treatment over time.
In conclusion, increased production of alpha-defensins 1-3 by dendritic cells in HIV-infected individuals is associated with a slower disease progression.

La Sclerosi Multipla (MS) è una patologia autoimmune cronica che colpisce il sistema nervoso centrale (SNC) determinando demielinizzazione. I principali fenotipi patologici sono Recidivante-Remittente (RRMS) e Primariamente Progressiva (PPMS) – quest’ultima è la forme più grave. Ajami B. et al. hanno dimostrato che nel modello murino affetto da Encefalomielite (EAE) - il modello animale di MS- vi è una forte correlazione tra la presenza di monociti a livello del SNC ed il peggioramento dei sintomi motori tipici della malattia. Partendo da queste evidenze , per il nostro studio sono stati prelevati campioni di sangue da HC, RRMS e PPMS -tutte femmine- per poi isolare i monociti CD14+. Su tali campioni sono stati condotti esperimenti microarray e successivamente qRT-PCR. L’analisi bioinformatica ha evidenziato che gli RRMS si distribuivano formando due gruppi: un gruppo (RR1) si distribuiva similmente agli HC, l’altro (RR2) si distribuiva similmente ai PPMS. Dalla Gene Ontology è emerso che i processi biologici maggiormente deregolati erano quelli di Infiammazione e Colesterolo. In aggiunta a questi pazienti, è stata validata tramite qRT-PCR una seconda coorte di RRMS e PPMS. La validazione tramite qRT-PCR ha confermato che i geni coinvolti nella biosintesi del colesterolo sono deregolati nei pazienti di entrambe le coorti, ma con specifiche differenze basate sul paziente. Infatti, sia per gli RRMS che per i PPMS si sono potute apprezzare differenze nei livelli di espressione dello stesso gene anche tra i pazienti con lo stesso fenotipo clinico. Questa deregolazione a livello metabolico, ha permesso di ipotizzare che i monociti di questi pazienti possano avere un fenotipo concordante con la Trained Immunity (TI), recentemente scoperta. Per TI si intende la memoria dell’immunità innata: monociti venuti in contatto con uno stimolo primario conserverebbero memoria di tale “incontro” reagendo in maniera più violenta ad una seconda stimolazione come potrebbe essere uno stimolo infiammatorio. Il primo stimolo può essere indifferentemente un vaccino o molecole come mevalonato (intermedio della biosintesi del colesterolo), β-Glucano e LDL ossidato (oxLDL). Per verificare questa ipotesi abbiamo testato l’espressione di geni che possono essere coinvolti nella TI, tra cui CD36, SR-A, OLR1 – collegati all’oxLDL- NLRP3, DECTIN-1 (codifica per il recettore del β-Glucano) e KDM6B (legato a modificazioni epigenetiche). I più deregolati sono risultati essere OLR1 e DECTIN-1, ma in modo diverso tra le due coorti. In particolare, la coorte 1 risulta più deregolata. Per quanto riguarda il pathway infiammatorio, invece, non è stata osservata la stessa deregolazione nella coorte 1 e nella coorte 2. La coorte 1 è risultata tendenzialmente più infiammata rispetto alla coorte 2, in particolare per i geni TNFα, CXCL2, CXCL3 e CXCL8. A seguito di questi risultati molecolari, si è proceduto con la messa a punto di un possibile modello in vitro. Stimolando ThP1 (monociti umani immortalizzati) con LPC (componente principale dell’oxLDL) si è osservata una corrispondente up-regolazione sia dei geni del colesterolo che dei geni infiammatori (NLRP3, TNFα), confermando di fatto che Infiammazione e Colesterolo viaggiano di pari passo. Per finire, sui pazienti della coorte 1 analizzati con microarray, è stata effettuata l’analisi del miRnoma che ha identificato miRNA collegati ai geni del colesterolo. Alla luce di questi risultati, dove è stato possibile caratterizzare meglio la coorte 1 che la coorte 2, e date le differenze riscontrate anche tra pazienti dello stesso fenotipo clinico di MS, si suggerisce un approccio di tipo personalizzato partendo da una signature molecolare, in modo da definire il profilo di ogni paziente. Inoltre, questo studio suggerisce che per almeno dei sottotipi di pazienti con MS, un trattamento con statine potrebbe essere un importante aiuto nel miglioramento dei sintomi.
Multiple Sclerosis (MS) is a chronic autoimmune disease that affects the central nervous system (CNS) leading to demyelination. The main pathological phenotypes are Relapsing-Remitting (RRMS) and Primary Progressive (PPMS) - the latter being the most severe form. Ajami B. et al. have shown that in the mouse model affected by Encephalomyelitis (EAE) - the animal model of MS- there is a strong correlation between the presence of monocytes at the level of the CNS and the worsening of the motor symptoms typical of the disease. Based on this evidence, blood samples from HC, RRMS and PPMS -all female- were collected for our study and CD14+ monocytes were isolated. Microarray experiments were conducted on these samples and subsequently qRT-PCR was performed. Bioinformatics analysis showed that RRMS distributed in two groups: one group (RR1) had similar trend to HC, the other group (RR2) had similar trend to PPMS. Gene Ontology showed that the most deregulated biological processes were those of Inflammation and Cholesterol. In addition to these patients, a second cohort of RRMS and PPMS was validated by qRT-PCR. Validation by qRT-PCR confirmed that the genes involved in cholesterol biosynthesis were deregulated in patients of both cohorts, but with specific patient-based differences. In fact, for both RRMS and PPMS, differences in expression levels of the same gene could be appreciated even among patients with the same clinical phenotype. This deregulation at the metabolic level, allowed us to hypothesize that the monocytes of these patients may have a phenotype consistent with the recently discovered Trained Immunity (TI). By TI is meant the memory of innate immunity: monocytes come into contact with a primary stimulus would retain memory of such "encounter", reacting more violently - and in the case of Multiple Sclerosis in an autoimmune way - to a second stimulation as could be an inflammatory stimulus. The first stimulus may be either a vaccine or molecules such as mevalonate (intermediate cholesterol biosynthesis), β-Glucan, and oxidized LDL (oxLDL). To verify this hypothesis, we tested the expression of genes that may be involved in IT, including CD36, SR-A, OLR1 - linked to oxLDL- NLRP3 and DECTIN-1 (the latter is the β-Glucan receptor). The most deregulated were OLR1 and DECTIN-1, but in a different way between the two cohorts. In particular, cohort 1 is more deregulated. For inflammatory pathways, however, the same deregulation was not observed in cohort 1 and cohort 2. Cohort 1 tended to be more inflamed than cohort 2, particularly for TNFα, CXCL2, CXCL3 and CXCL8 genes. Following these molecular results, a possible in vitro model was developed. By stimulating Thp1 (immortalized human monocytes) with LPC (main component of oxLDL) a corresponding up-regulation of both cholesterol genes and inflammatory genes (NLRP3, TNFα) was observed, confirming that inflammation and cholesterol travel hand in hand. Finally, on cohort 1 patients analyzed with microarrays, miRNome analysis was carried out and identified miRNAs related to cholesterol genes. In view of these findings, where cohort 1 has been better characterized than cohort 2, it is suggested a personalized approach starting from a molecular signature, in order to define the profile of each patient. In addition, this study suggests that for at least subtypes of patients with MS, statin treatment could be an important aid in improving symptoms.

La cirrosis ha sido definida como una etapa final de la enfermedad hepática, donde convergen diferentes fenómenos que afectan a la supervivencia de los individuos que la padecen. Dentro de estos fenómenos encontramos el paso de productos bacterianos al torrente sanguíneo, definido como translocación bacteriana patológica. Estos productos bacterianos al ser reconocidos por los Toll-like receptors de las células del sistema inmune, generan un estado pro-inflamatorio crónico. Este estado altera la respuesta inmune sistémica y predispone al desarrollo de infecciones bacterianas y complicaciones de la cirrosis, como la peritonitis bacteriana espontanea. Los probióticos pueden prevenir la translocación bacteriana patológica modulando la microbiota intestinal y mejorando la barrera intestinal. Sin embargo, se desconocen los mecanismos por los cuales mejoran estas condiciones. El primer objetivo de este trabajo ha sido evaluar como el polimorfismo en los TLRs influye en el estado pro-inflamatorio crónico y en concreto en la respuesta inmune sistémica de los pacientes con cirrosis. El segundo objetivo ha sido caracterizar la respuesta inmune innata local en los líquidos ascíticos de los pacientes con cirrosis. Por ultimo, se ha evaluado la influencia de los probióticos en el manejo de la cirrosis inducida con CCl4 en un modelo experimental de rata. A continuación se resumen los resultados de cada publicación. Cytokine production in patients with cirrhosis and TLR4 polymorphisms.% Nieto JC, Sánchez E, Román E, Vidal S, Oliva L, Guarner- Argente C, Poca M, Torras X, Juárez C, Guarner C, Soriano G. World Journal of Gastroenterology. 2014; 20(46): 17516-24. Se analizó la producción de citocinas de las células de sangre perférica de pacientes con cirrosis con o sin el polimorfismo de TLR4 D299G y/o T399I. Los pacientes con alguno de los polimosrfismos de TLR4 tuvieron una mayor incidencia de encefalopatía hepática previa que los pacientes wlid-type (78% vs 20%, P = 0.02). La producción espontanea de IL-6 e IL-10 fue menor en los pacientes con polimorfismos de TLR4 que en los pacientes wild-type [IL-6: 888.7 (172.0-2119.3) pg/mL vs. 5540.4 (1159.2-26053.9) pg/mL, P < 0.001; IL-10: 28.7 (6.5-177.1) pg/mL vs 117.8 (6.5-318.1) pg/mL, P = 0.02]. Sin embargo la producción de TNF-α, IL-6 e IL-10 después de la estimulación con LPS y LTA fue similar en los dos grupos. La presencia de alguno de los polimorfismos de TLR4 en los pacientes con cirrosis se asoció con un patrón distinto en la producción de citocinas, sugiriendo que los polimorfismos en TLR4 tienen un papel importante en el desarrollo de las complicaciones de la cirrosis. Impaired innate immune response of leukocytes from ascitic fluid of patients with spontaneous bacterial peritonitis Juan Camilo Nieto, Elisabet Sánchez, Cristina Romero, Eva Román, Maria Poca, Carlos Guarner, Cándido Juárez, Germán Soriano, Silvia Vidal. Journal of Leukocyte Biology. Epub ahead of print August 7, 2015 - doi:10.1189/jlb.3AB0315-106R Se analizó la respuesta inmune en el líquido ascítico de pacientes con cirrosis y ascitis que presentaban: ascitis estéril, PBE de cultivo positivo bacteriano ó PBE de cultivo negativo, antes y después del tratamiento con antibióticos. En el momento del diagnóstico, se observaron altos niveles de IL-6 e IL-10 en el líquido ascítico de los pacientes con PBE con cultivo positivo y PBE con cultivo negativo. Además había una correlación de los niveles de IL-6 con el porcentaje de neutrófilos (R = 0.686, P , 0.001). En este entorno, los neutrófilos de PBE con cultivo positivo y negativo tenían un estallido respiratorio deficiente, y, después del tratamiento solamente los neutrófilos de PBE con cultivo negativo recuperaron su función completa. Las altas concentraciones de IL-6 e IL-10 se correlacionaban con macrófagos de baja granularidad y niveles bajos de la expresión de CD14 (R = - 0.436, P = 0.005 and R = 0.414, P = 0.007, respectivamente). Los macrófagos de pacientes con PBE con cultivo positivo expresaban niveles bajos de CD16, CD86, CD11b, CD206 y HLA-DR, sugiriendo una función global deficiente. El tratamiento con antibióticos incrementó todos los marcadores antigénicos analizados en los macrófagos de PBE con cultivo positivo. El tratamiento con antibióticos también aumentó la expresión de CD11b y CD86 en los macrófagos de PBE con cultivo negativo. En estos macrófagos, el incremento estuvo acompañado por la recuperación de la función fagocítica. En resumen, los antibióticos revirtieron los niveles de expresión de los marcadores analizados en los macrófagos de PBE con cultivo positivo y negativo a niveles de los macrófagos de la ascitis estéril y restauraron la función de las células de PBE con cultivo negativo. VSL#3 probiotic treatment decreases bacterial translocation in rats with carbon tetrachloride-induced cirrhosis Elisabet Sánchez*, Juan C. Nieto*, Ana Boullosa, Silvia Vidal, Francesc J. Sancho, Giacomo Rossi, Pau Sancho-Bru, Rosa Homs, Beatriz Mirelis, Caándido Juárez, Carlos Guarner, Germán Soriano. Liver International. 2015 Mar;35(3):735-45. *Los autores contribuyeron de igual manera en la publicación. Se estudió como el tratamiento con VSL#3 modificaba la translocación bacteriana, la barrera intestinal y la respuesta inmune en el modelo experimental de rata con cirrosis inducida con CCl4. La mortalidad fue similar en el grupo de ratas que habían tomado VSL#3 (10/22, 45%) y el grupo que solo había bebido agua (10/24, 42%). La incidencia de translocación bacteriana fue de 1/12 (8%) en el grupo VSL#3, 7/14 (50%) en el grupo que solo bebía agua (P=0.03 grupo agua vs. grupo VSL#3) y 0/11 (0%) en el grupo control (no cirrosis) (P=0.008 grupo control vs. grupo agua). La concentración de enterobacterias y enterococos ileal y cecal fue similar en los dos grupos de ratas con cirrosis. La concentración de ocludina fue mayor y los niveles de malondialdheido y TNF-α en el suero fueron menores en el grupo VSL#3 comparado con el grupo que solo bebió agua (P<0.05). En resumen, el VSL#3 disminuye la translocación bacteriana, el estado pro-inflamatorio y el daño oxidativo ileal, además incrementa la expresión de ocludinas en el modelo experimental de ratas con cirrosis.
Cirrhosis has been defined as end-stage of liver disease that invariably leads to death. Liver damage can originate pathological bacterial translocation and the passage of bacteria and/or bacterial products to blood flow. Bacterial products can be recognized by immune cells with TLRs and subsequently develop a chronic proinflammatory state. This state alter immune responses predispose to bacterial infections and frequent complications such as spontaneous bacterial peritonitis (SBP). Probiotics can prevent pathological bacterial translocation by modulating intestinal microbiota, improving intestinal barrier and immune disturbances. However, mechanisms involved in better responses in cirrhosis are still unknown. The first objective of this study was to analyze how TLR polymorphisms influence the chronic pro-inflammatory state and specifically in the systemic immune response of patients with cirrhosis. The second objective was to characterize the local innate immune response in ascitic fluid of patients with cirrhosis. Finally, we evaluated the influence of probiotics in the management of cirrhosis induced in an experimental model rats with CCl4. We summarize the results of each publication in the next paragraphs. Cytokine production in patients with cirrhosis and TLR4 polymorphisms.% Nieto JC, Sánchez E, Román E, Vidal S, Oliva L, Guarner- Argente C, Poca M, Torras X, Juárez C, Guarner C, Soriano G. World Journal of Gastroenterology. 2014; 20(46): 17516-24. We analyze the cytokine production by peripheral blood cells from cirrhotic patients with and without TLR4 D299G and/or T399I polymorphisms. Patients with TLR4 polymorphisms had a higher incidence of previous hepatic encephalopathy than wild-type patients (78% vs. 20%, P = 0.02). Spontaneous production of interleukin (IL)-6 and IL-10 was lower in patients with TLR4 polymorphisms than in wild-type patients [IL-6: 888.7 (172.0-2119.3) pg/mL vs. 5540.4 (1159.2-26053.9) pg/mL, P < 0.001; IL-10: 28.7 (6.5-177.1) pg/mL vs. 117.8 (6.5-318.1) pg/mL, P = 0.02]. However, the production of tumor necrosis factor-α, IL-6 and IL-10 after LPS and LTA stimulation was similar in the two groups. TLR4 polymorphisms were associated with a distinctive pattern of cytokine production in cirrhotic patients, suggesting that they play a role in the development of cirrhosis complications. Impaired innate immune response of leukocytes from ascitic fluid of patients with spontaneous bacterial peritonitis Juan Camilo Nieto, Elisabet Sánchez, Cristina Romero, Eva Román, Maria Poca, Carlos Guarner, Cándido Juárez, Germán Soriano, Silvia Vidal. Journal of Leukocyte Biology. Epub ahead of print August 7, 2015 - doi:10.1189/jlb.3AB0315-106R Immune response was analyzed in patients with cirrhosis and ascities with sterile ascites, bacterial positive culture SBP or negative culture SBP before and after antibiotic treatment. At diagnosis, a high concentration of IL-6 and IL-10 was found in the ascitic fluid from negative and positive bacteriological culture. The IL-6 concentration correlated with the percentage of neutrophils (R = 0.686, P = 0.001). In this context, positive and negative culture neutrophils had an impaired oxidative burst, and, after the antibiotic, the negative culture spontaneous bacterial peritonitis burst was fully recovered. Higher concentrations of IL-6 and IL-10 correlated with the presence of low granular CD14low macrophages (R = -0.436, P = 0.005 and R = 0.414, P = 0.007, respectively). Positive culture spontaneous bacterial peritonitis macrophages expressed the lowest levels of CD16, CD86, CD11b, and CD206, and HLA-DR, suggesting an impaired global function. Treatment increased all markers on the positive culture macrophages and CD11b and CD86 on negative culture macrophages. In negative culture spontaneous bacterial peritonitis, this increase was accompanied by phagocytic function recovery. The antibiotics then reverted the marker levels on positive and negative culture macrophages to the levels on sterile ascitis macrophages and restored ascitic negative culture cell function. VSL#3 probiotic treatment decreases bacterial translocation in rats with carbon tetrachloride-induced cirrosis Elisabet Sánchez*, Juan C. Nieto*, Ana Boullosa, Silvia Vidal, Francesc J. Sancho, Giacomo Rossi, Pau Sancho-Bru, Rosa Homs, Beatriz Mirelis, Caándido Juárez, Carlos Guarner, Germán Soriano. Liver International 2014 *Both authors contributed equally to the study. Bacterial translocation, Intestinal barrier and immune response were assessed in rats with CCl4 induced cirrhosis. Mortality during this study was similar in the VSL#3 group (10/22, 45%) and the water group (10/24, 42%). The incidence of bacterial translocation was 1/12 (8%) in the VSL#3 group, 7/14 (50%) in the water group (P = 0.03 vs. VSL#3 group) and 0/11 in the control group (P = 0.008 vs. water group). The concentration of ileal and caecal enterobacteria and enterococci was similar in the two groups of cirrhotic rats. The ileal occludin concentration was higher and ileal malondialdehyde and serum levels of TNF-α were lower in the VSL#3 group than in the water group (P < 0.05). VSL#3 decreases bacterial translocation, the proinflammatory state and ileal oxidative damage and increases ileal occludin expression in rats with experimental cirrhosis.

