Dissertations / Theses on the topic 'Lungs Immunology'

Lung transplantation is the only therapeutic approach for patients presenting end-stage pulmonary failure. Despite progress made in organ preservation and immunosuppression, primary graft dysfunction and obliterative bronchiolitis still hamper short-term and long-term outcomes, respectively. Interleukin-17 recently emerged as a major actor in several immuno-inflammatory disorders. Clinical and experimental evidence also suggest the implication of interleukin-17 or type 17 CD4+ T cells in lung rejection. We therefore investigated the contribution of this cytokine to graft pathology in a murine model of tracheal transplantation that recapitulates pathological features of lung rejection including the development of obliterative airway disease.

We first demonstrated that interleukin-17 contributes to inflammatory lesions in the early phase post-transplantation. Interleukin-17 was found to be produced by &61543;&61540;+ T cells and CD4+ T cells infiltrating the graft and interleukin-17 neutralization significantly reduced the development of epithelial lesions together with inhibition of interleukin-6 and heat-shock-protein 70 gene transcription.

We then investigated the contribution of interleukin-17 to obliterative airway disease. Although interleukin-17 did not play a dominant role in absence of immunosuppression, it was found to contribute to airway pathology in animals receiving cyclosporin A. Under this treatment, we first observed dramatic changes in the composition of the lymphocyte populations infiltrating the graft: the numbers of CD8+ T cells producing interferon-&61543; and type 1 CD4+ T cells were dramatically decreased while the numbers of type 17, and also type 2 CD4+ T cells were unaffected. The pathological relevance of these findings was first demonstrated by the prolongation of graft survival afforded by the depletion of CD4+ T cells in cyclosporin A-treated animals. Furthermore, graft rejection was also delayed in mice genetically deficient in either interleukin-17 or interleukin-4, providing evidence that type 17 and type 2 CD4+ T cells actively contribute to graft rejection in cyclosporin A-treated recipients. On the other hand, parallel experiments in interferon-&61543;-deficient mice revealed that interferon-&
Doctorat en Sciences médicales
info:eu-repo/semantics/nonPublished

Amongst respiratory diseases, inflammatory lung diseases constitute a major part of public

health problem. As a consequence, investigating the immune mechanisms that contribute to

the pathogenesis of these diseases is essential to identify candidate targets for the

development of new therapeutic drugs. Furthermore, over the past 20 years, the growing awareness

that purinergic signalling events shape the immune and inflammatory responses to infection and

allergic reactions warranted the development of animal models to assess their importance in vivo in

acute lung injury and chronic airway diseases. The field of purinergic inflammation formulated the

unifying concept that ATP is released as a «danger signal» to induce inflammatory responses upon

binding purinergic receptors.

According to these elements, we began in 2007 to evaluate lung inflammation in mice deficient for

the P2Y2 purinergic receptor in TH2 and TH1 models. The most convincing evidence that the P2Y2

receptor is engaged during alarm situations comes from studies related to cystic fibrosis and asthma.

Indeed, chronic respiratory diseases are commonly associated with elevated airway ATP

concentrations, as reported in cystic fibrosis, but also in idiopathic pulmonary fibrosis and chronic

obstructive pulmonary disease (COPD) patients, and they are raised by allergens in asthmatic

patients.

First, we demonstrated a significant role of the P2Y2R in a TH2-ovalbumin(OVA)-induced asthma

model. We observed that eosinophil accumulation, a distinctive feature of lung allergic inflammation,

was defective in OVA-treated P2Y2-deficient mice compared with OVA-treated wild type animals.

Interestingly, the upregulation of VCAM-1 was lower on lung endothelial cells of OVA-treated P2Y2

knockout mice compared with OVA-treated wild type animals. Adhesion assays demonstrated that

the action of UTP on leukocyte adhesion through the regulation of endothelial VCAM-1 was

abolished in P2Y2-deficient lung endothelial cells. Additionally, the level of soluble VCAM-1, reported

as an inducer of eosinophil chemotaxis, was strongly reduced in the bronchoalveolar lavage fluid of

P2Y2-deficient mice.

Secondly, we studied the consequences of P2Y2R loss in lung inflammation initiated after pneumonia

virus of mice (PVM) infection in collaboration with the group of Pr. Daniel Desmecht (ULg). We

demonstrated here that P2Y2

-/-

mice display a severe increase in morbidity and mortality rate in

response to PVM. Lower survival of P2Y2

-/-

mice was not correlated with excessive inflammation

despite the higher level of neutrophil recruiters in their broncho-alveolar fluids. Interestingly, we

observed lower numbers of dendritic cells, CD4

+

T cells and CD8

+

T cells in P2Y2

-/-

mice compared to

P2Y2

+/+

infected lungs. Lower level of IL-12 and higher level of IL-6 in broncho-alveolar fluid support

an inhibition of Th1 response in P2Y2

-/-

mice. Quantification of DC recruiter expression revealed

comparable IP-10 and MIP-3&
Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Se ha establecido que un subconjunto de pacientes con cáncer debuta con infiltrados inmunes que se organizan en agregados en el nicho tumoral y sus alrededores. Estas estructuras linfoides se desarrollan en respuesta a estímulos inflamatorios a través de un proceso estrechamente regulado. Además de su valor pronóstico, las TLSs pueden representar una nueva vía para las estrategias terapéuticas, pero actualmente se encuentra en etapas iniciales. En contraste con el papel en la activación inmune de las TLSs, en ciertos tumores, su efecto puede apuntar hacia la progresión tumoral como consecuencia de las condiciones altamente inmunosupresoras mediadas por las células malignas presentes en el nicho tumoral. Los datos preliminares proporcionados por la siguiente investigación sugieren que los pacientes con cáncer de pulmón podrían presentar un perfil inmunitario diferencial respecto a estos mismos que además subyacen EPOC. Este hecho podría presentar un impacto potencial en el pronóstico y la terapia de estos pacientes. Por otra parte, resultados procedentes de la actual investigación también revelan que ciertos marcadores cruciales presentes en diferentes vías de señalización involucradas con el estrés oxidativo, la apoptosis y la autofagia podría sobre expresarse en respuesta a la administración de inmunomoduladores. Estos datos ponen de manifiesto el interés de mecanismos adicionales relacionados con la inmunidad que podrían ser manipulados para asistir a la inmunidad contra el cáncer.
It is well established that a subset of cancer patients debuts with immune infiltrates, which organize into aggregates in the tumor niche and its vicinity. These lymphoid structures develop in response to inflammatory stimuli through a tightly regulated process. Besides the prognostic value of TLSs, they also may represent a novel avenue for therapeutic strategies, but it is currently still in its early stages. In contrast with the immune activator role of TLSs, in certain cancers, its effect may point towards tumor progression as a consequence of highly tumor-mediated immunosuppressive conditions present in the tumor niche. Preliminary data provided by the current investigation suggests that a differential immune profile may be present between LC patients and LC patients underlying COPD. This fact could present a potential impact in the prognosis and therapy of these patients. Moreover, crucial markers targeting different signaling pathways involved with oxidative stress, apoptosis, and autophagy were found to be overexpressed in response to immunomodulators administration in the current thesis. These data puts into manifest the interest of additional immunity-related mechanisms that could be targeted in order to assist immunity against cancer.

Asthma, a chronic TH2-mediated inflammatory disease of the airways, is the most common form of allergy in the Western world, affecting 300 million people worldwide. Epidemiological studies have shown that asthma is associated with mood disorders, such as anxiety and depression, and numerous experiments have reported that asthma induces functional changes in neuronal fibres of the peripheral nervous system (PNS), which innervate the brain. It is unknown, however, how allergic lung inflammation impacts on the central nervous system (CNS). The ability for peripheral inflammation to impact on the brain, altering behaviour and neuronal activity in the CNS, is a well-recognised and physiological phenomenon, known as immune to brain communication, but has, until now, only focused on how innate pro-inflammatory and TH1, but not TH2, type immune responses impact on the brain. Critically, immunomodulatory therapeutics, which involve stimulation of an innate pro-inflammatory immune response, are currently being developed for the treatment of asthma, highlighting the importance of understanding the effect of allergic lung inflammation and its treatment on the brain. Consequently, using acute and chronic localised TH2 models of inflammation, we investigated how allergic lung inflammation impacted on the CNS and subsequently determined the secondary impact of immunomodulation with the Toll-like receptor 7 (TLR7) agonist resiquimod. Acute TH2 inflammation in the peritoneum and lung was found to communicate with the brain, via a vagal route of communication. Crucially, it led to a distinct pattern of neuronal activity, with no changes in sickness behaviour or CNS inflammation, changes widely different to those known to occur following systemic TH1 inflammation. At chronic stages of lung inflammation, changes in genes associated with synaptic plasticity in the brainstem and altered expression of the GABAB receptor and brain-derived neurotrophic factor in the hippocampus were observed, firstly providing a CNS-dependent biological explanation for airway hyperresponsiveness, a critical pathological symptom of asthma, and secondly offering a biological justification for the prevalence of mood disorders in asthmatic patients. Resiquimod treatment in allergic animals was associated with attenuated central inflammatory responses, as compared to treatment in healthy animals, encouraging and reassuring in terms of patient well-being and, critically, also insinuating that safety of therapeutics differs in diseased, as opposed to healthy individuals. The results in this thesis are some of the first to identify that physiological inflammatory diseases impact on the CNS, highlighting the importance of immune to brain communication on pathological and psychopathological symptoms of a disease, and additionally demonstrating how inflammatory conditions can modify the off-target effects of a drug. Not only do these results provide a foundation for the future of immune to brain communication research, namely understanding how physiological inflammatory diseases impact on the CNS, but also have the potential to be translational and emulated in a clinical setting.

