Tesis sobre el tema "Immune response"

Abstract Heavy alcohol consumption places a substantial burden on health all over the world. Metabolites of alcohol evoke alterations that lead to tissue damage in many organs. Phosphatidylethanol (PEth) is a unique phospholipid formed in the cellular membranes during the metabolism of ethanol after alcohol consumption. PEth has attracted special attention as it is postulated to be a reliable marker of long term heavy alcohol consumption. The aims of present study were to investigate the immunogenicity of phosphatidylethanol in mice and to analyze the plasma antibodies binding to phosphatidylethanol in humans. In this study a clear immune response was generated in mice immunized with PEth in human low density lipoprotein (LDL) carrier. Mouse monoclonal IgM antibodies binding specifically to phosphoethyl head group of PEth were generated using hybridoma technology. Since PEth was shown to be immunogenic in mice, plasma was analyzed for the presence of antibodies also in humans. PEth-specific antibodies of IgG, IgA and IgM isotypes in plasma were detected in heavy drinkers of alcohol with or without pancreatitis as well as in the controls. The plasma levels of the antibodies binding to PEth were significantly lower in the study subjects with heavy alcohol use and in this present study sample the low IgA levels to PEth were better indicators of heavy alcohol consumption as compared to the some of the traditional markers of heavy alcohol use. The antibody levels to PEth associated significantly to plasma antibodies binding to malondialdehyde-acetaldehyde adducts that are known to be formed during alcohol metabolism but not to antibodies binding to phosphocholine which is generated by lipid oxidation in humans. In conclusion, this study demonstrates that phosphatidylethanol is immunogenic in mice when using carriers such as human LDL in the immunization process. The binding of the monoclonal antibodies specifically to the PEth head group suggests that it would be feasible to develop a diagnostic immunoassay to PEth. The presence of antibodies binding to PEth in plasma indicates that PEth may be a target of humoral immunity in humans<br>Tiivistelmä Runsas alkoholinkulutus aiheuttaa maailmanlaajuisesti merkittäviä terveydellisiä haittoja. Alkoholin aineenvaihduntatuotteet muuttavat kudoksien rakenteita ja aiheuttavat kudosvaurioita. Fosfatidyylietanoli on alkoholin aineenvaihdunnan tuloksena solukalvoilla syntyvä fosfolipidi, jota on tutkittu kahdenkymmenen vuoden ajan lupaavana alkoholin suurkulutuksen merkkiaineena. Tutkimuksen tavoitteena oli selvittää fosfatidyylietanolin immunisoinnin aiheuttamaa vasta-aineiden muodostumista koe-eläinmallina käytetyissä hiirissä sekä määrittää ihmisten plasmanäytteistä vasta-aineita, jotka sitoutuvat fosfatidyylietanoliin. Tutkimuksessa havaittiin immuunivasteen muodostuminen hiirissä, jotka immunisoitiin ihmisen LDL hiukkasiin liitetyllä fosfatidyylietanolilla. Hiiren monoklonaalisia fosfatidyylietanoliin sitoutuvia IgM-luokan vasta-aineita tuotettiin tutkimuksessa soluviljelyn avulla. Fosfatidyylietanolin aiheuttama vasta-aineiden muodostuminen hiirillä johdatti mittaamaan fosfatidyylietanoliin sitoutuvia vasta-aineita myös ihmisiltä. Tutkimuksessa havaittiin fosfatidyylietanoliin sitoutuvia IgG-, IgA- ja IgM-luokan vasta-aineita alkoholin suurkuluttajilla, alkoholihaimatulehdusta sairastavilla ja verrokkihenkilöillä. Vasta-aineiden pitoisuudet olivat alkoholia runsaasti käyttävillä koehenkilöillä merkitsevästi pienemmät kuin verrokkiryhmällä. Matalat IgA-vasta-ainepitoisuudet osoittautuivat aineistossa paremmaksi alkoholin suurkulutuksen osoittajiksi kuin eräät tavanomaisesti käytetyt alkoholinkäytön merkkiaineet. Plasman fosfatidyylietanoli-vasta-aineiden ja alkoholin aineenvaihdunnan seurauksena syntyvien malondialdehydi-asetaldehydi-addukteihin sitoutuvien vasta-aineiden määrän välillä havaittiin merkitsevä yhteys, jota ei havaittu rasvojen hapettumisen seurauksena syntyvien fosfokoliini-vasta-aineiden ja fosfatidyylietanoli-vasta-aineiden välillä. Tutkimus osoittaa, että hiirillä voidaan aikaansaada vasta-ainevälitteinen immuunivaste, kun ne rokotetaan ihmisen LDL-hiukkaseen liitetyllä fosfatidyylietanolilla. Fosfatidyylietanoliin spesifisesti sitoutuvien monoklonaalisten vasta-aineiden tuottaminen voi tulevaisuudessa johtaa immunologisen diagnostisen määritysmenetelmän kehittämiseen. Fosfatidyylietanoliin sitoutuvien plasman vasta-aineiden havaitseminen viittaa siihen, että fosfatidyylietanoli on vasta-ainevälitteisen immuunivasteen kohde myös ihmisillä

The immunogenicity of breast cancer (BC) is quite heterogeneous among the clinical subtypes, with immune responses identified most frequently in triple negative (TNBC) and HER2-positive tumors. The extent, spatial localization, distribution patterns, organization and phenotype of the BC immune infiltrate are currently being widely investigated but require standardization before they can be used clinically. One highly relevant unmet clinical need is to understand how immune features are linked to prognosis and potential benefit from treatments, particularly immunotherapy. The present work investigated tumor-infiltrating lymphocytes (TIL), tertiary lymphoid structures (TLS), the expression of multiple targetable inhibitory immune checkpoint molecules (PD-1, PD-L1 and PD-L2, CTLA-4, LAG3 and TIM3) and their clinical relevance in primary BC. Different technical approaches were employed including: flow cytometry (FC) on fresh tissue homogenates; immunohistochemistry (IHC) and immunofluorescence (IF) on formalin-fixed paraffin embedded (FFPE) tissue blocks from untreated primary tumors; and gene expression on a large dataset of BC patients with available long-term survival data. Flow cytometric analysis of PD-1 expression and its principal ligands PD-L1 and PD-L2 together with CTLA-4, LAG3 and TIM3 on TIL in fresh untreated primary tumors revealed that PD-1 and CTLA-4 are most highly expressed on BC TIL and PD-L1 is the principal PD-1 ligand in BC. Immune checkpoint molecule expression parallels the extent of TIL infiltration and TLS presence and number, with the patterns detected similar to that observed in secondary lymphoid organs. Significantly improved disease-specific survival (DSS) has been associated with PD-1hi HER2-enriched and PD-L1hi, PD-L2hi and CTLA-4hi basal-like BC; however there is significant heterogeneity between individual tumors even within the same subtype. These observations suggest that determining expression levels of multiple targetable inhibitory immune checkpoint molecules in patients might help to successfully target them in BC patients most likely to respond.We examined the concordance between two experienced immuno-pathologists who read 800 IHC-stained slides from five independent series over a period of four years to determine the reproducibility of assessing multiple immune biomarkers. This included scoring TIL, TLS, PD-1 and PD-L1 together with detailed information on the spatial localization and cell types expressing these molecules in the tumor microenvironment (TME). The interobserver reproducibility for the assessment of TIL and TLS was consistently good to excellent overtime, while the concordance for PD-L1 evaluation ranged from fair to excellent when it was only expressed on tumor cells (TC); and the concordance for PD-1 evaluation was fair to excellent when it was expressed in TLS and evaluated in primary tumors. Neither PD-L1 expression by TC, nor PD-1 expression within a TLS was significantly associated with prognosis in our datasets.The extent of TIL, TLS and PD-1 and PD-L1 expression were studied in a cohort of TNBC patients who underwent genetic counseling for their personal/familial history of BC or ovarian cancer (OC). This study revealed a remarkable similarity in patterns of immune infiltration between the two cohorts. Interestingly, a higher prevalence of TIL intermediate cases (≥10% and <50% TIL) was detected in the BRCA-mutated cohort, suggesting that this group may be more immunogenic.We next investigated whether the extent and presence of these immune parameters were associated with prognosis in the most highly infiltrated, aggressive BC subtypes (TNBC and HER2-positive). We determined the ideal cut-off for each subtype (TNBC and HER2-positive) to use TIL as a categorical variable. This study found a consistent prognostic impact from TIL (in any tumor compartment including stromal, intratumoral and global areas) and a novel association between PD-L1 expression within TLS and better survival in TNBC. This last effect was driven by baseline stromal TIL, strengthening the importance of reliably quantifying the levels of TIL in BC. Overall, our analyses show that among the targetable inhibitory immune checkpoint molecules investigated in BC, PD-1 and CTLA-4 are most highly expressed by BC TIL and are associated with higher infiltration of TIL; PD-L1 is the principal ligand for PD-1; TIL and TLS are reproducibly scored on IHC-stained tissues; and TIL levels are associated with a better prognosis in TNBC independent of their location in the TME at optimal cut-offs. Our data also provide new insight on targetable inhibitory immune checkpoint molecule expression and location as well as showing a prognostic role for TIL assessed by IHC in primary BC, which identifies these biomarkers as ideal candidates for further investigation.<br>Doctorat en Sciences médicales (Médecine)<br>info:eu-repo/semantics/nonPublished