Determinades situacions patològiques en el cervell així com estímuls inflamatoris provoquen un augment en l’expressió de la COX-2. En el context de la isquèmia cerebral, l’augment en l’expressió de la COX-2 està relacionada amb efectes perjudicials. Les cèl•lules del sistema nerviós central (SNC) disposen de sistemes de reconeixement que els permeten detectar i reconèixer agents infecciosos i iniciar una resposta immune per a fer front a una intrusió. Els astròcits expressen receptors tipus Toll-Like, responen als agonistes d’aquests receptors amb l'alliberació de molècules, i són capaços de modular la resposta immune innata. Els astròcits tenen una ubicació estratègica a la interfase entre els vasos sanguinis i el parènquima cerebral, participant així en la funció de la BHE, en la regulació del flux sanguini cerebral, mitjançant l'alliberament de mediadors vasoactius i expressant molècules implicades en la patogènesi d'algunes malalties autoimmunes que afecten la permeabilitat de la BHE. A més, els astròcits formen una intricada xarxa intercomunicativa amb altres cèl•lules nervioses. La senyalització iniciada en els astròcits pot afectar la funció sinàptica cerebral, i tenir un gran impacte en la unitat neurovascular. En aquesta tesi ens hem plantejat l’ objectiu d’esbrinar les vies de senyalització intracel•lular i els mediadors inflamatoris que participen en la resposta dels astròcits a l’activació dels receptors d’immunitat innata, concretament TLR-4, i determinar el paper de la Cox-2 astroglial en la resposta inflamatòria induïda per aquest receptor. L’activació de TLR-4 indueix en els astròcits senyalització a través de NFκB, les vies MAPK i JAK1/STAT1, que al seu torn regulen l'expressió d'una àmplia gamma de molècules proinflamatòries. Malgrat que el LPS no sembla induir IFN-β en els astròcits, sí que promou l'activació JAK1/STAT1 de manera independent de MyD88, i STAT1 pot regular negativament l'expressió de gens que s'indueixen a través de la via MyD88. Aquests resultats mostren l’activació d’una complexa xarxa de senyalització iniciada per l’activació del receptor TLR-4 en astròcits. Les cèl•lules astroglials són sensibles a la immunitat innata, desencadenant una resposta específica del tipus cel•lular i generant un ambient pro-inflamatòri que podria afectar la resposta o viabilitat de les cèl•lules circumdants. El LPS augmenta l’expressió de la COX-2 en astròcits, i també provoca un augment en la producció i alliberació de prostanoïds dependent de l'expressió de la COX-2. El paper clau de la Cox-2 en la producció de prostanoids després de LPS i l'evidència que la manca de l’enzim Cox-2 exacerba l'expressió de diversos mediadors inflamatoris. Aquest efecte es deu, almenys en part, a la manca de mecanismes naturals de regulació negativa induïts per la PGE2. Els mecanismes de retroalimentació d'aquest tipus podrien estar implicats en la resolució natural del procés inflamatori.
Brain pathological conditions and pro-inflammatory stimuli induce cyclooxygenase-2 (Cox-2). Cox-2 is regarded as an inflammatory mediator but Cox-2 deficiency exaggerates the cerebral inflammatory response to lipopolysaccharide (LPS). Here we investigate the role of Cox-2 in LPS-induced prostanoids and inflammation in glia. Intracerebral administration of LPS in rodents induced Cox-2 mainly in astroglia and microglia, while Cox-1 expression was constitutive and predominant in microglia, and it did not increase after LPS. In cultured astrocytes, LPS induced Cox-2 and microsomal prostaglandin-E2 (PGE2) synthase-1 expression. Cox-2 fully mediated LPS-induced production of PGE2 and other prostanoids, as demonstrated in Cox-2-deficient cells and using Cox inhibitors. However, Cox-2-deficient astrocytes produced more TNF-alpha and other inflammatory mediators after LPS than the corresponding wild-type cells. Treatment with PGE2 prevented the exacerbated production of TNF-alpha after LPS in Cox-2 KO cells, and an EP4 agonist also attenuated LPS-induced TNF-alpha, supporting that PGE2/EP4 exerted selective feedback regulatory functions. However, Cox-2 inhibitor NS-398 only caused a very mild increase in LPS-induced TNF-alpha in wild-type astrocytes. This difference might be due to certain Cox-2-independent actions of NS-398 since it decreased TNF-alpha in Cox-2 KO cells, and enhanced the reduction of TNFalpha induced by treatment with PGE2. Therefore, Cox-2 deficiency exacerbates LPS-induced TNF-alpha by preventing a natural negative feedback regulation mediated by the Cox-2/PGE2/EP4 axis. In addition to this effect, NS-398 exerts Cox-2-independent actions becoming apparent in the absence of Cox-2 or in the presence of PGE2. The balance between these different effects determines how Cox-2 inhibition modulates inflammatory responses in astrocytes.

Background IL32 is a recently described proinflammatory cytokine produced by T lymphocytes, natural killer cells, monocytes and epithelial cells. Several studies have demonstrated the importance of IL32 in the regulation of the innate and adaptive immune responses, moreover, it has been found over expressed in inflammatory disorders and in autoimmune diseases. In this project, we focused our attention on studying Chronic Obstructive Pulmonary Disease (COPD) because we think it is a good model for studying the role of IL32 in the innate immunity. Infact, IL32 expression was found to be increased in alveolar macrophages and in lung epithelial cells of smokers with COPD compared to non-smokers. COPD is a chronic respiratory disease characterized by an irreversible limitation on pulmonary airflow associated with chronic inflammation due to several etiological factors such as cigarette smoke. Alveolar epithelial cells and macrophages are primarily involved in its pathogenesis because they secrete cytokines and chemokines recruiting inflammatory cells which sustain the phlogosis. The final effect is lung parenchyma destruction and remodeling . Aim of the study The aim of this study was to investigate the role of IL32 in response to lung injury and in inflammation induced by cigarette smoke. Firstly, ee evaluated if cigarette smoke extract (CSE) was cytotoxic for the lung epithelial cells and macrophages by inducing their apoptosis; then, we investigated if CSE activated epithelial cells by increasing the expression of some proinflammatory cytokines and growth factors. Secondly, we studied if CSE induced IL32 expression by epithelial cells and macrophages and we search for a synergic effect between CSE and other proinflammatory stimuli. Methods For our experiments we employed an alveolar epithelial cell line, A549, and macrophages obtained by THP1 differentiation. Cells were treated with CSE, with PAMPs and IL1β. The PAMPs we used were: polyI:C, imiquimod, MDP and ieDAP.. We evaluated cell viability with Annexin V flow cytometric assay. Moreover, we quantified the mRNA levels of IL32 and other cytokines such as CCL2/MCP1, RAGE and TNF-α with Real Time-PCR. Finally, we investigated IL32 protein expression by Western Blotting and we analyzed its secretion in the conditioned medium from cell cultures with ELISA test. Results We demonstrated that CSE, at low concentration (5%), was able to induce apoptosis of the A549. Moreover, it concurred to their activation by inducing the expression of the proinflammatory CCL2/MCP1 and RAGE and, more importantly, we found that it increased IL32 transcript levels, particularly those of isoform β. In order to test if CSE made epithelial cells more susceptible to the action of other proinflammatory stimuli, cells were treated with CSE together with PAMPs or IL1β. We observed that cells treated with IL1β and CSE showed higher levels of IL32β mRNA compared to those treated with the single stimuli. On the other hand, macrophages resulted to be more resistant to apoptosis induced by CSE dying only at high concentration (20%). Moreover, CSE induced a significant increase of TNF-α expression. More importantly, macrophages treated with CSE showed higher levels of IL32 compared with non-treated cells and this effect resulted to be amplified when they were co-stimulated with IL1β. Discussion These data suggest that cigarette smoke represents an important cytotoxic factor for the epithelium which seems to have a key role in the initial lung injury response. Alveolar epithelial cells activation leads to the production of some factors which recruit macrophages whom trigger the immune response. IL32 seems to be strongly involved in these processes because it is produced both by epithelial cells and macrophages and its expression seems to be enforced by the inflammatory setting
Introduzione L’Interleuchina 32 (IL32) è una citochina proinfiammatoria di recente identificazione prodotta dai linfociti T, dalle cellule natural killer (NK), dai monociti e dalle cellule epiteliali. Essa svolge un ruolo chiave nella regolazione della risposta immunitaria ed è espressa in maniera significativa in alcune patologie infiammatorie croniche e a componente autoimmune. In questo studio, abbiamo identificato nella Broncopneumopatia Cronica Ostruttiva (BPCO) un possibile modello per indagare il ruolo di IL32 nell’infiammazione. Infatti, è stato dimostrato che la sua espressione è elevata nei macrofagi alveolari e nell’epitelio polmonare di soggetti fumatori affetti da BPCO rispetto a quelli non fumatori. La BPCO è una malattia caratterizzata dall’ostruzione irreversibile delle piccole vie aeree e da uno stato di infiammazione cronica del tessuto polmonare dovuto all’azione di vari fattori eziologici tra cui il fumo di sigaretta. Le cellule epiteliali alveolari e i macrofagi alveolari sono primariamente implicati nella patogenesi della BPCO, poiché, in risposta al danno, secernono fattori reclutanti le cellule del sistema immunitario che, in seguito, sostengono la flogosi portando, come conseguenza ultima, alla distruzione e al rimodellamento del parenchima polmonare. Scopo della Tesi Lo scopo di questo studio è stato quello di indagare il ruolo di IL32 nei meccanismi di risposta al danno polmonare e nell’innesco della risposta infiammatoria causati dal fumo. Inizialmente, abbiamo valutato se l’estratto di fumo (CSE) era un fattore citotossico per le cellule epiteliali e per i macrofagi e se induceva la loro apoptosi; inoltre, abbiamo indagato se il fumo contribuiva all’attivazione delle cellule modulando l’espressione, da parte di esse, di citochine e di fattori di crescita proinfiammatori. In seguito, abbiamo studiato se il CSE induceva la produzione di IL32 da parte di queste cellule e se esisteva un effetto sinergico tra la loro azione e quella di altri stimoli proinfiammatori nella modulazione dell’espressione di questa citochina. Materiali e metodi Per gli esperimenti abbiamo utilizzato le cellule epiteliali alveolari di tipo II, A549 e i macrofagi ottenuti dal differenziamento della linea monocitica THP1. Le cellule sono state trattate con il CSE, con i PAMPs e con l’IL1β. I PAMPs utilizzati sono: il polyI:C, l’imiquimod, l’MDP e l’ieDAP. La vitalità cellulare è stata valutata mediante test citofluorimetrico con Annessina V; l'espressione genica di CCL2/MCP1, RAGE, TNF-α e di IL32 è stata effettuata mediante Real Time-PCR. L’espressione proteica di IL32 è stata studiata mediante Western Blotting, mentre la presenza della proteina secreta è stata valutata nel sovranatante delle colture cellulari per mezzo del test ELISA. Risultati: Abbiamo dimostrato che il CSE a basse dosi (5%) era in grado di indurre l’apoptosi delle A549. Oltre a ciò, esso contribuiva alla loro attivazione inducendo un aumento dell’espressione di CCL2/MCP1 e di RAGE e, soprattutto, era in grado di aumentare i livelli di espressione genica di IL32, in particolare, quelli dell’isoforma β. Al fine di testare se il CSE rendeva le A549 più sensibili all’azione di altri fattori proinfiammatori, abbiamo co-trattato le cellule con CSE, con i PAMPs o con IL1β. Abbiamo osservato che le cellule trattate con CSE e IL1β avevano livelli di espressione di IL32β più alti rispetto a quelle trattate con gli stimoli da soli. Dall’altra parte, gli esperimenti condotti sui macrofagi hanno evidenziato che essi erano più resistenti all’apoptosi indotta da CSE, infatti, morivano solo ad alte concentrazioni (20%). Inoltre, il trattamento con CSE induceva un significativo aumento dell’espressione di TNF-α. Oltre a ciò, basse dosi di CSE aumentavano significativamente i livelli di espressione di IL32 e questo effetto risultava amplificato dal co-trattamento con CSE e IL1β. Discussione I dati ottenuti suggeriscono che il fumo rappresenta un importante fattore di citotossicità per l’epitelio il quale sembra avere un ruolo cruciale nelle fasi iniziali di risposta al danno. L’attivazione delle cellule alveolari porta alla produzione di mediatori dell’infiammazione che hanno il compito di reclutare i macrofagi i quali innescano la risposta immunitaria. IL32, sembra essere pesantemente coinvolta nei processi descritti poiché viene prodotta sia dalle cellule epiteliali che dai macrofagi e il contesto infiammatorio contribuisce ad amplificarne l’espressione

En la isquemia cerebral se desarrolla un proceso inflamatorio que contribuye a incrementar la muerte neural. La presencia de células necróticas promueve la activación de la respuesta inmune innata que contribuye a iniciar el proceso inflamatorio. Las citoquinas actúan de mediadoras de la respuesta inmunitaria e inflamatoria. Durante la reoxigenación después de la isquemia, se generan especies reactivas de oxígeno (ERO) que aumentan el daño por reperfusión. En respuesta a la isquemia cerebral, se activan proteínas de la via de señalización JAK/STAT. Esta vía participa en la respuesta celular a los ERO y a las citoquinas. De modo que la inflamación, la respuesta inmune innata y el estrés oxidativo son los procesos derivados de la isquemia/reperfusión que potencialmente pueden activar la vía JAK/STAT. La vía JAK/STAT utiliza un mecanismo en el cual los factores de transcripción STAT que se hayan en estado latente en el citoplasma son fosforilados en tirosina por las quinasas de la familia Janus, dando lugar a la dimerización y translocación al núcleo de las STATs. Las proteínas STAT modulan la expresión de genes relacionados con la diferenciación, la inflamación y la supervivencia. El objetivo de esta tesis es el estudio de la activación de la vía JAK/STAT en respuesta al estrés oxidativo, la inflamación y la inmunidad innata en astrocitios. La vía Jak2/Stat1 se activa en respuesta a citoquinas proinflamatorias, como IFN-gamma y interleuquina-6, y peróxido de hidrógeno en astrocitos, y promueve la muerte celular a las 24 horas. El tratamiento con el inhibidor de Jak2, AG490, previene de la muerte demostrando la implicación de la vía Jak2/Stat1 en la muerte. La activación de la vía Jak2/Stat1 es específica de peróxidos, ya que tratamientos con otros agentes proxidantes, tales como el sulfato de hierro (donador de iones hidroxilo), el nitroprusiato (donador de óxido nítrico) o el paraquato (donador de aniones superóxido) inducen la generación de EROs pero no activan a Stat1. De manera que el peróxido de hidrógeno induce específicamente la activación de la vía Jak2/Stat1. Paralelamente, los tratamientos con peróxido de hidrógeno y los compuestos antioxidantes trolox y propilgallato, inhiben la generación de EROs pero no detienen la activación de Stat1, mientras que el tratamiento de peróxido de hidrógeno con el antioxidante N-acetil-cisteína no reduce la producción de EROs y si inhibe la activación de Stat1. Estos resultados demuestran que la activación de Stat1 y la generación de EROs son efectos independientes.
En el siguiente trabajo se demuestra que los siRNAs (por small interfering RNAs) inducen efectos inespecíficos en determinados tipos celulares de manera dependiente de secuencia e independiente del efecto silenciador. En cultivo glial mixto, el tratamiento con siRNAs aumenta
la expresión de Stat1 y de otras moléculas proinflamatorias como iNOS, la citoquina IL-6 y las quimiocinas IP-10 e IP-9. La adición de un grupo O-metilo en la cadena sense del siRNA no impide el silenciamiento de la proteína para la cual es específico, y si inhibe el incremento de la expresión de los marcadores inflamatorios que se han estudiado. La activación de la inmunidad innata se observa en cultivo de glía mixta y en cultivo puro de astrocitos, pero no tiene en cultivo de microglía pura ni en la línea celular no glial 3T3, demostrando que los astrocitos son particularmente sensibles a desarrollar respuestas inmunes innatas frente al tratamiento con siRNAs. En este trabajo se demuestra que los siRNA activan la respuesta inmune innata en astrocitos activando el receptor TLR3, puesto que el tratamiento con siRNA, del mismo modo que el tratamiento con el agonista de TLR3, poly di dC, induce un aumento de la expresión de este receptor. En esta tesis, se pone de manifiesto el papel de la vía JAK/STAT en la modulación del estrés oxidativo, la inflamación y la inmunidad innata.

Objetivos. Primero: determinación de la susceptibilidad de macrófagos derivados de monocitos activados por citocinas a ser infectados por diferentes cepas del genotipo 1 y 2 de PRRSV. Segundo: evaluación de la modulación de la respuesta inmune por la cepa 2982 de PRRSV-1 en pulmón, tonsila y nódulos linfáticos de credos infectados. Tercero: caracterización de la respuesta inmune inducida por diferentes cepas de PRRSV-1 que varían en virulencia en distintos compartimentos de nódulo linfático mediastínico (Med-LN) de cerdos infectados. Metodología. En el experimento in vitro se seleccionaron monocitos de PBMC de cerdos SPF y se incubaron durante 72 h para permitir la diferenciación en macrófagos. Entonces esas células se estimularon con citocinas durante 24 h: IFN-γ (M1), IL-4 (M2), IFN-β o no estimulados. Después mediante citometría de flujo se evaluó el fenotipo de los diferentes macrófagos (CD16, MHC I, MHC II, CD86, CD16 and CD14). La infección de macrófagos por PRRSV se cuantificó por medición de la nucleocápside mediante citometría de flujo. En el sobrenadante se midió por ELISA IFN-α, TNF-α, and IL-10. Para el primer estudio in vivo 28 cerdos SPF se infectaron intramuscularmente con la cepa 2982 de PRRSV-1 y se eutanasiaron humanitariamente a 3, 7, 10, 14, 17, 21 y 24 dpi. Se dejaron 4 animales como grupo control negativo, que se eutanasiaron a los 24 dpi. Los niveles de transcripción de citocinas proinflamatorias (IFN-α, IFN-γ, IL-12p35, IL-12p40 y TNF-α) e inmunododuladoras (IL-10 y TGF-β) se midieron mediante RT-qPCR. Para el segundo experimento in vivo 76 lechones SPF fueron aleatoriamente agrupados en 3 grupos de 20 animales para la infección, y los restantes 16 se dejaron como control. Se utilizaron tres cepas de PRRSV-1 con distinta virulencia: LV como prototipo, 215-06 del subtipo 3, y la SU1-bel del subtipo 3. Los animales se eutanasiaron humanitariamente a los 3, 7 y 35 dpi. El folículo y área inter-folicular de Med-LN se tomaron separadamente mediante captura por microdisección láser. Los niveles de transcripción de citocinas (IFN-α, IFN-γ, TNF-α, IL-23p19, IL-10 and TGF-β) y de SOCS1 se evaluaron por RT-qPCR. Las poblaciones celulares de Med-LN se evaluaron por inmunohistoquímica. Resultados. Los macrófagos estimulados por citocinas desarrollan diferente fenotipo comparado con macrófagos no polarizados. Los cultivos celulares de macrófagos polarizados por citocinas pueden muestran diferencias importantes entre cepas de PRRSV. Mientras que los macrófagos no polarizados y M2 son sensibles a todas las cepas de PRRSV, el tratamiento de macrófagos con IFNs induce un claro estado antivírico en dichas células con una menor infección y replicación de PRRSV. Sólo los M1 infectados con LVP23 produjeron cierta cantidad de IFN-α. Este tipo de macrófagos representan un flexible modelo in vitro alternativo al empleo de líneas celulares derivadas de mono o a macrófagos alveolares porcinos. El primer estudio in vivo mostró que la cepa 2982 del PRRSV-1 es capaz de evadir el establecimiento de respuesta inmune innata efectiva, permitiendo una expresión génica de IFN- α 1 y TNF-α disminuida, y una respuesta inmune adquirida débil y retardada, por una expresión ineficiente de IL-12 e IFN-γ. El segundo experimento in vivo demostró que PRRSV permanece alojado en Med-LN al menos hasta 35 dpi, y la cepa SU1-bel es la que posee una mayor capacidad de replicación. Las cepas de PRRSV-1 evitaron el establecimiento correcto de la respuesta inmune en cerdos infectados mediante la depleción de células linfáticas y disminución de la expresión de IFN- α y TNF-α en los compartimentos de Med-LN. Finalmente, los resultados sugieren que la inducción de IL-10 por PRRSV con el propósito de retrasar la respuesta inmune del hospedador no es una estrategia común a todos los aislados de PRRSV-
1. Objectives. First: determination of the cytokine primed monocyte-derived macrophages susceptibility to be infected by different PRRSV-1 and 2 strains. Second: evaluation of the swine immune response modulation by PRRSV-1 2982 strain in lung, tonsil, tracheobronchial and retropharyngeal lymph nodes of infected pigs. Third: characterization of the immune response induced by several PRRSV-1 strains varying in virulence in distinct compartments of mediastinal lymph node (Med-LN) (follicle and inter-follicular areas) of infected pigs. Methodology. For the in vitro experiment, monocytes were isolated from PBMC of SPF pigs and incubated during 72 h to lead the macrophage differentiation. Then, these cells were stimulated during 24 h with cytokines: IFN-γ (M1), IL-4 (M2), IFN-β or no stimulated. After that the phenotype of the different macrophages were evaluated by flow cytometry (CD16, MHC I, MHC II, CD86, CD16 and CD14). Several PRRSV-1 and PRRSV-2 strains were used to infect macrophages. PRRSV infection of macrophages was evaluated by nucleocapsid protein acquisition by flow cytometry. In the supernatant IFN-α, TNF-α, and IL-10 were determined by ELISA. For the first in vivo experiment 28 SPF pigs were infected intramuscularly with PRRSV-1 2982 isolate and were humanely euthanized at 3, 7, 10, 14, 17, 21 and 24 dpi. Four pigs were kept as non-infected control group, being humanely euthanized at 24 dpi. The proinflammatory (IFN-α, IFN-γ, IL-12p35, IL-12p40 and TNF-α) and immunomodulatory (IL-10 and TGF-β) cytokines transcript levels were evaluated by means of RT-qPCR. For the second in vivo experiment, 76 SPF piglets were ramdonly allocated in groups of 20 animals for the viral infection and the 16 remaining animals as control group. Three PRRSV-1 isolates varying in virulence were selected in this study: LV as prototype, 215-06 from subtype 1, and SU1-bel from subtype 3. Animals were humanely euthanized at 3, 7 and 35 dpi. Med-LN follicle and inter-follicular areas were separately selected by Laser Microdissection Capture. Cytokines (IFN-α, IFN-γ, TNF-α, IL-23p19, IL-10 and TGF-β) and SOCS1 transcript levels were evaluated by means of RT-qPCR. The lymph node cell populations were evaluated by immunohistochemistry. Results. The in vitro experiment showed that stimulated macrophages developed different phenotype compared with non polarized macrophages. Cytokine-primed macrophages cultures can reveal important PRRSV strain differences. Whereas undifferentiated macrophages and M2 are sensitive to all PRRSV strains, IFN-γ and IFN-β treatment of macrophages induced a clear antiviral state in macrophages with reductions in virus infection and replication. Only IFN-α production was detected in the supernatant of M1 infected with LVP23 PRRSV-1 strain. These macrophages represent a flexible in vitro model alternative against monkey cell lines and porcine alveolar macrophages. The first in vivo experiment showed that PRRSV-1 strain 2982 is able to evade the onset of an effective innate immune response, leading to an impaired expression of IFN-α 1 and TNF-α gene expression, and a weak and delayed acquired immune response through an inefficient IL-12 and IFN-γ expression. This experiment showed that PRRSV remains allocated in follicle of lymph node during at least 35 dpi, and SU1-bel showed the higher ability of replication. PRRSV-1 stains showed to avoid the correct innate immune response in infected pigs through lymphatic cell depletion, IFN-α and TNF-α down-regulation in Med-LN compartments. Finally, the IL-10 induction by PRRSV was not a common strategy among the PRRSV-1 to delay of the host immune response.

