The cationic host defence peptide LL-37 is an immunomodulatory agent that plays an important role in epithelial innate immunity. Previously, concentrations of LL-37 thought to represent levels present during inflammation have been shown to elicit the production of cytokines and chemokines by epithelial cells. To investigate the potential of lower concentrations of LL-37 to alter epithelial cell responses, normal primary keratinocytes and bronchial epithelial cells were treated with pro-inflammatory stimuli in the presence or absence of 1 – 3 μg/ml LL-37. Low, physiologically relevant concentrations of LL-37 synergistically increased IL-8 production by both proliferating and differentiated keratinocytes in response to IL-1β and the TLR5 agonist flagellin, and synergistically increased IL-8 production by bronchial epithelial cells in response to IL-1β, flagellin, and the TLR2/1 agonist PAM3CSK4. Treatment of bronchial epithelial cells with LL-37 and the TLR3 agonist poly(I:C) resulted in synergistic increases in IL-8 release and cytotoxicity. The synergistic increase in IL-8 production observed when keratinocytes were co-stimulated with flagellin and LL-37 was suppressed by pretreatment with inhibitors of Src-family kinase signalling and NF-κB translocation. These data suggest that low concentrations of LL-37 may alter epithelial responses to microbes in vivo. Microarray analysis of keratinocyte transcriptional responses after LL-37 treatment suggest that LL-37 may alter the expression of growth factors and a number of genes important to innate immune responses. LL-37 may thus play a more important role than previously suspected in the regulation of epithelial inflammation; an improved understanding of the mechanisms by which LL-37 alters chemokine responses could lead to the development of novel anti-infective and anti-inflammatory therapeutics.

The aim of this thesis was to obtain some insights into mechanisms by which the immune system affects melanoma cells after anchoring agonists of phagocytic receptors (laminarin and f-MLF) to their surface. To verify the hypothesis that innate immune system plays a critical role, in vivo experiments were performed on SCID mice. To elucidate the importance of CR3, CD11b-deficient mice were used. In in vitro experiments production of inflammatory cytokines in tumor tissue was examined as well as the release of myeloperoxidase from neutrophil granules after incubation with malignant cells.

This study is focused on characterization of fibrinogen-related proteins (FREPs) from the tick Ixodes ricinus using molecular methods - PCR, cloning, qRT-PCR, RNA interference via dsRNA synthesis and injection, and also pathogen (Borrelia sp.) transmission on animal model.

- 4 - Abstract Introduction: Periodic fever syndromes are clinical entities classified as autoinflammatory diseases. The most of the periodic fever syndromes have genetic predisposition (monogenic periodic fever syndromes). PFAPA (Periodic Fever, Aphtous stomatitis, Pharyngitis a Adenitis) syndrome is an idiopathic disease with unknown aetiology. Results: In our study, we described the largest clinical series of patients with PFAPA syndrome from a single center. The laboratory results have confirmed uncomplicated course of PFAPA syndrome. In our measurements we observed significantly higher levels of serum cytokines (IL-1β and IFN-γ) during episodes of fever in PFAPA patients compared to the control group. Our measurements showed increased numbers of plasma cells in the peripheral blood of PFAPA patients. We have found increased levels of naïve CD4 and CD8 T cells and approximately 2-fold higher proportion of CD8 T cells in tonsils of PFAPA patients. Significant differences were also present at levels of IFN-γ, IL-1β, IL-6 and TNF-α in stimulated supernatants compared to supernatants from unstimulated peripheral blood from patients with PFAPA syndrome. Measurements of bacterial profile showed individual microbial profile in patients. Conclusion: Removal of the tonsillar tissue with the potential...

