Suite à un traumatisme cérébral, les microglies sont impliquées dans la réponse cellulaire inflammatoire. Le passage de la forme quiescente à la forme activée des microglies est associé à l’augmentation marquée du récepteur membranaire TLR2 (Toll-like receptor). Le rôle de ce récepteur sur la récupération fonctionnelle à court et à longs termes demeure incertain. C’est pourquoi cette thèse a pour but de clarifier la dynamique et le rôle de l’activation des microglies en utilisant le récepteur TLR2 comme marqueur suite à une ischémie cérébrale transitoire. Pour ce faire, nous avons entre autre utilisé un modèle murin généré dans le laboratoire, la souris TLR2-luc-GFP, pour étudier de façon longitudinale la réponse inflammatoire in-vivo. En premier lieu, l’activation microgliale, suite à la lésion, a été analysée chez une souris n’exprimant pas le récepteur TLR2. Les résultats de cette étude démontrent que l’absence du récepteur TLR2 retarde la réponse immunitaire et cause à long terme une augmentation du volume ischémique. De plus, la perte des récepteurs TLR2 corrèle avec la baisse des niveaux de IGF-1, un facteur anti-apoptotique et neurotrophique exprimé par les microglies dans la région ischémique. En deuxième lieu, nous avons évalué l’effet de l’environnement sur la réponse microgliale suite à une ischémie cérébrale induite par photothrombose. Une baisse d’activation microgliale fut mesurée chez les souris en milieu enrichi et cette diminution est accompagnée d’une meilleure récupération fonctionnelle. En troisième lieu, l’activation microgliale, en absence d’estrogène a été analysée chez une souris ErαKO à la suite d’une ischémie cérébrale transitoire. Les résultats de cette étude démontrent que l’absence du récepteur Erα diminue l’activation microgliale. De plus, la voie signalétique JAK/STAT, via la cytokine IL-6, serait préférentiellement utilisée chez ces souris au cours de la réponse inflammatoire. Finalement, cette thèse démontre l’importance de la microglie dans l’établissement de la réponse immunitaire ainsi que dans la récupération fonctionnelle suite à une ischémie cérébrale dans différents modèles. Grâce à une compréhension plus approfondie des mécanismes inflammatoires, la mise en place de stratégies thérapeutiques ne sera que plus efficace. / Cerebral ischemia is associated with a strong inflammatory response. Glial activation that ensues contributes to neuronal damage and/or provides trophic support to injured neurons. Activated microglia is one of the key components of the cellular response to CNS injuries and the passage from the quiescent to reactive microglia is associated with an upregulation of a membrane receptor called the toll-like receptor 2 (TLR2). Although upregulation of TLR2 his known to play a major role in the innate immune response, the functional significance and the dynamics of this response in a model of focal cerebral ischemia is still unclear. Therefore, to further clarify the role of TLR2 after a cerebral ischemia, we developed a model system for in vivo, non-invasive analysis of the TLR2 activation in the brain. This thesis is divided in three distinct chapters. In the first chapter the role of the TLR2 signaling and microglial activation in a TLR2KO mouse was investigated beyond 72 hours. This work clearly demonstrates that the lack of TLR2 affects the neuroinflammatory response causing a delayed exacerbation of the ischemic lesion. Furthermore, TLR2 deficient mice reduced levels of IGF-1, a neurotrophic and antiapoptotic factor express by microglial cells. In the second chapter, we evaluated the effect of environmental enrichement on the inflammatory response after phototrombotic stroke in our TLR2-luc-gfp mice. Study of microglial activation showed that multisensory stimulation attenuated TLR2-induced microglial activation, thus leading to a greater neurological outcome. In the third chapter, we crossed TLR2-luc-gfp mice with ErαKO mice to study the effect of a major oestrogen signalling pathway on microglial activation after cerebral ischemia. The lack of Erα receptor reduced microglial activation in normal female mice as well as oestrogen-deprived mice. Likewise a shift toward the JAK/STAT pathway was observed suggesting its implication in the inflammatory process. Finally this thesis illustrates the significant impact of the microglia on the immune response and the recovery after a cerebral ischemia in different mouse models. With this newly acquired knowledge on inflammatory process the establishment of better therapeutic strategy could only benefit from it.