El sistema immune innat és el primer en respondre davant patògens que amenacen l’hoste. La resposta immune innata es caracteritza per induir una activació immune innata, iniciar la senyalització d’IFNs i activar vies inflamatòries que generen un estat antiviral capaç de limitar la propagació de patògens i induir la mort cel·lular de cèl·lules afectades. La identificació de factors de restricció del VIH, com SAMHD1, ha proporcionat coneixements que han relacionat el metabolisme dels nucleòtids, l’activació immune innata i la patogènesi del virus. Per tant, aquesta tesi avalua la relació entre factors virals i de l’hoste que poden afectar la regulació del sistema immune innat i activació de vies d’interferó on potencialment poden controlar la replicació viral i progressió de malalties associades. Gràcies a l’estudi in-vitro de la infecció del VIH, hem identificat que el virus indueix un mecanisme antiviral innat associat a la producció d’interferó de tipus I i activació de gens estimulats per interferó (ISGs). D’altra banda, el VIH indueix una aturada en el cicle cel·lular a la fase G2/M, mediada per p21, que causa un augment en els nivells de mort cel·lular en macròfags derivats de monòcits (MDMs). D’altra banda, la proteïna accessòria Vpx de VIH-2/SIV, té com a objectiu disminuir l’expressió de SAMHD1 i TASOR. Al mateix temps, Vpx inicia una cascada de senyalització d’activació immune innata caracteritzada per la inducció d’IFNs i expressió d’ISGs que poden ser claus en el disseny d’estratègies dirigides a la reactivació latent del VIH. El descobriment que mutacions en SAMHD1 i TREX1 estan associades a la malaltia autoimmune del síndrome d’Aicardi-Goutières (AGS) i el fet que ambdós gens tinguin un paper en la replicació del VIH, suggereix que la avaluació de sensors d’àcid nucleic en base a AGS pot potencialment identificar factors de l’hoste que regulen la replicació viral. De fet, un cribratge de gens d’AGS en macròfags primaris ha identificat ADAR1 com un regulador negatiu de la via de senyalització RLR-MAVS que contribueix a l’activació immune innata i la producció d’IFNs. Les cèl·lules on s’ha disminuït l’expressió d’ADAR1 mostren un augment de l’expressió de sensors d’ARN com MDA5 i RIG-I, un augment en la fosforilació del factor de transcripció IRF7 i un augment en la producció d’IFNs de tipus I. L’avaluació in-vitro d’ADAR1, ha mostrat que ADAR1 té un efecte proviral davant el VIH o antiviral davant de VHC i VPH, suggerint que el rol d’ADAR1 depèn de la combinació específica entre virus i hoste. Finalment, les variacions genètiques d’ADAR1 in-vivo s’associen a mal pronòstic clínic i afecten a la resposta del tractament i a la progressió de la malaltia. En resum, aquesta tesi demostra que la disfunció en vies de detecció d’àcids nucleics intracel·lulars, que desencadenen a una forta activació immune innata i les signatures IFN, són prometedores pel disseny de noves estratègies terapèutiques que tenen com a objectiu modular la replicació viral, alterar la progressió de la malaltia i afectar el resultat final de la infecció.
El sistema inmune innato es el primero en responder ante patógenos que amenazan el huésped. La respuesta inmune innata se caracteriza por inducir una activación inmune innata, iniciar la señalización de IFNs y activar vías inflamatorias que generan un estado antiviral capaz de limitar la propagación del patógeno e inducir la muerte celular de células afectadas. La identificación de factores de restricción del VIH, como SAMHD1, ha proporcionado conocimientos que han relacionado el metabolismo de los nucleótidos, la activación inmune innata y la patogénesis del virus. Por lo tanto, esta tesis quiere evaluar la relación entre factores virales y del huésped que pueden afectar a la regulación del sistema inmune innato y activación de vías de interferón donde potencialmente pueden controlar la replicación viral y progresión de enfermedades asociadas. Gracias al estudio in-vitro de la infección del VIH, hemos identificado que el virus induce un mecanismo antiviral innato asociado a la producción de interferón de tipo I y activación de genes estimulados por interferón (ISGs). Por otra parte, el VIH induce una parada en el ciclo celular en la fase G2 / M, mediada por p21, que causa un aumento en los niveles de muerte celular en macrófagos derivados de monocitos (MDMS). Por otra parte, la proteína accesoria Vpx de VIH-2/SIV, tiene como objetivo disminuir la expresión de SAMHD1 y TASOR. Al mismo tiempo, VPX inicia una cascada de señalización de activación inmune innata caracterizada por la inducción de IFNs y expresión de ISGs que pueden ser claves en el diseño de estrategias dirigidas a la reactivación latente del VIH. El descubrimiento que mutaciones en SAMHD1 y TREX1 están asociadas a la enfermedad autoinmune del síndrome de Aicardi-Goutières (AGS) y el hecho de que ambos tengan un papel en la replicación del VIH, sugiere que la evaluación de sensores de ácido nucleico en base a AGS puede potencialmente identificar factores del huésped que regulan la replicación viral. De hecho, un cribado de genes de AGS en macrófagos primarios ha identificado ADAR1 como un regulador negativo de la vía de señalización RLR-MAVS que contribuye a la activación inmune innata y la producción de IFNs. Las células donde se ha disminuido la expresión de ADAR1 muestran un aumento de la expresión de sensores de ARN como MDA5 y RIG-I, un aumento en la fosforilación del factor de transcripción IRF7 y un aumento en la producción de IFNs de tipo I. La evaluación in-vitro de ADAR1, ha mostrado que ADAR1 tiene un efecto proviral ante el VIH o antiviral ante VHC y VPH, sugiriendo que el rol de ADAR1 depende de la combinación específica entre virus y huésped. Finalmente, las variaciones genéticas de ADAR1 in-vivo se asocian a mal pronóstico clínico y afectan a la respuesta del tratamiento y la progresión de la enfermedad. En resumen, esta tesis demuestra que la disfunción en vías de detección de ácidos nucleicos intracelulares, que desencadenan una fuerte activación inmune innata y patrón de IFNs, son prometedoras para el diseño de nuevas estrategias terapéuticas que tienen como objetivo modular la replicación viral, alterar la progresión de la enfermedad y afectar el resultado final de la infección.
The innate immune system is the first to respond against a pathogen invasion that threatens the host. An innate immune response is characterized by inducing innate immune activation, IFN signaling and inflammatory pathways that generate an antiviral state able to limit pathogen spread and induce cell death of damaged cells. The identification of HIV host restriction factors, such as SAMHD1, has provided insights linking nucleotide metabolism, innate immune activation and viral pathogenesis. Hence, this thesis evaluates the relationship between viral and host factors that strongly affect innate immune modulation and IFN pathways that have the potential to alter viral outcome and progression of associated diseases. Through the study of HIV infection, we have identified that HIV induces an innate antiviral mechanism associated to IFN-I production and interferon stimulated gene activation. Moreover, HIV induces a cell cycle arrest at G2/M mediated by p21 leading to elevated levels of cell death in monocyte derived macrophages. On the other hand, accessory protein Vpx from HIV-2/SIV, which targets the downregulation of SAMHD1 and TASOR, also triggers innate immune activation signatures characterized by induction of IFNs and expression of ISGs which may hold key in designing strategies targeting reactivation of latent HIV. The discovery that mutations in SAMHD1 and TREX1 are associated to autoimmune disease Aicardi-Goutières syndrome (AGS) and the fact that both genes have a role in HIV replication, suggests that evaluation of nucleic acid sensors in AGS may have potential for identifying host factors regulating viral replication. Indeed, a screening of AGS genes in primary macrophages has identified ADAR1 as a negative regulator of RIG-I like receptor RLR-MAVS signaling pathway contributing to innate immune activation and IFN production. ADAR1 downregulated cells show increase expression of RNA sensors, MDA5 and RIG-I, enhanced phosphorylation of transcriptional factor IRF7 and increase type I IFN production. ADAR1 in-vitro evaluation have shown a proviral role in HIV, but an antiviral role in HCV and HPV, suggesting ADAR1 role depends on the specific virus host combination. Finally, ADAR1 in-vivo genetic variations are associated to poor clinical outcomes and affect treatment response and disease progression. In summary, this thesis demonstrates that dysfunction of intracellular nucleic acid sensing pathways that trigger strong innate immune activation and IFN signatures hold promising in the design of novel therapeutic strategies aiming to modulate viral replication and disease progression and infection outcomes.

Per secoli, i prodotti naturali e i loro derivati hanno fornito una ricca fonte di composti per lo sviluppo di nuovi immunomodulatori. Gli immunomodulatori naturali possono fornire la chiave per controllare e, infine, sconfiggere i disturbi che colpiscono il sistema immunitario, stimolando o inibendo la risposta immunitaria con pochi effetti collaterali negativi. Molti di questi composti sono attualmente in fase di sperimentazione clinica, in particolare come agenti antiossidanti, antimicrobici e antitumorali. Tuttavia, la funzione e il meccanismo di azione dei prodotti naturali, e il modo in cui interagiscono con il sistema immunitario, devono ancora essere ampiamente esplorati. Negli ultimi anni, la conoscenza del ruolo dei macrofagi nel contesto dell'infiammazione ha aperto numerose nuove strade, tra cui la possibilità di intervento terapeutico mirato sfruttando la plasticità intrinseca dei macrofagi per il trattamento di patologie infiammatorie acute e croniche. I recettori Toll-simili (TLR), incluso TLR4, svolgono un ruolo cruciale nelle malattie a base infiammatoria; pertanto, la via di segnalazione di TLR4 è stata identificata come bersaglio terapeutico per l'intervento farmacologico. Questo lavoro mira a valutare il potenziale dei fitoestratti e delle sostanze fitochimiche di influenzare la via di segnalazione di TLR4 in un modello cellulare di macrofago. Nello specifico, sono stati testati estratti ottenuti da chicchi di caffè verde (GCE) e tostato (RCE), nonché acido clorogenico puro (5-CQA). Inoltre, è stato valutato l'effetto antinfiammatorio della palmitoiletanolamide (PEA) e del suo analogo sintetico RePEA. Come modello in vitro sono stati impiegati macrofagi originati da monociti umani, derivanti dalla differenziazione di una linea cellulare (THP-1) e/o da monociti umani primari. Il saggio MTT è stato utilizzato per determinare gli effetti citotossici dei trattamenti. L'attivazione di TLR4 è stata stimolata mediante esposizione delle cellule all'endotossina batterica LPS (E. coli) in presenza o assenza di trattamento. Sono stati valutati diversi parametri: produzione di citochine pro-infiammatorie, ma anche attivazione e traslocazione nucleare dei mediatori di segnalazione intracellulare. Questi parametri sono stati misurati applicando differenti tecniche di biologia cellulare e molecolare, principalmente test di immunoassorbimento enzimatico (ELISA), immunofluorescenza e immunofissazione (Western blot). Inoltre, abbiamo impiegato diverse cellule transfettate stabilmente disponibili in commercio come strumenti per studiare il meccanismo d'azione del nostro estratto o della nostra molecola, rispettivamente le cellule THP1-XBlue™, RAW-Blue™ e HEK-Blue™. I risultati chiave includono un marcato effetto inibitorio, dose-dipendente, degli estratti di caffè verde e tostato verso il rilascio di interferone-β (IFN-β) dopo la stimolazione con LPS. Coerentemente, l'acido clorogenico, un importante componente polifenolico degli estratti di caffè, ha mostrato un'attività biologica comparabile. Complessivamente, i risultati ottenuti forniscono nuove prove sugli effetti immunomodulatori e sul meccanismo d'azione degli estratti di caffè e dell'acido clorogenico, in particolare come modulatori della via di segnalazione pro-infiammatoria mediata da TLR4. Inoltre, la capacità della PEA di ridurre il rilascio di TNF-α da parte delle cellule microgliali stimolate con LPS è stata corroborata anche nel nostro modello di macrofagi, confermando il suo potenziale antinfiammatorio. Inoltre, RePEA, un analogo sintetico della PEA progettato per essere degradato più lentamente, si è rivelato più attivo del suo composto originario. Questi risultati aiutano a convalidare le suddette molecole naturali, 5-CQA e PEA, ma anche RePEA, come potenziali nuovi candidati per ulteriori indagini precliniche per il trattamento delle malattie autoimmuni e infiammatorie.
For centuries, natural products and their derivatives have provided a rich source of compounds for the development of new immunomodulators in the treatment of human diseases. Natural immune modulators may provide the key to control and ultimately defeat disorders affecting the immune system, by either up- or down-regulating the immune response with few adverse side effects. Many of these compounds are currently undergoing clinical trials, particularly as anti-oxidative, anti-microbial, and anti-cancer agents. However, the functions and mechanisms of action of natural products, and how they interact with the immune system, has yet to be extensively explored. In recent years, the increasing body of knowledge regarding the role of macrophages in the steady-state and in the context of inflammation has opened diverse new avenues of investigation and possibilities for therapeutic intervention targeting the inherent plasticity of macrophages for the treatment of acute and chronic inflammatory disease. Toll-Like Receptors (TLRs), including TLR4, play a crucial role in inflammatory-based diseases, therefore TLR4 signalling has been identified as a therapeutic target for pharmacological intervention. This work aims to screen and investigate the potential of phytoextracts and phytochemicals to affect TLR4 signalling in a macrophage-like cell model. Specifically, extracts from green (GCE) and roasted (RCE) coffee beans as well as pure chlorogenic acid (5-CQA) were tested. Also, the anti-inflammatory effect of palmitoylethanolamide (PEA), and its synthetic analogue RePEA, was assessed. Human monocyte-derived macrophages, deriving from the differentiation of a monocytic cell line (THP-1) and/or from human primary CD14+ monocytes, were employed as an in vitro model. MTT was used to determine cytotoxic effects of the treatments. TLR4 activation was stimulated by exposure of cells to bacterial endotoxin LPS (E. coli), in presence or absence of treatments. Different readouts were evaluated: endpoint pro-inflammatory cytokines production, as well as phosphorylation and nuclear translocation of intracellular signalling mediators. These parameters were measured applying different cellular and molecular techniques, mainly enzyme-linked immunosorbent assay (ELISA), High Content Analysis (immunofluorescence microscopy), and Western blot. Alongside, we employed different commercially available stable transfected cells as tools to investigate the mechanism of action of our tested extract or molecule, THP1-XBlue™, RAW-Blue™ and HEK-Blue™ cells, respectively. Key findings include a dramatic, dose-dependent, inhibitory effect of both green and roasted coffee extracts towards interferon-β (IFN-β) release, upon LPS stimulation. Consistently, chlorogenic acid, a major polyphenolic component of coffee extracts, showed a comparable biological activity. Additionally, novel evidence towards the immunomodulatory effects and mechanism of action of coffee extracts and chlorogenic acid as modulators of TLR4-related pro-inflammatory signalling have been provided. Alongside, PEA capability of reducing TNF-α release from LPS-stimulated microglial cells, was corroborated in our macrophage model also, confirming its anti-inflammatory potential. Moreover, RePEA, a synthetic PEA analogue designed to be degraded slower, was revealed to be more active than its parent compound. These experimental data demonstrate that natural products may act as lead molecules for the development of safe and effective immunomodulators. Taken together, these findings help to validate the above-mentioned natural molecules, 5-CQA and PEA, but also RePEA, as potential novel candidates for further preclinical investigations for the treatment of inflammatory-based disorders.

La superfamília de receptors scavenger rics en cisteïna (SF-SRCR) és un grup de proteïnes de membrana i/o secretades molt antic i conservat al llarg de l’evolució. Els membres de la SF-SRCR es caracteritzen per la presència d’una o més repeticions d’un domini anomenat SRCR, d’aproximadament 100 aminoàcids (aa) i ric en cisteïna. Malgrat l’alt grau de conservació del domini SRCR, no s’ha descrit una funció o lligand comú per tots els membres de la SF-SRCR. Els estudis realitzats a llarg d’aquesta tesi han permès caracteritzar molecular i funcionalment dos membres solubles relativament nous de la SF-SRCR, S5D-SRCRB (soluble 5 domains-SRCR of group B) i S4D-SRCRB (soluble 4 domains-SRCR of group B). A més, s’ha aprofundit en la descripció del paper que juga DMBT1/Hensin en la diferenciació terminal de les cèl•lules intercalades de ronyó. S5D-SRCRB és una glicoproteïna secretòria altament glicosilada, formada per cinc dominis SRCR, espaiats per pèptids rics en els aa Pro, Ser, Thr (PST), a més d’un domini C terminal de tipus mucina. Les glicosilacions que S5D-SRCRB presenta són del tipus N i O. Al seu torn, S4D-SRCRB és una glicoproteïna formada per quatre dominis SRCR també espaiats per pèptids PST, però únicament conté glicosilacions de tipus O. A més de la caracterització bioquímica dels dos membres, la generació d’anticossos monoclonals contra S5D-SRCRB va permetre estudiar el patró d’expressió tissular d’aquesta proteïna. L’estudi va desvetllar que S5D-SRCRB s’expressava principalment als epitelis, on el tracte urogenital presentava els nivells d’expressió més importants a nivell protèic i d’ARN. Pel que fa la funció de S5D-SRCRB i S4D-SRCRB, es va observar que ambdues proteïnes s’unien de manera dosi-depenent a proteïnes de la matriu extracel•lular (fibronectina i laminina). S5D-SRCRB, a més, interaccionava amb galectina-1 i galectina-3, mitjançant els seus dominis d’unió a lectines. D’altra banda, S5D-SRCRB i S4D-SRCRB interactuaven amb components de la paret bacteriana (peptidoglicà i lipopolisacàrids) i fúngica (glucans lineals), actuant com a reconeixedors dels patrons moleculars associats a patògens (pathogen-associated molecular patterns, PAMPs). Les dues proteïnes eren també capaces d’agregar cultius bacterians (E. coli i S. aureus) i fúngics (C. albicans). Per tant, S5D- i S4D-SRCRB podien actuar com a receptors duals capaços de reconèixer estructures endògens i exògenes, i participar en el manteniment de la homeòstasi de l’organisme. La possibilitat que S5D-SRCRB jugués un paper en la defensa dels epitelis contra infeccions es va estudiar amb més detall en el tracte urogenital, emprant models in vitro i in vivo. Els resultats van revelar que l’expressió de S5D-SRCRB augmentava tant en cultius de la línia cel•lular clone C tipus ß expossats a components de la paret bacteriana, com en els ronyons de ratolins a un model d’infecció en el tracte urinari (urinary tract infection, UTI). El patró d’expressió de S5D-SRCRB també canviava durant el procés de diferenciació terminal de les cèl•lules intercalades de ronyó, i durant l’espermatogènesi en els túbuls seminífres. Per aquest motiu s’ha proposat que S5D-SRCRB pot jugar un paper en la diferènciació cel•lular, com s’ha demostrat que passa en el cas de DMBT1/Hensin. La secreció de DMBT1/Hensin és clau en el procés de diferenciació terminal que pateixen les cèl•lules intercalades en condicions d’acidosi, i pel qual passen d’expressar un fenotip de cèl•lula intercalada tipus ß a un de tipus α.
The scavenger receptor cysteine-rich superfamily (SRCR-SF) members are transmembrane and/or secreted receptors exhibiting one or several repeats of a cysteine-rich protein module of +/-100 aa, named scavenger receptor cysteine-rich (SRCR). Despite the high degree of conservation of SRCR domain, non a shared function neither a ligand has been described for all the SRCR-SF members. Studies conducted on this work characterized two relatively new members of the SRCR-SF: S5D-SRCRB (soluble 5-SRCR domains of group B) and S4D-SRCRB (soluble 4-SRCR domains of group B). In addition, we better described the role of DMBT1/Hensin in terminal differentiation of renal intercalated cells. S5D-SRCRB is a highly glycosylated secretory glycoprotein, consisting of five SRCR domains, spaced by peptides rich in Pro, Ser, Thr (PST), and a mucin-like domain at the C terminal extreme. S5D- SRCRB contains N- and O- glycosylations. On the other hand, S4D-SRCRB is a glycoprotein consisting of four SRCR domains also spaced by PST peptides, but only contains O-glycosylations. S4D- and S5D-SRCRB were shown to bind endogenous extracellular matrix proteins (laminin fibronectin), as well as Pathogen-associated molecular patterns (PAMPs) present on Gram-positive and Gram-negative bacteria and fungi. PAMP binding by S4D- and S5D-SRCRB induced microbial aggregation and changes in PAMP-induced cytokine release. These abilities suggest that S4d- and S5D-SRCRB might play a role in the innate defense and homeostasis of certain specialized epithelial surfaces. Immunohistochemical and real-time PCR analysis showed significant S5D-SRCRB expression in murine genitourinary and digestive tracts. S5D-SRCRB expression increased when collecting duct cells were infected. S5D-SRCRB expression pattern also changed during terminal differentiation of renal intercalated, and during spermatogenesis in the seminiferous tubule. It has been proposed that S5D-SRCRB may play a role in cell differentiation, in a similar way it does DMBT1/Hensin. This work has shown that DMBT1/Hensin secretion is required during terminal differentiation carried out by intercalated cells in conditions of acidosis, which drives type ß- intercalated cells to α- intercalated cells.