<p>Circulating immune complexes play an important role in the modulation of antibody responses and in the pathogenesis of immune diseases. This thesis deals with the <i>in vivo </i>regulatory properties of antibodies and their specific Fc receptors.</p><p>The immunosuppressive function of IgG is used clinically, to prevent rhesus-negative women from becoming sensitized to rhesus-positive erythrocytes from the fetus. The mechanism behind this regulation is poorly understood but involvement of a receptor for IgG, FcγRII, has been suggested. It is shown in this thesis that IgG and also IgE induce immunosuppression against sheep erythrocytes to a similar extent both in mice lacking all the known Fc receptors as in wild-type animals. These findings imply that antibody-mediated suppression of humoral responses against particulate antigens is Fc-independent and that the major operating mechanism is masking of epitopes.</p><p>Immunization with soluble antigens in complex with specific IgG leads to an augmentation of antibody production. The cellular mechanism behind this control is examined here and it is found that the capture of IgG2a immune complexes by a bone marrow-derived cell expressing FcγRI (and FcγRIII) is essential. An analysis of the ability of IgG3 to mediate this regulation indicated that, in contrast, this subclass of IgG augments antibody responses independently of FcγRI (and FcγRIII). These findings suggest that distinct mechanisms mediate the enhancing effect of different subclasses of antibodies.</p><p>Finally, the contribution of FcγRIII was studied in the development of collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis in humans. It was discovered that while DBA/1 wild-type control mice frequently developed severe CIA, with high incidence, FcγRIII-deficient mice were almost completely protected, indicating a crucial role for FcγRIII in CIA.</p><p>The results presented here help to understand how immune complexes regulate immune responses <i>in vivo</i> and show that Fc receptors for IgG, if involved, could be new targets for the treatment of immune complex-related disorders.</p>

Microglia activity has been detected in Parkinson’s disease (PD) post-mortem brains and experimental animal models; however the precise interplay between microglia and dopamine neurons of the SNpc is not well understood. In the blood plasma of PD patients, our laboratory found elevated levels of interferon-gamma (IFN-γ), a proinflammatory cytokine and potent activator of microglia. Given this, we sought to untangle the immune responses relevant to PD in mice, examining IFN-γ’s involvement and signaling mechanism using an inflammatory co-culture model of microglia and midbrain neurons treated with rotenone. By means of RT-PCR, we discovered IFN-γ mRNA transcripts are produced by microglia, and this expression increases upon exposure to rotenone. We delineated IFN-γ’s signaling mechanism in co-cultures using different IFN-γ receptor deficient cells, and showed it engages receptors in an autocrine (not paracrine) manner to further microgliosis and dopamine cell loss. After exploring the innate immune response in a model of PD, we subsequently shifted focus to an in vivo system to better investigate any involvement of the delayed humoral arm of the adaptive immune system. Needing a time appropriate death paradigm, we developed a protracted low dose regimen of MPTP, which elicits dopaminergic cell death after 2 weeks of treatment. Subjected to this paradigm, Rag 2 mutant mice (deficient in both T and B cells) exhibit resistance to dopamine cell loss, microglia activation and motor impairments. Further evidence in support of immune involvement came with the resensitization of Rag2 mice to MPTP after reconstitution with WT splenocytes. Additionally, mice deficient in Fcγ receptors exhibited neuroprotection in our protracted degeneration model. Taken together, these data indicate the innate and humoral arm can modulate the microglial response to dopaminergic degeneration and may participate in Parkinson's disease.

The innate immune system has a variety of ways of recognizing infection from pathogens such as viruses and bacteria. One of its first lines of defence is to detect Pattern Associated Molecular Patterns (PAMPs) using Pattern Recognition Receptors (PRRs) such as the Toll-Like Receptors (TLRs), the RIG-Like Helicases (RLHs) and the Nod-Like Receptors (NLRs). These receptors recognize certain molecular structures from the pathogens and lead to first line of defence which includes increased cytokines and IFNs. This study delineate the human body’s innate immune responses to human respiratory viruses such as HRV (Human Rhinovirus), RSV (Respiratory Syncytial Virus) and IAV (Influenza A virus). In Vitro experiments carried out on various kinds of lung tissues suggest that respiratory disease pathogenesis is related to inflammatory mediators including interleukins and cytokines produced by the host’s innate immune system. Virus induced respiratory tract infection are known to trigger bronchiolitis, wheezing and acute asthma exacerbations, as a result of inflammation of lung tissues and excessive release of pro- inflammatory cytokines such as IL-1β. This study identifies that intracellular macromolecular complexes called inflammasomes assemble as a result of viral trigger. Inflammasomes convert the inactive forms of the pro-inflammatory cytokines to their active forms. Although the exact mechanism of activations of these complexes was unknown. This study identified that Virus encoded proteins such as the 2B protein of HRV, the SH protein from RSV and the Influenza M2 which are also termed viroporins can activate the inflammasome by causing ion imbalance (across cells membranes and organelles). Thus providing a trigger for inflammasome assemblage. v Drugs which act as Ion channel blockers have been shown to block viroporin activity and hence reduce IL-1β production. Therefore in the future the use of ion inhibitors could be a possible therapeutic intervention in order to reduce lung inflammation and prevent associated respiratory disease such as COPD and Asthma.

Breast cancer is a highly heterogenous disease and current methods of treatment selection in the clinic do not take account of this heterogeneity. In order to improve outcomes in breast cancer, a personalised approach is required. Previously we identifed a DNA damage response deficient (DDRD) molecular subgroup within breast cancer. A 44-gene expression assay prospectively identifying this subgroup was validated as predicting benefit from DNA-damaging chemotherapy. This subgroup was defined by interferon signalling. In this study the biology identified by the subgroup is addressed, identifying activation of the cytosolic DNA-sensing innate immune pathway cGAS/STING/TBK1/IRF3 in DNA damage response deficient cancer cells. This study highlights the importance of this pathway in cancer cell chemokine production. Importantly, activation of this pathway is cell-cycle related and can be induced by exogenous S-phase DNA damage. We characterise the immune infiltrate in DNA damage response deficient breast tumours, identifying both anti-tumourigenic and pro-tumourigenic immune cell populations. Also, expression of the immune checkpoint protein PD-L1 is identified as upregulated in DDRD assay positive breast tumours. These data indicate that tumours with an endogenous DNA damage response deficiency employ expression of immune checkpoints as a means of immune escape. Therefore, immune checkpoints are important therapeutic targets in this subgroup of breast cancer. In addition, we present preliminary analysis of a clinical trial designed to assess the feasibility of using the 44-gene expression assay in predicting response to neoadjuvant chemotherapy in breast cancer. Given our findings, we identify potential targets in DNA damage response deficient tumours, combination strategies with anti-PD-1/PD-L1 targeted therapies and provide evidence for the prospective utility of the DDRD gene expression assay in predicting response to DNA damaging chemotherapy.

Nuclear bone morphogenetic protein 2 (nBMP2) is a nuclear variant of the secreted growth factor BMP2. Experiments in nBmp2NLStm mutant mice, which lack nBMP2 in the nucleus, have shown that nBMP2 affects intracellular calcium transport in skeletal muscle and hippocampal neurons. The objective of this study was to determine whether nBMP2 affects the immune system, since activation of lymphocytes and other immune cells depends on intracellular calcium transport. We found that spleens in nBmp2NLStm mutant mice were 24% smaller than in wild type mice. The white pulp of the spleen contains many immune cells, particularly B and T lymphocytes and reduced spleen size in the nBmp2NLStm mutant mice could be caused by a reduced number of lymphocytes migrating to the spleen. When mutants and wild types were challenged with an intravenous infection of 10^7 CFU of S. aureus, they showed similar immune responses. Samples of blood, liver, spleen, kidney and lymph nodes cultured three days after infection showed no difference in post infection bacterial load between mutant and wild type. Likewise, post-infection weight loss and percent survival were similar between mutant and wild type, suggesting that the innate immune response is functional in nBmp2NLStm mice. However, when mice were challenged with a secondary infection, immune response and spleen function were severely impaired. Mutant mice showed higher levels of bacteria remaining in the blood and had lower rate of survival to day 3 after secondary infection. In addition, CD4+ and CD8+ T-cell levels within mutant lymph nodes were significantly reduced, indicating that nBMP2 is involved in the secondary immune response.

