Les cellules lymphoïdes innées (ILC) représentent une famille de cellules hématopoïétiques récemment identifiée, qui joue un rôle essentiel dans la réponse immunitaire précoce via la production rapide de cytokines. Trois groupes - ou types - d’ILC ont été définis selon l’expression de certaines molécules membranaires ou intracellulaires, ainsi que la production différentielle de cytokines. Les ILC du groupe 1 (ILC1) expriment le facteur de transcription(FT) T-BET et sécrètent des cytokines inflammatoires de la réponse immune de type 1, l’IFN-? et le TNF-?. Les ILC2 sécrètent des cytokines associées à la réponse immune de type 2,notamment l’IL-5 et l’IL-13, et ce de façon dépendante du FT GATA-3. Enfin, les ILC3 se caractérisent par la production de cytokines telles que l’IL-17 et l’IL-22, et expriment le FTROR?t. J’ai étudié en utilisant des techniques de biologie moléculaire et cellulaire, et à partir d’échantillons sanguins et tissulaire de donneurs sains ou de patients atteints de maladies inflammatoires chroniques, la fonction de ces trois groupes d’ILC chez l’homme. Ces travaux ont permis la construction d’un nouveau modèle de développement de ces cellules à partir de précurseurs. / Innate lymphoid cells (ILC) represent a novel family of hematopoietic effectors that serve essential roles in early immune response by rapid cytokines production. Three distinct groups of ILC subsets have been described. Group 1 ILC include cytotoxic natural killer (NK) cells and other type-1 cytokines (IFN-? and TNF-?) producing cells that regulated by T-BET. Group 2 ILC (ILC2) express GATA-3 and ROR?, secrete type-2 cytokines, IL-5 and IL-13. Group 3 ILC (ILC3) utilize ROR?t to drive production of the TH17-associated cytokines, IL-17 and/or IL-22. In this thesis, I have performed series of experiments to uncover the developmental pathway and function of human ILC that may allow us to harness ILC in diverse clinical settings. First, I analyzed the phenotypic and functional heterogeneity of human peripheral blood ILC2. I found human IL-13+ ILC2 can acquire the capacity to produce IFN-?, thereby generating ÔplasticÕ ILC2. ILC2 cultures demonstrated that IFN-?+ ILC2 clones could be derived and were stably associated with increased T-BET expression. The inductive mechanism for ILC2 plasticity was mapped to the IL-12/IL-12R signaling pathway and was confirmed through analysis of patients with Mendelian susceptibility to mycobacterial disease (MSMD) due to IL-12R?1 deficiencies that failed to generate plastic ILC2. This IL-13+IFN-?+ ILC2 are detected ex vivo in gut tissues from CrohnÕs patients. Second, I identified and isolated ILC precursors (ILCP) in peripheral blood of healthy donors. This circulating ILCP can give rise to four lineages of mature ILC including cytotoxic NK cells and helper ILC1, 2 and 3 in vitro and in vivo. Transcirptomic and epigenetic analysis showed ILCP have ILC-committed transcription factor profiles but have mature ILC signature locus at the epigenetics poised states. We further identified ILCP in various tissues including fetal liver, cord blood, postnatal lung and tonsil. Our result proposed a new model of ÒILC-poiesisÓ where circulating ILCP serve as cellular substrates to generate mature ILC subsets in tissues. Understanding the role of IL-12 on driving ILC2 to ILC1 plasticity may allow us to target plastic ILC2 in various diseases. The identification and isolation of ILCP from circulating blood allow further transfer into clinical setting for cellular therapy, especially for various diseases that ILC has been shown to be importance including infection, allergy, cancer and metabolic diseases.