Gli interferoni (IFN) sono mediatori e regolatori fondamentali della risposta immunitaria dell'ospite a virus e ad altri agenti microbici. Gli IFN di tipo I e di tipo III (o IFN-λ) sono tra le prime citochine ad essere indotte in seguito a infezioni virali. Il legame tra gli IFN e i rispettivi recettori attiva vie di trasduzione del segnale simili tra loro che inducono l'espressione di geni stimolati dagli IFN (ISG) con funzioni antivirali. La caratteristica principale che rende ciascuna di queste famiglie di IFN unica e non ridondante è l'esistenza di recettori distinti che fanno sì che gli IFN-I attivino una risposta ubiquitaria e che gli IFN-III agiscano esclusivamente sulle cellule epiteliali e su un sottoinsieme di cellule immunitarie. Ulteriori distinzioni riguardano la natura meno infiammatoria degli IFN-III e la loro induzione solitamente anticipata rispetto a quella degli IFN-I. Pertanto gli IFN-III sono considerati i difensori di prima linea delle mucose con la capacità di attivare una risposta antivirale precoce senza causare danno tissutale. Se la loro azione risulta insufficiente a contenere l’infezione, il sistema passa all’induzione degli IFN-I, i quali generano una risposta antivirale e infiammatoria più potente e a livello sistemico, che tuttavia può portare ad immunopatologia. Nel corso della mia tesi ho verificato l'ipotesi secondo cui anche gli IFN-III possano causare immunopatologia, in particolare durante infezioni virali delle vie respiratorie e in contesti di danno all’epitelio gastrointestinale in malattie infiammatorie croniche intestinali e lesioni da radiazioni. In primo luogo, io e i miei colleghi abbiamo dimostrato che in un polmone in cui è stata indotta una risposta antivirale, gli IFN-III prodotti dalle cellule dendritiche inibiscono la proliferazione delle cellule epiteliali portando ad una compromissione del rigenerazione della barriera e ad un aumento della suscettibilità ad infezioni batteriche. In seguito abbiamo analizzato la produzione di IFN lungo il tratto respiratorio di pazienti affetti da COVID-19. Abbiamo trovato che, nelle alte vie aeree, l'espressione di IFN-I/III correla con la carica virale e che negli anziani, che presentano un maggiore rischio di sviluppare una patologia severa, questa correlazione è più debole o assente. Una forte espressione di IFN-λ1, IFN-λ3 e ISG caratterizza le alte vie aeree di pazienti con sintomatologia lieve, mentre risultano fortemente espressi gli IFN-I, IFN-λ2 e un insieme di geni antiproliferativi e proapoptotici lungo tutto il tratto respiratorio di pazienti ospedalizzati, suggerendo che possano ostacolare il processo di riparazione dell’epitelio. Infine, abbiamo dimostrato che gli IFN-III ritardano la rigenerazione dell'intestino tenue e del colon in seguito a danno da radiazioni o da colite indotta da destrano sodio solfato, poiché contribuiscono a indurre la morte cellulare delle cellule epiteliali tramite la formazione di un complesso proteico costituito da Z-DNA binding protein (ZBP1) e gasdermin C (GSDMC). I nostri risultati mettono in discussione il ruolo degli IFN-III come protettori delle mucose poiché indicano che quando non propriamente regolati possono causare immunopatologia. Queste evidenze portano alla necessità di progettare l’uso clinico degli IFN di tipo III in modo da evitare le loro funzioni dannose per i tessuti e massimizzarne gli effetti benefici.
Interferons (IFNs) are fundamental mediators and regulators of the host immune response to viruses and other microbial agents. Type I and type III IFNs (also known as IFN-λ) are some of the first cytokines to be induced upon detection of viral infections. Signaling through their specific receptors leads to the activation of a similar signaling cascade that triggers the expression of a common set of IFN-stimulated genes (ISGs) with antiviral effector functions. The main feature that makes each of these families of IFNs unique and nonredundant is the existence of distinct receptors that differentiate them in their ability to act on virtually every cell type (type I IFNs) or exclusively on epithelial cells and a subset of immune cells (type III IFNs). Despite inducing a widely overlapping set of genes, IFN-I can mount a stronger proinflammatory response compared to IFN-III. This, coupled with the earlier induction of IFN-III upon infection, has led to the classification of IFN-III as front-line defenders of mucosal surfaces with the ability to initiate an early antiviral response with minimal tissue-damaging effects. If their response is insufficient the system shifts to the more potent and broader-acting antiviral and inflammatory IFN-I response that can cause immunopathology. In the course of my thesis, I have tested the hypothesis that also IFN-III contribute to immunopathology at barrier sites such as the respiratory and gastrointestinal epithelia during viral infections and inflammatory bowel disease/radiation-induced injury respectively. First, my colleagues and I found that in a mouse model where we mimicked the induction of antiviral responses in the respiratory tract, IFN-III produced by lung dendritic cells inhibited the proliferation of lung epithelial cells leading to an impairment in barrier restoration and an increase in susceptibility to bacterial infections. Then we measured IFN responses along the respiratory tract of COVID-19 patients. We uncovered that in the upper airways expression of IFN-I/III correlated with viral load and elderly patients, that have a higher risk of developing severe COVID-19, had a dysregulation in the IFN response. A strong expression of IFN-λ1, IFN-λ3 and ISGs characterized the upper airways of mild patients. IFN-I and IFN-λ2 together with antiproliferative and proapoptotic genes were upregulated along all the respiratory tract of severe COVID-19 patients, suggesting that they might contribute to the impairment of epithelium restitution. Finally, we demonstrated that IFN-III delayed colon and small intestine repair after dextran sulfate sodium-induced colitis and radiation-induced injury by triggering cell death of epithelial cells via the formation of a novel protein complex that includes Z-DNA binding protein (ZBP1) and gasdermin C (GSDMC). Our findings challenge the role of IFN-III as protectors of mucosal barriers as they indicate that a dysregulated IFN-III response holds the potential to contribute to immunopathology. Therefore, the clinical use of type III IFNs should be designed in such a way that their tissue-damaging functions are avoided and their beneficial effects are maximized.

Se ha observado en nuestro grupo de investigación que tanto las células 3T3L1 como el tejido adiposo blanco in vivo son capaces de liberar, a partir de la glucosa, grandes cantidades de lactato en condiciones aeróbicas y glicerol en mayor cantidad que la que puede justificarse por lipólisis. Para investigar por qué los adipocitos actúan principalmente de manera glucolítica produciendo fragmentos de 3C sin verse afectados por la hipoxia desarrollamos la metodología necesaria aplicada a cultivos primarios. Puesto que el tejido adiposo blanco está compuesto por otros tipos celulares, comprobamos también si la hipoxia afecta de la misma manera a las células de la fracción estromal del tejido. El tejido responde a señales que el organismo produce como hormonas, por ello nos propusimos estudiar en adipocitos y en la fracción estromal, la modulación del metabolismo provocada por la insulina y la respuesta innata del tejido, es decir sin ningún estimulo externo más que el de la glucosa. Los resultados obtenidos señalan que el tejido adiposo blanco tiene una actividad metabólica elevada en términos relativos ya que a pesar de su escasa cantidad de masa “viva” (sólo el 1,3% del total), las células adiposas producen y liberan grandes cantidades de fragmentos de 3C. En los adipocitos, el lactato es producido a partir de la glucosa, independientemente de la presencia de oxígeno e insulina, para ser exportado a otras células y utilizado como fuente de energía. Esta idea se ve reforzada por el hecho que a concentraciones más elevadas de glucosa se produce más cantidad de lactato. Sin embargo, aunque las células de la fracción estromal también producen gran cantidad de lactato, este proceso no se ve influenciado por las concentraciones de glucosa. Por otra parte, el glicerol es producido solo por los adipocitos, siendo regulado por la insulina y constante en el tiempo. No obstante, al inicio el glicerol proviene de la vía glucolítica cuando la concentración de glucosa es elevada, pero cuando esta es baja proviene de la lipolisis. A las 48 h su origen cambia a glucolítico-lipolítico, excepto a 3,5mM de glucosa. Mientras que los ácidos grasos libres se reciclan para formar triacilgliceroles, muy poca glucosa 14C es incorporada, lo que indica una lipogénesis limitada. Sin embargo, la insulina promueve esta lipogénesis, aunque al mismo tiempo limita, al inicio, la incorporación de glicerol-3P para la síntesis de triacilgliceroles. La coexistencia de estos dos procesos, lipogénico y glucolítico a la vez, es probablemente una consecuencia para evitar la acumulación del exceso de energía, como un mecanismo de defensa contra la “glucolipotoxicidad” y una manera de suministrar energía en forma de fragmentos de 3C, fácilmente utilizable por otros tejidos.
Our research group has observed that both 3T3L1 cells and white adipose tissue in vivo are able to release, from glucose, large amounts of lactate under aerobic conditions and glycerol in greater quantity than which can be justified by lipolysis. To study why adipocytes, act mainly in a glycolytic manner producing 3C fragments without being affected by hypoxia, we developed the necessary methodology applied to primary cultures. Since white adipose tissue is composed by other cell types, we also check whether hypoxia affects the stromal fraction cells in the same way. White adipose tissue responds to signals produced by the body as hormones, so we decided to study in adipocytes and the stromal fraction, how insulin can affect the adipocytes metabolism and their innate response, that is without any external stimulus. The results indicate that white adipose tissue has a high metabolic activity in relative terms since, despite its small amount of "living" mass (only 1.3% of the total), adipose cells produce and release large amounts of 3C fragments. In adipocytes, lactate is produced from glucose, independently of the presence of oxygen and insulin, to be exported to other cells and used as an energy source. This idea is reinforced by the fact that at higher concentrations of glucose, more lactate is produced. However, although the stromal fraction cells also produce large amounts of lactate, this process is not influenced by glucose concentrations. On the other hand, glycerol is produced only by adipocytes, being regulated by insulin and constant over time. However, initially glycerol comes from the glycolytic pathway, when the glucose concentration is high, but when it is low it comes from lipolysis. At 48 h, its origin changes to glycolytic-lipolytic, except at 3.5 mM glucose. While free fatty acids are recycled to form triacyclglycerols, very little 14C glucose is incorporated over time, indicating limited lipogenesis. But when insulin is added, this lipogenesis is promoted, even though at the same time the hormone limits, at the beginning, the incorporation of glycerol-3P for the synthesis of triacylglycerols. The coexistence of these two processes, lipogenic and glycolytic simultaneously, is probably a consequence to prevent the accumulation of energy excess as a defense mechanism against "glycolipotoxicity", and a way to supply easily usable energy by other tissues in the form of 3C fragments.

La tubercolosi (TB) è la malattia infettiva, causata da un singolo agente infettivo, più diffusa a livello mondiale ed è responsabile di più di due milioni di decessi ogni anno. L’agente eziologico della tubercolosi è il Mycobacterium tuberculosis (MTB), un patogeno intracellulare aerobio obbligato che infetta l’uomo, preferibilmente per via aereosolica, riuscendo a sopravvivere a livello intracellulare. Un meccanismo d’evasione che MTB attua per sfuggire all’azione micobattericida del macrofago è rappresentato dalla maturazione del fagolisosoma e dall’inibizione dell’enzima fosfolipasi D (PLD), il cui prodotto metabolicamente attivo è l’acido fosfatidico (PA). In questa Tesi abbiamo studiato il ruolo di ligandi d’origine microbica, quali le sequenze CpG, e ligandi naturali, come Acido Lisofosfatidico (LPA) e Sfingosina 1-fosfato (S1P), nella risposta immunitaria innata antimicobatterica in cellule epiteliali alveolari e in cellule della linea monocito/macrofagica. In questo contesto, abbiamo dimostrato che, in entrambi i tipi cellulari, la stimolazione con CpG, LPA o S1P può i) inibire la crescita intracellulare di MTB attraverso l’azione della PLD, ii) indurre l’attivazione della PLD Ca++-dipendente, iii) promuovere la maturazione del fagolisosoma mediate il coinvolgimento della PLD in cellule infettate con MTB. Questi risultati indicano che le cellule epiteliali alveolari, oltre ai monociti-macrofagi, possono svolgere un ruolo attivo nell’ambito della risposta immunitaria innata antitubercolare e che l’attivazione della PLD con la conseguente produzione di PA, è cruciale per l’attivazione della risposta antimicrobica basata sulla maturazione fagolisosomale. Su queste basi, abbiamo ideato liposomi in grado di veicolare secondi messaggeri lipidici, come il PA la cui produzione è soppressa in corso di infezione con MTB, in grado di ristabilire o potenziare la risposta immunitaria innata micobattericida. Abbiamo, quindi, prodotto liposomi asimmetrici simili a corpi apoptotici caratterizzati dalla presenza di fosfatidilserina (PS) sul foglietto lipidico esterno e PA nel foglietto interno. I nostri risultati indicano che tali liposomi possono essere efficacemente internalizzati nei macrofagi senza indurre segnali proinfiammatori. Inoltre, la stimolazione di macrofagi umani con questi liposomi i) induce un incremento dei flussi di Ca++, ii) promuove la maturazione del fagolisosoma Ca++-dipendente e iii) riduce la crescita intracellulare micobatterica in maniera dipendente dalla maturazione fagolisosomale. In conclusione, questi risultati suggeriscono la possibilità di usare i liposomi come “cavalli di Troia” per veicolare secondi messaggeri lipidici e ripristinare quei segnali molecolari frequentemente inibiti dai patogeni intracellulari come strategia di sopravvivenza nella cellula ospite.
Tuberculosis (TB) is a worldwide infection disease, due to a single pathogen infection, which is estimated to kill over 2 million people annually. The causative agent of TB is Mycobacterium tuberculosis (MTB), an intracellular pathogen which infects human host and interferes with host antimicrobial pathways to allow intracellular survival, inhibiting Phospholipase D (PLD) dependent Phosphatidic Acid (PA) generation. The present study shows a novel role of synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN), Lysophosphatidic Acid (LPA) and Sphingosine 1-phosphate (S1P) in the activation of antimicrobial immune response by innate immune cells, such as human monocytes/macrophages and type II human alveolar epithelial cells alveolar. In this context, we found that CpG, LPA and S1P stimulation i) induce Ca++-dependent PLD activation, ii) promote PLD-dependent phagolysosome maturation, and iii) enhance a PLD dependent intracellular mycobacterial killing. These results show that alveolar epithelial cells may represent efficient effecter cells of innate antimycobacterial immune response and that PLD activation i) is crucial for the activation of phagolysosome maturation-based antimicrobial response, ii) is conserved among cell types and iii) is at the intersection of different metabolic pathways, independent of the upstream stimulus received. In order to deliver these second lipid messengers able to recover or enhance host antimicrobial innate immune response to infected cells, a technological platform has been developed. In this context, apoptotic body-like liposomes characterized by the presence of phosphatidylserine (PS) in the outer lipid leaflet were engineered to contain PA in the inner lipid surface. We demonstrate that these liposomes can be internalized by macrophages and reduce production of proinfiammatory cytokines (IL-beta, TNF-alpha, IL-12, IL-23) by simultaneously inducing the production of IL-27. Moreover, the stimulation of human macrophages with apoptotic body like liposomes lead to Ca++ mobilization, Ca++-dependent phagolysosome maturation, and phagolysosome maturation dependent intracellular mycobacterial killing. Altogether, these results suggest the possibility to use apoptotic-like vesicles as Trojan particles to deliver second lipid messengers to restore those molecular pathways inhibited by intracellular pathogens as an intracellular survival strategy.

Evalúa el efecto inmunomodulador del fucoidan de Lessonia trabeculata en un modelo murino. Se trataron ratones por vía oral con fucoidan de L.trabeculata y se inmunizaron con glóbulos rojos de carnero (GRC). Un grupo de ratones recibió una dosis de 5 y otro, de 10 mg/kg p.c. Se emplearon 2 grupos controles: uno fue tratado con fucoidan de Fucus vesiculosus a la dosis de 5 mg/kg p.c. y otro, con solución salina (0.9%). En los 4 grupos se evaluó tanto la respuesta inmune primaria, como la secundaria. Concluidos los tratamientos se extrajeron los macrófagos peritoneales para evaluar parámetros de la inmunidad innata; Especies reactivas de oxígeno (ERO), especies reactivas de nitrógeno (ERN) y fagocitosis; y parámetros de la inmunidad humoral: presencia de anticuerpos hemaglutinantes (IgM) y opsonizantes (IgG). Los resultados obtenidos para los citados parámetros fueron contrastados con la presencia de mRNA transcripcional para IL-4, IL-5, IL-6 e IL-10 en esplenocitos. Adicionalmente se determinó la presencia de IL-10 sérico por ELISA. Los resultados indican que el fucoidan puede aumentar la producción de ERO en la respuesta inmune primaria (RIP), no detectándose diferencias en la respuesta inmune secundaria (RIS). La producción de ERN aumentó de forma dosis dependiente en la RIP y RIS. La fagocitosis mediada por anticuerpos en la RIS aumentó significativamente solo en el grupo tratado con fucoidan de L. trabeculata con dosis 10 mg/kg p.c. También se observó que la expresión transcripcional de IL-4, IL-5, IL-6 e IL-10 por esplenocitos fue modulada y dependiente del tratamiento, dosis y tipo de respuesta inmune. En contraste, no se encontraron diferencias estadísticamente significativas entre L.trabeculata y F.vesiculosus, y dosis en la producción de hemaglutininas anti-GRC, ni en la concentración de IL-10 en suero. Se concluye que el tratamiento con fucoidan de L. trabeculata por vía oral en ratones inmunizados posee capacidad de estimular diferentes parámetros inmunes como la producción de ERO y ERN, fagocitosis mediada por anticuerpos y expresión transcripcional de IL-4, IL-5, IL-6 e IL-10.
Tesis