Amphibian metamorphosis is facilitated solely by thyroid hormones (THs), L-thyroxine (T4) and 3,5,3’-triiodothyronine (T3). TH modulates the remodeling of many different organs and systems in the body of developing tadpoles, including the immune system. Previous research found evidence of T4 action on direct-response genes in outer ring deiodinase-poor premetamorphic tadpole tail fin and liver without the required conversion to T3 described by current TH dogma. The mechanisms of environmental endocrine disrupting chemicals (EDCs) may be better understood by expanding our understanding of the transcriptomic effects of both forms of THs and how they relate to estrogen signaling. Furthermore, analysis of TH-modulation of the immune system may enable a greater understanding of the devastating effects of amphibian pathogens such as Ranavirus. Premetamorphic Rana (Lithobates) catesbeiana tadpoles were exposed to physiological concentrations of T4, T3, or 17-beta-estradiol (E2) through water bath immersion. qPCR analysis was performed to assess the response of canonical TH-responsive genes thra, thrb, and thibz to these hormones in the liver and tail fin tissues of bullfrog tadpoles. E2 treatment did not elicit a response in these gene transcripts in either tissue. T3 treatment in the tail fin elicited an overall stronger response than T4, while T4 treatment in the liver recapitulated results consistent with non-genomic mechanisms of T4 signaling for thrb and thibz transcripts. Illumina Hiseq2500 was used to sequence RNA isolated from hormone-treated premetamorphic tadpole liver and tail fin tissues to assess differential transcriptomic responses and identify TH-responsive immune system-associated transcripts. The impact of TH-treatment on the general immune system in the liver and tail fin transcriptomes was also analyzed using RNA-seq data. We found that E2 modulates at least some shared TH pathways in the liver, but none in the tail fin and that the tail fin transcriptome is more affected by T3, while the liver transcriptome is more affected by T4. Additionally, evidence of immune system modulation by both THs was found in both the liver and tail fin transcriptomes. Antimicrobial peptides (AMPs) are an important component of the amphibian immune response. Details regarding the regulation, synthesis, and expression of AMPs remain obscure, although evidence of TH-modulation of specific AMPs has been identified, as well as evidence of increased expression of AMPs throughout metamorphosis. Frog skin is a prolific source of AMPs that may prove useful in the quest for alternative antimicrobial agents in the face of antibiotic resistance. Identification of new AMPs is hindered by the practical limitations of classical protein-based discovery approaches. By using known AMP characteristics and common ¬AMP properties, we developed a high throughput bioinformatics approach predicated on the use of R. catesbeiana genome resources. We mined these resources and identified novel and known AMPs that exhibited verified antimicrobial activity against various bacterial organisms. This thesis sought to elucidate the differential and modulatory effects of both forms of TH on a transcriptomic level and in the context of immunity, and to examine the utility of the bullfrog transcriptome and genomics resources in identifying and characterizing novel bullfrog-derived AMPs and elucidating aspects of AMP expression. / Graduate / 2018-12-08

Majorer la tolérance de l’hôte à l’inflammation est un facteur qui pourrait diminuer l’ampleur des lésions tissulaires au cours des pathologies inflammatoires. Dans ce travail, nous rapportons que la molécule clusterine, une protéine chaperonne extracellulaire très conservée et présente dans la plupart des tissus et fluides biologiques, neutralise les effets cytotoxiques de molécules associées à l’inflammation. Le taux sérique de clusterine est diminué chez les patients atteints de certaines maladies inflammatoires, et les souris déficientes en clusterine ont une sensibilité plus importante à l’inflammation. Clusterine interagit avec certaines molécules associées à l’inflammation et forme des complexes qui sont détectés dans le sérum des patients. In vitro, clusterine inhibe la mort cellulaire induite par les molécules associées à l’inflammation. Bien que l’inflammation induise la libération rapide d’un stock préformé contenu dans les polynucléaires neutrophiles et les plaquettes, les taux de clusterine sérique sont abaissés chez l’homme et la souris en contexte inflammatoire. Démontrant la non redondance et le rôle protecteur de clusterine, l’inflammation est majorée chez la souris déficiente en clusterine.Nous concluons que clusterine augmente la tolérance de l’hôte à l’inflammation. / Increasing host tolerance to inflammation may lower the intensity of tissular injury in inflammatory diseases. We report here that clusterin, a conserved extracellular chaperone, present in most tissues and fluids, neutralizes the in vitro and in vivo cytotoxic properties of inflammation-induced molecules. Serum clusterin levels are decreased in patients with some inflammatory diseases and clusterin deficient mice have higher sensibility to inflammation.Clusterin interacts with some inflammation-associated molecules and form complexes that are detected in human serum and, consequently, prevents in vitro cell death induced by these molecules. Although inflammation triggers the rapid release of preformed clusterin stock by neutrophils and platelets, serum clusterin concentrations get down in inflammatory conditions in human and mice. Inflammation is boosted, in vivo and in vitro, using clusterin-deficient mice, showing the non-redundant and major protective roles of clusterin. We conclude that clusterin enhances host tolerance to inflammation.