Death Receptor 3 (DR3) is a death domain containing member of the TNF Receptor Superfamily (TNFRSF), refereeing a range of cellular responses from differentiation and proliferation to cell death, depending upon the context of receptor activation. DR3 has been reported to have a role in many inflammatory diseases, including inflammatory arthritis and inflammatory bowel disease. The aim of this study was to determine the contribution of DR3 in a mouse model of acute and chronic allergic lung inflammation. Mice genetically deficient in the DR3 gene (DR3ko) were resistant to cellular accumulation within the lungs and bronchoalveolar lavage following acute lung inflammation, induced by priming with ovalbumin (OVA) and the adjuvant aluminium hydroxide (Alum) prior to 2 OVA aerosol exposures. To discern the role of DR3 in a more physiologically relevant chronic model of allergic lung inflammation, mice underwent repeated inhalation challenges with OVA subsequent to priming with OVA and Alum. Whilst cellular accumulation did not differ, DR3ko mice displayed reduced immuno-histopathology, and goblet cell hyperplasia, hallmarks of the asthmatic phenotype. Intriguingly, DR3ko mice exhibited reduced accumulation of various cell types into the spleen in both models. Early priming events were therefore investigated, prior to aerosolised antigenic challenge to decipher the effects of DR3. One sensitisation injection was sufficient to induce decreased DR3ko splenocyte accumulation, though T and B cell responses were observed to be comparable between DR3ko and DR3wt controls. DR3ko mice had depleted CXCL10 levels, suggesting cellular recruitment in response to inflammation is DR3 dependent. The underlying DR3 dependent mechanisms concerning the DR3ko splenic defects are under further investigation and may have impact on the use of the DR3/TL1A pathway as a therapeutic target, either as an anti-inflammatory or as a booster of the immune response to pathogens.

IIn the first study, we investigated the functional implications of prolonged TLR signalling on IFN-γ mediated killing of mycobacteria by murine macrophages in vitro. TLR2, but not TLR4 ligation interfered with IFN-γ mediated killing of mycobacteria in macrophages. In terms of mechanisms, neither TNF nor nitric oxide (NO) production was significantly affected, and the refractoriness induced could be reversed with increasing amounts of IFN-γ In the second study, we aimed to identify immunological markers of diagnostic importance in both the respiratory tract and serum during pulmonary mycobacterial infection in mice. We found that increased levels of immunological markers in the respiratory tract, but not serum, correlated better with active mycobacterial infection in the lungs, suggesting that the immune response in the respiratory tract is more reflective of the infection status and pathology than the systemic response. Finally, we investigated the level and nature of immune responses to pulmonary mycobacterial infection in BALB/c and C57BL/6 mice, two mouse strains known to exhibit different susceptibilities to infection with several intracellular pathogens, including mycobacteria. We showed that increased susceptibility of BALB/c mice to early mycobacterial infection was associated with reduced Th1 immune responses, and increased sTNFR secretion in the lung. Moreover, BALB/c mice recruited fewer monocytes/macrophages to the lung, and although IFN-γ stimulation of infected bone marrow derived macrophages in both mouse strains resulted in induction of antimycobacterial activity, BALB/c mice had a reduced capacity to kill ingested bacteria. The work presented in this thesis provide further insight into the mechanisms involved in the host-pathogen interaction; from persistence, to the immunological processes induced by the pathogen, to susceptibility of the host to infection.

Early life is a period of increased vulnerability to infection, particularly of the respiratory tract. The neonatal adaptive immune system is immature, with a bias towards Th2/Th17 responses and against Th1. Very little is understood about the innate response in early life, especially at mucosal surfaces such as the lung. To address this, Toll-like receptor (TLR) ligands were administered intranasally to neonatal and adult mice. In neonates, pulmonary neutrophilic influx was barely detectable, and expression of inflammatory chemokines greatly attenuated. Administration of exogenous CXCL1 elicited a strong neutrophilic response, indicating that diminished chemokine production is a limiting factor for cellular inflammation in early life. An unbiased microarray approach revealed that whilst expression of immune-related genes was mostly suppressed in the naïve neonatal lung, antimicrobial peptides such as cathelicidin were over-expressed. These novel findings challenge the perception that the infant immune system is simply an ‘immature’ version of the adult system, with implications for development of vaccines and adjuvants for this vulnerable population. An important respiratory pathogen of infants is respiratory syncytial virus (RSV), which can cause bronchiolitis, and in clinical studies is linked with lung dysfunction such as asthma and wheezing later in life. However, the mechanisms by which early life RSV infection could cause these delayed sequelae are unknown, and the effect on the innate immune response has not been explored. The long-term consequences of early life RSV infection were investigated using a murine model. Following neonatal RSV infection, allergen challenge resulted in an exaggerated inflammatory response in the adult murine lung, indicating that pulmonary innate immunity was dysregulated. Further, expression of various immune response, apoptosis-related and circadian rhythm genes was found to be altered. These data provide new evidence that infantile RSV has a causative effect on later lung dysfunction and identify gene targets for further investigation.

The primary muscle of respiration is the diaphragm. Diaphragm muscle dysfunction and ventilatory pump failure are well documented phenomena in animal models of sepsis. However, the primary cellular mechanisms underlying respiratory muscle dysfunction in sepsis are poorly understood. In addition, most investigations of respiratory muscle dysfunction in sepsis have been performed in models involving high doses of bacterial endotoxin and these investigations have been criticized on the basis of questionable relevance to human sepsis. Therefore, the objective in the first study of this thesis was to study respiratory muscle dysfunction in a more clinically relevant animal model, namely, the Pseudomonas aeruginosa pulmonary infection model. Remote inflammatory processes in different diseases, such as cancer, arthritis, sepsis, and cystic fibrosis are known to contribute to muscle wasting and weakness through more widespread systemic effects. In keeping with the above notion, we hypothesized that sustained P. aeruginosa lung infection would cause diaphragmatic and limb muscle weakness. In this thesis, we demonstrate for the first time that persistent pulmonary infection with P. aeruginosa induces significant dose- and time-dependent contractile dysfunction of the diaphragm. By comparison, prototypical slow- and fast-twitch hindlimb muscles were not influenced by pulmonary P. aeruginosa infection.
Because skeletal muscles can express a variety of immune modulating molecules such as cytokines, chemokines, adhesion molecules, and major histocompatibility molecules, the objective of the second study in this thesis was to study the possible role of pro-inflammatory cytokines in diaphragm muscle dysfunction in our animal model. Our results indicate for the first time that intra-diaphragmatic pro-inflammatory cytokine gene expression (TNF-alpha, IL-1alpha, IL-1beta, IL-6, and IL-18) is highly up-regulated in infected animals and the magnitude of such upregulation is dependent upon the dose of P. aeruginosa lung infection. Parallel to the absence of muscle contractile dysfunction in hindlimb muscle under the same conditions, P. aeruginosa infection did not alter the levels of pro-inflammatory gene expression within the hindlimb muscle. To further address the involvement of muscle-derived pro-inflammatory cytokines in diaphragmatic contractile dysfunction, we have employed recombinant adenovirus (Ad) as a vehicle for systemic delivery of the anti-inflammatory cytokine IL-10, in order to shift the balance between pro- and anti-inflammatory cytokines within the diaphragm toward a more anti-inflammatory profile. We report here that systemic delivery of Ad-IL-10 suppresses pro-inflammatory gene expression and improves force generating capacity of the diaphragm in P. aeruginosa infected animals. This finding emphasizes the role of anti-inflammatory cytokines as beneficial immune modulators in respiratory muscle failure caused by pro-inflammatory cytokines.
P. aeruginosa lung infection is a major cause of morbidity and mortality among cystic fibrosis (CF) patients and many patients with CF have weak peripheral and respiratory muscles. Although the role of pro-inflammatory cytokines has been extensively studied within the lungs of CF patients, the involvement of these cytokines in skeletal muscle dysfunction in animal models of CF or in human CF patients has not been studied. Therefore, in the third study of this thesis we have used mice sharing the same genetic defect as CF patients (Cftr knockout mice), in combination with our model of P. aeruginosa lung infection, to address several fundamental questions related to muscle function in CF. Our first objective in this portion of the thesis was to determine if diaphragmatic skeletal muscle cells express the CFTR mRNA. Our second objective was to ascertain whether intrinsic differences between CF and wild-type muscle cells could be detected in vitro, which might differentially affect the regulation of pro-inflammatory mediators in the setting of infection/inflammation. Our third objective was to evaluate possible differences in the ability of respiratory muscles to generate force prior to and after P. aeruginosa lung infection in Cftr knockout mice, as compared to their wild-type littermates. Finally, we aimed to determine if the absence of CFTR expression would predispose to muscle dysfunction triggered by up-regulation of intra-diaphragmatic pro-inflammatory gene expression. Our major results indicate that: First, in vitro stimulation with pro-inflammatory cytokines (TNF-alpha, IL-1alpha, and IFN-gamma) and LPS (extracted from Pseudomonas aeruginosa) triggered increased expression of pro-inflammatory mediators (iNOS, RANTES, MIP-1alpha, MIP-1beta, MIP-2 and KC) in both Cftr -/- and wild-type diaphragmatic myotubes, but the magnitude of cytokine/chemokine upregulation was significantly greater in CF than in wild-type diaphragm muscle cells. Sec
In the final study of this thesis, we sought to test the hypothesis that increased diaphragm muscle activation would lead to increased production of intra-diaphragmatic cytokine expression, since this could possibly explain the greater susceptibility of the diaphragm to express pro-inflammatory cytokines in response to pulmonary P. aeruginosa infection as compared with the hindlimb muscle. To test this hypothesis, we subjected rats to inspiratory resistive loading (IRL), corresponding to 45-50% of the maximum inspiratory pressure, and described that mRNA levels of IL-1beta, IL-6, and to a lesser extent, IL-4, IL-10, TNF-alpha, and IFN-gamma were all significantly increased in a time-dependent fashion in the diaphragm but not hindlimb muscle (gastrocnemius) of loaded animals. In addition, elevated protein levels of IL-1beta and IL-6 in response to loading were confirmed with immunoblotting and immunostaining. We also detected significant IL-6 protein to be localized inside diaphragmatic muscle fibers of loaded animals. We conclude that increased diaphragm muscle activity during resistive loading induces upregulation of pro-inflammatory cytokine gene expression in the diaphragm, which could also provide an explanation for the greater cytokine expression observed in the diaphragms of animals with P. aeruginosa lung infection.