Le cellule fagocitiche; monociti, macrofagi e neutrofili, sono i maggiori componenti della immunità innata. Tali cellule rispondono a stimoli infiammatori e/o immunitari attraverso meccanismi di difesa che si esplicano tramite la fagocitosi, la produzione di citochine e la generazione di specie reattive dell’ossigeno (ROS). I meccanismi di difesa operati dai fagociti sono selettivamente influenzati dal pH intracellulare (pHi) che è in genere regolato da differenti trasportatori di membrana tra cui lo scambiatore sodio/idrogeno (NHE). L’NHE opera l’influsso di ioni sodio ed il contemporaneo efflusso di ioni idrogeno in accordo con i rispettivi gradienti di concentrazione. Le funzioni di NHE non sono ristrette alla regolazione del pHi: lo scambiatore esercita anche un importante ruolo in diverse funzioni biologiche tra cui la proliferazione e il differenziamento cellulare, l’apoptosi e la riorganizzazione citoscheletrica. Inoltre, in diversi tumori, il microambiente extracellulare è più acido di quello che caratterizza i tessuti normali, e in queste condizioni l’NHE rappresenta l’unico sistema in grado di assicurare l’omeostasi del pHi. A questo riguardo, nel modello di carcinoma epatico umano (HepG2), i livelli di mRNA di NHE come l’attività dello scambiatore sono 10 e 3 volte superiori rispetto a quanto riportato per i normali epatociti. Variazioni del pHi in risposta a differenti agonisti possono rappresentare dei segnali precoci capaci di contribuire alla regolazione dell’attività delle fosfolipasi; una famiglia di enzimi in grado di generare dei lipidi bioattivi tra cui il diacilglicerolo (DAG) e l’acido fosfatidico (PA). Il DAG e il PA possono attivare l’enzima NADPH ossidasi, che rappresenta una importante sorgente di ROS nei fagociti. La NADPH ossidasi svolge un importante ruolo anche in altri sistemi cellulari tra cui le HepG2, dove la sua attivazione appare essere positivamente correlata all’inibizione della proliferazione cellulare. I monociti ed i macrogafi in risposta a degli stimoli infiammatori sono in grado di rilasciare il peptide natriuretico atriale (ANP), un piccolo ormone principalmente sintetizzato dai cardiomiociti atriali capace di promuovere la natriuresi, la diuresi e di contribuire alla regolazione della pressione sanguigna. L’ANP può anche regolare alcune funzioni immunitarie in quanto capace di contrastare l’espressione di alcuni mediatori infiammatori tra cui la ciclossigenasi-2 (COX2), l’ossido nitrico (NO) e il fattore di necrosi tumorale (TNF)-α.Sulla base dell’importante ruolo del pHi e della possibile relazione tra NHE e produzione di ROS, il presente studio è stato incentrato nel valutare l’effetto dell’ANP sul pHi, sull’attività di alcune fosfolipasi (Ce D) e sulla produzione di ROS nei monociti/macrofagi umani e nelle cellule HepG2. I risultati ottenuti hanno evidenziato un significativo decremento del pHi dovuto all’inibizione di NHE dopo stimolazione con ANP nei macrofagi e nelle cellule HepG2. Sia i monociti che i macrofagi si siano rivelati in grado di esprimere l’intero set dei recettori per i peptidi natriuretici (NPR-A,NPR-B e NPR-C), ma nessuna significativa variazione del pHi è stata evidenziata nei monociti stimolati con ANP. Tuttavia, il trattamento dei monociti con 5-(etil-N-isopropil)amiloride, uno specifico inibitore di NHE è stato in grado di riprodurre il modello di acidificazione osservato nei macrofagi dopo stimolazione con ANP. Nei macrofagi il decremento ANP-dipendente del pHi è apparso parallelo ad un incremento dell’attività della PLD e della PLC, mentre nelle cellule HepG2 l’acidificazione intracellulare è stata correlata unicamente dell’attivazione della PLD. Il decremento ANP-dipendente del pHi, è stato correlato alla produzione di messaggeri lipidici bioattivi quali il DAG e il PA che si sono dimostrati responsabili dell’attivazione della NADPH ossidasi sia nei macrofagi che nelle cellule HepG2. Infine, tutti gli effetti mediati dall’ANP sono stati riprodotti utilizzando un analogo troncato dell’ormone denominato cANF specifico per i recettori NPR-C.
Phagocytes, namely monocytes, macrophages, and neutrophils, are major components of innate immunity. They respond to inflammatory/immune insults by up-regulating their host defense functions, including phagocytosis, cytokine production, and generation of reactive oxygen species (ROS). The host defense functions of phagocytes are selectively influenced by intracellular pH (pHi) that is controlled by which several plasmamembrane acid-base transporters, including the Na+/H+ exchanger (NHE) which operate the exchange of extracellular Na+ with cytoplasmic H+ ions according to the concentration gradient. The functions of NHE are not only restricted to pHi homeostasis: the exchenger plays also an important role a variety of downstream events, including cell proliferation, cell differentiation, apoptosis, and cytoskeletal organization. Moreover, in several tumors, the extracellular microenviroment is more acidic with respect to normal tissues and, in these conditions, the NHE represents the only system able to regulate pHi homeostasis. In this contest, it was reported , in hepatocellular carcinoma (HepG2 cells), that NHE mRNA levels as well as the exchanger activity are respectively 10-and-3 fold higher than in normal hepatocytes, Changes in pHi in response to a variety of ligands may represent a signalling event for the regulation of phospholipase activities: a family of enzymes able to generate bioactive lipids such as diacyglycerol (DAG) and phosphatidic acid (PA). DAG as well as PA can activate the enzyme NADPHoxidase, an important source of ROS in phagocytes. The NADPHoxidase plays an important role also in other cell systems including HepG2, where its activation appears positively coupled to the inhibition cell proliferation. It is well known that monocytes and macrophages in response to inflammatory insults can release the atrial natriuretic peptide (ANP), an hormone mainly secreted by the heart atria able to induce natriuresis, vasodilation and contribute to the regulation of blood pressure. The ANP can also regulate several immune functions since its able to reduce production of proinflammatory mediators by inhibition of nitric oxide (NO), and cyclooxygenase-2 (COX2) as well as tumor necrosis factor (TNF)-α synthesis. On the basis of both the important role of pHi and the possible relationship between NHE and ROS generation, the present study was aimed to evaluate the effects of atrial natriuretic peptide (ANP) on intracellular pH (pHi), phospholipase (C and D) activities and (ROS) production in human monocytes, macrophages and HepG2 cells. A significant pHi decrease due to the NHE inhibition was observed in ANP-stimulated macrophages as well as in HepG2 cells. Conversely, even if both monocytes and macrophages were show to express all three natriuretic peptide receptors (NPR-A, NPR-B, and NPR-C), no significant effect on pHi was observed in monocytes stimulated with ANP. Nevertheless, the treatment of monocytes with 5-(N-ethyl-N-isopropyl)amiloride, a specific inhibitor of NHE was able to determine a decrease of pHi which was similar to the one observed in macrophages after ANP stimulation. In human macrophages the ANP-induced pHi decrease was paralleled by an increased activity of both phospholipase D (PLD) and phospholipase C (PLC), whereas in HepG2 cells the intracellular acidification was correlated only to an increased PLD activity. Our results suggest that second lipid messengers produced after ANP-induced pHi decrease, such DAG and PA, were able to promote the NADPH oxidase activation in human macrophages as well as in HepG2 cells. Finally, all ANP-effects were mediated by NPR-C receptors.

Introducción: La enfermedad cardiovascular se presenta de forma muy precoz en los pacientes con enfermedad renal crónica (ERC) y trasplantados renales, siendo la principal causa de mortalidad en estas poblaciones. Los factores de riesgo clásicos de enfermedad cardiovascular como la hipertensión y la diabetes son muy prevalentes en la enfermedad renal crónica pero esto no explica el enorme incremento del riesgo cardiovascular. Los factores de riesgo no clásicos como la disfunción endotelial, inflamación subclínica, las alteraciones de la Mannose binding lectin (MBL) que es un componente de la inmunidad innata, son un hecho importante en la ERC y trasplante. La presencia y progresión de aterosclerosis subclínica evaluada mediante ecografía carotidea, permite la mejor estratificación del riesgo cardiovascular. Dicho esto, el objetivo de esta tesis es evaluar si el trasplante modifica de forma independiente del grado de insuficiencia renal la presencia y progresión aterosclerosis subclínica. Material y métodos: En dos cohortes de pacientes con enfermedad renal crónica y trasplantados renales con filtrado glomerular estimado (FG-e) <60 ml/min/1.73 m2 no en diálisis, se determinaron en la visita basal, niveles de ADMA, VEGF, ICAM-1, IL-6, TNFR2, MCP-1 y MBL, se cuantificaron el número de células progenitoras endoteliales y células circulantes endoteliales. Se realizó monitorización ambulatoria de presión arterial de 24 horas (MAPA), índice tobillo brazo, velocidad de onda de pulso (VOP) y ecografía carotidea con la cuantificación del grosor de intima media y número de placas carotideas. Se realizó seguimiento de los pacientes a los 18 meses con realización de VOP y ecografía carotidea y a los 36 meses, se realizaron MAPA 24 horas, ITB, VOP y ecografía carotidea, y se registraron los eventos renales y cardiovasculares. Resultados: la presión arterial en la consulta fue similar en ambos grupos. La presión arterial por MAPA de 24h (133.9±14.3 vs. 126.2±16.1, P=0.014), PAS diurna (135.6±15.2 vs. 128.7±16.2, P=0.042), y PAS nocturna (131.2±16.2 vs. 120.2±17.9, P=0.0014) fue más elevada en los trasplantados renales. Además, presentaban una mayor proporción de pacientes con hipertensión arterial nocturna y non dipper (68.5% vs. 47.4%,P=0.03). Para estudiar la contribución del trasplante a esta diferencia, se incluyó en el modelo multivariado el hecho de ser trasplantado como una variable independiente, resultando que para la PAS 24h, diurna y nocturna, el trasplante era un predictor independiente para un incremento de PAS. Se observó que los trasplantados renales presentaban unos niveles de IL-6 más elevados así como una mayor proporción de pacientes con placas carotideas (55.4% vs 30%, P=.016) con respecto a los pacientes con ERC. Las variables que se asociaban a la PAS 24 horas fueron la proteinuria y los monocitos circulantes (P=.001), mientras que IL-6, creatinina sérica y el número total de placas se asociaban de forma independiente a la ausencia de caída de la presión arterial nocturna (P=.0001). Conclusiones: La MAPA de 24 horas está más elevada en los trasplantados, a expensas de la hipertensión nocturna y presentan una mayor proporción de pacientes con hipertensión arterial nocturna y patrón "non dipper", lo que sugiere un peor perfil de riesgo cardiovascular. Existe un mayor grado de inflamación subclínica en el paciente trasplantado renal evaluado mediante el número de monocitos circulantes y los niveles de IL-6. Los pacientes trasplantados tienen más aterosclerosis subclínica que los pacientes con ERC a igualdad de función renal y proteinuria. La ausencia de caída de presión arterial o patrón "non dipper" en el trasplante se asocia a la inflamación sistémica medida mediante la IL-6 y a la carga aterosclerótica.
Introduction: Cardiovascular disease is prevalent in patients with chronic kidney disease (CKD) and kidney transplants and constitutes a main cause of death in these patients. The classical cardiovascular risk factors (i.e. hypertension and diabetes) are very prevalent but they do not explain the increase of cardiovascular risk and mortality. Moreover, non-traditional cardiovascular risk factors such as endothelial dysfunction, subclinical inflammation, mannose binding lectin alterations, are important in CKD and kidney transplants recipients. The presence and progression of subclinical atherosclerosis evaluated by carotid ultrasound, allows a better stratification of cardiovascular risk in general populations and populations at risk. Thus, the aim of this doctoral thesis is evaluate if kidney transplants modify the presence and progression of subclinical atherosclerosis independently of renal function. Material and methods: Two cohorts of patients with CKD and kidney transplants recipients with glomerular filtration rate (GFR-e) < 60 ml/min/1.73 m2 not on dialysis were included. In basal visit levels of ADMA, VEGF, ICAM-1, IL-6, TNFR2, MCP-1 and MBL were determined, number of endothelial progenitor cells and circulating endothelial cells were quantified. Ambulatory blood pressure monitoring (ABPM), ankle-brachial index, pulse wave velocity (PWV) and carotid ultrasound were performed at basal visit and 36 months of follow-up. At 18 months of follow up, a PWV and carotid ultrasound were recorded. In each visit, cardiovascular and renal events were registered. Results: Office BP was not different between transplants and CKD patients (139.5±14.3 vs. 135.2±19.3, P=1.00, respectively). ABPM 24-hr systolic blood pressure (SBP) (133.9±14.3 vs. 126.2±16.1, P=0.014), awake SBP (135.6±15.2 vs. 128.7±16.2, P=0.042), and sleep SBP (131.2±16.2 vs. 120.2±17.9, P=0.0014) were higher in renal transplants. When patients were classified according to BP patterns associated with highest cardiovascular risk, the proportion of patients with both nocturnal hypertension and non-dipper pattern was higher in transplants (68.5% vs. 47.4%,P=0.03). In the multivariate regression analysis, transplantation was an independent predictor of 24-hr, awake, and sleep SBP. Log IL-6 (P=.011), and total number of carotid plaques (P=.013) were higher, while the percentage decline of SBP from day to night was lower in kidney transplant recipients (P=.003). Independent predictors of 24-hour SBP were urinary protein/creatinine ratio and circulating monocytes (P=.001), while Log IL-6, serum creatinine, and total number of carotid plaques (P=.0001) were independent predictors of percentage decline of SBP from day to night. Conclusions: Office BP is similar in kidney transplants and CKD patients with similar renal function. On the contrary, hypertension is more severe in kidney transplants when evaluated with ABPM mainly as a result of increased sleep systolic BP. Thus, precise evaluation of hypertension in kidney transplants requires ABPM. Kidney transplants presents a higher levels of IL-6 and more subclinical atherosclerosis, and subclinical atherosclerosis and systemic inflammation are associated with hypertension after transplantation.

Le infezioni fungine invasive rappresentano un'importante causa di morbilità e mortalità nei pazienti affetti da malattie linfoproliferative. Negli ultimi anni, l'introduzione di nuovi farmaci, in particolare gli inibitori della Bruton tirosin-chinasi (BTK), ha permesso di migliorare drasticamente la prognosi dei pazienti affetti da leucemia linfatica cronica (LLC) e da altri disordini clonali delle cellule B. Sebbene queste molecole fossero inizialmente considerate meno immunosoppressive rispetto alla classica chemio-immunoterapia, un numero crescente di segnalazioni hanno descritto l'insorgenza di infezioni fungine opportunistiche inattese, in particolare l'Aspergillosi invasiva, nei primi 6 mesi di trattamento. La BTK rappresenta una molecola cruciale nella trasmissione della cascata del segnale da diversi recettori, come i pathogen-associated molecular patterns (PAMP), cioè β-glucani, chitina e mannani, CD11b/CD18, triggering receptor expressed on myeloid cells 1 (TREM-1) e Toll-like receptors (TLR), che permettono il riconoscimento dei funghi da parte del braccio innato dell'immunità. Tuttavia, i meccanismi immunopatogenetici alla base dello sviluppo di infezioni fungine invasive in pazienti trattati con inibitori della BTK non sono stati ancora completamente delucidati. Il nostro studio, basato su diversi saggi funzionali immunologici in vitro, mira a caratterizzare gli effetti specifici degli inibitori della BTK sulla risposta immunitaria innata contro i funghi mediata da neutrofili, monociti, macrofagi e piastrine, ottenuti da pazienti con linfomi a cellule B. L'inibizione farmacologica della BTK si è associata a una ridotta trasmissione delle vie di segnale attivate da Aspergillus fumigatus, determinante un difetto significativo nella secrezione di citochine infiammatorie nei neutrofili, nei macrofagi e nei monociti, isolati da pazienti e donatori sani. Nei neutrofili, l'inibizione della BTK ha ridotto in modo significativo l'attività fagocitaria (decremento medio del 51,6% e del 37,6% con due diversi farmaci inibitori della BTK). Allo stesso modo, si è riscontrata una notevole riduzione della funzione fagocitaria dei macrofagi associati alla LLC (dette nurse-like cells, NLC), così come dei monociti circolanti CD14+ (sia nei pazienti affetti da LLC che nei volontari sani). Concordemente, la secrezione di TNF-α da parte delle NLC, valutata con il saggio di secrezione citochinica (cytokine secretion assay, CSA), è stata fortemente indotta dalla stimolazione con i conidi di Aspergillus fumigatus, mentre è risultata notevolmente soppressa da due diversi inibitori della BTK. Inoltre, per la prima volta, abbiamo dimostrato che l'esposizione agli inibitori della BTK compromette diverse funzioni immunitarie delle piastrine, quali l'attivazione (come indicato dai bassi livelli di espressione della P-selectina), l'attività di uccisione diretta e la capacità di aderire ai conidi. Il test di danno ifale (hyphal damage test), eseguito su neutrofili, monociti, macrofagi e piastrine in presenza di inibizione farmacologica della BTK, ha documentato una notevole riduzione dell'attività litica antimicotica in tutti i suddetti tipi di cellule, sia nei pazienti che nei volontari sani. Nel complesso, questi effetti concorrono all’inefficacia nel contrastare completamente la germinazione dei conidi. I nostri risultati hanno rivelato specifiche modifiche nella risposta innata indotte dall'inibizione della BTK in pazienti con neoplasie delle cellule B, dimostrando il ruolo di questa chinasi come "guardiano" dell'immunità innata.
Invasive fungal diseases (IFDs) represent an important cause of morbidity and mortality in patients affected by lymphoproliferative diseases. In the recent years, the introduction of new drugs, in particular those targeting Bruton tyrosine kinase (BTK), has allowed dramatic improvement in the prognosis of patients with chronic lymphocytic leukemia (CLL) and other B-cell clonal disorders. Although these small molecules were initially considered less immunosuppressive than chemo-immunotherapy, an increasing number of reports have described the occurrence of unexpected opportunistic fungal infections, in particular invasive aspergillosis, in the first 6 months of treatment. BTK represents a crucial molecule in the transmission of signaling cascade from several immune receptors, such as pathogen-associated molecular patterns (PAMPs, i.e. β-glucans, chitin and mannans), CD11b/CD18, triggering receptor expressed on myeloid cells 1 (TREM-1) and Toll-like receptors (TLRs), that allow the recognition of fungi by the innate arm of the immunity. However, the immunopathogenetic mechanisms underlying the development of IFDs in patients treated with BTK inhibitors are not fully elucidated, and multiple pathways might be involved. Our in vitro study, based on different immunological functional assays, aimed at characterize the specific off-target effects of BTK inhibitors on anti-mold innate immune response mediated by neutrophils, monocytes, macrophages, as well as platelets, obtained from patients with B-cell neoplasms. Pharmacological inhibition of BTK reduced signaling pathways activated by Aspergillus fumigatus, determining an exacerbation of an immunosuppressive signature and a significant defect in the secretion of inflammatory cytokines, either in neutrophils, macrophages and monocytes isolated from patients and healthy donors. In neutrophils, the inhibition of BTK significantly hampered the phagocytic activity (mean reduction of 51,6% and 37,6%, with two different BTK-inhibitors). Similarly, we found a remarkable reduction in the phagocytic function of CLL-associated macrophages (nurse-like cells, NLC), as well as of CD14+ circulating monocytes (either in CLL patients and healthy volunteers). Concordantly, secretion of TNF-α by NLC, assessed by cytokine secretion assay (CSA), was strongly induced by pulsing the cells with Aspergillus fumigatus conidia and was markedly reduced by two different drugs targeting BTK. Furthermore, we demonstrated, for the first time, that the exposure to BTK-inhibitors impairs several immune functions of platelets, i.e. activation (as indicated by the low levels of P-selectin expression), direct killing activity and ability to adhere to conidia. Hyphal damage test, performed on neutrophils, monocytes, macrophages and platelets in the presence of pharmacological BTK-inhibition, documented a striking reduction of the anti-hyphal lytic activity in all aforementioned cell types, either in patients and healthy volunteers. Overall, these effects concur to a failure in completely counteracting conidia germination. Our results revealed specific modifications in the innate response in patients with B-cell neoplasms induced by the inhibition of the BTK pathway, underlying the importance of this targetable kinase as a “guardian” of the innate immunity.