Abstract Chlamydia pneumoniae is an obligate intracellular gram-negative bacterium, which causes respiratory infections in humans and may participate in the development of chronic diseases like atherosclerosis, chronic obstructive lung disease, adult-onset asthma and late-onset Alzheimer’s disease. It can infect various cell types, e.g. vascular endothelial cells, smooth muscle cells and monocyte-derived macrophages in vitro. It has been speculated that circulating macrophages disseminate the infection in the body, and that genetically susceptible individuals become chronically infected. Quantification of C. pneumoniae growth inside cultured cells is needed when studying e.g. the effect of drugs or host cell factors on infectivity and replication. Conventionally this has been done by immunofluorescence staining and microscopic counting of chlamydial inclusions. However, this method is usable only if the cell numbers do not fluctuate in cell culture vials and the inclusions are uniform. In macrophages, inclusions are often aberrant, their sizes vary and multiple inclusions are also seen. Therefore we developed a new method based on the real-time PCR quantification of chlamydial genomes adjusted to the number of human genomes and used it to quantify the exact amounts of C. pneumoniae in infected cells. The susceptibility of monocyte-macrophages from healthy individuals to C. pneumoniae infection in vitro was studied first. Intracellular growth of C. pneumoniae was used as an indicator of susceptibility to infection, and it was compared to serum levels of CRP, soluble CD14, human HSP-IgG, human HSP-IgA, C. pneumoniae IgG and IgA antibodies. The growth of C. pneumoniae in infected macrophages was highly variable, ranging from 0 to 638 chlamydial genomes per human genome. C. pneumoniae growth associated positively with serum C. pneumoniae IgA (titer ≥10) and hHSP-IgG and negatively with soluble CD14 concentration. The association between chlamydial IgA antibodies, hHSP-IgG and C. pneumoniae growth was statistically significant only among men. Age did not correlate with the growth. Therefore we hypothesize that persons whose macrophages cannot restrict the growth of C. pneumoniae are more prone to chronic infection by this agent. In the next study, we evaluated the effects of innate immunity genes CD14 -260 C>T, TLR2 Arg753Gln, TLR4 Asp299Gly, LBP Phe436Leu and IL-6 -174 G>C polymorphisms on C. pneumoniae growth in human macrophages in vitro. The growth of C. pneumoniae was highest in CD14 -260 C>T TT genotype cells and the difference to CC or CT genotype was statistically significant. The G-allele of the IL6 -174 G>C polymorphism had a positive influence on chlamydial growth; the difference was statistically significant only between CC and GC genotypes. TLR2 Arg753Gln, TLR4 Asp299Gly, LBP Phe436Leu polymorphisms showed no effect on chlamydial growth.

La complexité de la prise en charge des hépatites B chroniques est en partie liée à la persistance virale. La clairance du virus est compliquée du fait du mode de réplication du virus et de l'interaction de HBV avec les acteurs de la réponse immunitaire innée. On distingue plusieurs stades d'infection chronique à HBV, qui se caractérisent par un équilibre variable entre réplication virale et réponse immunitaire de l'hôte. La définition clinique de ces différentes phases est insuffisante pour discriminer le risque évolutif des patients. Des études de cohorte sont nécessaires pour mieux appréhender les relations entre réplication virale et immunité lors des infections chroniques à HBV. Le premier travail est une étude de cohorte rétrospective s'intéressant aux relations entre marqueurs virologiques sériques et intrahépatiques et expression intrahépatique des gènes de l'immunité innée. Les gènes de l'immunité innée étudiés sont moins exprimés au cours des hépatites B chroniques en comparaison à des contrôles sains, sans influence de la réplication virale. Le taux d'AgHBs sérique pourrait refléter l'importance de la réponse immunitaire innée intrahépatique, notamment la réponse IFN de type I pour les patients négatifs pour l'AgHBe. Le deuxième travail est une étude de cohorte prospective, qui ne retrouve pas de bénéfice à l'addition d'un traitement par PEG-IFN chez des patients co-infectés HBV/HIV et répondant au traitement par NUCS. Cette étude souligne de façon intéressante que le taux d'AgHBs à l'initiation du traitement pourrait être prédictif de la réponse au traitement, des taux plus faibles d'AgHBs étant favorables. L'ensemble de ces résultats démontre l'importance de la relation entre réponse immunitaire et réplication virale pour expliquer les différentes phases de l'hépatite B chronique. Ces résultats pourraient se révéler intéressants pour le développement de nouvelles prises en charge thérapeutiques de patients infectés chroniquement par HBV / Chronic HBV infections (CHB) are difficult to treat diseases because of viral persistence. It’s explained by the particular replication of Hepatitis B Virus (HBV) and its interplays with host immunity. CHB is characterized by different stages, which reflect a balance between viral replication and immune response. However, our knowledge regarding the natural history of CHB is insufficient to allow us to predict patients’ prognosis. Further clinical studies are needed to improve our understanding of interplays between HBV replication and host immunity. The first study is a retrospective one about interplays between serological and intrahepatic viral markers and intrahepatic innate immunity genes expression. Immunity genes seem to be down-regulated during CHB in comparison to healthy controls, without impact of the level of viral replication. HBsAg levels in blood may reflect the intrahepatic innate immune response and especially the type I IFN response for HBeAg negative patients. The second part is a prospective study which shows any relevance of adding PEG-IFN to HIV/HBV co-infected responders to NUCS therapy patients. The results highlight the potential interest of baseline HBsAg level to predict PEG-IFN response (low HBsAg levels being more favorable). Finally, these results highlight the role of interplays between HBV replication and innate immune response during the natural history of CHB. They may be interesting in the context of the development of new antiviral strategies