Les virus herpétiques sont des pathogènes ubiquitaires caractérisés, entre autres, par la persistance de leur génome au sein de l'hôte. Parmi ceux-ci, le virus Epstein-Barr (EBV) est associé à la mononucléose infectieuse, au carcinome du nasopharynx et au lymphome de Burkitt. L'homologie génomique qui existe entre certains virus herpétiques, principalement entre le gammaherpèsvirus murin 68 (MHV-68) et EBV, est établie depuis plusieurs années. Cette homologie est d'autant plus importante que ces virus sont reconnus par le système immunitaire de manière similaire chez leurs hôtes respectifs. Ce système se décline en deux volets complémentaires, soit l'immunité innée et l'immunité acquise. Les "Toll-like receptors" (TLRs) composent l'une des principales familles de récepteurs impliquées dans l'immunité innée. C'est donc sur cet aspect que le projet s'est concentré, et plus précisément sur l'activation du récepteur membranaire TLR2 par le virus MHV-68. Dans un premier temps, nous avons démontré que l'activation du TLR2 par MHV-68 mène à une augmentation de l'activité NF-KB. À l'aide de cellules fibroblastiques embryonnaires (MEFs) provenant de souris C57BL6 TLR2⁻/⁻ ou MyD88⁻/⁻, nous avons prouvé que la synthèse d'IL-6 et d'IFN-α induit par MHV-68 est partiellement dépendante du TLR2 et entièrement dépendante de MyD88. L'induction du gène TLR2 par MHV-68 a pu être établie in vivo à l'aide de souris transgéniques exprimant le gène de la luciférase sous le contrôle du promoteur du TLR2. L'utilisation de MHV-68 irradiée aux UV a donné des résultats similaires suggérant que la transcription génique et la replication virale ne sont pas essentielles à l'activation ni à l'induction du TLR2. Nous avons par la suite mis en évidence par cytometric de flux que le TLR2 était induit à la suite d'une infection par MHV-68 chez les granulocytes, les monocytes circulants et les monocytes inflammatoires recrutés au poumon. Nous avons finalement observé une diminution de la sécrétion d'IFN-α, d'IFN-β et d'IL-6 dans les poumons et la rate des souris TLR2⁻/⁻ et MyD88⁻/⁻ infectées, comparativement aux témoins (WT), et ce, de façon concomitante avec une augmentation de la charge virale. 11 s'agit de la première étude qui prouve que le virus MHV-68 est reconnu par le récepteur membranaire TLR2 murin.

Aim: The aim of this study is to determine the frequency of single nucleotide polymorphisms (SNPs) in various pattern recognition receptor (PRR) genes, including Toll like receptors (TLR) -2, -4, -9, and CD14 in chronic (CP), localized (LAP) and generalized aggressive (GAP) periodontitis and periodontally healthy (NP) patients in an African American population. Methods: A total of 205 subjects were involved in the study. The LAP group consists of 25 subjects, the GAP group 50 subjects, the CP group 73 subjects and the NP group 57subjects. Genotyping was performed in TLR2 (G2408A), TLR4 (A896G),TLR9 (T1486C) and CD14 (C260T) genes by TaqMan® allelic discrimination using Assay-by-DesignSM SNP Genotyping Assays (Applied Biosystems). Accuracy of genotyping was confirmed by known DNA samples of each genotype and by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analyses on selected samples. Fisher’s exact test and chi-square analyses were performed to compare genotype and allele frequencies. Within disease groups, we investigated whether SNPs were related to disease severity by step-wise logistic regression adjusted for age, gender, and smoking status. Results: There was a significant difference in the distribution of specific TLR9 (T1486C) genotypes between diseased-groups versus reference group. Expression of TT genotype was more prevelant in periodontally-diseased individuals compared to periodontally-healthy subjects (p<0.0001) whereas individuals expressing C allele of the TLR9 SNP (CC&CT) were more frequently found in healthy group after adjusting for age, gender, and smoking status (p<0.0001) There was no statistically significant difference in the distribution of genotypes between groups for any other TLRs or CD 14 polymorphism. Conclusion: Based on findings of this study, homozygocity for the T allele of TLR 9 polymorphism was related to the periodontal disease susceptibility in African Americans. Additionally, presence of C allele at TLR-9 appeared to confer resistance to periodontal destruction. Our results showed that specific SNPs in TLR-2, -4 and CD 14 genes are not related to periodontitis in African Americans. However, low copy number of certain alleles warrants further investigations with increased sample size to explore the role of SNPs in periodontal disease. This study was supported by the Alexander Fellowship.