2008/2009
We are exposed daily to a myriad of potential pathogens, mainly through dermal contact, ingestion and inhalation. The innate immune system is of crucial importance in preventing pathogens from colonizing and growing to a point where they can cause life-threatening infections. Different types of cationic antimicrobial peptides (AMP), also known as host defense peptides (HDP), are among the innate immune mechanisms involved in this protective activity. -defensins are an important class of antimicrobial peptides that are present in human beings and have been widely reported to display a direct, salt and medium sensitive bactericidal activity, in vitro, against a broad spectrum of bacteria and fungi. Moreover, there is increasing evidence that they may play a significant role in alerting and enhancing other components of innate and adaptive immunity, thus also playing an indirect role in defense against microbes. Human -defensins are induced in vivo at the sites of microbial invasion, where they are thought to provide a bactericidal barrier, and to form chemotactic gradients that contribute to the recruitment of immune cells to the site of infection. Their interaction with the biological membranes of both microbial and host cells, appears to play a central role in both these types of activities. In this thesis, I was interested in characterizing the role of -defensin hBD2 in modulating activities of important immune host cells, such as monocytes, macrophages and immature dendritic cells, and its ability to bind to and/or be internalized within those cells, in relation to a possible immunomodulatory role. Initially, hBD2 was chosen amongst the human -defensins, and the native peptide synthesized, folded and characterized for antimicrobial activity and cytotoxicity. A fluorescent conjugate, as well as several structural analogues were also designed and prepared, and were respectively used to probe internalization and structure/activity relationships for this defensin. Subsequently, the immunomodulatory potential of hBD2 was probed, considering its possible effects in modulating important processes such as chemotaxis, degranulation, phagocytosis and reactive oxygen species (ROS) production of immune cells it comes into contact with. From this investigation, it emerged that hBD2 displays different effects depending on the cell type which comes into contact with it, and the exposure time. In particular, we confirmed the ability of hBD2 to induced chemotaxis of myeloid derived immature dendritic cells (iDC), contributing to recruitment of these cells to site of infection, although, from our studies, the mechanism of action turned out to be rather more complex than that which had been previously been proposed, and our observations may help reconcile apparently conflicting literature reports. Some novel additional properties of hBD2 were then described. In particular, a short exposure of iDC to hBD2 causes a cell type-dependent degranulation process, which could be involved in the tolerogenic/immunogenic roles of these cells, supporting a further immunomodulatory role for the defensin. A longer term exposure to hBD2 seems to augment the ability iDC to recognize bacteria and interact with them, possibly improving their ability to present antigens and activate adaptive immune responses. An equivalent treatment of macrophages resulted in a significant increase in their phagocytic activity and release of ROS, thus allowing these immune cells to better respond against a bacterial invasion. All these activities were evidenced in vitro at relatively low (micromolar) and sub-cytotoxic concentrations, which are compatible with the likely hBD2 concentration at sites of infection. Moreover, contrary to the direct bactericidal activity, these activities were not found to be salt or medium sensitive, but occur in conditions closely resembling the physiological ones. The activities of hBD2 were also compared to those of the structural analogues to study whether specific structural features of this defensin are required for its activity on immune cells. Results were somewhat puzzling because the evolutionarily conserved defensin scaffold seems to be quite important for exerting some biological function, such as the antimicrobial activity and phagocytosis, but not others, such as chemotaxis. This observation and the time and cell-type dependent activity may point to the concurrent existence of multiple modes of action. In the last part of this thesis, I describe the interaction of fluorescently labelled hBD2 with immune cells, with the aim of determining whether a cellular internalization could play a part for its biological effects. hBD2 was able to interact in a cell specific manner with different types of immune cells, upon short term exposure, leading to differentiated binding and cellular uptake, in a process devoid of cytotoxic or permeabilizing consequences. Macrophages were the most efficient in peptide internalization, whereas iDC seemed to be more avid for peptide surface binding than peptide internalization. The mechanism by which this occurs is as yet unknown, but our preliminary flow-cytometric data indicated that uptake of the defensin was an energy-dependent and temperature-sensitive process, which depends on actin fibers and can reasonably be supposed to involve lipid rafts, and/or clathrin-mediated endocytosis. Moreover, confocal microscopy of macrophages treated with labelled peptide showed that it may interact with specific patches on the membrane and, on binding, the peptide rapidly re-localized in the cytoplasm of cells with a characteristic punctate distribution. Taken together, these data could suggest that the peptide selectively binds to specific sites on membrane, such as lipid rafts, and that endocytosis could be a general mechanism of hBD2 internalization. These observations may thus indicate that hBD2 is capable of modulating activities of host cells not only by interacting with the membrane and surface receptors, but also with cytoplasmic targets. Noi siamo costantemente esposti a una miriade di potenziali patogeni, principalmente attraverso il contatto, l’ingestione e l’inalazione. Il sistema immunitario innato è di importanza cruciale nel prevenire la colonizzazione e la proliferazione dei patogeni che possono causare infezioni pericolose per la sopravvivenza. Tra i diversi meccanismi del sistema immunitario innato coinvolti in questa azione protettiva si contano diversi tipi di peptidi antimicrobici di natura cationica (AMP), anche noti come peptidi di difesa dell’ospite (HDP). Le -defensine sono un’importante classe di peptidi antimicrobici presenti anche nell’uomo che hanno dimostrato di possedere in vitro un’azione battericida diretta sensibile alla concentrazione salina nei confronti di un largo spettro di batteri e funghi. Inoltre è stato dimostrato che esse giocano un ruolo significativo nell’allertare e potenziare altri componenti dell’immunità innata e adattativa, esercitando così anche un’azione indiretta nella difesa contro i patogeni. Le -defensine umane vengono indotte in vivo nei siti dove si verifica un’invasione batterica, formando così una barriera battericida e creando un gradiente chemiotattico che contribuisce a reclutare cellule immunitarie nel sito d’infezione. La loro interazione con le membrane biologiche, sia dei microbi che delle cellule dell’ospite, sembra essere centrale in entrambe le attività. In questa tesi l’interesse principale è caratterizzare il ruolo della -defensina hBD2 nel modulare l’attività di importanti cellule immunitarie, quali monociti, macrofagi e cellule dendritiche immature, e la sua abilità di legarsi a tali cellule e/o di essere internalizzata da esse. Inizialmente, tra le -defensine umane è stata scelta hBD2 e il peptide naturale è stato sintetizzato, “foldato” e caratterizzato per l’attività antimicrobica e la citotossicità. Un coniugato fluorescente e molti analoghi strutturali sono stati inoltre progettati e sintetizzati e sono stati utilizzati, rispettivamente, per indagare l’eventuale internalizzazione e per fare studi di relazione fra struttura e attività. Successivamente è stato analizzato il potenziale immunomodulatorio di hBD2, considerando i suoi possibili effetti nel regolare importanti processi delle cellule immunitarie con cui il peptide viene in contatto, quali ad esempio la chemiotassi, la degranulazione, la fagocitosi e la produzione di specie reattive dell’ossigeno (ROS). Da questa analisi è emerso che hBD2 esercita diverse azioni a seconda del tipo cellulare con cui viene a contatto e del tempo di esposizione delle cellule alla defensina. In particolare, è stata confermata l’abilità di hBD2 a indurre la chemiotassi di cellule dendritiche immature (iDC), contribuendo a reclutare tali cellule al sito d’infezione. Negli studi condotti è però emerso che il meccanismo d’azione potrebbe essere più complesso di quanto proposto fino ad ora; tuttavia le osservazioni proposte in questa tesi appaiono in grado di conciliare i dati contrastanti riportati in letteratura. Sono inoltre emerse alcune nuove proprietà per hBD2. In particolare, un breve tempo di esposizione delle iDC alla defensina causa un processo di degranulazione che risulta dipendente dal tipo cellulare e che potrebbe essere coinvolto nel ruolo immunogenico/tolerogenico di queste cellule, supportando un ruolo immunomodulatorio per hBD2. Un’esposizione per tempi più lunghi a hBD2 sembra incrementare l’abilità delle iDC di riconoscere i batteri e interagire con loro, probabilmente aumentando la loro capacità di presentare gli antigeni e attivare le risposte immunitarie. Un trattamento equivalente sui macrofagi risulta invece in un aumento significativo della loro attività fagocitaria e della produzione di ROS, permettendo così a queste cellule di rispondere meglio ad un’invasione batterica. Tutte queste attività sono state evidenziate in vitro a concentrazioni relativamente basse (micromolari) e sub-citotossiche, compatibili con quelle che hBD2 raggiunge ai siti d’infezione. Inoltre, contrariamente all’attività battericida diretta, l’attività immunomodulatoria non è risultata sensibile alla concentrazione salina, ma si esplica in condizioni simili a quelle fisiologiche. L’attività di hBD2 è stata anche paragonata a quella di analoghi strutturali per studiare quali siano le caratteristiche di questa defensina che sono richieste per le attività sulle cellule immunitarie. I risultati sono per certi versi inspiegabili in quanto la struttura evolutivamente conservata delle defensine sembra essere importante per alcune attività biologiche, come l’attività antimicrobica diretta o la fagocitosi, mentre non lo è in altre, come la chemiotassi. Questa osservazione e la dipendenza dell’attività da tempo di esposizione e dal tipo cellulare considerato potrebbero indicare che esistono diversi meccanismi d’azione. Nell’ultima parte della tesi vengono descritti gli studi effettuati con il coniugato fluorescente di hBD2 con l’obiettivo di determinare se l’internalizzazione del peptide può giocare un ruolo nella sua attività biologica nei confronti delle cellule immunitarie. È emerso che hBD2 è capace di interagire in modo specifico già dopo una breve esposizione con i diversi tipi di cellule immunitarie considerati in questa tesi. Si è evidenziata una diversa capacità di legame e di internalizzazione a seconda del tipo cellulare, ma il processo è risultato sempre non accompagnato da effetti citotossici o permeabilizzazione delle cellule. In particolare, i macrofagi sono risultati i più efficienti nell’internalizzare del peptide, mentre le iDC sembrano più avide nel legare il peptide sulla superficie che nell’internalizzazione. Il meccanismo attraverso cui avvengono legame e internalizzazione è ancora ignoto, ma i nostri dati al citofluorimetro indicano che l’uptake della defensina è un processo che dipendente dall’energia e dalla temperatura, che è legato alla polimerizzazione delle fibre di actina e probabilmente coinvolge un processo di endocitosi mediata dai lipid rafts e/o dalla clatrina. Inoltre, la microscopia confocale di macrofagi trattati con il peptide fluorescente ha mostrato che esso interagisce con specifiche zone della membrana e, dopo il legame, si localizza rapidamente nel citoplasma della cellula con una caratteristica distribuzione puntata. Nell’insieme, questi dati suggeriscono che il peptide si leghi selettivamente a zone specifiche della membrana, come ad esempio i lipid rafts, e che l’endocitosi potrebbe essere un meccanismo generale per l’internalizzazione di hBD2. Tutti queste osservazioni suggeriscono quindi che hBD2 è capace di modulare le attività delle cellule dell’ospite non solo interagendo con le membrane o i recettori di superficie, ma anche con target citoplasmatici.
XXI Ciclo
1981

Memoria para optar el título de Bioquímico
Los receptores tipo Toll (TLR) son proteínas de transmembrana que reconocen patrones moleculares asociados a patógenos (PAMPs). El TLR2 reconoce PAMPs de bacterias gram positivas, los cuales activan una vía de señalización clásica que involucra el reclutamiento de la proteína MyD88 y una alternativa donde participa la Fosfoinositol 3-Quinasa (PI3K). A su vez, los glucocorticoides (GCs) son moléculas que suprimen la inflamación y la inmunidad adaptativa, que recientemente han sido reportadas como potenciadoras de la expresión de ciertas moléculas de la inmunidad innata, tales como el TLR2. La importancia de PI3K y los GC en la regulación de la vía alternativa del TLR2 no ha sido estudiada aún. El objetivo general fue determinar la participación de PI3K en la producción de TNFα inducida por agonistas del TLR2 y GCs. Los objetivos específicos fueron: 1) Analizar el efecto de Pam3Cys-Ser-Lys4 un lipopéptido sintético análogo a la porción NH2-terminal de lipoproteínas de bacterias gram positivas, y Dexametasona, sobre la secreción de TNFα y la activación transcripcional de NFκB, en células A549. 2) Estudiar la participación de PI3K-Akt en la ruta de señalización del TLR2 inducida por Pam3Cys-Ser-Lys4 y Dexametasona. 3) Determinar la interacción entre TLR2-PI3K y GR-PI3K. Los resultados obtenidos muestran que la activación del TLR2 por Pam3Cys-Ser- Lys4 provocó un aumento en la expresión de TNFα y la activación transcripcional del TLR2, sin embargo el co-tratamiento con Dexametasona no alteró el efecto del agonista del TLR2. Al mismo tiempo, PI3K aumenta la expresión de TNFα e inhibe la transcripción del TLR2, al usar un dominante negativo para PI3K (p85-DN). PI3K activa la transcripción de genes que responden a la movilización de NFκB y AP-1, ya que células expuestas a Pam3Cys-Ser-Lys4 y que expresan el constructo p85-DN, presentan una disminución significativa de la activación transcripcional de NFκB y APLos resultados indicaron que Pam3Cys-Ser-Lys4 aumentó significativamente la fosforilación de Akt, como un indicador indirecto de la actividad de PI3K, y Dexametasona la revirtió. Por otra parte, se determinó la interacción entre p85 con el GR o el TLR2, siendo la primera modulada exclusivamente por Dexametasona. Estos resultados demuestran que PI3K presenta diversos mecanismos de regulación en la vía de señalización del TLR2 y además proponen mecanismos por los que los GCs intervienen en la ruta transduccional
Toll-like receptors (TLRs) are transmembrane proteins that recognize pathogenassociated molecular patterns (PAMPs). TLR2 identify PAMPs from gram positive bacteria, which activate a classical signaling pathway involving MyD88 recruitment to the receptor intracellular domain, and an alternative signaling pathway, which comprises the activation of the phosphoinositol 3-kinase (PI3K). Moreover, glucocorticoids (GCs) suppress the inflammation and adaptive immune responses, and have been recently reported to enhance the TNFα-induced expression of TLR2. The relevance of GCs in the regulation of the alternative pathway has not yet been described. The general aim of this thesis was to determine PI3K participation on TNFα production induced by TLR2 agonists in the presence or absence of GCs. The specific aims were: 1) To analyze the effect of Pam3Cys-Ser-Lys4, a specific synthetic ligand analogue to the NH2-terminal portion of gram positive bacterial lipoproteins, and Dexamethasone, a synthetic GC, on TNFα expression and NFκB transcriptional activation,in A549 cells. 2) To study the role of PI3K-Akt in the TLR2 signaling pathway induced by Pam3Cys-Ser-Lys4 in the presence or absence of Dexamethasone. 3) To determine the interaction between TLR2-PI3K and GR-PI3K. The results show that TLR2 activation by Pam3Cys-Ser-Lys4 promotes TNFα expression and TLR2 transcriptional activity, however co-treatment of cells with Pam3Cys-Ser-Lys4 and Dexamethasone did not revert TNFα increase. In relation to this pathway, we investigated if PI3K was involved in TNFα expression and TLR2 transcription activity regulation. Pam3Cys-Ser-Lys4 significantly increased Akt phosphorylation, as an indicator of PI3K activity, and Dexamethasone reverted to it. Moreover, by using a PI3K regulatory subunit dominant negative mutant cDNA (p85-DN) transiently expressed in cells, a remarkably decrease in NFκB and AP-1 transcriptional activation was observed. On the other hand, we determined p85 regulatory PI3K subunit interaction with GR or TLR2, and we were able to identify interaction between p85-GR in the presence of Dexamethasone. These results demonstrate that PI3K shows different mechanism of regulation in the TLR2 signaling and propose pathways in which GCs may be participating

2010/2011
L’avvento delle tecniche di sequenziamento di nuova generazione ha reso recentemente disponibile l’opportunità di analizzare su scala genomica e trascrittomica organismi non modello, anche nel caso in cui virtualmente non sia disponibile alcuna informazione pregressa. Il mitilo mediterraneo Mytilus galloprovincialis è un organismo di grande importanza economica ed è considerato un utile bioindicatore, ma nonostante ciò fino a questo momento gli studi molecolari sono stati fortemente limitati proprio dalla limitata conoscenza genomica di questo importante bivalve. In questa tesi sono state utilizzate tecniche di sequenziamento di nuova generazione per analizzare la risposta del mitilo a biotossine algali paralitiche (PSP) su scala trascrittomica a livello della ghiandola digestiva. L’enorme mole di dati di sequenza ottenuti ha permesso di studiare in modo approfondito alcune famiglie di geni di grande importanza nella risposta immune del mitilo. In particolare sono state individuate e descritte l’ampia famiglia di lectine C1q-like, coinvolte nel riconoscimento dei patogeni, e due nuove famiglie di peptidi antimicrobici, le big defensine e le mitimacine.
XXIV Ciclo
1984

In vitro assessment of bovine mammary epithelial cells response to infectious stimuli Non-specific (innate) immune response plays a major role in defending the udder from bacterial invasion. Moreover, recent investigations suggest that mammary gland epithelial cells could have a large and important role as a source of soluble components of immune defenses. An experimental model was developed based on BME-UV cell line, Staphylococcus aureus as a challenge to assess the interactions among epithelial cells and bacteria by measuring cytokine and three lysosomal enzymes, lysozyme lactoferrin and N-acetyl-b-D-glucosaminidase (NAGase), in S. aureus-stimulated MGEC. S.aureus strains invading epithelial cells elicited an immunological response with levels related to strain characteristics. We found that BME-UV cells didn’t produce lactoferrin. NAGase and Lysozyme have a similar substrate as a target, but our results suggest different secretion mechanisms . These data confirms that epithelial cells could play an important role in udder innate defenses. Besides we investigate the expression of the bovine cathelicidins BMAP-27, BMAP-28, Bac5, and indolicidin in lipopolysaccharide (LPS)-treated cells to examine their potentials to trigger defense responses in bovine mammary cells. Our results indicate multiple roles for the bovine cathelicidins with complementary and mutually enhanced antimicrobial activities. We also investigate the potential effects of tylosin, a macrolide antimicrobial on the immune system. The results of this study confirmed that tylosin could have a significant effect on the release of Lysozyme, NAGase and cytokines by bovine epithelial cells without affecting cell functions. This study showed that an in-vitro model based on S. aureus challenge on epithelial cells could be helpful in assessing both the intracellular and extracellular activity of antimicrobials and their influence on epithelial cell immune and inflammatory response.

INTRODUCCIÓ: La malaltia pneumocòccica invasiva (MPI) pot tenir una elevada morbimortalitat, condicionada pel pneumococ i per factors de l’hoste com els receptors Toll-like (TLRs) i la seva via de senyalització intracel·lular comuna Toll-IL1R (TIR). El desenvolupament d’una MPI greu (MPIG) podria estar condicionat per polimorfismes genètics (SNPs) en regions crítiques de la via de senyalització comuna TIR. Aquests SNPs podrien influenciar també l’evolució i el pronòstic de la MPI. La determinació de la pèrdua d’expressió de la L-selectina (CD62L) “in vivo” en la superfície dels granulòcits podria ser útil en l’avaluació de la via de senyalització TIR en pacients amb MPIG. OBJECTIUS: Descriure els SNPs en proteïnes de la via de senyalització TIR (IRAK1, IRAK4, IRAKM i MyD88) en pacients amb MPIG. Determinar si aquests SNPs s’associen a la presència de MPIG. Determinar si aquests SNPs condicionen l’evolució dels pacients amb MPIG. Analitzar si la determinació de CD62L “in vivo” és útil com a cribratge inicial de les variacions de funció de la via de senyalització TIR en pacients amb MPIG. METODOLOGIA: Estudi prospectiu observacional de casos i controls. Casos: 60 pacients amb MPI i síndrome de resposta inflamatòria sistèmica. Controls: 120 pacients sense infecció aguda ni antecedent d’infecció greu. Criteris d’exclusió: immunodeficiència coneguda. Variables independents: 1) freqüències genotípiques i al·lèliques dels SNPs rs1059701, rs1059702, rs1059703 (IRAK1); rs1624395, rs1370128 (IRAKM); rs1141168, rs4251513, rs1461567 (IRAK4); rs7744, rs6853 (MyD88). 2) Nivell d’expressió de CD62L en superfície de neutròfils, limfòcits i monòcits en fase aguda de la infecció i en fase basal. Altres variables: demogràfiques, antecedents personals i dades evolutives clíniques, analítiques i microbiològiques de la MPIG. RESULTATS: Associació significativa entre la presència de MPIG i: rs1059701-CC (IRAK1) [p=0,0067, OR 1,430 (IC95%: 1,140-1,795)], rs4251513-CC (IRAK4) [p<0,0001, OR 2,183 (IC95%: 1,578-3,019)], rs4251513-C (IRAK4) [p<0,0001, OR 1,468 (IC95%: 1,404-1,535)], rs1461567-T (IRAK4) [p=0,0158, OR 1,500 (IC 95%: 1,132-1,987)], rs6853-AA (MyD88) [p<0,0001, OR 2,125 (IC95%: 1,787-2,526)] i rs6853-A (MyD88) [p<0,0001, OR 1,935 (IC95%: 1,462- 2,563)]. En els pacients amb MPIG: associació significativa entre leucocitosi >15000/mmc i rs1059702- noTT (IRAK1) [p=0,0460, OR 7,500 (IC95%: 1,862-30,214)], pleuropneumònia i rs1624395-G (IRAKM) [p=0,0147, OR 1,834 (IC95%: 1,230-2,736)] i rs1370128-C (IRAKM) [p=0,0055, OR 2,060 (IC95%: 1,367-3,105)], seqüeles i rs4251513-noGG (IRAK4) [p=0,0010, OR 7,066 (IC95%: 2,645-18,872)], exitus i rs6853-noAA (MyD88) [p=0,0054, OR 16,086 (3,336- 77,574)] i rs6853-G (MyD88) [p=0,0064, OR 8,388 (IC95%: 2,472-28,455)]. Estudi d’expressió de CD62L : només s’han obtingut dades completes en 21 pacients. Entre els 3 grups cel·lulars, diferències significatives en el grup de monòcits entre fase aguda i basal (p=0,0343). En els monòcits, diferències significatives en rs4251513-CG (IRAK4) entre fase aguda i basal (p=0,0391). CONCLUSIONS: Alguns SNPs d’IRAK1, IRAK4 i MyD88 estan associats a un risc incrementat de desenvolupar MPIG en comparació amb la població general. Altres SNPS d’IRAK1, IRAKM, IRAK4 y MyD88 condicionen l’evolució de la MPIG. La identificació de SNPs que predisposin a malalties infeccioses greus com la MPIG podria ajudar a estratificar els pacients i dissenyar tractaments específics. Nous estudis amb mida mostral major podrien ajudar a entendre les conseqüències funcionals d’aquestes associacions a través de la determinació dels nivells de CD62L en monòcits abans i després de la MPIG.
INTRODUCTION: Invasive pneumococcal disease (IPD) may have high morbidity and mortality, conditioned by pneumococcus and host factors such as Toll-like (TLRs) receptors andtheir common intracellular signaling pathway Toll-IL1R (TIR). Development of severe IPD (SIPD) could be conditioned by genetic polymorphisms (SNPs) in critical regions of the common signaling pathway TIR. These SNPs could also have influence on the evolution and prognosis of IPD. Determination "in vivo" of loss of expression of L-selectin (CD62L) on surface of granulocytes could be useful in the evaluation of the TIR signaling pathway in patients with SIPD. OBJECTIVES: To describe SNPs in proteins of the TIR signaling pathway (IRAK1, IRAK4, IRAKM and MyD88) in patients with SIPD. To determine whether these SNPs are associated with presence of SIPD. To determine whether these SNPs condition the evolution of patients with SIPD. To analyze if determination of CD62L "in vivo" is useful as an initial screening of variations in function of TIR signaling pathway in patients with SIPD. METHODS: Observational prospective case-control study. Cases: 60 patients with IPD and systemic inflammatory response syndrome. Controls: 120 patients without acute infection nor history of severe infection. Exclusion criteria: known immunodeficiency. Independent variables: 1) Genotypic and allelic frequencies of SNPs rs1059701, rs1059702, rs1059703 (IRAK1); rs1624395, rs1370128 (IRAKM); rs1141168, rs4251513, rs1461567 (IRAK4); rs7744, rs6853 (MyD88). 2) Level of expression of CD62L on surface of neutrophils, lymphocytes and monocytes in acute phase of infection and in basal phase. Other variables: demographic, personal history and clinical, analytical and microbiological evolutionary data of SIPD. RESULTS: Significant association between presence of SIPD and: rs1059701-CC (IRAK1) [p = 0.0067, OR 1,430 (IC95%: 1,140-1,795)], rs4251513-CC (IRAK4) [p <0.0001, OR 2,183 ( IC95%: 1,578-3,019)], rs4251513-C (IRAK4) [p <0.0001, OR 1,468 (IC95%: 1,404-1,535)], rs1461567-T (IRAK4) [p = 0.0158, OR 1,500 ( IC 95%: 1,132-1,987)], rs6853-AA (MyD88) [p <0.0001, OR 2,125 (IC95%: 1,787-2,526)] and rs6853-A (MyD88) [p <0.0001, OR 1,935 (IC95%: 1,462-2,563)]. In patients with SIPD: significant association between leukocytosis> 15000 / mmc and rs1059702- noTT (IRAK1) [p = 0.0460, OR 7,500 (IC95%: 1,862-30,214)], pleuropneumonia and rs1624395-G (IRAKM) [p = 0.0147, OR 1,834 (IC95%: 1,230-2,736)] and rs1370128-C (IRAKM) [p = 0.0055, OR 2,060 (IC95%: 1,367-3,105)], sequelae and rs4251513-noGG (IRAK4) [p = 0.0010, OR 7,066 (IC95%: 2,645-18,872)], exitus and rs6853-noAA (MyD88) [p = 0.0054, OR 16,086 (3,336-77,574)] and rs6853-G (MyD88) [ p = 0.0064, OR 8,388 (IC95%: 2,472-28,455)]. CD62L expression study: complete data were obtained in 21 patients. Among the 3 cell groups, significant differences in group of monocytes between acute and basal phase (p = 0.0343). In monocytes, significant differences in rs4251513-CG (IRAK4) between acute phase and basal phase (p = 0.0391). CONCLUSIONS: Some SNPs from IRAK1, IRAK4 and MyD88 are associated with an increased risk of developing SIPD compared to general population. Other SNPS from IRAK1, IRAKM, IRAK4 and MyD88 condition the evolution of SIPD. Identification of SNPs that predispose to serious infectious diseases such as SIPD could help stratify patients and design specific treatments. New studies with a larger sample size could help to understand functional consequences of these associations through the determination of CD62L levels in monocytes before and after SIPD.