Despite the convergence of rapid technological advances in genomics and the maturing field of ecoimmunology, our understanding of the genes that regulate immunity in wild populations is still nascent. Previous work to assess immune function has relied upon relatively crude measures of immunocompetence. However, with next-generation RNA-sequencing, it is now possible to create a profile of gene expression in response to an immune challenge. In this study, captive zebra finch (Taeniopygia guttata; adult males) were challenged with bacterial lipopolysaccharide (2 mg/Kg BW; dissolved in 0.9% saline) or vehicle (0.9% saline) to stimulate the immune system. Two hours after injection, birds were euthanized and hypothalami, spleen, and red blood cells (RBCs) were collected. Taking advantage of the fully sequenced genome of zebra finch, total RNA was isolated, sequenced, and partially annotated in these tissue/cells. The data show 628 significantly upregulated transcripts in the hypothalamus, as well as 439 and 121 in the spleen and RBCs, respectively, relative to controls. Also, 134 transcripts in the hypothalamus, 517 in the spleen, and 61 in the RBCs were significantly downregulated. More specifically, a number of immunity-related transcripts (e.g., IL-1β, RSAD2, SOCS3) were upregulated among tissues/cells. Additionally, transcripts involved in metabolic processes (APOD, LRAT, RBP4) were downregulated, suggesting a potential trade-off in expression of genes that regulate immunity and metabolism. Unlike mammals, birds have nucleated RBCs, and these results suggest a novel transcriptomic response of RBCs to immune challenge. Lastly, molecular biomarkers could be developed to rapidly screen bird populations by simple blood sampling in the field.

Mastitis represents one of the major economical and animal welfare concerns within the dairy industry. Animals affected with this disease can experience a range of clinical symptoms from mild discomfort and swelling of the udder to a severe systemic inflammatory response that could result in the death of the animal. This range of responses is due to differences in pathogen, environment, and inter-animal differences in their innate immune response. A dermal fibroblast model was used to predict the magnitude of an animal's innate immune response towards an intra-mammary S. aureus challenge. Animals whose fibroblasts exhibited a low response phenotype, characterized by lower levels of IL-8 following in vitro immune stimulation, suffered less mammary tissue damage and a less severe reduction in milk quality following the in vivo S. aureus challenge as compared to animals classified as high responders. Furthermore, the heightened inflammatory response of the high responders offered no advantage in bacterial clearance. For a S. aureus infection, the lower response phenotype is preferred. To further explore inter-animal variation in the innate immune response, fibroblast cultures were established and challenged with LPS from two breeds of cattle, Holsteins, a dairy breed and Angus, a beef breed. Cultures from Holstein animals exhibited a higher responding phenotype than cultures from Angus animals. As these two breeds undergo selection for different traits and are reared differently as calves, whole transcriptome analysis (RNA-Seq) and DNA methylation analysis (Methylated CpG Island Recovery Assay; MIRA-Seq) of their fibroblasts was completed to examine the genetic and epigenetic basis for the contrasting responses. RNA-Seq revealed several immune associated genes that were expressed at higher levels in Holstein cultures compared to Angus cultures, including TLR4, IL-8, CCL5, and TNF-α, both basally and following LPS exposure. Although MIRA-Seq analysis revealed 49 regions with differential methylation between the Holstein and Angus cultures, overall, the methylation of the fibroblast genome was similar between these breeds. A combination of genetic and epigenetic factors seems to contribute to the breed-dependent differences observed between Holstein and Angus fibroblasts. Early life exposure to bacterial compounds or inflammatory mediators can have long-term effects on the magnitude of an animal's innate immune response, and may contribute to inter-animal variation in this response. To determine if an early life exposure to LPS would modify the response to a subsequent LPS challenge in dairy animals, neonatal Holstein calves were treated with LPS or saline at 7 days of age and subsequently challenged with LPS 25 days later. Calves that received LPS at 7 days of age had greatly elevated levels of plasma IL-6 and TNF-α compared to calves that received saline, indicating a substantial inflammatory response. However, following the subsequent LPS challenge completed on all calves, there were no differences in plasma IL-6 and TNF-α between the LPS- and saline- treated calves. Alternative exposure strategies in calves may generate the long-term effects observed in other model systems. There is a wide range in the responses observed in the innate immune response of the bovine. Animals with a lower innate immune response effectively clear the infection, but avoid the collateral tissue damage from excessive inflammation. Therefore, it seems that a reduced innate immune response would be more beneficial to the dairy cow.