Production diseases of dairy cows include several pathologies. These are considered man-made problems, mostly reported in the post-calving period of negative energy balance (NEB), when high-yielding dairy cows are unable to achieve a feed energy intake matching their high production requirements (Mulligan et al., 2008). A correct management of production diseases demands an early diagnostic approach and prognostic parameters. In ruminants, metabolic stress greatly influences the forestomachs physiology, including their immune response ability. Therefore a proper evaluation of production diseases in ruminants should also include markers of the innate immune response to metabolic stress (Amadori, 2016). Since forestomach walls express immune receptors and cytokines, and the rumen liquor is infiltrated by leukocytes, it is reasonable that they play an important role in the local immune response to metabolic stress (Ingvartsen et al., 2003; Trevisi et al., 2014a). My PhD hypothesis implies that ruminal fluids could be an important source of diagnostic information for the identification of herds at risk for production diseases, in addition to the traditional analyses. Accordingly, we further characterized the leukocytes subpopulations in the rumen liquor, highlighting the presence of B cells (the most frequent leukocyte population in the rumen liquor), T cells, and myeloid cells. We also compared the leukocyte composition in rumenocentesis versus esophageal probe samples, and we did not observe any significant statistical difference between the two sample collection techniques. We investigated the origin of the leukocytes of the rumen fluid and demonstrated that they partly derive from the oral cavity and reach the rumen through the saliva. On the basis of these findings, we carried out a field survey of innate immune parameters in rumen fluids of 128 animals from 12 farms, along with clinical inspections, assessment of milk yield, rumen pH and volatile fatty acids (VFA), and evaluation of major metabolic and hematologic parameters. Significant statistical correlations were found between immune markers in rumen fluids and biochemical parameters of dairy cows. In particular, a significant negative correlation was found between CD45 gene expression in rumen fluids (leukocyte infiltration) and ruminal pH. The infiltration of B cells was negatively affected by ruminal pH and high concentrations of volatile fatty acids (VFA). The same type of regulation was also exerted on the concentrations of IgM and total Ig. Interestingly, total Ig and IgM in rumen fluids showed a strong positive correlation with urea levels in blood, to some extent correlated with feed intake. We also investigated how the alteration of the ruminal microbiota could influence the immunologic profile of the rumen liquor. The use of a low-dose antibiotic (Monensin) led to significant differences in metabolic and immunological parameters in the rumen, and this resulted in a reduction of leukocyte colonization and immunoglobulin concentration in the ruminal fluid. Our procedures were included into a kit and tested in a group of 10 animals, half of them suffering from ruminal stasis; we could detect differences between the two groups in terms of leukocytes infiltration and Ig concentration. At a molecular level, the group with overt pathology showed a marked reduction of IGLC and KRT5 gene expression. Our data suggest that forestomach immune responses could be directed to “dangers” arising within the forestomach environment (diet unbalance, abnormal fermentations), but also arise as reporter system of disease conditions elsewhere in the body. The immune markers could integrate consolidated diagnostic parameters (e.g. rumen pH and VFA, milk cell counts, blood, fecal analytes) and contribute to robust, early diagnosis of production diseases of dairy cattle.

New understanding of immune system biology has led a paradigm shift in the development of biomaterials away from classically ‘inert’, to ‘immuno-modulatory’ biomaterials that have the potential to stimulate an immune response able to promote constructive and functional tissue remodeling responses as opposed to persistent inflammation and scar tissue formation. This project aims to examine macrophage behavior and in particular integrin expression at the cell–biomaterial interface in vitro, in order to delineate the underlying molecular events occurring during biomaterial-mediated osseous healing. After reviewing the current literature to identify research gaps that this thesis should focus on, the activation of macrophages with controlled timing, and modulation of their interactions with other cell types involved in wound healing, emerged as key strategies to improve biomaterial efficacy. Careful design of biomaterial structure and controlled release of immunomodulators can be employed to manipulate macrophage phenotype for the maximization of the wound healing response with enhanced tissue integration and repair, however, to elicit predictable immune responses there is a need for a thorough understanding of how the biomaterial properties can be tuned to harness a designed immunological outcome. Our systematic review of in vitro studies suggested that the initial immune response of macrophages to titanium may be modulated by its surface characteristics both topographically and physiochemically. We therefore assessed the potential effects of three novel titanium surfaces; Machined, Blasted and Fluoride-treated, on macrophage phenotype. We chose a monocytic cell-line as our macrophage source rather than using primary macrophages, as the inflammatory status of primary cells could not be readily defined nor reproduced between experiments. This was crucial to the overall study if we were to clearly assess any changes in macrophage phenotype as a result of their II interaction with the titanium surfaces. We subsequently analysed in some detail the experimental processes involved in the optimization of macrophage cell culture, macrophage differentiation and phenotype characterization. We used microscopy, profilometry and elemental analysis to understand how the roughness of the titanium surfaces affected macrophage viability and proliferation in both non-activated ‘M0’ and activated inflammatory ‘M1’ macrophages. Subsequent analysis of one of the key mediators of cell attachment to biomaterials showed for the first time that αM, β1, β2 and α2 integrins play an important role in the adhesion of non-polarised M0 macrophages onto both smooth and rough titanium surfaces. Moreover, the distinct temporal expression profile of αM integrin expression correlated broadly with the subsequent secretion profiles of inflammatory cytokines IL-1β and TNF-α in these cells. Specific integrin expression also played an important role in the adhesion of polarised inflammatory M1 macrophages onto surface-modified titanium surfaces. In these cells, early integrin αM and β1 in particular, along with integrin α2 was associated with modulation of the subsequent cytokine response in these cells. Furthermore, the surface induced differential expression of integrins on the different titanium surfaces showed the Fluoride-modified surface in general induced greater cytokine and integrin modulation than either the Machined or Blasted surfaces. While now biologically plausible, further studies to fully determine how integrin-derived modulation of the macrophage-associated inflammatory response following macrophage attachment as suggested by these studies, can enhance wound healing and osteogenesis are required. Despite this, ‘harnessing the immune response’ is clearly a potential goal of engineered titanium implant surfaces in order ‘to enhance osseointegration’.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>School of Medicine & Dentistry<br>Griffith Health<br>Full Text

Vaccination and the use of immune stimulants are two important ways to mitigate the costs of disease in fish aquaculture. A vaccine to Enteric redmouth disease (ERM) was the first licensed fish vaccine in the world. Although effective in protecting fish from the motile bacterial (Yersinia ruckeri) infection, ERM can occur in ERM vaccinated fish due to the rise of non-motile Y. ruckeri that does not express flagellin. This highlighted the need for continual improvement of vaccine efficacy and the importance of flagellin in fish immune responses. In this thesis the immune response to the ERM vaccine was studied first to give insights for vaccine development. A recombinant flagellin from Y. ruckeri (YRF) was then produced and its bioactivities were investigated in vitro and in vivo. The immune response to ERM vaccination was studied in rainbow trout in two major and relevant immune organs, the spleen and gills. Intraperitoneal injection of the ERM vaccine induces an early balanced expression of pro- and anti-inflammatory cytokines and adaptive cytokines in the spleen, with a heightened expression of acute phase proteins (APPs) and anti-microbial peptides (AMPs) in both spleen and gills. The analysis suggests that ERM vaccination activates host innate immunity and the expression of specific IL-12 and IL-23 isoforms leading to a Th1 and Th17 biased immune responses. This study has increased our understanding of the host immune response to ERM vaccination and the adaptive pathways involved. The early responses of a set of genes established in this study may prove useful as biomarkers in future vaccine development in aquaculture. YRF was next produced in a bacterial system, and purified. Its bioactivity was investigated first in the trout macrophage cell line RTS-11 and head kidney primary cell cultures. YRF is a potent activator of pro-inflammatory cytokines, APPs, AMPs and subunits of the IL-12 cytokine family in vitro. This property was further confirm in vivo in multiple tissues after intraperitoneal injection of YRF. These results suggest that flagellins are important pathogen-associated molecular patterns (PAMPs) that can activate an inflammatory response in fish not only in vitro but also in vivo. Furthermore, YRF was shown to be the most potent PAMP in vitro, in terms of activation of an inflammatory response, compared to pure LPS and peptidoglycan. In addition, YRF mixed with complete Freund's adjuvant can induce YRF-specific IgM antibodies in rainbow trout. These antibodies are able to neutralize YRF bioactivity, and react against the middle domain of YRF, as assessed in Western blot analysis. When YRF was fused with a protein antigen, it increased the antigen-specific IgM antibody response. This analysis reveals that YRF is a potent activator of host immune responses and can be used as an immune stimulant and adjuvant to improve vaccine efficacy.