Tuberculosis (TB) is a global health problem, which is proving extremely difficult to control in the absence of an effective vaccine. Bacille Calmette-Guérin (BCG), the only vaccine currently licensed against TB, demonstrates variable efficacy in humans and cattle. A greater understanding of what constitutes a protective host immune response is required in order to aid the development of improved vaccines. Tissue-resident memory T cells (T_RM) are a recently-identified subset of T cells, which may represent an important aspect of protective immunity to TB. This thesis aims to characterise the role of lung T_RM in BCG-induced protection against TB. In a mouse model, intravascular staining allowed discrimination between lung-vascular and lung-parenchymal T cells. Experiments demonstrated that BCG vaccination induced a population of antigen-specific lung-parenchymal CD4⁺ T cells, a putative tissue-resident population. This lung-parenchymal population was significantly increased in frequency following mucosal BCG vaccination, compared to systemic BCG vaccination. This correlated with enhanced protection against Mycobacterium tuberculosis (M.tb) infection in the lungs of mice receiving mucosal BCG, compared to those receiving systemic BCG. Mucosal BCG induced lung-parenchymal CD4⁺ T cells with enhanced proliferative capacity and a PD1⁺KLRG1^- cell-surface phenotype, a memory-like phenotype associated with improved protection against M.tb infection. These cells may represent a BCG-induced lung T_RM population responsible for the enhanced protection observed following mucosal BCG. Overall, this thesis highlights the potential of mucosal vaccination to elicit lung T_RM and identifies this as a possible immunological mechanism underlying enhanced protection against M.tb infection. These cells may constitute an important target for future vaccination strategies.

Includes bibliographical references (leaves 108-126).
Infection by the parasitic nematode Nippostrongylus brasiliensis involves migration through the lungs, causing significant damage and generating chronic lung pathology. The resolution of N. brasiliensis infection and also the induction of pulmonary pathology, including goblet cell hyperplasia and acute airway inflammation, depend on IL-4Rá signalling. A key feature of IL-4Rá signalling is the induction of a strong TH2 response which induces the development of alternatively activated macrophages (AAMs). AAMs are associated with tissue remodelling and the control of exacerbated inflammation. In order to investigate potential roles for IL-4Rá in N. brasiliensis' induced lung pathology, we infected mice deficient for IL-4Rá on macrophages and neutrophils (LysMCreIL-4Rá-/lox), IL-4Rá -/- and control mice (IL-4Rá-/lox) with N. brasiliensis and examined lung pathology at days 5, 42 and 180 post infection (p.i.).All three mice strains showed similar emphysemic-like pathology (alveolar dilatation) and airway hyperresponsiveness (AHR) which was well developed by day 42 p.i. and remained chronic. However, LysMCreIL-4Rá-/lox mice consistently demonstrated earlier and increased pulmonary inflammation when compared to IL-4Rá-/lox control mice and IL-4Rá-/- mice. Immunological studies at day 5 p.i. revealed that there were increased CD4+ and CD8+ T-cell numbers and increased CD4+ IL-4 and IL-13 production in the lungs of LysMCreIL-4Rá-/lox mice when compared to control and IL- 4Rá-/- mice. LysMCreIL-4Rá-/lox mice also showed decreased pulmonary arginase activity, indicative of a reduction of AAMs. RNA transcript analysis of isolated alveolar macrophages showed a strong association with promoting inflammation in LysMCreIL-4Rá-/lox mice. Together these data demonstrate that IL-4Rá-responsive macrophages control pulmonary inflammation and play an important protective role in the lung following N. brasiliensis infection.