La acuicultura es una de las áreas de mayor prioridad para el desarrollo de nuestro país, sin embargo, existen muchos aspectos en los cuales no se ha investigado y otros en los que se está iniciando, como es el caso del uso de inmunoestimulantes para lograr mejores resultados en la producción de algunas especies de importancia económica. Uno de los inmunoestimulantes investigados en peces dulceacuícolas es la quitina, que administrada como suplemento dietético potencia la respuesta inmune previniéndoles del ataque de agentes patógenos como Flavobacterium psychrophilum. El objetivo de la investigación fue evaluar la actividad inmunoestimulante de la quitina, administrada por vía oral a juveniles de trucha arco iris (Oncorhynchus mykiss) en condiciones de inmunocompetencia e inmunosupresión y posteriormente desafiados con la cepa estándar F. psychrophilum 1947T causante de la “enfermedad del agua fría”. Para demostrar los efectos de la quitina se evaluaron parámetros de la inmunidad innata celular (actividad fagocitaria y producción de óxido nítrico) y humoral (complemento por vía alternativa y lisozima sérica). Además, se determinó el tiempo necesario de tratamiento con quitina para lograr una adecuada inmunoestimulación. Las truchas fueron alimentadas con pienso suplementado con quitina y sin ella (n=20 en cada caso) durante 2 y 4 semanas. La quitina se incorporó a la dosis de 100g/Kg de alimento, el mismo que fue suministrado a la proporción del 1% de la biomasa. Luego del tratamiento los dos grupos fueron inmunosuprimidos con ciclofosfamida y desafiados por vía intramuscular. Se concluye que existe una mejora significativa en la producción de óxido nítrico y la actividad de lisozima sérica de los peces inmunosuprimidos y tratados con quitina en comparación con los peces inmunocompetentes y los no tratados. El complemento por vía alternativa y la actividad fagocitaria in vitro no mostraron variaciones significativas para ambos grupos desde las dos semanas de tratamiento.
The aquaculture is a field major priority than other for the development in our country; however, there exist many aspects in which it has not been investigated and others in which is beginning, as is the case of the use of immunoestimulants for to achieve better results in the production of the many species from economic importance. One of the stimulants investigated in other species of fish is the chitin that administered as dietary supplement enhances the immune response, providing them of the assault of pathogenic agents as Flavobacterium psychrophilum. The aim of the investigation was to evaluate immunostimulant activity of chitin administered by oral route to rainbow trout’s youths (Oncorhynchus mykiss) in immunocompetent and immunosupressed conditions e infected with a test strain, Flavobacterium psychrophilum 1947T, causing of the “cold water disease”. To demonstrate the immunostimulating with chitin there were evaluated parameters of the cellular (phagocytary activity and production of nitric oxide) and humoral (complement’s activity by alternative route and lysozyme serum’s) innate immunity. In addition it decided the necessary time of treatment with the immunostimulant to achieve the suitable one immunostimulation. Trouts were fed by pienso with and without chitin (n=20 in each case) for 2 and 4 weeks. The chitin was added to the dose of 100g/Kg of food and was supplied a proportion of 1% of biomass. Immediately of treatment both groups were immunosupressed with ciclophosphamide and infected by intramuscular route. I concluded that there exists a significant improvement of the production of nitric oxide and lysozyme serum’s activity in the immunosupressed fishes and treated with chitin in comparison with the immunocompetents fishes and not treated. The complement by alternative route and in vitro phagocytary activity did not show significant variations for both groups from two weeks of treatment.
Tesis

Las ribonucleases humanas son un grupo heterogéneo de proteínas pertenecientes a la superfamilia de la Ribonucleasa A. Estas proteínas se caracterizan por su capacidad de hidrolizar ácidos ribonucleicos y por la presencia de actividad antimicrobiana frente diversos organismos patógenos como bacterias, hongos, parásitos y virus. El primer objetivo del presente estudio doctoral se centra en la caracterización de la actividad antimicrobiana y en modelos de membrana de las formas nativas de la ribonucleasa 3 purificadas a partir de eosinófilos. Las distintas formas nativas presentan modificaciones postraduccionales dadas por diversos grados de glicosilación que se correlacionan con la activación de los eosinófilos durante los procesos inflamatorios. El estudio establece la capacidad antimicrobiana de las formas nativas y su actividad en modelos de membrana. Los resultados indican que las modificaciones postrasduccionales modulan la actividad biológica de la RNasa 3, sugiriendo una contribución in vivo en su función fisiológica. Como segundo objetivo de esta tesis, se evaluó por primera vez en nuestro grupo de investigación la actividad antimicótica de las ribonucleasas 3 y 7 frente al hongo Candida albicans, el cual fue elegido como modelo patógeno eucariota. Se determinó y caracterizó la presencia de actividad frente a Candida por parte de ambas ribonucleasas humanas. Por último, el tercer objetivo de esta tesis se centra en la purificación y caracterización de la ribonucleasa 8, la más reciente ribonucleasa humana descrita, identificada inicialmente en placenta. La RNasa 8 presenta un patrón inusual de enlaces disulfuro respecto a sus proteínas homólogas. Este cambio estructural modifica la estabilidad de la proteína y expone regiones que facilitan el proceso de agregación proteica. Fue necesaria la previa optimización de un protocolo alternativo de purificación. Se analizaron sus propiedades antimicrobianas, sugiriendo su posible participación en la respuesta inmunitaria innata. Los resultados del presente estudio corroboran las propiedades antimicrobianas de diversas ribonucleasas humanas miembros de la familia de la RNasa A, sugiriendo una función ancestral en el sistema de defensa innato. El estudio contribuye a la comprensión de su mecanismo de acción y plantea su potencial uso como herramientas terapéuticas.
Human ribonucleases are a heterogeneous group of proteins belonging to the superfamily of RNase A. These proteins are characterized by their ability to hydrolyse ribonucleic acids and the presence of antimicrobial activity against various pathogens including bacteria, fungi, parasites and viruses. The first objective of this doctoral study is focused on the antimicrobial characterization of native Ribonuclease 3 forms purified from eosinophils. Native forms present posttranslational modifications giving different glycosylation grades that modulate their activity during inflammatory processes. This study aims to establish the antimicrobial properties of native forms purified from eosinophils and their activity in a membrane model system. Results indicate that post-translational modifications contribute to the the protein biological activities, suggesting a related physiological role. As a second objective, we evaluated for the first time the antifungal activity of the antimicrobial RNase 3 and RNase 7 against Candida albicans, an eukaryotic pathogen selected as a simple model to test the antimicrobial mechanism of action. Both human ribonucleases displayed a high antifungal activity. Results highlighted a dual mechanism of action, where cell lysis takes place at high protein concentration, while depolarization, cell internalization and cellular RNA degradation is achieved at sublethal doses. Finally, the last objective is focused on the characterization of ribonuclease 8, also called the placental RNase, the most recent human ribonuclease described. RNase 8 has gained and lost one cysteine residue in non-conserved positions in a mechanism called "disulphide shuffling". The protein tendency to aggregate required the design of an alternative purification protocol. We analysed its antimicrobial abilities, suggesting a possible role in innate defence. The results of this study confirmed the high antimicrobial activity of several human ribonucleases from the RNase A superfamily suggesting an ancestral role in the host immune defence response. The study contributed to the understanding of their mechanism of action and set the basis for applied drug design.

Esta es una tesis de Maestría que se realizó para probar la hipótesis de que exise la inmunidad innata en insectos triatominos
El estudio del sistema inmune de los insectos frente a diversos patógenos, ha sido un tema de interés en los últimos años. Este tema es particularmente importante en insectos vectores de algún patógeno para el humano, como en el caso de los insectos triatominos que son vectores del parásito T. cruzi, agente causal de la Enfermedad de Chagas. Entender la interacción parásito-vector, podría generar nuevas estrategias para disminuir el número de infectados por estos parásitos en los humanos. Este trabajo intenta dilucidar si existe memoria inmunitaria innata en los triatominos Meccus pallidipennis y Rhodnius prolixus frente a los parásitos T. rangeli y T. cruzi. También permitió conocer si los triatominos, ante un reto dual específico contra los parásitos (grupo de Memoria), poseían mejor respuesta inmunitaria y supervivencia que los triatominos sin reto dual (grupo Control). El primer objetivo de la tesis fue proponer, después de una revisión exhaustiva, que los triatominos podrían tener memoria inmunitaria innata. El segundo objetivo fue determinar la respuesta inmunitaria (número de hemocitos y sistema de proFenoloxidasa –proPO, por sus siglas en inglés) de Meccus pallidipennis, después de su activación, alimentando a las chinches con sangre con parásitos muertos fijados con glutaraldehido, y después de una segunda alimentación con parásitos vivos (grupo de Memoria). El grupo Control consistió en triatominos que recibieron sangre sin parásitos en la primera alimentación y con parásitos vivos en la segunda alimentación. El tercer objetivo fue saber si Rhodnius prolixus del grupo de Memoria, en comparación con el grupo Control, tenía mejor probabilidad de vivir aunque esto implicara un costo en términos de un retardo en el crecimiento. Respecto al primer objetivo, propusimos realizar estudios de memoria inmunitaria en triatominos. Los resultados del segundo objetivo no mostraron diferencias significativas en el número de hemocitos ni en la actividad proPO en Meccus pallidipennis, sin embargo, es necesario realizar más experimentos porque el tamaño de muestra fue pequeño. Finalmente, en el tercer objetivo, Rhodnius prolixus, mostro evidencia de memoria inmunitaria en términos de supervivencia aunque el grupo de Memoria, tuvo un retraso en su crecimiento, respecto al grupo Control. Esto sugiere la existencia de memoria innata en triatominos pero con un costo en el crecimiento. En conclusión, de manera empírica y experimental, sugerimos que existe memoria inmunitaria en triatominos, pero se requiere analizar los mecanismos fisiológicos y moleculares que están detrás de esta memoria.
CONACYT beca a la estudiante de maestria CONACYT Proyecto número 156701 PAPIIT- UNAM Proyecto número IA205318

Evalúa la expresión transcripcional de citoquinas moduladoras de la respuesta inmune innata y adaptativa en células mononucleares humanas (PBMC) tratadas con fucoidan del alga parda Lessonia trabeculata. El fucoidan de Lessonia trabeculata fue proporcionado por la empresa PSW S.A y caracterizado fitoquímicamente por el Laboratorio de Bioquímica y Biología Molecular de la Universidad Nacional Agraria La Molina. Las PBMC se trataron con diferentes concentraciones de fucoidan de Lessonia trabeculata (FLt, 80.84% pureza) y como controles se emplearon fucoidan de Fucus vesiculosus (FFv, 95% pureza) y lipopolisacárido de E. coli. La citotoxicidad se evaluó mediante el ensayo de reducción metabólica del bromuro de 3-(4,5-dimetiltiazol-2-ilo)-2,5-difeniltetrazol (MTT). Para evaluar la expresión transcripcional de las citoquinas de la inmunidad innata: IL-8, IL-12 (p35 y p40) y TGF-􀈕 y las citoquinas de inmunidad adaptativa; IL-2 e IL-10 se empleó la reacción en cadena de la polimerasa convencional (PCR convencional). El tratamiento de cultivos de PBMC a las concentraciones de 10 y 100 μg/ml de FLt y FFv no ocasionó efecto citotóxico; por el contrario, FLt a 100 μg/ml incrementó significativamente el porcentaje de viabilidad celular respecto al control sin fucoidan (p<0.05). Respecto a la expresión transcripcional, el tratamiento con FLt a 10 y 100 μg/ml incrementó el ARNm IL-10 (p<0.0001) y ARNm IL-12p40 (p<0.05) respectivamente en relación al control sin fucoidan. Se concluye que el fucoidan de L. trabeculata a 10 y 100 μg/ml modula la expresión transcripcional de IL-12 de inmunidad innata e IL-10 de inmunidad adaptativa.
Universidad Nacional Mayor de San Marcos (Lima). Vicerrectorado de Investigación y Posgrado
Tesis

Klebsiella pneumoniae és un bacil Gram negatiu que causa infeccions del tracte urinari així com pneumònies adquirides en la comunitat i nosocomials. La membrana externa de les bactèries Gram negatives està formada pel lipopolisacàrid (LPS). El LPS és una molècula formada per un polisacàrid, amb càrrega negativa, ancorat a una regió hidrofòbica, el lípid A, que posseeix característiques d’endotoxina i és, per tant, immunoestimuladora. Els sistemes de transducció de senyals de dos components en bactèries detecten canvies en l’ambient i regulen l’expressió de gens que modifiquen l’estructura del lípid A per tal de mantenir l’homeòstasi de la cèl•lula bacteriana, augmentar la resistència en front als pèptids catiònics o modular la resposta immune de l’hoste. En resultats previs del nostre laboratori va posar-se de manifest que la xaperona Hfq regula les modificacions del lípid A a Yersinia enterocolitica O8. En aquesta tesi doctoral posem de manifest que aquest fet no sols es dóna a Y. enterocolitica si no que Hfq també participa en la regulació de les modificacions dels lípid A tant a Salmonella Typhimurium com a K. pneumoniae. En la segona part d’aquesta tesi doctoral hem caracteritzat l’estructura de lípid A que K. pneumoniae expressa in vivo, durant una infecció, sense la necessitat de realitzar un pas previ d’aïllament bacterià en medis de cultiu tradicionals. Durant una infecció pulmonar, K. pneumoniae expressa un lípid A que es caracteritza per la presència d’una cadena de 2-hidroximiristat com acilació secundària. Les nostres observacions indiquen que el lípid A que K. pneumoniae expressa en el pulmó és menys inflamatori que el lípid A que la bactèria expressa quan creix en medis de cultiu tradicionals. A més a més, el lípid A que K. pneumoniae expressa in vivo juga un importantíssim paper conferint a la bactèria resistència en front als pèptids antimicrobians.
K. pneumoniae is a Gram negative bacterium which causes urinary tract infections as well as community-acquired and nosocomial pneumonia. Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram negative bacteria. LPS is a negatively charged polysaccharide anchored to a hydrophobic region, the endotoxic lipid A. In bacteria, two component transduction systems detect environmental changes and trigger the expression of genes responsible for lipid A modifications. Lipid A remodelling helps maintaining cellular integrity, confers resistance to many antimicrobial peptides as well as modulates host’s immune response. Previous results from our laboratory showed that Hfq regulates lipid A modifications in Yersinia enterocolitica O8. In this doctoral thesis we demonstrate that Hfq also regulates lipid A modifications in other Enterobacteriaceae such as Salmonella Typhimurium and K. pneumoniae. In a second study, we have characterised for the first time the lipid A structure expressed by K. pneumoniae in vivo during a lung infection without the need of a previous isolation step on traditional culture media. During the course of a lung infection, K. pneumoniae expresses a lipid A characterised by the presence of a 2-hydroxymiristate as a secondary acylation. Our results show that the lipid A expressed by K. pneumoniae in vivo in the lung is less inflammatory than the lipid A expressed when cultured on traditional culture media. Furthermore, this lipid A found in the lung plays a major role conferring resistance to antimicrobial peptides.
Klebsiella pneumoniae es un bacilo Gram negativo que causa infecciones del tracto urinario así como neumonías adquiridas en la comunidad y nosocomiales. La membrana externa de las bacterias Gram negativas se compone del lipopolisacárido (LPS). El LPS es una molécula formada por un polisacárido, cargado negativamente, anclado a un dominio hidrofóbico, el lípido A, que posee propiedades de endotoxina y, por tanto, es inmunoestimulador. Los sistemas de transducción de señales de dos componentes en bacterias detectan cambios en el ambiente y regulan la expresión de genes que remodelan la estructura del lípido A para mantener la homeóstasis de la célula bacteriana, aumentar su resistencia a péptidos catiónicos o modular la respuesta inmune del huésped. En resultados previos de nuestro laboratorio se puso de manifiesto que la chaperona Hfq participa en la regulación de las modificaciones del lípido A en Yersinia enterocolitica. En esta tesis doctoral se ha puesto de manifiesto que este hecho no sólo ocurre en Y. enterocolitica si no que también Hfq participa en la regulación de las modificaciones del lípido A tanto en Salmonella Typhimurium como en K. pneumoniae. En la segunda parte de esta tesis doctoral se ha caracterizado la estructura de lípido A que expresa K. pneumoniae in vivo durante una infección sin la necesidad de aislar la bacteria previamente en medios de cultivo tradicionales. Durante una infección pulmonar, K. pneumoniae expresa una estructura de lípido A caracterizada por la presencia de una cadena de 2-hidroximiristato como acilación secundaria. Nuestras observaciones indican que el lípido A expresado en el pulmón es menos inflamatorio que el expresado cuando la bacteria crece en medios de cultivo tradicionales. Además, el lípido A expresado por K. pneumoniae in vivo juega un importante papel en la resistencia de la bacteria frente a péptidos antimicrobianos.