Allergic manifestations are increasingly common in infants and children. Accumulating evidence suggests that the ‘epidemic’ increase of childhood allergy may be associated with environmental factors such as stress. Although the impact of stress on the manifestation and exacerbation of allergy has been demonstrated, the underlying mechanisms of stress-induced exacerbation are still obscure. A growing number of studies have suggested an altered hypothalamus-pituitary-adrenal (HPA) axis function to stress in allergic children. It is speculated that a dysfunctional HPA axis in response to stress may facilitate and/or consolidate immunological aberrations and thus, may increase the risk for allergic sensitization and exacerbation especially under stressful conditions. In the present review the potential impact of a hyporesponsive as well as a hyperresponsive HPA axis on the onset and chronification of childhood allergy is summarized. Moreover, potential factors that may contribute to the development of an aberrant HPA axis responsiveness in allergy are discussed<br>Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Allergic manifestations are increasingly common in infants and children. Accumulating evidence suggests that the ‘epidemic’ increase of childhood allergy may be associated with environmental factors such as stress. Although the impact of stress on the manifestation and exacerbation of allergy has been demonstrated, the underlying mechanisms of stress-induced exacerbation are still obscure. A growing number of studies have suggested an altered hypothalamus-pituitary-adrenal (HPA) axis function to stress in allergic children. It is speculated that a dysfunctional HPA axis in response to stress may facilitate and/or consolidate immunological aberrations and thus, may increase the risk for allergic sensitization and exacerbation especially under stressful conditions. In the present review the potential impact of a hyporesponsive as well as a hyperresponsive HPA axis on the onset and chronification of childhood allergy is summarized. Moreover, potential factors that may contribute to the development of an aberrant HPA axis responsiveness in allergy are discussed.<br>Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Els virus de la influença tipus A (VIA) són patògens zoonòtics que poden infectar un ampli nombre d’hostes incloent-hi les aus, els porcs i els homes, entre altres. Anualment es documenten milions d’infeccions en humans causades per virus de la influença estacionals. Les pandemies causades pel virus influença també tenen una elevada repercussió pel que fa a la sanitat i l’economia. Tot i que determinats subtipus de VIA s’adapten millor en espècies d’aus que en humans, hi ha hagut casos d’infeccions en humans per virus de la influença de tipus aviars. La susceptibilitat dels porcs per infectar-se amb virus de la influença tant d’origen aviar com humà és també important pel que fa a la salut pública. El genoma del virus influença és segmentat in consta de vuit molècules de ARN de cadena senzilla i sentit negatiu que codifiquen per 11 o 12 proteïnes. Per tant, si una cèl·lula s’infecta simultàniament per dos VIA diferents, pot succeir un reagrupament amb la conseqüent generació d’una nova soca de virus. A més, mutacions a les glicoproteïnes de superfícies (sobretot a l’hemaglutinina, HA) són les responsables de l’elevada variabilitat de VIA. Tot i que les vacunes front a les epidèmies estacionals són eficaces, no produeixen resposta immunològica front una amplia varietat de VIA. És a dir, les vacunes estacionals només protegeixen front a les soques virals circulants durant una determinada estació. Aquest fet, junt amb el risc de possibles pandèmies, han fet encara més important i urgent el desenvolupament d’una vacuna universal capaç de produir immunitat front a múltiples subtipus virals. En la present tesis s’ha estudiat la resposta immunitària front a la infecció i vacunació del VIA en el context de VIA d’alta patogenicitat (vIAAP) A/H5N1 i A/H7N1 i el virus pandèmic A/H1N1 (pH1N1). El treball s’ha dividit en tres parts i cada part s’ha subdividit en capítols. Part I (capítols 1 i 2), conté la introducció general i els objectius de la tesi doctoral. L’objectiu d’aquesta primera part és donar una visió global i introduir informació per entendre (i) la infecció pel virus de la influença, (ii) la resposta immunològica provocada després de la infecció per VIA i (iii) un breu resum de les vacunes actuals front a influença. A continuació, s’exposen els objectius a aconseguir. Part II, és el cos de la tesis i conté els quatre treballs (del capítol 3 al 6) duts a terme durant els quatre anys que ha durat el programa de doctorat. Tots els capítols presentats han estat publicats o sotmesos a publicació en revistes indexades internacionals. Per tant, cada estudi manté l’estructura estàndard de: resum, introducció específica, materials i mètodes, resultats i breu discussió. Estudiar el paper dels determinants virals i caracteritzar la infecció pel VIA en diversos hostes pot ser de gran interès a l’hora de dissenyar vacunes òptimes. S’ha descrit la proteïna NS1 com a un dels principals determinants de virulència en mamífers, però no s’ha estudiat gaire el paper d’aquesta en aus. En el capítol 3 es va avaluar la implicació de la proteïna NS1 en la patogenicitat viral en pollets. Es van infectar pollets amb vIAAP H7N1 que contenien el segment NS de vIAAP H5N1. Les manifestacions patològiques i la resposta immunològica conseqüència de la infecció amb cada un dels virus van ser avaluades. També és molt important el paper de la immunitat prèvia durant un brot perquè pot ser determinant de la mort o supervivència de l’animal. En el capítol 4 es van exposar pollets a un virus H7N2 de baixa patogenicitat (vIABP) i a continuació es van infectar amb un vIAAP H7N1. Posteriorment es van infectar amb un vIAAP H5N1. Els animals que havien estat infectats prèviament amb vIABP quedaven protegits a la posterior infecció letal amb el vIAAP H7N1. No obstant, la resposta immunitària produïda no era suficient per a protegir els pollets front a la infecció amb un virus heterosubtípic (vIAAP H5N1). La presència o absència d’anticossos inhibitoris front a H7- i H5- correlacionaven amb la presència o absència de protecció, respectivament. Conèixer els programes de vacunació actuals i la seva eficàcia és útil per a planificar i dissenyar futures estratègies de vacunació. Les aus aquàtiques són el reservori dels VIA; per tant, són extremadament importants pel que fa a l’ecologia del virus. Aprofitant els programes de vacunació es va testar el sèrum de diverses espècies d’aus de zoològics i centres de recuperació d’Espanya (capítol 5). Els sèrums es van utilitzar per a l’avaluació de la resposta humoral deguda a la vacuna. El principal objectiu del treball era determinar l’eficàcia de vacunes disponibles (inactivades en suspensió oliosa) en diverses espècies d’aus i comparar la variabilitat inter- i intra-espècie. Finalment, i tenint en compte el potencial risc del VIA, els esforços es van focalitzar en desenvolupar una vacuna capaç de protegir a un ampli nombre de subtipus de VIA. La pandèmia de 2009 amb el virus H1N1 (pH1N1) és un clar exemple que els porcs poden actuar com a “coctelera” i generar nous virus. En el capítol 6 es van immunitzar porcs amb pèptids derivats de l’HA i a continuació es van infectar amb el virus pH1N1. Tot i que els pèptids-HA produïen una molt bona resposta humoral i cel·lular, no es va detectar activitat neutralitzant i només es va obtenir un efecte parcial en l’eliminació del virus. Part III (Capítols 7 i 8), és la secció on es discuteixen les implicacions dels resultats obtinguts en els diferents estudis i on s’enumeren les conclusions principals. En una secció a part, s’han inclòs totes les referències bibliogràfiques utilitzades per a l’elaboració de la tesi. S’ha inclòs també un apèndix per afegir informació addicional.<br>Influenza A viruses (IAV) are zoonotic pathogens that can replicate in a wide range of hosts, including birds, pigs and humans, among others. Millions of human infections caused by seasonal influenza virus are reported annually. Influenza pandemics have also a significant health and economic repercussions. Although certain subtypes of IAV are better selected in avian species than in humans, there are reports that evidence cases of human infections with avian influenza viruses (AIV). The susceptibility of pigs to infection with influenza viruses of both avian and human origins is also important for public health. The genome of influenza virus is segmented and consists of eight single-stranded negative-sense ribonucleic acid (RNA) molecules encoding 11 or 12 proteins. Thus, if a single cell is simultaneously infected by two distinct influenza viruses, a reassortment can occur resulting in the generation of a novel virus strain. Moreover, mutations in the surface glycoproteins (mainly in the hemagglutinin, HA) are the responsible of the high variability of IAV. Influenza vaccines against seasonal epidemics, although have good efficacy do not elicit immune response against a wide variety of IAV. Thus, seasonal vaccines only confer protection against the circulating viral strains. This, together with the risk of potential pandemics, has highlighted the importance of developing a universal vaccine able to elicit heterosubtypic immunity against multiple viral subtypes. In this thesis the immune response to IAV infection and vaccination was evaluated in the light of the risk of highly pathogenic AIV (HPAIV) A/H5N1 and A/H7N1, and the pandemic IAV A/H1N1. The work is divided into three parts and each one is further divided into chapters. Part I (chapters 1 and 2) contains the general introduction and the objectives of the thesis. The aim of this first part is to give a global overview and to introduce information to understand (i) the influenza infection, (ii) the immune responses elicited after IAV infection and (iii) a brief summary of current vaccines against influenza. Afterwards, the initial objectives to be achieved are exposed. Part II is the body of the thesis and it contains four studies (from chapter 3 to 6) developed during the four-year period comprising the PhD program. All the chapters are published or submitted to publish in international peer-reviewed journals. Thus, each study contains an abstract, a specific introduction, the materials and methods section, the obtained results and a discussion. To study the role of IAV determinants and to characterize the influenza infection in different hosts could be of great importance to direct the efforts to the formulation of more efficient vaccines. The non structural 1 (NS1) protein is known to be a major determinant of virulence in mammals but little is known about its role in avian species. In chapter 3, the involvement of NS1 in viral pathogenicity was evaluated in chickens. Birds were challenged with two reassortant AIV carrying the NS-segment of H5N1 HPAIV in the genetic background of an H7N1 HPAIV. The pathological manifestations, together with the immunological outcome were evaluated. The role of pre-existing immunity during an outbreak is also important and can determine whether the animals succumbed to infection or not. In chapter 4, chickens pre-exposed to H7N2 low pathogenic AIV (LPAIV) were challenged with H7N1 HPAIV and subsequently infected with H5N1 HPAIV. Pre-exposed animals were protected against the lethal H7N1-challenge whereas naïve animals succumbed. However, pre-existing immunity did not provide protection against HA-heterosubtypic virus (H5N1 HPAIV). The presence or absence of H7- and H5-inhibitory antibodies correlate with the protection (or lack of it) afforded. The control of current vaccination programs and their efficacy is useful to plan and design better vaccines. It is well known that wildfowl are the reservoirs of IAV; thus they are extremely important concerning the ecology of the virus. Sera from several avian species from Spanish zoos and wildlife centers were collected during two successive vaccination programs and were tested to evaluate the vaccine-elicited humoral response (chapter 5). The main objective of this work was to determine the efficacy of current vaccines (inactivated water-in-oil) in several avian species and to compare the differences inter- and intra-specie. Finally, and taking into account the potential risk that IAV represent to our society, the efforts were focused on developing a broadly protective influenza vaccine. The 2009 human H1N1 pandemic (pH1N1) is a clear example that pigs can act as a vehicle for mixing and generating new assortments of viruses. In chapter 6 pigs were immunized with HA-derived peptides and subsequently infected with pH1N1 virus. Although the HA-peptides induced broad humoral and cellular responses no neutralization activity was detected and only a partial effect on virus clearance was observed. Part III (chapters 7 and 8) is where the implications of all the findings from the studies are discussed and the major conclusions are listed. A list of all the references used to develop the thesis is listed after the three parts, in an independent section. An appendix section is also included to give further information.