El trasplantament pulmonar (TP) és el tractament d’elecció per a malalties respiratòries terminals. No obstant, el desenvolupament de la disfunció crònica de l’empelt pulmonar (DCEP) representa el principal factor limitant per assolir la supervivència a llarg termini després del TP. El tractament immunosupressor de per vida en pacients trasplantats d’òrgan sòlid comporta una sèrie de complicacions (nefrotoxicitat, infeccions, aparició de càncer, desordres metabòlics) que influeixen negativament en la supervivència a llarg termini. Un dels principals objectius en el trasplantament d’òrgans és aconseguir una manca de resposta immunològica del receptor en absència de teràpies immunosupressores sostingudes. En la majoria de casos, la retirada de la immunosupressió comporta el rebuig de l’empelt però un petit grup de pacients manté una bona funció a llarg termini malgrat la interrupció del tractament (tolerància operacional). Aquest estat es infreqüent i únicament s’ha en pacients trasplantats de ronyó i fetge. En altres trasplantaments d’òrgan sòlid (pulmó, cor o intestí) només s’han reportat casos anecdòtics d’aquest fenomen. Donada la falta de pacients trasplantats pulmonars amb tolerància operacional, els supervivents a llarg termini (SLT) amb bona funció de l’empelt després del TP són el grup més semblant als tolerants operacionals després del trasplantament de ronyó o fetge. La identificació de biomarcadors de tolerància operacional permetria la precisa selecció d’aquells pacients candidats a la minimització i potencial retirada del tractament immunosupressor. A més, aquests potencials biomarcadors podrien proporcionar coneixement sobre els mecanismes biològics subjacents a la tolerància i usar-se en el desenvolupament de noves teràpies tolerogèniques. S’ha observat que les firmes de tolerància en ronyó i fetge difereixen, suggerint que els mecanismes són òrgan-específics i que els potencials biomarcadors de tolerància no poden ser extrapolats al cas del pulmó. Donada l’escassetat d’estudis publicats sobre la supervivència a llarg termini amb bona funció de l’empelt després del TP, l’objectiu d’aquest tesi és analitzar el major nombre de paràmetres clínics i immunològics a través de diferents plataformes per a la identificació de potencials biomarcadors, i així proporcionar nou coneixement sobre els mecanismes biològics responsables de la supervivència a llarg termini amb bona funció de l’empelt en comparació amb pacients amb DCEP. El microbioma de les vies respiratòries altes i el complet perfil transcriptòmic i immunofenotipat en sang perifèrica van ser analitzats per avaluar les característiques dels pacients SLT. Els resultats derivats dels anàlisis bioinformàtics corresponents a l’expressió gènica i microRNAs van proporcionar noves dades respectes als mecanismes implicats en la supervivència a llarg termini. A més, es va aconseguir classificar els pacients trasplantats pulmonars amb alta precisió mitjançant perfils d’expressió gènica i d’expressió combinada amb diversos biomarcadors. Aquests descobriments han demostrat la utilitat de l’ús del perfil transcriptòmic i de les mostres de sang perifèrica per tal de discriminar als pacients SLT i amb DCEP, i per identificar els potencials mecanismes responsables de l’acceptació de l’empelt a llarg termini. En conclusió, els estudis inclosos en la present tesi proporcionen nous coneixements sobre els mecanismes subjacents a l’acceptació de l’empelt després del TP, suggerint una complexa interacció entre diferents mecanismes immunològics i proporcionant noves perspectives per a futures investigacions en la immunologia del TP.
El trasplante pulmonar (TP) es el tratamiento de elección en enfermedades respiratorias terminales. La supervivencia a corto plazo tras el mismo ha ido aumentando progresivamente debido a las mejoras en las técnicas quirúrgicas, la preservación de órganos, las terapias inmunosupresoras y el manejo perioperatorio. Sin embargo, el desarrollo de la disfunción crónica del injerto pulmonar (DCIP) representa el principal factor limitante para alcanzar la supervivencia a largo plazo tras el TP, siendo la tasa de supervivencia a diez años aproximadamente de un 34.3%. El tratamiento inmunosupresor de por vida en los pacientes trasplantados de órgano sólido conlleva una serie de complicaciones (nefrotoxicidad, infecciones, aparición de cáncer y/o desórdenes metabólicos) que influyen negativamente en su supervivencia a largo plazo. Uno de los principales objetivos en el trasplante de órganos es conseguir la falta de respuesta inmunológica del receptor frente al injerto en ausencia de terapias inmunosupresoras sostenidas. En la mayoría de los casos, la retirada de la inmunosupresión conlleva el rechazo del injerto. Sin embargo, se ha observado que un pequeño grupo de pacientes mantiene una buena función del injerto a largo plazo a pesar de la interrupción del tratamiento (tolerancia operacional). Este estado es infrecuente y únicamente se han observado casos en pacientes trasplantados renales y de hígado. En otros trasplantes de órgano sólido (pulmón, corazón o intestino) solo se han reportado casos anecdóticos de este fenómeno. Dada la falta de pacientes trasplantados pulmonares con tolerancia operacional, los supervivientes a largo plazo (SLP) con una buena función del injerto tras el TP son el grupo que más se asemeja a los tolerantes operacionales tras el trasplante de riñón o de hígado. La identificación de biomarcadores de tolerancia operacional permitiría la precisa selección de aquellos pacientes candidatos a la minimización y potencial retirada del tratamiento inmunosupresor. Además, estos potenciales biomarcadores podrían proporcionar conocimiento acerca de los mecanismos biológicos que subyacen a la tolerancia y podrían usarse en el desarrollo de nuevas terapias tolerogénicas. Se ha observado que las firmas de tolerancia en riñón e hígado difieren, sugiriendo que los mecanismos responsables de la tolerancia operacional son órgano-específicos y que los potenciales biomarcadores de tolerancia no pueden extrapolarse al caso del pulmón. Dado los escasos estudios publicados sobre la supervivencia a largo plazo con buena función del injerto tras el TP, el objetivo de esta tesis es analizar el mayor número de parámetros clínicos e inmunológicos a través de diferentes plataformas para la identificación de potenciales biomarcadores, y así, proporcionar nuevo conocimiento sobre los mecanismos biológicos responsables de la supervivencia a largo plazo con buena función del injerto en comparación con pacientes con DCIP. El microbioma de las vías respiratorias altas y el completo perfil transcriptómico e inmunofenotipado en sangre periférica fue analizado para evaluar las características de los pacientes SLP. Los resultados derivados de los análisis bioinformáticos correspondientes a la expresión génica y de microRNAs proporcionaron nuevos datos referentes a los mecanismos implicados en la supervivencia a largo plazo. Además, se consiguió clasificar a los pacientes trasplantados pulmonares con alta precisión empleándose para ello perfiles de expresión génica y de expresión combinada de varios biomarcadores. En conclusión, los estudios incluidos en la presente tesis han demostrado la utilidad del uso del perfil transcriptómico y de las muestras de sangre periférica para discriminar a los pacientes SLP y con DCIP y para identificar potenciales mecanismos responsables de la aceptación del injerto a largo plazo, proporcionando nuevas perspectivas para futuras investigaciones en la inmunología del TP.
Lung transplantation (LT) is an established treatment for end-stage respiratory diseases. Short-term survival has progressively improved due to advancements in surgical techniques, donor preservation, immunosuppressive agents and perioperative management. However, the development of chronic lung allograft dysfunction (CLAD) is the main limiting factor of long-term success after LT, with an approximate ten-year survival rate of 34.3%. The lifelong immunosuppression of solid organ transplant patients leads to severe complications, such as nephrotoxicity, infectious diseases, malignancies, and metabolic disorders, which poorly affect their long-term survival. For this reason, one of the main goals in organ transplantation is achieving an alloantigen-specific unresponsiveness state in the sustained absence of toxic immunosuppressive therapies. In most cases, immunosuppression withdrawal leads to transplant rejection. However, a small group of transplant patients maintains long-term stable graft function despite interrupted treatment (operational tolerance state). This phenomenon is infrequent and varies according to the type of allograft; excluding kidney and liver transplant fields, there are only anecdotal cases in lung, heart and intestine transplantation. Due to the lack of operationally tolerant lung transplant recipients, long-term survivors with normal allograft function (LTS) after LT are the closest group to “operational tolerance” of kidney or liver transplant patients. Identifying biomarkers of operational tolerance would serve to accurately identify candidate patients for minimization and potential withdrawal of immunosuppression. Furthermore, these potential tolerance biomarkers could provide knowledge of the underlying biological mechanism of tolerance for new tolerogenic therapies. It has been observed that tolerance fingerprints from kidney and liver transplant recipients differ, suggesting that the underlying mechanisms of operational tolerance, not fully elucidated, are organ-specific and consequently, potential renal and liver tolerance biomarkers cannot be extrapolated to lung. Since no extensive studies examining this LT state have been published, the main objective of this thesis was to screen the greatest number of clinical and immunological parameters to identify potential biomarkers across different platforms in order to provide a better understanding of the biological mechanisms underlying LTS with normal allograft function after LT in comparison with CLAD patients. The microbiome of the upper respiratory tract and the full transcriptomic expression profile and extensive cell immunophenotyping of peripheral blood samples were studied to assess the particular characteristics of LTS patients. The results derived from bioinformatics analyses of gene and miRNA expression provided a better understanding of the mechanisms involved in long-term survival. Moreover, they proved to be highly accurate in the classification of LT patients by employing gene and multi-biomarker expression profiling using different transcriptional platforms, including microarrays and RT-qPCR arrays. The findings obtained demonstrated the usefulness of global transcriptome profile and peripheral blood samples to differentiate between LTS and CLAD patients and to identify some of the potential mechanisms responsible for graft acceptance. Overall, the studies included in this thesis shed light on the biology underlying graft acceptance after LT, suggesting a complex interaction of several immunological mechanisms and opening up new perspectives for future research in LT immunology.

Lung surfactant protein D (SP-D) is a soluble pattern recognition and innate immune molecule, which has been shown to be protective against lung infection, allergy, asthma and inflammation. SP-D is composed of an N-terminal collagen region and a homotrimeric, C-terminal carbohydrate binding domain (CRD). A recombinant form of trimeric CRD region (rhSP-D) has been shown to offer protection against asthma and inflammation in murine models by bringing down IgE levels, eosinophilia, and causing T helper cell polarisation from a pathogenic Th2 to a protective Th1 phenotype. Thus, rhSP-D can provide a therapeutic effect by dampening asthmatic symptoms in mice. The therapeutic mechanisms include inhibition of allergen-IgE binding and histamine release by sensitized mast cells, downregulation of allergen/antigen-specific IgG and IgE antibodies, pulmonary and peripheral eosinophilia, a shift from Th2 to Th1 cytokine response, interference with airway remodelling processes, and apoptosis- induction in sensitised eosinophils from allergic patients. The majority of the ex vivo and in vivo studies where a therapeutic effect of rhSP-D has been reported can not be explained by hitherto described candidate receptor involvement, especially CD91-calreticulin complex that requires collagen region for its cellular response. Thus, it is pertinent to examine at the cellular and molecular level how a trimeric lectin domain of human SP-D modulates immune cells. This was achieved by firstly expressing, purifying and characterising the recombinant rhSP-D and examining the interaction of rhSP-D with various immune cells such as macrophages, which are potent antigen presenting cells and play a crucial role in the maintenance of the inflammatory and humoral response to allergens. The highlight of this study is the demonstration that rhSP-D interferes with the co-operative binding of allergen-IgE complexes to B cells, and also downregulates expression of CD23, a low affinity IgE receptor (FcεRII), found on B cells. This suggests that inhibition of IgE-facilitated antigen presentation may represent a mechanism whereby SP-D suppresses Th2-driven allergic inflammation. In addition, this study is also the first to establish the calcium-dependent interactions between rhSP-D, CD23 and CD21. The possibility of formation of a trimolecular complex on the B cell surface may account for the suppression of IgE in therapeutic murine models since rhSP-D may interfere with CD21-CD23 mediated IgE production by primed B cells.

Parmi les manifestations cliniques du paludisme où P. falciparum est le principal responsable des formes graves, les manifestations pulmonaires sont souvent sous-estimées. Malgré de nombreuses études tentant de mieux comprendre les mécanismes inflammatoires mis en jeu dans l'aggravation de la maladie, des zones d'ombres persistent. Pour étudier les interactions hôte-parasite et identifier des biomarqueurs de classification et d'évolutivité de l'accès palustre, nous avons effectué une étude de cohorte longitudinale prospective à l'Hôpital Monkole de Kinshasa, par une approche combinée immunologique et transcriptomique sur 30 jours. L'étude devant porter initialement sur les atteintes pulmonaires a été recentrée sur l'étude de la sévérité de l'accès palustre devant le peu de patients présentant des atteintes pulmonaires.A travers l'approche immunologique, une réponse pro-inflammatoire de type Th1 modérée est activée à l'admission des patients impaludés. L'approche transcriptomique a permis de le relier à une activation modérée des monocytes/macrophages médiée par la voie de signalisation de l'IL-12, toutefois plus importante chez les accès graves. L'infection palustre, et plus particulièrement l'accès grave paraissent être fortement sous dépendance de la réponse Th17 et plus particulièrement à l'activité des neutrophiles. La mise en place d'une réponse Th2 est également observée chez les patients impaludés. A J7, la réponse Th2 est majoritaire, associée à une importante activité hématopoïétique. A J30, les patients sont guéris et présentent globalement un profil similaire aux témoins.Ces résultats sont à confirmer et à approfondir par une augmentation de notre cohorte
Among complications of the malaria access, mostly due to P. falciparum, the pulmonary injuries are often underestimated. Despite numerous studies trying to better understand the inflammatory mechanisms involved in disease worsening, they are still poorly understood. In view to study the host-parasite interactions and identify biomarkers according to severity of malaria, a prospective longitudinal cohort study was carried out in Monkole Hospital (Kinshasa) through a combined immunological and transcriptomic approach, with a follow up on 30 days. Initially, the study concerned the pulmonary injuries, but was focused on the the severity of malaria because of a weak patients number with lung damages.The immunological approach highlighted a moderate Th1 pro-inflammatory response at admission. The transcriptomic approach allowed to connect it with a moderate activation of monocytes/macrophages, mediated by IL-12 signaling pathway, higher in severe malaria. Malaria infection, particularly severe malaria, appears to be strongly dependent on Th17 response, especially to neutrophil activity. The establishment of a Th2 response is also observed in malaria patients. On day 7, Th2 response is predominant, associated with significant hematopoietic activity. On day 30, patients are cured and present a similar profile to healthy controls.These results have to be confirmed with an increased cohort