The cationic host defence peptide LL-37 is an immunomodulatory agent that plays an important role in epithelial innate immunity. Previously, concentrations of LL-37 thought to represent levels present during inflammation have been shown to elicit the production of cytokines and chemokines by epithelial cells. To investigate the potential of lower concentrations of LL-37 to alter epithelial cell responses, normal primary keratinocytes and bronchial epithelial cells were treated with pro-inflammatory stimuli in the presence or absence of 1 – 3 μg/ml LL-37. Low, physiologically relevant concentrations of LL-37 synergistically increased IL-8 production by both proliferating and differentiated keratinocytes in response to IL-1β and the TLR5 agonist flagellin, and synergistically increased IL-8 production by bronchial epithelial cells in response to IL-1β, flagellin, and the TLR2/1 agonist PAM3CSK4. Treatment of bronchial epithelial cells with LL-37 and the TLR3 agonist poly(I:C) resulted in synergistic increases in IL-8 release and cytotoxicity. The synergistic increase in IL-8 production observed when keratinocytes were co-stimulated with flagellin and LL-37 was suppressed by pretreatment with inhibitors of Src-family kinase signalling and NF-κB translocation. These data suggest that low concentrations of LL-37 may alter epithelial responses to microbes in vivo. Microarray analysis of keratinocyte transcriptional responses after LL-37 treatment suggest that LL-37 may alter the expression of growth factors and a number of genes important to innate immune responses. LL-37 may thus play a more important role than previously suspected in the regulation of epithelial inflammation; an improved understanding of the mechanisms by which LL-37 alters chemokine responses could lead to the development of novel anti-infective and anti-inflammatory therapeutics. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Human APOBEC3 proteins are host-encoded intrinsic restriction factors that can prevent the replication of a broad range of human and animal retroviruses such as HIV, SIV, FIV, MLVs and XMRV. The main pathway of the restriction is believed to occur as a result of the cytidine deaminase activity of these proteins that converts cytidines into uridines in single-stranded DNA retroviral replication intermediates. Uridines in these DNA intermediates disrupt the viral replication cycle and also alter retrovirus infectivity because of the C-to-T transition mutations generated as a result of the deaminase activity on the minus strand DNA. In addition, human APOBEC3 proteins also exhibit a deamination-independent pathway to restrict retroviruses that is not currently well understood. Although the restriction of retroviruses by human APOBEC3 proteins has been intensely studied in vitro, our understanding of how the murine APOBEC3 (mA3) protein restricts retroviruses and/or prevents zoonotic infections in vivo is very limited. In contrast to humans and primates that have 7 APOBEC3 genes, mice have but a single copy. My study of the function and structure of mA3 revealed that it has an inverted functional organization for cytidine deamination in comparison to the human A3G catalytic sites. I have also found that disruption of the integrity of either of these catalytic sites substantially impedes restriction of HIV and MLV. Interestingly, our data shows that mA3 induces a significant decrease in retroviral activity of HIV and MLVs by exploiting both deamination-dependent and -independent pathways. However, the deaminase activity of mA3 is essential to confer long-term restriction of retroviral infection. My observations suggest that mA3 has dual activities, both deamination-dependent and -independent, that work cooperatively to restrict a broad range of human and animal retroviral pathogens. In the context of the intrinsic immune system, APOBEC3 proteins provide a powerful block to the transmission of retroviral pathogens that very few have found ways to evade.