PARV4 is a novel human parvovirus initially identified in an intravenous drug user at risk of HIV infection. PARV4 is a small single stranded DNA virus principally absent from the general population, but common in HCV- and HIV-infected individuals. Until 2009, most published PARV4 studies related to the prevalence of PARV4 in various risk groups. PARV4 has been detected in the liver of HCV-patients and the bone marrow of HIV-patients. Parvovirus B19, the closest related virus, elicits a strong immune response and can cause serious disease. Thus, this project was initiated to characterise the immune response to PARV4, and investigate the clinical significance of this virus. Cohorts of HCV-infected, HIV-infected, HCV-HIV co-infected, healthy and acute parvovirus B19-infected individuals were screened for humoral and cellular responses in both acute and chronic PARV4 infection. HCV- and HIV-related disease progression was also assessed relative to PARV4 infection. This study demonstrates that the highest prevalence of PARV4 infection is found in HCV-HIV co-infected intravenous drug users, and provides additional evidence for parenteral transmission. I present here the first data on the cellular immune response to PARV4 in acute and chronic infection and define PARV4 as a persistent virus. Although no clear correlation could be found between PARV4 and HCV or HIV disease progression, the high prevalence rates emphasize the importance of investigations into emerging infections such as PARV4.

La réponse immunitaire de la Drosophile implique l’activation des voies de signalisations Toll et IMD contrôlant l’expression de gènes codant des peptides antimicrobiens. Ces deux voies présentent de fortes similarités avec les voies NF-kB régulant la réponse immunitaire innée chez les mammifères. Durant ma thèse, j’ai étudié les mécanismes d’activation de la voie Toll. Mon travail a mené à une nouvelle perception des mécanismes de reconnaissances impliquant la detection des activités microbiennes. J’ai également participé à l’identification de nouveaux composants des voies Toll et IMD. Grâce à un crible par ARNi, nous avons identifié un nouveau gène, Akirin dont la fonction dans les voies NF-kB est conservée de la Drosophile aux mammifères. Lors d’un crible à l’EMS nous avons identifié deux nouveaux mutants des voies Toll et IMD respectivement. Ce travail procure de nouveaux outils pour la compréhension de la réponse immunitaire innée chez la Drosophile et les mammifères<br>The hallmark of Drosophila immune response is the activation of the Toll and IMD pathways, which control the expression of antimicrobial peptides genes. Both pathways share similarities with NF-B pathways involved in mammalian immunity. During my thesis, I performed an analysis of the mechanisms of Toll pathway activation. In addition to the classical microbial motifs sensing, my work points to the detection of the invaders through a sensor system monitoring microbial activities. In a second project, I participated to the identification of new components in the Toll and IMD pathways. During an RNAi screen, we identified a new gene, Akirin, whose function in NF-B activation is conserved in drosophila and mammals. By an EMS mutagenesis screen we isolated two new mutants involved in the Toll and IMD pathways respectively. Characterization of these mutants is underway. Both projects provided new tools or concepts for a better understanding of innate immunity in drosophila and mammals

Measles virus belongs to the Morbillivirus genus, Paramixoviridae family. It is the causative agent of a highly contagious acute infective disease, typical of infancy, characterized by fever, skin rash, cough and conjunctivitis, and a generalized immune suppression (Griffin, 2013). The virus is transmitted through the respiratory tract, multiplicates in its upper part and in regional lymph nodes, thus resulting in lymphatic and hematic dissemination with appearance of first clinical signs after 9-19 days (de Vries et al., 2015). In 30% of the cases, complications in the lower respiratory tract or the central nervous system (CNS) can occur. The first sign of infection is represented by an early immune depression, due to the loss of B and T immune memory cells (Mina et al., 2015), resulting in an increased susceptibility to opportunistic infections and to life-threatening complications such as pneumonia and/or gastro-intestinal disease (de Vries et al., 2015). However, this type of disease is paradoxically associated with the induction of a strong and specific immune response to the virus, which is usually permanent (Laksono et al., 2016). There is no specific treatment against measles, and this is the reason why vaccination is considered the best strategy against the virus. Furthermore, the monotypic nature of the virus and the lack of an animal reservoir, make measles a considerable candidate for eradication (Rota et al., 2016). Although a combined vaccine, called MMR (measles, mumps and rubella) is used in routinely vaccination schedule, measles remains a significative cause of morbidity and mortality, particularly during infancy (Moss &amp; Griffin, 2012; Wolfson et al., 2009; Nandy et al., 2003). MMR live attenuated vaccine is very efficacious in protecting people against measles, mumps, and rubella, and preventing the complications caused by these diseases. The measles virus contained in the vaccine is represented by the live attenuated Edmoston B strain. The World Health Organization recommends two doses of vaccine for all children and adults; the first dose should be given at 13-15 months of age. The second dose is often done at 5 - 6 years, in Italy. About 3 out of 100 people who get two doses of MMR vaccine will get measles if exposed to the virus. However, they are more likely to have a milder illness, and are also less likely to spread the disease to other people (Centers for Disease Control and Prevention, 2018). Epidemiologic studies have shown that the level of neutralizing antibodies at the time of exposure to wild type (WT) virus in the community is a good indicator of protection from infection, with higher titers necessary to prevent infection than to prevent disease (rash) (Chen et al., 1990). High avidity antibodies are required to neutralize CD150-mediated WT MeV infection of lymphoid cells (Polack et al., 2003). However, levels of circulating anti-measles neutralizing antibody tend to reduce or even to fade during lifetime, especially among vaccinated subjects (Kennedy et al., 2019; Davidkin et al., 2008; Carryn et al., 2019; Seagle et al., 2018; Gonçalves et al., 2015; Le Baron et al., 2007). Because CD4+ T cell help is required for isotype and affinity maturation of antibody-secreting cells, B cell memory and maturation of CD8+ T cell memory, cellular immune response is also important for the induction of protective immunity (Laksono et al., 2018). All these things highlight the necessity to invest on studies focused on the correlates of protection against Measles virus. Recent evaluation systems for vaccines point towards the measurement of Tcell quality with regards to cytokine secretion as a protective correlate in addition to antibody titers in serum during the course of an immune response. Although the generation of immune memory supports the concept of vaccine efficacy, direct assessment of immune memory cells and their precursors has not yet been established as a correlate of protection. With the growing knowledge on the phenotype, function and localization of the immune memory cells in the body, researchers think that these cells may provide a novel correlate of protection for evaluation of more efficacious vaccines. Finally, transcriptome-level characterization (mRNA-Seq data) of responses to measles virus stimulation in antibody responders (either vaccinated or naturally infected) and those who have not responded to the vaccine, could help to identify plausible regulators (genes/pathways) that drive the observed differences among these subjects. Such study may help to develop a panel of biomarkers to monitor, besides the antibody response, the immune response to measles vaccine with the aim to protect, in case of outbreaks, not only the fragile subjects, but also the vaccinated subjects who eventually become seronegative along the time, with a booster composed of specific, immunogenic MeV proteins.