Le polynucléaire neutrophile (PN) humain est une cellule phagocytaire qui constitue une des premières barrières de défense de l’organisme contre les agents pathogènes. Sa stimulation par des facteurs chimioattractants, provoque sa migration de la circulation sanguine vers le foyer inflammatoire. Dans le site inflammatoire, les PN reconnaissent l’agent pathogène par l'intermédiaire d'opsonines, des fractions résultant de l'activation du complément et par l’intermédiaire de motifs de reconnaissance conservés au cours de l’évolution des agents pathogènes qui se lient à des récepteurs de la famille Toll (Toll-like receptors ; TLR). Le contact du pathogène avec le PN va provoquer sa phagocytose et sa destruction par la libération de molécules contenues dans les granules du PN et par la production de formes réactives de l’oxygène (FRO) par un complexe enzymatique la NADPH phagocytaire composée au repos de de protéines cytosoliques (p40phox, p47phox, p67phox et Rac 2) et membranaires (gp91phox et p22phox formant le cytochrome b558). Un des événements majeur de l’activation de la NADPH oxydase est la phosphorylation de certains composants cytosoliques comme la p47phox ou la p67phox ce qui conduit à la translocation de ces protéines vers le cytochrome b558 membranaire et permet d’activer l’enzyme pour la production de FRO. L’hyperactivation de cette enzyme ou son « priming » consiste en une pré-activation du PN par des agents dit « primants » tels que des cytokines (TNFα, GM-CSF, IL-1), des chimiokines comme l’IL-8, des molécules lipidiques (PAF et LTB4), ou encore des endotoxines bactériennes LPS, agoniste de TLR4. Les TLR sont des récepteurs exprimés à la surface de nombreuses cellules dont les cellules immunitaires ; ils détectent des motifs conservés au cours de l’évolution des agents pathogènes appelés PAMPs pour "pathogen-associated molecular patterns", des protéines modifiées reconnues comme étrangères, des lipides oxydés, des ligands endogènes. Quelques agonistes des TLR comme le LPS ont été décrits pour induire un priming de la production des FRO par les PN. D’autres ont été connus par leur pouvoir activateur de la NADPH oxydase des PN. Le CL097 (Imidazoquinoline : agoniste des TLR7/8) était l’agoniste des TLR induisant le plus fort effet de « priming » par les PN stimulés par le fMLP. Le CL097 induit la phosphorylation de la p47phox sur la sérine 345. Cette phosphorylation implique des MAPKinases ERK1/2 et de la p38MAPK. La phosphorylation de ce site induit le changement de conformation de la p47phox sous l’action d’une proline isomérase Pin1. Ce changement de conformation favorise la phosphorylation des autres sites (Ser-315, Ser-328) et par conséquent l’activation de la NADPH oxydase. La comparaison de l’effet du CL097 à deux agonistes reconnaissant l’un le TLR7, l’autre le TLR 8 a montré que l’action du CL097 dépendait du TLR8. Le zymosan non opsonisé (agoniste de TLR2) stimule l’activation de la NADPH oxydase des neutrophiles. IL induit la phosphorylation de la p47phox au niveau des Ser-345, -315 et -328. Ces phosphorylations font intervenir respectivement les MAPK ERK1/2 et p38, une protéine tyrosine kinase et les PKC. En plus cet agoniste active la petite protéine cytosolique Rac2, nécessaire à l’activation de la NADPH oxydase des PN. Ces données permettraient d’identifier de nouvelles cibles thérapeutiques de première importance afin de moduler les réponses inflammatoires pathologiques. / Superoxide anion production by the neutrophil NADPH oxidase plays a key role in host defense; however, excessive superoxide production is believed to participate to inflammatory reactions. Neutrophils express several TLR that recognize a variety of microbial motifs or agonists. The interaction between TLR and their agonists is believed to help neutrophils to recognize and to eliminate the pathogen. However, the effects of some TLR agonists on the NADPH oxidase activation and the mechanisms controlling these effects have not been elucidated. In this study, we show that the TLR7/8 agonist CL097 by itself did not induce NADPH oxidase activation in human neutrophils, but induced a dramatic increase of fMLF-stimulated activation. Interestingly, CL097 induced cytochrome b558 translocation to the plasma membrane and the phosphorylation of the NADPH oxidase cytosolic component p47phox on Ser345, Ser328 and