Periodontal diseases (PD) affect 46% of American adults over age 30. These diseases cause symptoms including bleeding and swelling of the gums, bone resorption, and tooth loss, that affect quality of life and have a high economic burden. Periodontal diseases are caused by an imbalance in the oral microbiome, from a healthy state that contains anti-inflammatory commensals like Streptococcus gordonii and mitis, to a diseased state that has pro-inflammatory anaerobic pathogens including Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Tannerella forsythia. The latter initiate disease progression in the oral cavity. However, it’s the host immune response that causes a majority of the symptoms. Ideally, treatment for PD would be approached from both sides to reduce the numbers of pro-inflammatory bacterial cells in the oral cavity but also reduce the host immune response. A novel therapeutic, amixicile, has been created, which specifically targets anaerobes through the pyruvate:ferredoxin oxidoreductase (PFOR) system, the mechanism of energy metabolism found in anaerobic organisms. Our studies show that amixicile inhibits in vitro growth of oral anaerobes in monospecies cultures at concentrations as low as 0.5 µg/mL in broth and 1 µg/mL in biofilms, without affecting the Gram-positive commensal species. In multispecies cultures, amixicile specifically inhibited anaerobes, even in biofilms, with the concentration as low as 5 µg/mL in broth and 10 µg/mL in biofilms. By not affecting the commensal bacteria, we think this treatment could restore a healthy oral microbiome. Aside from the bacterial presence, the host response, particularly the innate immune response is not well understood. Using a Drosophila melanogaster infection model, we elucidated the innate immune response to both mono- and multispecies infections. The 7-Species infection included bacteria mentioned above and Aggregatibacter actinomycetemcomitans in order to replicate in vivo-like disease conditions. We determined that both Drosophila Toll and Imd pathways, which mimic TLR/IL-1 and TNF signaling pathways of mammalian innate immunity respectively, respond to the 7-Species challenge. We also verified virulent bacteria in Drosophila, including P. gingivalis and P. intermedia. Future directions include RNA sequencing to determine the full scope of immune gene expression and using human immune cells to further clarify the response.

Neutrophils (PMN) and group IIA phospholipase A2 (gIIA PLA2) are components of the innate immune system mobilized to sites of invasion by microorganisms such as Staphylococcus aureus. Although accumulating coincidentally in vivo, the in vitro anti-staphylococcal activities of PMN and gIIA PLA2 have thus far been separately studied. The goal of this thesis was to study the collaborative activity of PMN and gIIA PLA2 against S. aureus. We have identified and characterized the collaboration of PMN and gIIA PLA2 against S. aureus ingested by PMN. PMN induced conversion of bacterial phosphatidylglycerol into cardiolipin, but were unable to degrade S. aureus phospholipids without gIIA PLA2. PMN reduced by 10-fold the concentration of gIIA PLA2 needed to digest bacterial phospholipids alone. In addition to increased phospholipid degradation, collaboration of PMN and gIIA PLA2 caused greater bacterial killing and greater loss of bacterial green fluorescent protein fluorescence. The collaboration of PMN and gIIA PLA2 against S. aureus is dependent on catalytic activity and is specific to gIIA PLA2 as related secretory PLA2, groups IB, V, and X, show little or no phospholipid degradation of S. aureus either alone or in the presence of PMN. Synergy of PMN and gIIA PLA2 requires a functional NADPH oxidase and phagocytosis. Although addition of gIIA PLA2 after phagocytosis causes some bacterial phospholipid degradation, the greatest effect is observed when gIIA PLA2 is added before phagocytosis. An extracellular source of H2O2 can partially restore antibacterial activities to NADPH oxidase deficient PMN including the ability to collaborate with gIIA PLA2, supporting a role for reactive oxygen species in NADPH oxidase dependent antimicrobial functions of PMN. In contrast, iberiotoxin, an inhibitor of BK potassium channels had no effect of PMN antibacterial activities. Although H2O2 partially restored antibacterial activity to NADPH oxidase deficient PMN, extracellular H2O2 was not sufficient to increase S. aureus to gIIA PLA2 activity. In summary, PMN and gIIA PLA2 collaborate against S. aureus. These findings revealed collaboration between cellular oxygen-dependent and extracellular oxygen-independent host defense systems that may be important in the ultimate resolution of S. aureus infections.