Im Kampf gegen eine Virusinfektion spielen CD8+ T Zellen des adaptiven Immunsystems eine besondere Rolle. Sie patroullieren im Körper und entdecken spezifische Virusepitope, welche mittels MHC Klasse I Molekülen auf der Oberfläche infizierter Zellen präsentiert werden. Wird eine virus-infizierte Zelle erkannt, kann diese schnell und effizient eliminiert. Für die Generierung viraler Peptide, welche auf MHC Klasse I Komplexe geladen werden, ist das Ubiquitin-Proteasom-System von essentieller Bedeutung. Kürzlich wurden weitere Funktionen des Immunoproteasoms aufgedeckt wie zum Beispiel der Schutz gegen oxidativen Stress. Innerhalb der vorliegenden Arbeit konnte die Fähigkeit des Immunoproteasoms gegen eine Akkumulation oxidativ geschädigter Proteine zu schützen mit der Generierung von MHC Klasse I Liganden kombiniert und neu interpretiert werden. Es konnte gezeigt werden, dass während einer Virusinfektion in Nicht-Immunzellen die Produktion reaktiver Sauerstoffspezies durch die alternative NADPH Oxidase Nox4 eine bedeutende Rolle spielt. Die Aktivierung von Nox4 resultiert in der Akkumulation oxidativ geschädigter Proteine. Innerhalb von zwei Stunden nach dem Eintreten von Viruspartikeln in die Zellen wurden strukturelle Virusproteine oxidiert und anschließend ubiquityliert. Die gleichzeitige, virus-induzierte Expression von Immunoproteasomen führte zu einem schnellen und effizienten Abbau ubiquitylierter Virusantigene. Infolgedessen konnten immundominante Virusepitope vermehrt freigesetzt werden. Folglich wurde ein soweit unbekannter Mechanismus gefunden, welcher Substrate für das Proteasom zur Generierung von MHC Klasse I Liganden bereitstellt. Zusammenfassend konnte innerhalb dieser Arbeit gezeigt werden, dass das Immunoproteasom den Schutz vor oxidativen Stress mit der Generierung antigener Peptide verbindet, wodurch eine effektive adaptive Immunantwort etabliert werden kann.<br>An efficient immune control of virus infection is predominantly mediated by CD8+ T cells which patrol through the body and eliminate infected cells. Infected cells are recognized when they present viral antigenic peptides on their surface via MHC class I molecules. To make antigenic peptides available for loading on MHC class I complexes, the ubiquitin proteasome system plays a crucial role. Moreover, the induction of the i-proteasome is known to support the generation of MHC class I ligands. Recently, new functions of the i-proteasome have been discovered. Evidence is increasing that the i-proteasome is involved in the protection of cells against oxidative stress. Within this thesis the characteristic of the i-proteasome to protect cells against the accumulation of oxidant-damaged proteins could be linked to its role in improving the generation of MHC class I ligands. It could be demonstrated that during a virus infection in non-immune cells the production of reactive oxygen species by the alternative NADPH oxidase Nox4 is of critical importance resulting in the accumulation of potentially toxic oxidant-damaged proteins. Indeed, within two hours of infection structural virus proteins were oxidized and subsequently poly-ubiquitylated. The concomitant formation of i-proteasomes led to a rapid and efficient degradation of ubiquitylated virus antigens thereby improving the liberation of immunodominant viral epitopes. In conclusion, a so far unknown mechanism to fuel proteasomal substrates into the MHC class I antigen presentation pathway has been revealed. A new protein pool consisting of exogenously delivered viral proteins provides proteasomal substrates in the very early phase of a virus infection. Within the scope of this thesis the i-proteasome has been shown to link the protection against oxidative stress, initiated directly by pathogen recognition, with the generation of antigenic peptides. Together, an effective adaptive immune response is triggered.

Le projet Milieu Intérieur a pour but d'identifier les facteurs génétiques et environnementaux ayant un impact sur la variabilité immunitaire naturelle. Cette analyse multiparamétrique requière néanmoins d'utiliser des outils standardisés. Afin d'étudier la réponse immune induite, nous avons utilisé un système optimisé prêt à l'emploi de stimulation du sang et développé un protocole unique de quantification de l'ARN afin d'étudier la signature transcriptionnelle en réponse à des immuno-modulateurs. Testant l'hypothèse que la réponse à des composés complexes peut être définie par la signature ARN de cytokines clefs et utilisant une méthode statistique robuste, nous avons identifié 44 gènes capables d'optimiser la capture de la réponse à des stimulations plus complexes aidant à la réduction dimensionnelle pour la décomposition de réponses innées. Dans une seconde étude, l'analyse semi-automatisée par cytomètrie en flux des cellules du sang a été associée à l'analyse épidémiologique et génotypique pour les 1,000 donneurs inclus dans la cohorte. Nous avons observé que le tabac, l'âge, le genre et l'infection latente par le cytomégalovirus sont les facteurs impactant le plus la variabilité immunitaire. Cette étude a montré que les paramètres des cellules innées sont contrôlés par des facteurs génétiques alors que ceux des cellules adaptatives le sont plutôt par des expositions environnementales tout au long de la vie. Des outils interactifs incluant ces données de référence accompagnent ces études. Ces analyses montrent que nous avons développé des outils performants pour une étude intégrative du modèle humain constituant une approche innovante vers une médecine personnalisée<br>The project Milieu Intérieur aims to study the genetic and environmental factors that can have a major impact on occurring immunological variance in healthy human population. This characterization requires the use of standardized immunophenotyping technologies for integrating diverse, complex datasets. With this goal in mind, we used an optimized suite of standardized whole-blood stimulation systems to study the human induced immune response in physiological condition and developed a unique standardized protocol to analyze the ARN signatures upon whole-blood stimulation to test the hypothesis that responses to complex stimuli can be defined by the transcriptional signatures of key cytokines. We found 44 genes, identified using Support Vector Machine learning, which captured the diversity of complex innate immune responses with improved segregation between distinct stimuli. This provides new strategies for dimension reduction of large datasets and for deconvolution of innate immune responses, applicable for characterizing novel immunomodulatory molecules.In a second related study, we aimed to identify the environmental and genetic factors driving innate and adaptive immune cell parameters in homeostatic conditions. To do so, we combined semi-automated flow cytometric analysis of blood leukocytes and genome-wide DNA genotyping in the 1,000 healthy donors included in the collection. We show that smoking, age, gender and latent cytomegalovirus infection, are main drivers of human variation (i.e. numbers of Treg and MAIT cells). These results demonstrated that innate cell parameters are strongly controlled by genetic factors, whereas adaptive cells are driven by life-long environmental exposures. In addition, to help on the public data mining, we developed interactive R-Shiny application including healthy donor reference values for both studies.All together, these results indicate that we developed powerful tools for human system biology approaches to support personalized medecine

Early Growth Response Genes (EGR) is a family of four transcription factors containing a unique zinc finger domain. EGR-2 and EGR-3 are important for hindbrain development and myelination. These transcription factors are also necessary for lymphocyte function however, the mechanisms are still unclear. Previous findings have shown that EGR-2cKO mice develop lupus-like autoimmune disease with high levels of pro-inflammatory cytokines despite showing normal T and B cell proliferation after mitogenic stimulation. Therefore we established the CD2-EGR-2-/-EGR-3-/- mouse model to explore the phenotype, susceptibility to autoimmune disease and relevant lymphocyte function. We discovered that CD2-EGR-2-/-EGR-3-/- mice developed severe systemic autoimmune disease and expressed high levels of inflammatory cytokines. More importantly we discovered a novel finding that CD2-EGR-2-/-EGR-3-/- T and B cells had impaired cell proliferation after mitogenic stimulation. Further investigations revealed that the molecular mechanism defected in the T cell receptor signalling pathway is due to a dysfunction in Activator Protein-1 (AP-1). AP-1 is a heterodimeric protein composed of AP-1 family members including Jun, Atf and Fos. Our data shows that EGR-2 and EGR-3 directly bind with the Atf family member Batf, which prevents Batf’s inhibitory function on AP-1 activation. This research demonstrates that EGR-2 and EGR-3 intrinsically regulate chronic inflammation and also positively regulate antigen receptor activation. In conclusion EGR-2 and EGR-3 are essential for providing optimal immune responses, whilst limiting inflammatory immunopathology. We propose that this new model could be used for studying autoimmune disease.