The recent identification of two novel influenza-like viruses in bats, H17N10 and H18N11 virus, and the discovery of serologically positive Eidolon helvum bats in Ghana for avian H9 virus prompted my hypothesis that, in addition to the large repertoire of zoonotic viruses hosted, bats may serve as asymptomatic reservoir species to conventional influenza A viruses found in birds and mammals. To begin to test this hypothesis, the susceptibility of three bat cell lines, derived from lung epithelial cells of Eidolon helvum, Carollia perspicillata and Tadarida brasiliensis (TB1-Lu), to low pathogenicity avian viruses (H2N3 [A/mallard duck/England/7277/06] virus and H6N1 virus [A/turkey/England/198/09] virus), and human viruses (USSR H1N1 virus [A/USSR/77] and pandemic H1N1 2009 virus [A/California/07/2009]) was determined. All three species of bat epithelial cells were found to be more robust and resistant to influenza virus infections than permissive MDCK cells. Infected bat cells produced lower levels of viral RNA and viral progeny, and were more viable than correspondingly treated MDCK cells. Interestingly, bat cells were more susceptible and replication permissive to avian than human influenza viruses. Among the bat cells, TB1-Lu cells were the least susceptible to influenza virus infection and that appears to be related to a lack of sialic acid α2,6-Gal receptors, mammalian virus-preferred host receptors, which were present in the other two bat species. The innate mechanisms underlying resistance to influenza virus infection in bats remains to be determined, however, inhibition of the JAK/STAT pathway was found not to affect virus production from infected bat cells suggesting that JAK/STAT signalling may not have a major role in influenza virus resistance in bat cells. Modulation of NF-κB signalling was found to affect virus production suggesting that tight regulation of NF-κB may be key in controlling the pro-inflammatory response to influenza infection in bat cells and warrants further investigation.

Neonates are more susceptible to influenza virus infection than adults, resulting in increased morbidity and mortality as well as delayed clearance of the virus. Efforts to improve influenza infection outcomes in neonates typically center on prevention, although current vaccines fall short of complete protection and can only be administered in humans after 6 months of life. We propose that as the neonatal immune system responds differently than the adult immune system, interventions that are efficacious or tolerable in adults cannot be guaranteed to perform the same in neonates. T cell vaccines that target conserved influenza virus epitopes have been proposed for conferring protection to multiple influenza virus strains, but if T cell vaccines will be used in infants and adults, neonates must be able to respond to the same T cell antigens as adults. Mouse pups responded to influenza virus peptide PA224-233 but not NP366-374 during influenza virus infection in contrast to the codominant adult response. Mice infected as pups also generated diminished T cell memory compared to mice infected as adults and displayed skewed immunodominance during secondary infection. Adult bone marrow derived dendritic cells (BMDCs) improved viral clearance when loaded with influenza virus and promoted NP366-374-specific CD8+ T cell responses in infected pups. BMDC peptide vaccination could stimulate PA224-233-specific but not NP366-374-specific CD8+ T cell responses in pups, but, PA224-233 vaccination offered no protection to pups during lethal infection. These data suggest that altered immunodominance must be considered when stimulating CD8+ T cell responses in adults and neonates. Immaturity and active suppression of immune responses are both factors in neonatal vulnerability to disease. Specifically, active suppression of neonatal immunity by regulatory T cells (Tregs) has been proposed as a driving factor in diminished neonatal immunity, but removing these cells can compromise viral defense or increase deleterious inflammation. Mice that lacked Tregs displayed compromised anti-influenza antibody responses and decreased lymph node responses during influenza virus infection. A high proportion of pup Tregs also expressed Gata3. Transgenic pups with a Treg specific Gata3 knockout displayed an increase in Tbet expression in both conventional and regulatory T cells and an increase in IFNγ producing CD4+ T cells in the lungs during infection. These data suggest that Tregs are required for effective humoral responses to influenza virus and that Gata3 expression influences Treg suppressive function in neonates.

Lung transplantation is a therapeutic option for patients affected with end-stage lung diseases. However, several complications may arise following the procedure, such as Bronchiolitis Obliterans (BO). This condition, characterized by small airway fibrosis, remains a major cause of morbidity and mortality in patients following lung transplantation. It is thought to be a manifestation of chronic rejection within the airways, with hallmark inflammation and fibroproliferation. TGF-beta and other cytokines, including IL-1, IFN-gamma and PDGF, have been implicated in the pathogenesis of fibrosis, mostly in animal models. IL-11 and IL-17 are novel profibrotic cytokines that induce fibroblasts and epithelial cells to produce excess extracellular matrix. They have recently been identified as having a role in tissue remodelling and induction of tissue fibrosis. We hypothesize that IL-11 and IL-17 are involved in chronic lung rejection (Bronchiolitis Obliterans) and that their expression could be a predictive and prognostic marker of chronic lung rejection.
The objectives of the study were to investigate the expression of IL-11 and IL-17 (mRNA and protein) in endobronchial biopsies from lung transplant patients and to define the correlation between the expression of IL-11 and IL-17 and the development of chronic rejection.

Influenza is an important human pathogen that causes fatal disease in 250,000-500,000 people worldwide each year. Because of high levels of variation between influenza strains, vaccines are not always effective and must be administered annually. Influenza virus, which replicates primarily in the lung epithelium, encodes only 10 proteins and relies heavily on host products to replicate. Determining which cellular factors are important for influenza replication represents an important area of virology and cellular biology research, and could elucidate proteins or pathways to target for antiviral therapies. We developed a high throughput screening method in primary human bronchial epithelium (HBECs) to identify novel regulators of influenza replication. We first used this method to functionally examine 1745 genes that were identified as potential influenza regulators due to transcriptional regulation by virus or viral products, direct interaction with viral proteins via yeast two-hybrid, or through computational analysis. This screen confirmed some known regulators of influenza replication while identifying novel viral interactors as influenza regulators (e.g. USHBP1, ZMAT4). We also found that the WNT, p53, and ER stress pathways, among others, affect viral replication and interferon production. The life cycle of influenza involves extensive intracellular trafficking of viral components. We again used RNAi to systematically examine the roles of vesicle, RNA, and protein trafficking genes in the production of infectious influenza A virus in primary lung cells. Among the factors that significantly impact viral infection, we identify a set of five genes with strong antiviral effects that are mutated in patients with Hermansky-Pudlak syndrome (HPS). Depletion of HPS genes leads to elevated viral RNA at an early stage of influenza infection prior to transcription. In contrast, depletion of these genes does not alter the innate immune response to virus or interferon. Using an HPS-1 patient cell line, we find an increase in viral fusion to endosomal compartments but no change in viral binding to the cell surface or entry into the early endosome. Our studies uncover a potential role for many trafficking factors in the influenza life cycle, and point to an HPS1-dependent process that inhibits viral entry prior to viral membrane fusion.

La sclérodermie systémique (ScS) est une maladie autoimmune rare qui se caractérise par une fibrose cutanée et parfois pulmonaire. Nous avons tout d’abord évalué le rôle de BAFF, une cytokine impliquée dans le développement des lymphocytes B (LB), dans la fibrose pulmonaire induite par la bléomycine chez la souris. Nous avons démontré que BAFF était augmenté en réponse à la bléomycine et que les souris BAFF-/- ou traitées par le BAFF-R-Ig sont protégées de la fibrose pulmonaire. Ensuite, nous avons évalué si les LB et BAFF pouvaient moduler la production de collagène par des fibroblastes de peau isolés de patients atteints de ScS. Nous avons démontré que les LB augmentent la production de collagène et de cytokines impliquées dans la fibrose cutanée et l’ajout de BAFF augmente cet effet des LB sur les fibroblastes. Enfin, nous avons étudié la régulation de l’expression de BAFF par les microARNs. Nos résultats montrent que les miR-30a*, d* et e* ciblent directement l’ARNm de BAFF
Systemic sclerosis (SSc) is a rare autoimmune disease characterized by skin fibrosis and occasionally pulmonary fibrosis. We first assessed the role of BAFF, a cytokine involved in B cell maturation, in bleomycin-induced pulmonary fibrosis in mice. We showed that BAFF was increased in response to bleomycin and that BAFF-/- mice or BAFF-R-Igtreated mice are protected from pulmonary fibrosis. Then, we assessed whether B cells and BAFF could regulate collagen production by skin fibroblasts isolated from SSc patients. We demonstrated that B cells increase collagen production and cytokines involved in skin fibrosis. The addition of BAFF increases the effect of B cells on fibroblasts. Lastly, we studied the regulation of BAFF expression by microRNAs. Our results show that miR-30a*, d* and e* directly target the BAFF mRNA