Ser315. Phosphorylations of Ser328 and Ser315 were significantly increased in CL097-primed and fMLF-stimulated neutrophils. Phosphorylation of Ser345, Ser328 and Ser315 was decreased by inhibitors of p38MAPK and the ERK1/2-pathway. Phosphorylation of Ser328 was decreased by a PKC inhibitor. Genistein, a braod range protein tyrosine kinase inhibitor, inhibited the phosphorylation of these serines. Our results also show that CL097 induced proline isomerase (Pin1) activation and that juglone, a Pin1 inhibitor, inhibited CL097-mediated priming of fMLF-induced p47phox phosphorylation and superoxide production. These results show that activation of TLR7/8 in human neutrophils induces hyper-activation of the NADPH oxidase by stimulating the phosphorylation of p47phox on selective sites, and suggest that p38MAPK, ERK1/2, PKC and Pin1 control this process.Zymosan a cell-wall preparation from saccharomyces cerevisiae is largely used to activate neutrophils in its opsonized form. In this study, we show that non-opsonized zymosan induced ROS production by human neutrophils. Interestingly, zymosan induced the phosphorylation of the NADPH oxidase cytosolic component p47phox on Ser345, Ser328 and Ser315; and activation of the GTPase Rac2. Phosphorylation of p47phox as well as Rac2 activation were inhibited by genistein a broad range protein tyrosine kinase inhibitor. Wortmannin a PI3Kinase inhibitor, inhibited phosphorylation of p47phox on Ser328 and Ser315 and Rac2 activation. SB203580 and UO126, inhibitors of p38MAPK and ERK1/2-pathway respectively, inhibited phosphorylation of p47phox on Ser345. GF109203X a PKC inhibitor inhibited phosphorylation on Ser328 and Ser315. Zymosan-induced ROS production was inhibited by genistein, wortmannin, SB203580, UO126 and GF109203X. These results show that zymosan induced ROS production by NADPH oxidase in human neutrophils via the phosphorylation of p47phox and Rac2 activation. Our results also suggest that a protein tyrosine kinase and PI3Kinase control p47phox phosphorylation and Rac2 activation while p38MAPK, ERK1/2 and PKC are involved in zymosan-induced p47phox phosphorylation.

AIM: to determine if a relationship exists between TLR-2, TLR-4, TLR-9, or CD14 polymorphisms and risk for developing chronic periodontal disease in an African-American population. This is the first study conducted to determine role of SNPs in TLR genes and CD14 gene in a periodontally-diseased African-American population. Additionally, this is the first study to assess the role of TLR-9 polymorphism in periodontitis patients. METHODS: A total of 130 subjects were involved in the study. The chronic periodontitis (CP) group contained 73 subjects, and the healthy control (NP) group 57subjects. Genotyping was performed in TLR2 (G2408A), TLR4 (A896G),TLR9 (T1486C) and CD14 (C260T) genes by TaqMan® allelic discrimination using Assay-by-DesignSM SNP Genotyping Assays (Applied Biosystems). Accuracy of genotyping was confirmed by known DNA samples of each genotype and by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analyses on selected samples. Fisher’s exact test and chi-square analyses were performed to compare genotype and allele frequencies. Within disease groups, we investigated whether SNPs were related to disease severity by step-wise logistic regression adjusted for age, gender, and smoking status. RESULTS: There was a significant difference in the distribution of specific TLR9 (T1486C) genotypes between the periodontally diseased group versus the control group. Expression of TT genotype was more prevelant in periodontally-diseased individuals compared to periodontally-healthy subjects (p<0.0001) whereas individuals expressing C allele of the TLR9 SNP (CC&CT) were more frequently found in the control group after adjusting for age, gender, and smoking status (p<0.0001) There was no statistically significant difference in the distribution of genotypes between groups for any other TLRs or CD14 polymorphism. CONCLUSION: Based on findings of this study, homozygocity for the T allele of TLR 9 polymorphism was related to chronic periodontal disease susceptibility in African Americans. Additionally, presence of the C allele at TLR-9 appeared to confer resistance to periodontal destruction. Our results showed that specific SNPs in TLR-2, -4 and CD14 genes are not related to periodontitis in African Americans.