Almost 2 billion people world-wide are infected with parasitic helminths. These complex multicellular eukaryotic organisms are capable of establishing long-term infections even in the face of an intact immune response. Typically, in these settings regulatory components of the immune response, such as Foxp3+ T regulatory cells (Tregs), become dominant, limiting protective effector responses towards the parasite. Helminths are thought to have evolved mechanisms, including release of immunomodulatory molecules termed excretory-secretory products (ES), to sway the balance between the regulatory and effector arms of the immune response to favour their persistence. In this thesis both the development of a protective immune response toward, and the potential manipulation of the immune response by, the rodent gastrointestinal nematode Heligmosomoides polygyrus have been studied. Firstly, the effects of H. polygyrus ES (HES) on bone-marrow derived dendritic cells (DCs) were analysed. Although HES did not alter the phenotype of the DC it was found to be able to suppress the ability of the DC to respond to inflammatory stimuli. This activity was lost when HES was heat-inactivated (hiHES). After adoptive transfer, HES-pulsed DCs were able to induce a HESspecific T helper (Th)2-type response even if co-treated with an inflammatory stimulus. Th2-type responses are protective against H. polygyrus infection. Surprisingly, the ability of HES to generate a Th2-response in a co-treatment situation was not related to its anti-inflammatory properties; DCs co-treated with hiHES and an inflammatory stimulus were able to drive an equivalent Th2-response to HES in this situation. Next, making use of mouse strains with different susceptibility phenotypes to primary H. polygyrus infection, potential mechanisms of resistance were characterised. Development of granulomas in the gut wall were found to be associated with reduced worm burdens. Furthermore, in highly susceptible C57BL/6 mice, production of IL-23 was shown to be counter-regulatory to this process, as mice on the same background but deficient in this cytokine have increased numbers of granulomas and dramatically enhanced resistance. Susceptibility to H. polygyrus was also considered at the level of epigenetic regulation. A protein that binds specifically to methylated DNA, methyl-CpG binding domain protein (MBD)2, was found to affect the proportion of Foxp3+ Tregs within the CD4+ T cell population in vivo. Additionally, in vitro induction of Foxp3 in response to TGF-β was enhanced in MBD2-/- CD4+ T cells. MBD2-/- mice had a trend towards increased worm burdens when infected with H. polygyrus, suggesting that the difference in proportion of Tregs may limit generation of an effector response. Finally, the ability of HES to directly affect the regulatory arm of the immune response was focussed upon. It was found that HES was able to induce Foxp3 expression in naïve peripheral T cells, and that this was mediated by stimulation of the TGF-β pathway. The TGF-β mimic was of parasite origin as a pan-vertebrate TGF-β antibody was unable to block its effects but sera from H. polygyrus infected animals was competent to do this. Activity of this type was not limited to HES as ES from the ovine helminth Haemonchus contortus was found to have the same property. These data imply that some helminth parasites have evolved mechanisms to support generation of Foxp3+ Tregs, thus favouring the regulatory arm of the immune response and hence their own persistence.

Abstract Carbamylation of proteins in vivo occurs by cyanate, non-enzymatically when urea is dissociated, and by myeloperoxidase-catalyzed oxidation from thiocyanate. Carbamylation of low-density lipoprotein is suggested to enhance atherogenesis in patients with with chronic kidney disease and uremia. This thesis study assessed the questions of whether healthy humans or uremic patients under enhanced carbamylation have antibodies recognizing carbamyl-epitopes in plasma, and what is their role in vivo. Also, humoral immune response to carbamyl-LDL immunization and its impact on atherogenesis in LDLR-/- mice was investigated. In this thesis study, plasma antibodies to carbamylated proteins were detected in humans, and IgG antibodies to carbamylated proteins were associated with uremia and smoking, conditions with enhanced carbamylation. The human IgG and IgM antibodies binding to carbamyl-epitopes were associated with oxidation-specific epitopes in plasma. Monoclonal Fab antibodies with characteristics of a natural antibody and ability to bind both carbamyl- and malondialdehyde-derived epitopes were cloned from healthy humans. An investigated Fab antibody was able to bind epitopes found in atherosclerotic lesions and inhibit the uptake of modified LDL by macrophages. Human plasma antibodies and the monoclonal Fab bound to epitopes found on apoptotic cells. Human B-cells secreted antibodies with similar cross-reactive binding properties between carbamyl- and malondialdehyde adducts and apoptotic cells in vitro. Immunization with mouse carbamyl-LDL without adjuvant resulted in specific IgG immune response in LDLR-/- mice, but also a cross-reaction with malondialdehyde-adducts was observed. Carbamyl-LDL immunized mice had enhanced plasma antibody binding to apoptotic cells. Carbamyl-LDL immunization did not affect atherogenesis in mice. This thesis demonstrates that IgG antibodies to carbamyl-epitope might serve as a novel indicator of carbamylation in vivo in uremic patients or smokers. The cross-reactivity between antibodies binding to carbamylated and oxidation-specific epitopes, and apoptotic cells may have a role in explaining the link between enhanced atherogenesis and kidney disease<br>Tiivistelmä Proteiinien karbamylaatiota tapahtuu syanaatin vaikutuksesta. Sitä muodostuu urean hajotessa tai myeloperoksidaasin katalysoimana tiosyanaatin hapettuessa. Low-density lipoproteiinin eli LDL:n karbamylaation on esitetty edistävän valtimonkovettumataudin eli ateroskleroosin kehittymistä munuaisten vajaatoimintaa sairastavilla ureemisilla potilailla. Väitöskirjatyössä tutkittiin, onko terveillä ihmisillä ja ureemisilla potilailla karbamyyli-epitooppeja tunnistavia vasta-aineita, ja mikä niiden merkitys on elimistössä. Humoraalista immuunivastetta karbamyyli-LDL-immunisaation jälkeen sekä sen vaikutusta ateroskleroosin kehittymiseen tutkittiin LDL-reseptoripuutteellisilla hiirillä. Tutkimuksessa osoitettiin, että ihmisillä on plasmassa karbamyloituja proteiineja tunnistavia vasta-aineita. IgG-luokan vasta-aineet ovat yhteydessä uremiaan ja tupakointiin, joissa karbamylaatio on lisääntynyt. Karbamyyli- ja hapettuneita epitooppeja tunnistavien plasman IgG- ja IgM-vasta-aineiden välillä havaittiin olevan yhteys. Työssä kloonattiin terveistä ihmisistä monoklonaalisia Fab-vasta-aineita, joilla on luonnollisten vasta-aineiden kaltaisia ominaisuuksia ja kyky sitoutua sekä karbamyyli- että malonidialdehydi-epitooppeihin. Yksi tutkittu Fab-vasta-aine sitoutui valtimonkovettumataudin ateroomissa oleviin epitooppeihin ja esti muuntuneen LDL:n sisäänoton makrofagi-soluihin. Ihmisen plasman vasta-aineet ja monoklonaalinen Fab-vasta-aine sitoutuivat apoptoottisten solujen pinnalla oleviin rakenteisiin. Soluviljelyolosuhteissa ihmisen B-solut tuottivat vasta-aineita, joilla oli samanlaisia ristireaktio-ominaisuuksia karbamyyli- ja malonidialdehydi-epitooppeja sekä apoptoottisia soluja kohtaan. Karbamyyli-LDL-immunisaatio sai aikaan IgG-immuunivasteen hiirillä karbamyyli-LDL:a kohtaan, mutta myös ristireaktio malonidialdehydi-rakenteita sekä apoptoottisia soluja kohtaan havaittiin. Karbamyyli-LDL-immunisaatio ei vaikuttanut ateroskleroosin kehittymiseen hiirillä. Tutkimus osoittaa, että IgG-vasta-aineet karbamyyli-epitooppeja kohtaan voivat olla uudenlainen karbamylaation merkkiaine elimistössä ureemisilla potilailla ja tupakoitsijoilla. Karbamyloituneiden ja hapettuneiden epitooppien sekä apoptoottisten solujen välillä havaituilla vasta-aineiden ristireaktioilla voi olla merkitystä valtimonkovettumataudin etenemiseen munuaisten vajaatoiminnassa