Depuis peu, l’immunothérapie a émergé comme une nouvelle stratégie chez les patients atteint de Cancer Bronchique Non à Petites Cellules (CBNPC), confirmant ainsi le rôle du système immunitaire dans cette maladie. Malgré ces nouveaux traitements (thérapies ciblées, immunothérapie), les taux de réponses restent faibles avec un impact modeste sur la survie globale. Des biomarqueurs sont donc nécessaire pour définir les populations cibles de ces traitements. Une des pistes explorées est le statut immunitaire des patients, en effet celui-ci a un impact pronostic et pourrait influencer la réponse aux traitements standards tels que la chimiothérapie, les thérapies ciblées et même l’immunothérapie. Parmi les cellules du système immunitaire, les cellules Natural Killer (NK) auraient un rôle effecteur dans le CBNPC. Il est maintenant clairement établit que les cellules NK favorisent la mise en place d’une immunité adaptative fonctionnelle et efficace. Ainsi une altération des fonctions NK pourrait être un mécanisme associé à l’échappement à l’immunité adaptative de la tumeur. Dans notre première étude, nous avons mis en évidence que les exosomes de cellules dendritiques stimulaient les cellules NK via NKp30, cette activité étant associée à un gain de survie sans progression chez des malades inopérables porteurs d’un CBNPC avancé. Notre second projet a révélé, pour la première fois, un rôle pronostic des transcrits de NCR3 (gène de NKp30) chez des patients naïfs de tout traitement. L’activation des cellules NK via NKp30 pourrait être une stratégie efficace d’immunomodulation chez les patients atteints de CBNPC avancé. Ces travaux confirment le rôle important des cellules NK dans le CBNPC avancé
Recently, immunotherapy has emerged as a new strategy in Non-Small Cell Lung Cancer (NSCLC) patients, confirming the key role of the immune system in this disease. Despite these new treatments (targeted therapies, immunotherapy), response rates remain low with a modest impact on overall survival. Biomarkers are needed to define the target population of these treatments. One of the options explored is the immune status; indeed the immune status of cancer patients has a prognosis impact and may influence the response to standard treatments such as chemotherapy, targeted therapies and even immunotherapy. Among the immune cells, Natural Killer cells (NK) have an effector role in NSCLC. It is now established that NK cells can promote a functional and efficient adaptive immunity. Therefore, an impaired NK functions could be a mechanism associated with the escape from adaptive immunity of the tumor. In our first study, we demonstrated that exosomes from dendritic cells stimulated NK cells through NKp30, this activity is being associated with improved survival in advanced NSCLC. Our second project has revealed, for the first time, a independent prognostic role of NCR3 transcript (NKp30 gene) for naïve advanced NSCLC. Activation of NK cells via NKp30 could be an effective strategy for immunomodulation in advanced NSCLC patients. These studies confirm a major role of NK cells in advanced NSCLC

Asthma is a debilitating disease of the lungs affecting 235 million people worldwide. Fungus-associated asthma leads to a particularly severe type of disease, and exposure to environmental fungi and their products is unavoidable due to the ubiquitous nature of fungal species. Besides being allergenic, fungi are opportunistic pathogens, and anti-fungal and/or allergic pathways may be modified through repeated inhalation of immunomodulatory agents, affecting the outcome of fungus-induced asthma.

Our aim in this project was to investigate the extent to which repeated inhalation of immunomodulatory agents influence the lung mucosal responses in a naïve murine host or in one that had been sensitized to fungal proteins (allergic). The immunomodulatory substances chosen hold relevance to human inhalational exposure, and included live or irradiation-killed Aspergillus fumigatus (a fungi) spores, deoxyxnivalenol (a mycotoxin), and fluticasone propionate (an inhalationally administered corticosteroid, commonly prescribed for allergic asthma). In a naïve host, inhalation of live A. fumigatus spores showed pathological features of fungal asthma. However, in an allergen-sensitized lung, both dead and live A. fumigatus spores established fungal airway disease, albeit to different extents. Next, we tested the effect of deoxynivalenol in an allergic host and found that its repeated inhalation did not affect pulmonary disease pathology, but did lead to a dose- and time- dependent increase in mucosal and systemic total IgA. Finally, we tested the effect of fluticasone propionate, and found that it did not influence the development of fungal airway disease, but did induce dynamic changes in lung physiology and antibody titers.

Besides mimicking human inhalational exposures, inhalation ensures direct interaction of the inhaled substances with airway epithelium, which plays an important role in defense against inhaled substances and in asthma pathophysiology. By analyzing various mechanisms involved in murine lung-mucosal response to the inhaled substances, a critical involvement of airway epithelium as an orchestrator of immune responses is highlighted, and this would inform mechanism-based future studies. In conclusion, this project is likely to aid in establishing evidence based standards for fungus-related exposures and in making informed therapeutic decisions for fungus-associated diseases.

A lesão pulmonar aguda é caracterizada por inflamação pulmonar podendo ser induzida diretamente (LPD), por inalação de lipopolissacarídeo (LPS), ou indiretamente (LPI), por mediadores inflamatórios liberados por órgãos distantes após lesão de isquemia e reperfusão (IR). A Bradicinina, mediador inflamatório, pode agir em dois receptores, um constitutivo (B2R), e um induzido por citocinas inflamatórias (B1R). Neste trabalho verificamos o papel do B1R em modelos de LPD e LPI. Em camundongos C57bl/6, a LPD foi induzida por tratamento intra-nasal com LPS, e a LPI foi induzida por 45 minutos de IR renal. Observamos alterações pulmonares em ambos os modelos de lesão 24 horas após o insulto, como aumento de infiltrado celular, hiperreatividade pulmonar à metacolina, aumento de permeabilidade vascular, e citocinas pró-inflamatórias. Bloqueamos o B1R com antagonista e vimos que a lesão pulmonar foi diminuída em ambos os modelos de lesão. Assim, sugerimos que o B1R contribui tanto para a LPD, induzida por LPS, quanto para a LPI, induzida por IR renal.
The acute lung injury (ALI) is characterized by lung inflammation and can be induced directly by lipopolysaccharides inhalation, or indirectly, by systemic inflammatory mediators released from distant organs after and ischemia and reperfusion injury (IRI). Bradykinin, an inflammatory mediator, can act in two different receptors; one is constitutive (B2R), whereas the other is induced by inflammatory cytokines (B1R). We aimed to study the role of B1R in models of direct and indirect ALI. Direct ALI was induced by LPS instillation in C57bl/6 mice, while indirect ALI was induced by 45 minutes of renal IRI. In both injuries, 24 hours after insult, animals presented an increase in cellular infiltration, vascular permeability, hyperreactivity to methacholine, and an up-regulation of pro-inflammatory cytokines in lungs. We blocked the B1R using antagonist and observed that the lung injury was attenuated in both injury models. Thus, we suggest that B1R has an important role in the development of both, direct ALI, induced by LPS, and indirect ALI induced by renal IRI.

Acute Lung injury (ALI) and the more severe acute respiratory distress syndrome (ARDS) are respiratory maladies that present immense clinical challenges. ALI affects 200,000 individuals annually and features a 40% mortality rate. ALI can be initiated by both pathogenic and sterile insults originating locally in the lungs or systemically. While immense research has been poured into this disease in an effort to find a therapeutic strategy, the heterogeneously diffuse nature of the disease has not yielded a cure for the disease. Death from this disease is strongly attributed to reduced gas exchange from a severely compromised alveolar-capillary barrier. The only way currently to manage this disease is through enhanced ventilation and hyperoxic therapy. Hyperoxic therapy is a common treatment given to over 800,000 patients each year to treat respiratory maladies such as ALI. Prolonged exposure to oxygen at high concentrations results in the development of a condition known as hyperoxic acute lung injury (HALI). In this disease, the formation of reactive oxygen species damages healthy tissue and impairs gas exchange. Hyperoxia is also a well-documented murine sterile lung injury model that replicates the symptoms of ALI in lung injury patients. The ability of non-lethal dosages of hyperoxia to resolve without lung fibrosis also enables the study of molecules associated with ALI resolution and repair, a process not clearly understood. Inflammation in ALI is associated with disease progression, however pharmaceutical interventions aimed at targeting the inflammatory cascade have failed in clinical trials for ALI. Recent reports point to an aberrant injury resolution mechanisms that may be more strongly correlated with morbidity and mortality. There seems to be a homeostatic imbalance between endogenous inflammation progression and resolution initiation. This is especially the case with HALI, as significant ROS generation results in depletion of redox regulating antioxidants. Resolution mechanisms associated with ALI in the oxygen toxicity setting is poorly understood. Polyunsaturated fatty acids such eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are essential fatty acids that show immense antioxidant and anti-inflammatory action in cases of acute injury. The lung mucosa is rich in DHA and following inflammatory insult DHA is readily converted to resolution phase interaction products (resolvins), which have shown immense proresolutionary potential in recent reports of acute injury. In the presence of aspirin, more potent and longer-acting aspirin-triggered resolvins are formed. The effects of resolvins and their aspirin triggered epimers have not been studied in an oxygen toxicity setting and are the focus of this dissertation. For the first time, we show that one of these resolvin molecules, aspirin triggered resolvin d1 (AT-RvD1), can enhance resolution of hyperoxic acute lung injury. In vitro results reveals that AT-RvD1 treatment resulted in reduced interaction of two key players in the HALI inflammatory cascade, the macrophage and alveolar epithelium. AT-RvD1 was able to blunt macrophage cytokine secretion as well as inhibit epithelial cell cytokine secretion and adhesion molecule expression. More importantly, AT-RvD1 blunted cytokine mediated leukocyte-epithelial cell interaction in vitro. In a sublethal hyperoxic injury model, mice given AT-RvD1 following hyperoxia exposure displayed reduced HALI pathological severity. ATRvD1 treatment resulted in reduced alveolar-capillary permeability, tissue inflammation, proinflammatory mediator secretion, epithelial cell death, and leukocyte influx. Taken together these novel results demonstrate the therapeutic potential of resolvins in the oxygen toxicity setting. These results also arouse the idea that resolvins could be used to lessen the comorbidities associated with oxygen therapy and improve recovery times of ALI patients.