Notre laboratoire a démontré que le récepteur de chimiokine CCR7 joue un rôle majeur dans la migration des monocytes en réponse à ses ligands naturels, les chimiokines CCL19 et CCL21. Plusieurs études ont permis de mettre en évidence que les monocytes en réponse à des infections, des traumatismes ou des maladies neurologiques, migrent vers les tissus en inflammation du cerveau par un mécanisme jusqu’à présent inconnu. D’un autre côté, l’activation de certains récepteurs TLR (de l’anglais « Toll-like receptors ») par des composants bactériens est caractéristique aux infections bactériennes et tient un rôle primordial dans la réponse immunitaire. L’objectif principal de ce projet de maîtrise est de déterminer si l’activation des TLR bactériens module l’expression de CCR7 chez les monocytes, les rendant ainsi aptes à transmigrer à travers la barrière hémato-encéphalique (BHE). Pour ce faire, nous avons d’abord développé un modèle in vitro de la BHE à partir de co- culture d’astrocytes humains et de cellules endothéliales microvasculaires humaines. Les résultats de résistance électrique transendothéliale (de l’anglais « Transendothelial Electrical Resistance » (TEER)) démontrent que ce modèle est fonctionnel et possède des jonctions serrées rendant la BHE imperméable et plus près de la réalité. Des essais de transmigration de monocytes démontrent que le modèle de la BHE construite limite le passage des monocytes à travers la barrière. Ces résultats laissent supposer que la BHE limite donc le passage des monocytes à travers le SNC. Cette imperméabilité aux cellules, propre à la BHE in vivo, rend le modèle idéal à des essais chimiotaxiques de transmigration. Par la suite, nos travaux ont permis de déterminer si l’activation de certains TLR par des composants provenant de bactéries Gram + et Gram – sont capables de moduler l’expression iv et la fonctionnalité de CCR7 chez les monocytes. Des PCR quantitatives effectuées avec la lignée MONO-MAC-1 ont démontré que l’activation des TLR1/2 et TLR4 promeut l’expression de l’ARNm codant pour CCR7. Des essais de cytométrie en flux ont démontré que l’activation de TLR1/2 et TLR9 chez la lignée MONO-MAC-1 ainsi que l’activation de TLR4 chez les monocytes sanguins augmentait le pourcentage de cellules exprimant CCR7 à leur surface. Des essais de migration ont démontré que le récepteur CCR7 était fonctionnel puisque la lignée MONO-MAC-1 et les monocytes sanguins traités avec l’agoniste de TLR4 migraient en plus grand nombre en réponse aux agonistes de CCR7, les chimiokines CCL19 et CCL21. Nos travaux ont aussi démontré que le récepteur CCR7 exprimé par les MONO- MAC-1 et monocytes sanguins avait une plus grande affinité envers CCL19 que CCL21. L’activation de TLR4 promeut la migration CCR7 dépendante des MONO-MAC-1 et des monocytes sanguins à travers le modèle in vitro d’une barrière hémato-encéphalique en réponse à CCL19. Nos résultats suggèrent une implication importante des récepteurs TLR4 dans la migration CCR7 dépendante des monocytes à travers la BHE et donc qu’une infection bactérienne ou que la présence de LPS dans le sang peut influencer la migration des monocytes au cerveau exacerbant ainsi des conditions neurologiques pathologiques. Davantage d’études portant sur les rôles des TLR dans la migration cellulaire pourraient permettre de développer de nouvelles thérapies.