Tularemia is a potentially fatally illness caused by the facultative intracellular Gram-negative bacterium Francisella tularensis. Virulent strains of F. tularensis can cause a fatal disease after inhalation of a few as ten organisms. Due to the highly pathogenic features of Francisella, it has been designated as a Tier 1 select agent, meaning that its possession and handling is highly restricted. Macrophages are phagocytes that play a central role in the innate immune response to infection that can be used by certain pathogens, including Francisella, as a niche for bacterial replication and dissemination during infection. After infection of macrophages Francisella escapes from the phagosome and replicates in the cytosol, however the bacterial factors required for these aspects of virulence are incompletely defined. Here we describe the isolation and characterization of F. tularensis subspecies tularensis strain Schu S4 mutants in iglI, iglJ, and pdpC, three genes located in the Francisella Pathogenicity Island. Our data demonstrate that these mutants were unable to replicate in macrophages due to a defect in phagosome escape. However, a small percentage of pdpC mutants were able to reach the cytosol and replicate moderately. Both iglJ and pdpC mutants were highly attenuated for virulence in a mouse intranasal infection model, however pdpC but not iglJ mutants, were able to disseminate from the lung before eventual clearance. These data demonstrated that the FPI genes tested were essential for F. tularensis Schu S4 virulence, but suggest that they may have different functions due to the unique phenotype observed for pdpC mutants. Our studies also characterized the role of F. tularensis O-antigen and capsule to facilitate interactions with components of the serum complement system; demonstrating that the O-antigen is required for binding of IgM to the bacteria in order to initiate complement opsonization. IgM dependent complement opsonization of both F. tularensis Schu S4 and LVS strains facilitated enhanced phagocytosis of the bacteria by complement receptors 3 and 4 of human macrophages. In addition, we examined the mechanisms of macrophage cytotoxicity and proinflammatory cytokine secretion that was induced after infection with a Schu S4 LPS O-antigen and capsule mutant. The response to the mutant was dependent on phagosome escapes, suggesting a cytosolic pattern recognition receptor was involved in recognition of the bacteria. We found that the cytotoxic and proinflammatory responses had both similar and distinct requirements between human and murine macrophages. Infection with the O-antigen mutant induced robust proinflammatory cytokine secretion that was dependent on caspase-1, cathepsin B, and ASC while cytotoxicity was partially dependent on these molecules. Importantly, we demonstrated that wild-type Schu S4 predominately activated apoptotic caspases, and not inflammatory caspases, during infection and had a blunted cytotoxic response. This was in contrast to the robust cytotoxicity and activation of inflammatory caspases after infection with the non-virulent strain LVS. Together, these studies demonstrated that the Schu S4 LPS O-antigen and capsule are required for evasion of macrophage cytosolic host defense mechanisms.