La présence d’une immunité anti-tumorale est maintenant bien établie et ce, y compris dans le cancer du sein pourtant considéré comme peu immunogénique. Cette notion a été récemment renforcée dans différentes études qui ont mis en évidence une corrélation entre la présence d’un infiltrat immun au niveau des cancers du sein primaires et le pronostic des patientes. La réponse immune anti-tumorale n’est cependant pas assez efficace pour contrôler la survenue du cancer. En effet, différents mécanismes sont utilisés par les cellules tumorales pour échapper au système immunitaire comme l’expression de ligands inhibant les cellules immunes ou la production de médiateurs immunosuppresseurs dans le microenvironnement tumoral.Ces mécanismes sont la cible des thérapies immunomodulatrices visant à réactiver la réponse immune anti-tumorale. L’approche utilisant les inhibiteurs de points de contrôle a clairement montré des résultats très encourageants pour le traitement du mélanome et du cancer du poumon. De nombreux traitements immunomodulateurs sont actuellement à l’étude dans le cancer du sein. L’objectif de ce travail de thèse de doctorat est de caractériser la réponse immune dans le cancer du sein en s’intéressant plus particulièrement à l’infiltration lymphocytaire tumorale et à l’expression de marqueurs impliqués dans l’échappement au système immunitaire. La première partie de ce travail étudie différents aspects méthodologiques permettant la quantification histologique des cellules immunes. Il démontre que l’immunohistochimie offre des résultats très reproductibles et permet de distinguer des sous-types de lymphocytes ainsi que leur organisation en structures lymphoïdes tertiaires. Il démontre également que la quantification sur biopsies préthérapeutiques est représentative de la tumeur. Enfin, l’analyse d’images se révèle être un outil utile et fiable pour quantifier l’infiltration lymphocytaire tumorale.Le deuxième volet de cette thèse consiste en une caractérisation détaillée de la densité, de la composition, et de l’organisation de l’infiltrat lymphocytaire tumoral. L’analyse par cytométrie en flux montre que d’une part, les tumeurs sont plus infiltrées en lymphocytes que le tissu mammaire sain et d’autre part, cet infiltrat est composé d’un plus haut taux de lymphocytes T CD4 et B associé à une diminution du taux de CD8. Dans 60% des cas, des structures lymphoïdes tertiaires, composées d’un follicule delymphocytes B et de cellules dendritiques entourés de lymphocytes T, sont observées dans le stroma péri-tumoral. Le niveau d’infiltration lymphocytaire et son organisation en structures lymphoïdes sont positivement corrélées à l’expression de PD-1, un récepteur inhibiteur de l’activation des cellules immunes, et de son ligand PD-L1. La dernière partie de ce travail étudie l’expression de l’enzyme CD73 impliquée dans la production d’adénosine, un puissant médiateur immunosuppresseur dans le microenvironnement tumoral. L’expression de CD73 a été mesurée par immunofluorescence combinée à une analyse d’images dans une série de cancers du sein de type triple négatif. Celle-ci est significativement associée à un plus mauvais pronostic, et est inversement corrélée à l’infiltration immune. En conclusion, les résultats découlant des différents chapitres de cette thèse démontrent qu’une réponse immune est présente dans le cancer du sein et appuie la pertinence de développer des stratégies thérapeutiques pour renforcer la réponse immune anti-tumorale dans le cancer du sein. La combinaison de marqueurs immuns tels que ceux étudiés dans ce travail permettrait de mieux caractériser le type de réponse immune anti-tumorale afin de guider le choix de la thérapie immunomodulatrice et aussi de sélectionner plus adéquatement les patientes susceptibles d’en bénéficier.<br>Doctorat en Sciences médicales (Médecine)<br>info:eu-repo/semantics/nonPublished

Acute otitis media is the most common illness diagnosed during early childhood that can cause significant morbidity (Brook, 1994) and sometimes can cause irreversible sequelae such as a hearing defect and subsequent learning difficulties (Klein, 1994). The aims of the research presented here were to study some aspects of the middle ear defence mechanisms in both immune and non-immune rats following experimental otitis media (OM) with two pathogens nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (M. catarrhalis). This study also aimed at developing a suitable technique for preparing immunohistochemical staining of middle ear sections (chapter 2). A previous study has shown that a regime where rats received an IPP immunisation combined with an IT boost was effective in enhancing clearance of a middle ear infection with the same strain of NTHi and also in the presence of a concomitant viral infection (Moore et al, 2001). Results of this study have shown that for NTHi infection a distinct cellular influx to the middle ear in the immune rats was accompanied by an enhanced bacterial clearance compared to the non-immunised rats (chapter 3). This cellular influx was responsible for the remarkable reduction in the bacterial number. The sharp decline in PMNs numbers in the NTHi immunised rats that followed complete bacterial clearance at 72h post infection (Table 3.1) indicate a more effectively controlled down regulation of this cell infiltrate than the non-immunised rats. For M. catarrhalis infection, there was no difference in cell infiltrate between immune and non-immune rats, but enhanced clearance of the bacteria were observed for the immune animals. The histopathological changes in the middle ear mucosa of rats with experimentally induced infection were studied to provide a better understanding about the distribution of the inflammatory cells and changes in the mucosa during the first 24h post challenge with NTHi and M. catarrhalis (Chapter 4). These changes have not been previously studied for the two pathogens at 24h post challenge in rats. Induced infections with the two pathogens were found to produce similar histopathological changes but more inflammatory infiltration was observed within the infected mucosa with NTHi than that seen with M. catarrhalis. The infections were characterized by increased thickness of the middle ear mucosa, Eustachian tube mucosa, periosteum and tympanic membrane. There was also an increase in the number and size of small blood vessels at all sites, and these small blood vessels seem to be the source of the inflammatory infiltration into the middle ear mucosa and middle ear cavity during the infection. These findings provided an essential background to the immunohistochemical study. The effect of mucosal immunisation on the distribution of CD4+T cells and CD8+T cells has not been investigated previously. Results of the present study (Chapter 5) show the pattern of distribution of these cells during the first 48h post infection with NTHi in the rat. The number of CD4+and CD8+T cells peaked at 24h post infection in the nonimmunised animal and were highest at 48h post-infection in the immunised rats. The difference in response in the immunised rats may represent regulation of the inflammatory response by the immune system. The inflammatory response regulation is indicated by the difference in cellular influx into the immune rats and the response in the immune rats that corresponds to enhanced bacterial clearance prior to a decrease in numbers of inflammatory cells once the bacteria was no longer detected (Chapter 3). This resolution of the inflammatory mass would reduce the opportunity for continued damage to local tissue. These changes are also supported by the reduction in the thickness of the middle ear mucosa of the immunised rats especially at 24h and 48h post-infection (Chapter 5). This study has shown that there are distinct differences in the rate of bacterial clearance and cellular changes in the middle ear mucosa and tympanic bulla in immunised rats during a middle ear infection. Future studies are still required to gain a better understanding of differences in the inflammatory response for both pathogens, NTHi and M. catarrhalis.

The early steps of activation are crucial in deciding the fate of T-cells leading to the proliferation. These steps strongly depend on the initial conditions, especially the avidity of the T-cell receptor for the specific ligand and the concentration of this ligand. The recognition induces a rapid decrease of membrane TCR-CD3 complexes inside the T-cell, then the up-regulation of CD25 and then CD25-IL2 binding which down-regulates into the T-cell. This process can be monitored by flow cytometry technique. We propose several models based on the level of complexity by using population balance modeling technique to study the dynamics of T-cells population density during the activation process. These models provide us a relation between the population of T-cells with their intracellular and extracellular components. Moreover, the hypotheses are proposed for the activation process of daughter T-cells after proliferation. The corresponding population balance equations (PBEs) include reaction term (i.e. assimilated as growth term) and activation term (i.e. assimilated as nucleation term). Further the PBEs are solved by newly developed method that is validated against analytical method wherever possible and various approximate techniques available in the literature.

Despite the abundance of research conducted into the effects of exercise on mucosal immunity the results remain controversial. Much of the inconsistencies arise from the exercise protocols, the participants studied and their nutritional status, as well as methodological and analytical differences. The purpose of this thesis was to examine the influence of some of these factors, and to investigate potential means of enhancing the mucosal immune response to exercise. In study 1 (Chapter 3) it was shown that a fed or fasted state 2 h prior to exercise had no effect on the s-IgA concentration or secretion rate during prolonged exercise. However, when participants were fed during exercise (Chapter 4), the secretion rate of salivary antimicrobial proteins lysozyme and a-amylase increased, but sIgA remained unchanged. These changes were likely due to the activation of mechanical and gustatory receptors leading to a reflex stimulation of protein secretion via the autonomic nerves, rather than changes in stress hOnliones, since cortisol did not change significantly during exercise. Study 3 (Chapter 5) extended these findings where it was demonstrated that chewing flavoured gum during exercise enhanced lysozyme and a-amylase secretion but resulted in a small reduction in s-IgA secretion rate. Salivary antimicrobial proteins are affected by the exercise intensity since both s-IgA and lysozyme secretion rate increased post -exercise following an incremental test to exhaustion, but not after exercise at 50% Y02max. Moreover, lysozyme secretion rate was also elevated following exercise at 75% Y02mru<, whereas s-IgA remained unchanged. These effects are thought to be mediated by increased sympathetic nervous system activity reflected by the concomitant increases in (lamylase and chromogranin A, rather than the hypothalamic-pituitary-adrenal axis. Resting mucosal immunity exhibits significant gender differences. In study 1 (Chapter 3) s-IgA concentration, secretion rate and osmolality were found to be lower in females than in males at rest. In addition, saliva flow rate was found to be lower in females compared with males in study 5 (Chapter 7). However, these differences did not appear to influence the salivary responses to acute exercise or exercise training. Chronic exercise training in elite male and female swimmers resulted in lower levels of s-IgA secretion rate following periods of intense training prior to competition compared with post-competition (Chapter 7), but these levels were not directly associated with reported episodes of respiratory illness.

Imino sugars are carbohydrate-like alkaloids largely characterised over the last ten years that function as glycosidase inhibitors as they mimic the sugar moiety of the natural substrate. They have been reported to have immunomodulatory activity and the purpose of this study was to identify and characterise such activity. The ability of over sixty imino sugars to modify macrophage/dendritic cell cytokine production was analysed. The immunomodulatory activity of two imino sugars MNLP 462a and MNLP 24 with pro-inflammatory activity was studied in depth. They were found to upregulate in vitro macrophage IL-12 production and dendritic cell IL-12 and IL-2 production as well as co-stimulatory molecule expression particularly CD40, but in addition CD80 and CD86 on dendritic cells.