The effects of inhaled air pollution particles on lung clearance mechanisms is an important factor in understanding how the mammalian lung deals with such pollutants and, as such, how exposure to these pollutants can be regulated. The nanoparticle(diameter S lOOnm) and transition metal components of PMIO (particulate matter with a diameter less than lO~m) have been implicated as playing major roles in the impairment of alveolar macrophage function and the subsequent retention of particles in the respiratory system. The aim of this study was to investigate the effects of components of PMIO on macrophage functions both directly, by examining macrophage phagocytosis and migration, and indirectly, by studying peripheral factors affecting macrophagefunction such as recruitment by type II cells and complement based mechanisms. We hypothesised that the alveolar epithelial type II cell line would release leukocyte chemoattractants in response to particle exposure and that this could be measured by use of a macrophage migration assay. A sub-toxic dose (125 ~g/ml)of surrogate air pollutionparticles (fine and nanoparticle carbon black and titanium dioxide) was established by measuring LOH release from a murine alveolar macrophage cell line (1774.2) and an alveolar epithelial type II cell line (L-2) in response to particle exposure. Optimisation ofa chemotaxis assay and measurement of macrophage migration towards conditioned medium obtained from the particle-exposed type II cells was conducted and it was determined that carbon black nanoparticles induced type II cells to secrete a chemoattractant that resulted in significant increases in macrophage migration compared to the negative control. This was in contrast to other particle types tested in this study which did not induce any increases in macrophage migration. It was also hypothesised that complement proteins could be involved in macrophage recruitment to sites of particle deposition and, as such, the migration of macrophages towards particle exposed blood serum was examined in vitro. Foetal bovine serum (FBS) was exposed to fine and nanoparticle caroon black and titanium dioxide (l-Smg/ml) for 2 hours. It was found, in accord with the previous study involving type II cells, that carbon black nanoparticles could activate the generation of chemotactic factors in serum that could subsequently induce significant increases (p < 0.001) in macrophage migration when serum was diluted to 10% using serum-free RPMI 1640 culture medium. This effect could be ameliorated by co-incubating the particle-treated serum in the presence of the antioxidant Trolox suggesting that oxidative stress played a role in the generation of the chemoattractant molecules. However, incubation of the serum with a pure oxidant at a range of doses did not result in the generation of chemotactic molecules suggesting that another factor could be involved in the chemoattractant generation. Further investigation to determine the exact molecular mechanism behind the chemoattractant generation is warranted. In contrast to the previous studies, we have also found evidence that components of PM₁₀ can cause decreased efficacy of macrophage clearance mechanisms in vivo and in vitro. It was hypothesised that PM₁₀ instillation would result in a decrease in macrophage phagocytic potential and an increase in chemotactic potential ex vivo. Rats were instilled with 125 and 250μg of PM₁₀ collected from North Kensington, London or sterile saline (negative control). Post-instillation (18 hours), significantly elevated concentrations of TNFa were detected in the BAL fluid together with a significant increase in the number of BAL neutrophils. Phagocytosis and chemotaxis assays conducted with BAL macrophages ex vivo showed that macrophage migration towards a positive chemoattractant, Zymosan Activated Serum (ZAS), was significantly lower than the macrophages obtained from the negative control rats. Macrophage phagocytosis of latex beads ex vivo was also found to be significantly decreased when PM₁₀ was visible inside the cell. An in vitro study where a macrophage cell line (J774.Al) was exposed to a low dose of nanoparticle carbon black (31.25μg) together with varying concentrations (100μM - 100nM) of zinc chloride (ZnCl₂) was also conducted. Exposure of macrophages to nanoparticle carbon black and zinc chloride alone induced a decrease in macrophage phagocytosis. It was found that when macrophages were co-exposed to nanoparticle carbon black and ZnCl₂, there was an additive decrease in macrophage phagocytic potential. The results contained within this manuscript demonstrate that the components of PM₁₀ can induce adverse effects on specific aspects of macrophage clearance mechanisms, but that nanoparticles can also stimulate the production of chemoattractants to aid in the recruitment of phagocytes and subsequent particle clearance. Although a contrary relationship appears to exist between these findings, the recruitment of leukocytes in response to particulate exposure is a mechanism that supports particle clearance. However, the retardation of phagocytic and chemotactic mechanisms in particle exposed macrophages may help to explain the increased toxicity, inflammation and retention time observed with nanoparticle inhalation.

The current worldwide epidemic of Mycobacterium tuberculosis infection is a huge global health problem. Widespread BCG vaccination remains a useful tool in combating this epidemic; however, its variable efficacy requires urgent development of novel vaccines against Mycobacterium tuberculosis. Such a candidate vaccine is a serotype 5 adenovirus expressing antigen 85A from M. tuberculosis (Ad85A). In animal models Ad85A confers significant protection when administered intra-nasally. The work in this thesis demonstrates that intra-nasal immunisation with Ad85A results in inhibition of M. tuberculosis growth in the lung early after infection, in contrast to the late inhibition induced by parenterally administered vaccines. Early inhibition correlates with the presence in the lung of a highly activated population of antigen-specific CD8 T cells, maintained for at least 6 months post-immunisation by persistent antigen. For intra-nasal Ad85A to be effective, the vaccine must be delivered into the lower respiratory tract, as immunisation targeting only the nasal-associated lymphoid tissue (NALT) does not result in protection. Following a change of animal facility, the lung immune response to intra-dermal immunisation with Ad85A increased and this route of immunisation now induced protection, though growth of M. tuberculosis was inhibited only late after infection. However, this response and protection can be altered by exposure to environmental mycobacteria. Further experiments showed that simultaneous respiratory and parenteral immunisations (SIM) act additively, where local lung immunity inhibits the growth of M. tuberculosis early after infection and systemic immunity protects later. SIM regimes generate greatly improved protection over either immunisation alone and do not depend on priming and boosting.

Prostaglandins (PG) are ubiquitous lipid mediators that play key roles in pathophysiological responses to infections. They are considered to have both pro and anti-inflammatory roles depending upon the time of inflammation, the receptors that they bind to and the tissues that they act upon. Hence given their pleiotropic effects, a perfect balance between the pro and anti-inflammatory functions of PGs are required to ensure that a controlled timely immune response is elicited to mediate protection and to avoid immunopathology. PGD2 is one such PG that was reported to increase with age in the lungs of mice and to mediate an anti-inflammatory effect thereby blunting the immune response following Severe Acute Respiratory Syndrome – Coronavirus (SARS-CoV). Increase in PGD2 with age incapacitates respiratory dendritic cells (rDC) to migrate from lungs to the draining lymph node following SARS-CoV infection due to down regulation of CCR7 (a receptor for chemokines CCL19/21). Migration of rDCs to draining lymph nodes requires high expression of CCR7 and it's binding to CCL19/21, a chemokine that mediates migration of dendritic cells along its gradient. Although increase in levels of PGD2 might prove beneficial in high inflammatory conditions, it should be noted that high levels of such a potent anti-inflammatory mediator during the initiation of an immune response could prove detrimental. In chapter II of this thesis I show that age-related increases in oxidative stress result in the upregulation of a single phospholipase (PLA2) group II D (G2D) (PLA2G2D) with anti-inflammatory roles. PLA2G2D functions by releasing Arachidonic acid (AA) from the lipid membrane, which will be further metabolized to other pro-resolving/ anti-inflammatory lipid mediators including PGD2. I show that inducing oxidative stress in young mice as well as in human peripheral blood macrophages, results in the upregulation of PLA2G2D (probably as a counter mechanism against oxidative stress). Also increase in the expression levels of this gene during the course of SARS-CoV infection results in the upregulation of PGD2, which is completely abrogated in Pla2g2d-/- mice. I also show Pla2g2d/- middle-aged mice have low levels of PGD2 and that they are capable of mounting a strong immune response and survive the otherwise lethal SARS-CoV infection. PGD2 is also a major PG in the brain and its role has been investigated in many non-infectious setting such as stroke and Alzheimer' disease. The PGD2 binding to one of its receptors DP1 has been shown to have primarily a neuro-protective role. In chapter III, I show that PGD2/DP1 signaling has beneficial effects in the brain of mice infected with a neurotropic strain of murine hepatitis virus (MHV) (rj2.2). In agreement with the neuro-protective role of PGD2, at least 60% of DP1-/- mice succumb to a sublethal dose of rj2.2. rj2.2 infection in these mice is characterized by a delay in the induction of IFN I response and lower activation status of microglia and macrophages in the brain. I also show that abrogation of DP1 signaling results in global defects in the immune system response to infection. Notably, a genome wide expression analysis using microarray, shows that a gene, Pydc3 with putative inflammasome inhibiting function is upregulated in WT mice compared to DP1-/- mice in the CD11b population of cells which primarily comprises microglia and macrophages. In line with the predicted function of Pydc3, DP1-/- mice have higher frequency and number of IL-1β+ producing microglia in the brain. Studies are underway to determine the exact role of DP1 signaling in Pydc3 expression as well as the role of this gene in inflammasome function. Overall these studies emphasize the immuno-modulatory roles of PGs in the context of a viral infection. Thus, altering the levels of these lipid mediators at appropriate times during the course of infection might prove useful as an effective therapeutic strategy to decide the fate of an infection.