2008/2009
I peptidi in difesa dell’ospite (HDPs) esercitano molteplici ruoli nell’immunità, agendo sia come molecole antimicrobiche ad azione diretta sia come agenti immuno-moduatori. Il ruolo all’interfaccia tra immunità innata ed adattativa li rende molecole ideali per la futura applicazione nel trattamento di malattie infettive. Lo scopo di questo lavoro è stato quello di valutare le caratteristiche funzionali e strutturali di Catelicidine e Defensine selezionate al fine di correlare queste proprietà con l’ attività selettiva su cellule eucariotiche e procariotiche. Metodi biofisici e biochimici sono stati applicati alla catelicidina umana LL37 ed alcuni analoghi (ortologhi di primate e peptidi artificiali) con lo scopo di studiare la loro struttura e aggregazione in contatto con membrane biologiche e modello (i). Lo stesso approccio è stato anche applicato alle defensine umane hBD2 e 3 ed a loro analoghi. Inoltre, tecniche di microscopia, quali la microscopia a trasmissione elettronica (TEM), la microspettroscopia infrarossa in trasformata di fourier accoppiata ad una sorgente di sicrotrone (µSR-FTIR) e la citofluorimetria, sono state utilizzate in modo complementare al fine di studiare l’interazione a breve termine di hBD2 con cellule presentanti l’antigene, in particolare le cellule dendritiche immature (ii). (i) Lo studio strutturale e l’interazione di membrana di catelicidine ortologhe ci ha permesso di scoprire come l’evoluzione abbia lavorato sulla sequenza dei peptidi inducendo una diversa capacità di strutturare nei diversi ortologhi. Questo ha portato ad un’interazione differenziata e specifica con le membrane e a diversi meccanismi di lisi di membrana cellulare e probabilmente diversi modi di interagire con le cellule dell’ospite. (ii) Inoltre, abbiamo individuato una rapida interazione di hBD2 con le cellule presentanti l’antigene. hBD2 sembra indurre un riarrangiamento generale dei lipidi cellulari che sembra comportare un aumento nella fluidità di membrana e una ri- organizzazione del sistema endomembranoso. Queste variazioni potrebbero essere responsabili di un cambiamento morfologico delle cellule che promuoverebbe la mobilità cellulare in risposta a stimuli esterni. Questo studio dimostra l’esistenza di un posibile meccanismo alternativo di motilità cellulare rispetto alla chemotassi recettore-mediata, indicando un meccanimo di azione di hBD2 sulle iDC più complesso rispetto quanto riportato fino ad oggi.
XXII Ciclo
1980

L’aterosclerosi, a lungo considerata una patologia essenzialmente legata all’ accumulo di lipidi all’interno della tonaca intima, oggi è riconosciuta come un processo degenerativo infiammatorio cronico ad eziologia multifattoriale che, pur manifestandosi clinicamente nell’adulto, trova prodromi sin dalla giovane età. Il nostro studio, condotto sui pazienti arruolati dopo infarto STEMI e sottoposti a intervento di angioplastica coronarica primaria, è finalizzato a valutare il ruolo della modulazione del sistema immunitario in seguito alla rottura della placca aterosclerotica durante l’infarto. Lo studio ancora in corso, ha evidenziato una presenza massiva dell’ IFN-γ, dell’IL-17 e dell’IL- 21 nelle coronarie affette dalla lesione rispetto a quelle esenti e alle arterie periferiche. Gli elevati livelli di citochine osservati nelle arterie danneggiate non sono dovuti all'aumento del numero delle cellule leucocitarie presenti, ma sono riconducibile all’attivazione cellulare delle stesse e all’espressione del loro pattern citochinico. Nella fase acuta dell’infarto inoltre, i livelli sierici dei fattori proinfiammatori come IL-6 e antiinfiammatori come IL-10 sono aumentati, tuttavia, l'aumento di IL-6 è sensibilmente superiore a quello di IL-10. Dopo un mese dalla lesione i livelli sierici di IL-6 e IL-10 osservati risultano bilanciati. A nostro avviso, l’attivazione prevalente dei fattori proinfiammatori è rilevante nella progressione della lesione e nello sviluppo di complicanze associate all’infarto miocardico acuto.
Atherosclerosis, once considered a disease mainly linked to accumulation of lipids within the inner tunic, is now recognized as a chronic inflammatory degenerative process with a multifactorial etiology that, even if occurs clinically in adults, shows prognostic signs at an early age. Our study, performed on patients enrolled after acute myocardial infarction with ST elevation (STEMI) undergoing primary coronary angioplasty, aims to assess the role of the immune system modulation during the atherosclerotic plaque rupture due to myocardial infarction. Preliminary data, showed a massive presence of IFN-γ, IL-17 and IL-21 in coronary arteries affected by lesion compared to those exempt and to peripheral arteries. The high levels of cytokines observed in damaged arteries are not due to the increase in the number of leukocyte cells present, but are due to activation of the same cell and expression of their cytokine pattern. Moreover, in the acute myocardial infarction the serum levels of pro-inflammatory factors such as IL-6 and anti-inflammatory such as IL-10 increased. However, the increase of IL-6 was significantly higher than that of IL-10. After a month of the injury, the observed serum levels of IL-6 and IL-10 are balanced. In our view, the main activity of pro-inflammatory factors is relevant in the progression of the lesion and in the development of complications associated with acute myocardial infarction.

L'infiammazione è per definizione una reazione locale ad un danno tissutale, che tenta di risolvere gli effetti del trauma con esiti fisiologici o in rari casi patologici. I fibroblasti (FBs) sono le cellule chiave del processo di remodelling, interagendo sia con cellule infiammatorie che strutturali. I FBs attivati differenziano in myofibroblasti (myoFBs), caratterizzati dall'espressione del marker alpha-muscle actin (alpha-SMA). Tra i fattori pro-fibrogenici il Nerve Growth Factor (NGF), sembra essere associato al processo di remodelling tissutale attraverso i due recettori trkA e p75. Abbiamo studiato due patologie oculari con un differente decorso cronico infiammatorio associato a fibrosi: la Cheratocongiuntivite primaverile (VKC, un'infiammazione di tipo allergico, Th2) e il Pemfigoide Cicartriziale Oculare (OCP, una malattia autoimmune, Th1). Entrambe le patologie hanno in comune l'aumentata deposizione di matrice extracellulare (ECM). In questo studio in particolare abbiamo approfondito in vitro i seguenti punti: 1. gli effetti del NGF sul fenotipo FBs/myoFBs di VKC e OCP; 2. rapporto tra NGF e fenotipo FBs/myoFBs; 3. la possibile modulazione del NGF in questo processo di remodelling, in relazione al TGF-beta1 ed altre citochine profibrogeniche; 4. il potenziale contributo dell'NGF nell'immunità innata (TLRs). A tale fine sono state analizzate le biopsie congiuntivali di VKC, OCP e di soggetti sani. Sono state quindi sviluppate colture primarie di FBs, caratterizzate, esposte ad NGF ed infine valutati a livello cellulare (confocale), biochimico (Western Blot, ELISA, FACS) e molecolare (Real-Time PCR). I dati ottenuti su FBs sia di VKC che di OCP hanno evidenziato che una buona percentuale di queste cellule sono myoFBs, che esprimono entrambi i recettori dell'NGF e sono in grado di produrre alte quantità di NGF. In VKC l'esposizione ad NGF risulta in un aumento della produzione dell'ECM, ma non nell'espressione di alpha-SMA. In FBs di OCP l'espressione di alpha-SMA invece è strettamente collegata allo stadio della malattia. In accordo con i dati sulla VKC, questi FBs risultano anche p75 positivi, con una maggior espressione negli stadi avanzati della patologia. Diversamente trkA è presente nei primi stadi della malattia e cala in quelli avanzati. Il trattamento con NGF inverte questa tendenza nei FBs ottenuti da pazienti ad uno stadio precoce della patologia. E' stato quindi valutato l'effetto dell'NGF sulla sintesi ed il rilascio di citochine pro/anti-infiammatorie della risposta Th1 da parte dei Fbs in studio. I livelli di IL-2, IL-4 e il TGF-beta1, alti nella prima fase patologica diminuiscono in condizioni più severe della patologia e sono significativamente modulati dal NGF. Probabilmente l'NGF potrebbe agire sia sulla sopravvivenza/apoptosi dei linfociti che sull'attivazione dei FBs/myoFBs. Per valutare quindi se l'effetto dell'NGF potesse esser dipendente dal TGF-beta1 abbiamo sottoposto FBs non patologici a stimoli con NGF e/o TGF-beta1 o con anticorpi neutralizzanti, ed osservato che entrambi i fattori si stimolano reciprocamente sul fenotipo alpha-SMA. Si è valutato anche un possibile coinvolgimento nel processo di remodelling della microflora (biofilm) e quindi dei TLRs. La valutazione dell'espressione dei TLR4 e TLR9 in cellule di FBs/myoFBs di VKC ed OCP dimostra che l'espressione è diversa nelle due patologie: mentre nei FBs di OCP entrambi Toll risultano aumentati in quelli di VKC sono "down" regolati rispetto i normali controlli. Questo probabilmente dovuto ad una diversa storia immunitaria nelle due malattie. Il trattamento con NGF sembra modulare l'espressione dei suddetti recettori rendendo le cellule più o meno reattive nel riconoscimento dei batteri. Da questi studi è emerso che l'effetto pro-fibrogenico dell'NGF potrebbe esser parzialmente dipendente dal rapporto di espressione tra i recettori trkA e p75 sui FBs/myoFBs, dall'espressione del TGF-beta1, dei TLRs e dallo stadio patologico della malattia. In particolare nello studio dell'OCP è possibile che questi rapporti siano alterati e ciò spiegherebbe il diverso comportamento dei FBs/myoFBs sia ai diversi stimoli dell'NGF che ai diversi stadi della patologia.
Inflammation is by definition a local reaction against a tissue damage, trying to solve the traumatic effects with physiological or rarely pathological behaviours. The fibroblasts (FBs) are the key cells of tissue remodelling process, interacting both with inflammatory and structural cells. The activated FBs differentiate into myofibroblasts (myoFbs), characterized by the expression of the alpha-Smooth Muscle Actin (alpha-SMA) marker. In the fibrogenic factors set, the Nerve Growth Factor (NGF), seems to be associated with the tissue remodelling process via the two receptors trkA and p75. We have studied two ocular pathologies with different chronic inflammatory courses associated with fibrosis: the Vernal Keratocongiuntivitis (VKC, an allergic inflammation, Th2) and the Ocular Cicatricial Pemphigoid (OCP, an auto-immune disorder). Both these pathologies share the common feature of an increased deposition of Extra-Cellular Matrix (ECM). In this study in particular we have analyzed the following points: 1. the NGF effects on FBs/myoFBs phenotype of VKC and OCP; 2. the relation between NGF and FBs/myoFBs phenotypes; 3. the possible modulation effects by NGF in this remodelling process in relation with TGF-beta1 and other inflammatory cytokines (Th1/Th2); 4. The possible involvement of NGF in the innate immunity (TLRs). To this aim the we have analysed conjunctival biopsies of VKC, OCP and of healthy subjects. Primary FBs cultures have been created, characterized, NGF treated and finally evaluated at cellular (confocal), biochemical (Western Blot, ELISA, FACS) and molecular (Real-Time PCR) levels. Both VKC and OCP FBs data showed that a large part of these cells are myoFBs, expressing the NGF receptors and releasing high NGF levels. In VKC, the NGF treatment results in an increase of the ECM production but does not influence alpha-SMA expression. On the other hand, in OCP FBs the alpha-SMA expression is strictly connected with the pathological status of the disease. In accordance with VKC data, these FBs result also p75 positive, with a larger expression in the advanced stage of the pathology. Differently, TrkA is present in the in initial stage of the disease and decreases in the advanced one. The NGF treatment inverts this trend in FBs from patients at early stages of the disease. Moreover the NGF effect on synthesis and release of the pro/anti-inflammatory cytokines of the Th1 response, by FBs under exam, has been evaluated. The IL-2, IL-4 and TGF-beta1 levels, which were high in first pathological phase, decreased in the more severe conditions of the disease and resulted to be relevantly modulated by NGF. Probably, NGF might act both on survival/apoptosis of lymphocytes and on the activation of FBs/myoFBs. In order to evaluate if the NGF effect could be independent by TGF-beta1, we have stimulated non pathologic FBs with NGF and/or TGF-beta1 or neutralizing antibodies, and we have observed that both these factors stimulate each other on alpha-SMA phenotype. A possible role of microflora (biofilm) and TLRs in the remodelling process has been also studied. The evaluation of TLR4 and TLR9 expression in VKC and OCP FBs/myoFBs demonstrates that the expression is different in these two pathologies: while in FBs with OCP, being both Toll, these are augmented, in those of VKC these are down regulated with respect to normal controls. This fact is probably due to a different immunological history in the two pathologies. The NGF treatment seems to modulate the expression of these receptors making the cells more or less reactive in recognizing bacteria. From these studies it appeared that the NGF pro-fibrogenic effect could be partially dependent by the expression ratio of trkA and p75 on the FBs/myoFBs, by the TGF-beta1 and TLRs. In particular in the study of OCP it is possible that these ratio could be altered and this would explain the different FBs/myoFBs behaviour in relation with different NGF stimuli and with the different levels of the pathology.

Aquesta tesis doctoral està dividida en tres estudis on s’estudia la PNEUMÒNIA AGUDA DE LA COMUNITAT EN L’ADULT (PAC); des de l’etiologia fins als biomarcadors genètics de l’hoste que l’afavoreixen. ESTUDI 1: «Aetiology of community-acquired pneumonia among adults in an H1N1 pandemic year: the role of respiratori viruses» — El primer objectiu de la tesis doctoral s’avalua la capacitat de millora en el diagnòstic etiològic de la PAC per mitjà de l’ús sistemàtic d’una prova de PCR que permet identificar els principals virus patògens implicats en la PAC. Així mateix es vol descriure l’etiologia i la forma de presentació clínica de la PAC en pacients admesos en un hospital d’aguts, comparant les característiques de la pneumònia bacteriana i les de la pneumònia viral. La primera conclusió es que L’S. pneumoniae segueix sent la causa més freqüent de PAC en el nostre medi a més expliquem com la PCR múltiple augmenta significativament la proporció de diagnòstics microbiològics i permet la identificació d’una infecció viral en més d’un terç dels casos. I finalment definim diferències en la forma de presentació clínica, la gravetat i els valors de la procalcitonina entre els pacients amb pneumònia viral i els que tenen pneumònia bacteriana, fet que podria ajudar a dissenyar les pautes de tractament empíric. ESTUDI 2: «Impact of vaccination on invasive pneumococcal disease in adults with focus on the immunosuppresed» —L’objectiu del segon estudi d’aquesta tesis es estudiar l’evolució de la incidència global i per serotips de la malaltia pneumococicca invasora (MPI) en adults (en especial en els immunodeprimits) abans i després de la introducció de la vacuna conjugada en la població infantil a la nostra àrea. Descrivim que en el nostre medi, caracteritzat per una implementació de la vacunació de la població infantil lenta i progressiva existeix una disminució de la incidència de l’MPI en els adults, tant ens els immunosuprimits com en els no immunosuprimits. Finalment concloem que atesa l’evolució dels serotips vacunals, la cobertura vacunal que ofereix la PCV13 en la població immunosuprimida pot arribar a ser subóptima al llarg dels propers anys. ESTUDI 3: «Genetic susceptibility to invasive pneumococcal disease» — El darrer estudi de la tesis identifica possibles associacions entre polimorfismes genètics en gens involucrats en la resposta immune innata del hoste i la susceptibilitat a la malaltia pneumocòccica invasora en la població adulta. Descrivim una associació entre diferents variants genètiques de citocines clau en la resposta immune innata, com la IL1R1, la IL4 i la IL10, així com els gens de IKB (NFKBIE, NFKBIA i NFKBIZ), i el risc de malaltia pneumocòccica invasora.

El NFAT5 es un factor de transcripción de la familia Rel, la cual incluye a los NFATc y los NF-[kappa]B. Hasta ahora, el NFAT5 había sido caracterizado principalmente como un factor de respuesta a estrés osmótico ya que regula la expresión de genes osmoprotectores y citoquinas en respuesta a hipertonicidad. En este trabajo hemos identificado y caracterizado una nueva función del NFAT5, la regulación de genes (TNF[alfa], IL6, iNOS) activados por la vía de los receptores de tipo Toll (TLR), importantes para la respuesta inmunitaria innata y adaptativa a una amplia variedad de estímulos patogénicos. Nuestros resultados muestran que el NFAT5 es un regulador importante en la ruta de los TLR, jugando un papel en la actividad de varios miembros de esta familia de receptores, tal y como lo hacen los factores de transcripción NF[kappa]B e IRF5.
NFAT5 is a transcription factor of the Rel family, which includes NFATc and NF-[kappa]B. NFAT5 has been mainly characterized as a hypertonicity responsive factor, since it regulates the expression of osmoprotective genes and cytokines in response to osmotic stress. In this work we have identified and characterized a novel role for NFAT5, the regulation of genes (TNF[alfa], IL6, iNOS) activated by the Toll-like receptor pathway (TLR), important for innate and adaptive immunity responses to a wide variety of pathogenic molecules. Our results show that NFAT5 is an important transcription factor of the TLR pathway, playing a role in the activity of several members of this receptor family, like other transcription factors such as NF-[kappa]B and IRF5.

I often think of life as a tight rope stretching across an expanse. Our inner strength enables us to walk forward across it. When this fails us, we fall. But in those moments when we prevail, we soar and float as though weightless and timeless. As a gymnast I learned that control of one's insecurities results in a powerful and balanced presence of body. Give into them and the body becomes uncertain and clumsy. Rarely is life this transparent. Many forms of tension manifest themselves in physical, spiritual, and emotional unrest. How does the physical contour of the skin reflect the soul of a material body? Through the use of tension and balance, and with the aid of transparency, translucency, and opacity I alter the perception of surface, form, internal and external space. My work is a comment on the flux of my emotions and attitude towards daily life.

Although it is evident that physiological ageing is accompanied by marked alterations in the function of innate immune cells, little is known regarding the underlying mechanism(s). Furthermore, the effect of age on many novel aspects of innate immunity is unknown. This thesis has identified the mechanism(s) behind the well-documented age-related decline in natural killer (NK) cytotoxicity (NKCC) and demonstrated for the first time that human ageing is accompanied by a significant reduction in the generation of neutrophil extracellular traps (NETs). Following target cell recognition, it was found that NK cells from older adults secreted into the immunological synapse (IS) significantly lower levels of perforin, a pore-forming protein that plays a non-redundant role in NKCC. This impairment led to reduced perforin binding to the target cell surface, an event that correlated strongly with NKCC. Underlying the reduction in perforin secretion was defective polarisation of lytic granules to the IS, which was associated with delayed activation of extracellular signal-regulated kinase 1/2. Whilst no age-related difference was observed in NET production triggered by phorbol 12-myristate 13-acetate (PMA), neutrophils from older adults generated significantly fewer NETs when challenged with interleukin-8 or lipopolysaccharide, which was accompanied by a reduction in reactive oxygen species generation. As PMA activates cells independent of membrane receptors, aberrant intracellular signalling proximal to the neutrophil membrane may underlie the age-related impairment in NET production.

Viral infections affect millions of people worldwide and pose a major threat to human health. Therefore efforts to understand the host defences against viruses are timely and useful. There are specific receptors on the host cells such as Pattern Recognition Receptors (PRR), which are capable of sensing infectious viruses and initiate reactions collectively known as innate immune responses by detecting motifs or molecular signatures. These responses include activation of antiviral cytokines and initiation of the adaptive immune response, thus inhibiting virus replication. The main two families of PRR involved in virus recognition are the Toll like receptors and the RIG-1 like receptors (RLRs; also known as RIG-1 like proteins or RNA helicases). This study was aimed to clarify the innate immune responses and recognition pathways of Picornaviridae by the host. Picornaviridae are single-stranded RNA viruses that can infect many tissues and organs and produce a variety of symptoms and illnesses to the host. The results from this study have shown that TLRs and RLRs and more specifically TLR7, TLR8 and MDA5 are involved in the detection of Picornaviridae such as Coxsackievirus A9 (CAV-9) and Human Rhinovirus 6 (HRV6) leading to the activation of antiviral cytokines by the host cells.

The innate immune system is a generic response to infection or injury. Evidence shows the innate response has immunological memory capable of altering subsequent responses to stimuli. Fibroblasts are ubiquitous stromal cells capable of responding to inflammatory triggers, and of orchestrating endothelial cell and leukocyte behaviour during inflammation. Repeated challenge with cytokines (such as tumour necrosis factor (TNF) a) induced an augmented second response to stimulation. Fibroblasts from multiple anatomical locales significantly increased cytokine secretion upon second challenge with TNFa. The precise mediators augmented depended on fibroblast site of origin. Depending on site, memory was inherent, or only present in fibroblasts from chronically-inflamed tissue. This suggests a phenomenon intrinsic to some sites but pathological in others. The secreted mediators from the fibroblast initial or memory responses exerted differing effects on leukocytes, dependent upon fibroblast site of origin. Finally, examination of intracellular signalling showed the augmented response was at least partly due to prolonged activity of nuclear factor (NF) KB during the memory response. Innate immune memory exists in fibroblasts from multiple tissues, but may be pathologically acquired in some. The altered response to second challenge may represent a fibroblast mechanism for altering the recruitment and behaviour of the inflammatory infiltrate.