The efficacy of some vaccines is enhanced by the presence of adjuvants added to their formulations or, in the case of live attenuated or killed whole cell vaccines, because of their endogenous adjuvant activity. The immune system responds robustly to these endogenous adjuvants, which includes Pathogen Associated Molecular Patterns, which stimulate innate immune responses through Pattern Recognition Receptors, such as TOLL-like receptors (TLRs). The development of most vaccine adjuvants has occurred despite little understanding of their overall mechanisms of immune enhancement. We hypothesized that TLR-dependent adjuvant activities are mediated through TLR stimulation of antigen presenting cells (APCs), and each APC type may play a unique role in the immune-stimulating ability of these adjuvants, including effects on downstream T cell stimulation. We used a mouse model where TLR/MyD88 signaling is prevented in specific APC types, in vivo, using loxP/cre recombinase transgenic mice (B cells, dendritic cells and macrophages) to investigate its role in vaccine adjuvant activity. We found that intact MyD88 signaling is essential, separately, in all three APC types for optimal TLR-ligand based adjuvant (PorB, CpG, MPLA), but not for TLR-independent (Alum, MF59) adjuvant activity. However, the immune responses were reduced to the greatest extent in mice with macrophage specific MyD88 deletion (Mac-MyD88-/-). We demonstrated that TLR-dependent adjuvants are potent inducers of germinal center (GCs) formation needed for an effective and robust immune response. Interestingly, GCs are nearly absent in Mac-MyD88-/- mice upon immunization with TLR-dependent adjuvants, but not with TLR-independent adjuvants. Further investigations revealed a significant impairment in T cell cytokines important for GC formation in Mac-MyD88-/- mice when immunized with TLR-dependent adjuvants. Through these studies we discovered that vaccine formulated with PorB/OVA induced a robust and diverse T cell response including highly functional OVA-CD4 and CD8 T cells. These CD8 T cells are protective and significantly reduced the bacterial burden and increased survival in a Listeria mouse infection model. Our findings reveal that PorB has broad adjuvant activity, signaling through all three APC types, inducing strong and diverse humoral and cellular responses. These insights will allow for a more intelligent use of adjuvants in future vaccine development.

Rationale: Chronic obstructive pulmonary disease (COPD) is one of the leading causes of deaths worldwide. Patients with COPD have episodes of aggravated symptoms called exacerbations caused by pathogens or pollution. Respiratory viruses are associated with a significant number of COPD exacerbations with the most common virus being the rhinovirus (RV). The mechanisms by which RVs trigger COPD exacerbations are still unclear. Using human whole lung tissue explants (WTE), a novel model of RV-induced COPD exacerbations is proposed. Methods: WTE from COPD patients and smokers were initially stimulated with TLR ligands that are known to activate the same receptors as RV: poly(I:C) for TLR3 and R848 for TLR7/8 activation. Pro-inflammatory cytokines and type I and III IFN gene expression was measured by ELISA and qRT-PCR, respectively. A neutralising antibody against TNFα, a corticosteroid, and a panel of inhibitors targeting TLR pathway (p38 MAPK, IKK-2 and IRAK1/4) was applied to the tissue from COPD patients to establish which signalling pathways are responsible for the inflammatory response and IFN release. Explants from COPD patients and smokers was also exposed to two RV serotypes, RV-16 and RV-1B, in order to compare findings with a clinically relevant stimulant. Results: Poly(I:C) and R848 caused a significant increase of protein and gene expression of pro-inflammatory cytokines (TNFα, CCL5 and IL-6). Type I and III IFN gene expression was also significantly increased. Using the two ligands together caused a synergistic release of TNFα and CCL5. Tissue from COPD patients released more pro-inflammatory cytokines and expressed less IFNβ when compared to smokers. TNFα neutralisation inhibited subsequent release of CCL5 and IL-6. Dexamethasone and p38 MAPK inhibitor decreased TLR3- and TLR7/8-induced pro-inflammatory response whereas IKK-2 and IRAK1/4 inhibition had little effect on cytokine release. Dexamethasone and IKK-2 showed limited effect on IFN gene expression whereas p38 MAPK inhibitor significantly decreased and IRAK1/4 inhibition enhanced IFN expression. RV-16 induced modest but significant pro-inflammatory response in lung tissue, whereas RV-1B did not induce cytokine release. Both serotypes induced type I and III IFN gene expression. Tissue from COPD patients showed a lower expression of IFNβ and IFNλ when compared to smokers. Conclusion: This tissue explant was responsive to both synthetic TLR ligands and RV. The release of pro-inflammatory cytokines in response to TLR stimulation was partially inhibited by steroid. p38 MAPK is involved in TLR-induced inflammation but it also further decreases the already impaired IFN gene expression in COPD tissue. The role of IKK-2 and IRAK1/4 in TLR-induced innate immune response remains unclear. This model is a valuable system to study the mechanisms underlying RV-induced COPD exacerbations and also to test new inhibitors in the whole tissue environment.