The severe disease associated with seasonal epidemics of influenza A virus (IAV), as well as pandemic outbreaks, have highlighted the necessity for novel, broadly cross-reactive vaccination and therapeutic strategies against IAV. Our studies have focused on the contribution of IAV-specific CD8 T cells to mediating protection following IAV vaccination and infection as IAV-specific CD8 T cells are required for clearance of IAV. Further, IAV-specific CD8 T cells are typically cross-protective as they are generally directed at highly conserved areas of IAV. Recently, influenza virus-like particles (VLPs) have been developed from recombinant baculoviruses containing influenza proteins hemagglutinin (HA) and/or neuraminidase (NA) on the surface and matrix (M1) in the VLP core. Influenza VLPs induce potent antibody responses and have been shown to provide protection from morbidity and mortality during lethal homo- and hetero-subtypic IAV challenge. This suggests that conserved, VLP-induced CD8 T cell responses may also contribute to the overall protective ability of VLPs. However, whether influenza VLPs can induce influenza-specific CD8 T cell responses and if these T cells are protective during IAV challenge remains unknown. Here, I demonstrate that a single, intranasal vaccination with VLPs containing HA and M1 leads to a significant increase in HA533-specific CD8 T cells in the lungs and lung-draining lymph nodes. Our results also indicate that HA533-specific CD8 T cells primed by influenza VLP vaccination are significantly increased in the lungs following lethal IAV challenge. These VLP-induced memory CD8 T cells are critical in providing protection from lethality following subsequent challenge infections, as depletion of CD8 T cells leads to increased mortality, even when total, but not VLP-induced memory, CD8 T cell numbers have been allowed to recover prior to lethal dose IAV challenge. In addition, my studies also importantly demonstrate that these VLP-induced, HA533-specific CD8 T cells aid in protection from high-dose, heterosubtypic IAV infections where CD8 T cell epitopes are conserved, but the targets of neutralizing antibodies have been destroyed. This dissertation further elucidates the requirements for the regulation of the IAV-specific CD8 T cell response in the periphery (i.e. lung) by pDC and CD8α+ DC. Our studies have previously demonstrated that pDC or CD8α+ DC must present viral antigen in the context of MHC class I along with trans-presentation of IL-15 to effector, IAV-specific CD8 T cells in the lungs to protect the T cells from apoptosis and allow generation of the full magnitude CD8 T cell response needed to clear IAV infection. Herein, I demonstrate that in addition to antigen presentation and IL-15, costimulatory molecules on the surface of pDC and CD8α+ DC are also required. However, the specific costimulatory molecules required depends upon both the mouse strain utilized for IAV infection as well as DC subset. In addition to costimulatory molecules, I also demonstrate that the requirement for pDC and CD8α+ DC to be infected differs in order for them to participate in this pulmonary rescue of the IAV-specific CD8 T cell response. While CD8α+ DC are able to efficiently cross-present exogenous antigen, pDC must be directly infected and utilize the endogenous, direct antigen presentation pathway to present viral antigen to IAV-specific CD8 T cells in the lungs during IAV infection. These data suggest there are distinct differences between pDC and CD8α+ DC in their mechanism of regulating the pulmonary IAV-specific CD8 T cell response, which had not been previously appreciated. Together, the results presented herein further detail the mechanism of regulation of effector IAV-specific CD8 T cells by DC as well as the contribution of IAV-specific CD8 T cells to a novel, IAV VLP vaccination strategy. These findings highlight the importance of IAV-specific CD8 T cells in mediating protection following IAV vaccination and infection.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection and hospitalization in infants and young children, but no vaccine is currently available. CD4 and CD8 T cells are critical for mediating viral clearance but also contribute to immunopathology following an acute RSV infection. However, few RSV-derived CD4 and CD8 T cell epitopes in the commonly used C57BL/6 mouse strain have been described. I utilized an overlapping peptide library spanning the entire RSV proteome and intracellular cytokine staining for interferon-gamma (IFN-γ) to identify novel CD4 and CD8 T cell epitopes in C57BL/6 mice. I discovered and characterized two novel CD4 T cell epitopes and three novel CD8 T cell epitopes located within multiple RSV proteins. Overall, the novel RSV-derived CD4 and CD8 T cell epitopes identified in C57BL/6 mice will aid in future studies of RSV-specific T cell responses. While CD8 T cells are important for viral clearance following an acute RSV infection, the contribution of memory CD8 T cells in providing protection against reinfection with RSV remains unclear. I used a prime-boost immunization approach to induce robust, systemic memory CD8 T cell responses in the absence of RSV-specific CD4 T cells and antibodies. I determined that high magnitude, systemic memory CD8 T cell responses efficiently reduced lung viral titers following RSV infection, but unexpectedly did so at the expense of severe and fatal immunopathology. The exacerbated disease was mediated by the rapid and excessive production of IFN-γ by memory CD8 T cells in the lung and airways. In contrast, I found that local immunization generated a large population of tissue-resident memory CD8 T cells in the lung that efficiently reduced lung viral titers in the absence of exacerbated disease. Additionally, I observed that pre-existing RSV-specific neutralizing antibodies prevented the immunopathology induced by high magnitude, systemic memory CD8 T cell responses following RSV infection. Prophylactic treatment with neutralizing antibodies against RSV efficiently restricted early virus replication, which resulted in a significant decrease in lung IFN-γ levels, memory CD8 T cell activation, and the frequency of IFN-γ producing CD8 T cells. Thus, my results demonstrate that high magnitude, systemic memory CD8 T cells induce lethal immunopathology following RSV infection, which can be prevented by pre-existing RSV-specific neutralizing antibodies. Overall, my results have important implications for the development of future RSV vaccines. The development of a live-attenuated vaccine for RSV has been prevented by the inability to properly balance attenuation with immunogenicity and efficacy. Recently, a recombinant RSV strain lacking the gene that encodes the matrix (M) protein (RSV M-null) was developed. As the M protein is required for virion assembly following infection of a host cell, RSV M-null induces a single-cycle infection. I evaluated RSV M-null as a potential live-attenuated vaccine candidate by determining its pathogenicity, immunogenicity, and protective capacity in BALB/c mice compared to its recombinant wild-type control virus (RSV recWT). RSV M-null was sufficiently attenuated, as significantly reduced lung viral titers, weight loss, and pulmonary dysfunction were observed compared to mice infected with RSV recWT. Surprisingly, despite its attenuation, I found that RSV M-null infection induced effector T cell, germinal center B cell, serum antibody, and memory T cell responses of similar magnitude to that elicited by infection with RSV recWT. Importantly, RSV M-null immunization provided protection against secondary viral challenge by reducing lung viral titers as efficiently as immunization with RSV recWT. Overall, my results indicate that RSV M-null combines attenuation with high immunogenicity and efficacy and represents a promising novel live-attenuated RSV vaccine candidate.

Preclinical models of lupus indicate that T cell-B cell collaboration drives antinuclear antibody (ANA) production and sustains T cell activation. Autoreactive B lymphocytes are present in the normal repertoire but persist as ignorant or anergic cells. Mechanisms that normally limit T cell activation of autoreactive B cells remain incompletely resolved, but potentially include the absence of autoreactive effector T cell subsets and/or the presence of autoAgspecific regulatory T cells (Tregs). Several studies have addressed this issue by using experimental systems dependent on transgenic autoreactive B cells, but much less is known about the activation of autoreactive B cells present in a polyclonal repertoire. In the second chapter of this thesis, I have explored the role of effector T cells and Tregs using mice that express an inducible pseudoautoAg expressed on B cells and other antigen presenting cells (APCs). In this system, activated Th2 cells, but not naïve T cells, elicit the production of ANAs, but ANA production is severely limited by autoAg-specific Tregs. Bone marrow chimera experiments further demonstrated that this B cell activation is constrained by radioresistant autoantigen-expressing APCs (rAPC) present in the thymus as well as by non-hematopoietic stromal cells located in peripheral lymphoid tissue. Importantly, peripheral rAPC expression of autoAg induced the expansion of a highly effective subset of CD62L+CD69+ Tregs. The third chapter of this thesis focuses on the contribution of CD8+ T cells to fibrosis resulting from sterile lung injury. Type 2 effector production of IL-13 is v a demonstrated requirement in several models of fibrosis, and is routinely ascribed to CD4+ Th2 cells. However, we now demonstrate a major role for pulmonary CD8+ T cells, which mediate an exaggerated wound healing response and fibrosis through robust differentiation into IL-13-producing pro-fibrotic type 2 effectors (Tc2). Remarkably, differentiation of these Tc2 cells in the lung requires IL-21. We further show that the combination of IL-4 and IL-21 skews naïve CD8+ T cells to produce IL-21, which in turn acts in an autocrine manner to support robust IL-13 production. TGF-β negatively regulates production of IL-13 by suppressing CD8+ T cell responsiveness to IL-21. Our data illuminate a novel pathway involved in the onset and regulation of pulmonary fibrosis, and identify Tc2 cells as key mediators of fibrogenesis.