A memory response is established following primary antigen exposure that stays more or less constant. It appears to adopt a set-point in magnitude but upon re-exposure the response is quicker and better and there is an upward shift in memory frequency that varies with individuals based on the exposure pattern to other microbes or its components. Our investigations were designed to test such differences of non-specific stimulation by PAMPs in lowering the threshold of activation. Neonatal mice were pre-exposed to TLR-ligands intermittently and later analyzed for its resilience to challenge with virus during adult-life. Secondly, adult mice with pre-existing memory to virus were exposed to various TLR-ligands and analyzed for their quality of memory response. The TLR-ligands exposed animals were better responders to a new agent exposure compared to the animals kept in sterile surroundings. Moreover, immune memory recall and the viral specific CD8+ T cells response with TLR-ligands were comparable to the recall response with the cognate antigen. The results provide insights into the role of hyper-sanitized environment versus PAMPs mediated signaling in adaptive immunity and long-term immune memory.

The infections agent of Lyme disease, Borrelia Burgdorferi is a complex microorganism with a highly diverse genome. One of the most remarkable aspects of the B. burgdorferi genome is the large number of sequences encoding predicted or known lipoproteins, including outer-surface proteins. The B. burgdorferi genome encodes no recognizable toxins. Instead, this extracellular pathogen causes pathology by migration through tissues, adhesion to host cells, and evasion of immune clearance. Inflammation elicited by infection with B. burgdorferi depends on the ability of the spirochete to survive in the mammalian host, as well as the immune response that arises upon the interaction of the bacterium with phagocytic, T and other cell types. Innate immune responses are critical in recognition and clearance of pathogens, and also play an important role in the outcome of adaptive immune responses. The regulation of innate immune responses to pathogens occurs through the interaction of Toll-like receptors (TLRs) with pathogen-associated molecular patterns (PAMPs) and the activation of several signaling pathways whose contribution to the overall innate immune response to pathogens is poorly understood. In this study we demonstrate a mechanism of control of murine macrophage responses mediated by TLR1/2 heterodimers through c-Jun N-terminal kinase 1 (JNK1) activity. JNK also controls tumor necrosis factor production and TLR-mediated macrophage responses to B. burgdorferi. We also show that the proximal promoter region of the human tlr1 gene contains an AP-1 binding site that is subjected to regulation by the kinase and binds two complexes that involve the JNK substrates c-Jun, JunD, and ATF-2. These results demonstrate that JNK1 regulates the response to TLR1/2 ligands and suggest a positive feedback loop that may serve to increase the innate immune response to the spirochete. MCJ is a newly identified member of the DnaJ protein family of cochaperones that contains unique features different than the normally described DnaJ proteins. However, there is little known about its function and the role it plays in different cells and systems. It has been previously shown that MCJ is required for the repression of the ABCB1 drug transporter expression in breast cancer cells, and that this repression is mediated through the control of c-Jun protein stability. We were therefore interested in determining the role that MCJ plays in macrophages in response to B. burgdorferi antigens. We now provide evidence that MCJ controls inflammatory responses of macrophages through the regulation of c-Jun protein stability, and the expression and release of the inflammatory cytokine TNF through the regulation of the expression of TNF converting enzyme (TACE) inhibitor tissue inhibitor of metalloproteinase 3 (TIMP-3).