<p>All metazoans possess an epithelial barrier that protects them from their environment and prevents loss off body fluid. Insects, which have an open circulatory system, depend on fast mechanism to seal wounds to avoid excessive loss of body fluids. As in vertebrates, and non-insect arthropods such as horseshoe crab and crustaceans, insects form a clot as the first response to tissue damage. Insect hemolymph coagulation has not been characterized extensively at the molecular level before, and the aim of my studies was to gain more knowledge on this topic. Morphological characterization of the<i> Drosophila </i>hemolymph clot showed that it resembles the clots previously described in other larger bodied insects, such as <i>Galleria mellonella</i>. The <i>Drosophila</i> clot is a fibrous network of cross-linked proteins and incorporated blood cells. The proteins building up the clot are soluble in the hemolymph or released from hemocytes upon activation. Since bacteria are caught in the clot matrix and thereby prevented from spreading it is likely that the clot serves as a first line of defense against microbial intruders. The bacteria are not killed by the clot. What actually kills the bacteria is not known at this point, although the phenoloxidase cascade does not seem to be of major importance since bacteria died in the absence of phenoloxidase. We identified and characterized a new clot protein which we named gp150 (Eig71Ee). Eig71Ee is an ecdysone-regulated mucin-like protein that is expressed in salivary glands, the perithophic membrane of the gut and in hemocytes, and can be labeled with the lectin peanut agglutinin (PNA). Eig71Ee was found to interact with another clot protein (Fondue), and the reaction was catalyzed by the enzyme transglutaminase. This is the first direct functional confirmation that transglutaminase acts in <i>Drosophila </i>coagulation. A protein fusion construct containing Fondue tagged with GFP was created. The fusion construct labeled the cuticle and the clot, and will be a valuable tool in future studies. Functional characterization of the previously identified clotting factor Hemolectin (Hml) revealed redundancy in the clotting mechanism. Loss of Hml had strong effects on larval hemolymph clotting ex vivo, but only minor effects, such as larges scabs, <i>in vivo</i> when larvae were wounded. An immunological role of Hml was demonstrated only after sensitizing the genetic background of Hml mutant flies confirming the difficulty of studying such processes in a living system. Hemolectin was previously considered to contain C-type lectin domains. We reassessed the domain structure and did not find any Ctype lectin domains; instead we found two discoidin domains which we propose are responsible for the protein’s lectin activity. We also showed that lepidopterans, such as<i> Galleria</i> <i>mellonella</i> and <i>Ephestia kuehniella</i>, use silk proteins to form clots. This finding suggests that the formation of a clot matrix evolved in insects by the co-option of proteins already participated in the formation of extracellular formations.</p>

Eukaryotic initiation factor 2B (eIF2B) is a heteropentameric guanine nucleotide exchange factor (GEF) that converts inactive eIF2 GDP-bound binary complexes into active eIF2 GTP-bound complexes that can bind initiator t-RNA molecules and ribosomes to begin translation. eIF2B is functionally divided into two subcomplexes: the catalytic core comprised of eIF2B epsilon and eIF2B gamma, and the regulatory core comprised of eIF2B alpha, eIF2B beta and eIF2B delta. While the catalytic subunits are responsible for exerting GEF activity, the regulatory subunits recognize eIF2 and respond to eIF2 alpha phosphorylation. Cellular stress, such as virus infection, inhibits host protein synthesis by activating specific kinases that are capable of phosphorylating the alpha subunit of eIF2, which can then sequester eIF2B to stall guanine nucleotide exchange by a currently unresolved mechanism. Importantly, we demonstrate that loss of eIF2B alpha or expression of a variant of the human eIF2B alpha subunit harboring a single point mutation (T41A) is sufficient to neutralize the consequences of eIF2 alpha phosphorylation, and render primary MEFs significantly more susceptible to vesicular stomatitis virus infection. To extend this analysis, we further exhibit the vital function of eIF2B alpha in protein synthesis through phenotypic studies in yeast. Here, we report that this subunit can sufficiently substitute for its yeast counterpart, GCN3, and reproduce similar growth phenotypes under normal and amino acid deprived conditions. In addition, the human eIF2B alpha-T41A variant was unable derepress GCN4 translation in response to an inhibitor of amino acid biosynthesis in yeast, an activity that requires sensitivity to phosphorylation of the yeast eIF2 alpha homolog, SUI2. Previously, we have demonstrated that vesicular stomatitis virus can infect and replicate to high levels in tumor cells. Moreover, these cells appear to contain defects in eIF2 alpha-mediated translational control, plausibly due to disregulation of eIF2B activity, which overcomes the inhibitory effects of eIF2 alpha phosphorylation. Our data suggest a role for eIF2B, specifically eIF2B alpha, in suppression of translation following virus infection, and imply that this complex may contribute to oncogenic transformation. These results emphasize the importance of eIF2B alpha in mediating eIF2 kinase translation inhibitory activity and may provide insight into the complex nature of viral oncolysis and cellular transformation.

Blood was obtained from 5 adult horses and 16 juvenile horses (foals) at the time of birth and subsequently at 2-, 4-, and 8-weeks of age. Neutrophils from adult horses were purified and incubated for 2 h and 4 h with media, avirulent R. equi, virulent R. equi, or recombinant-human granulocyte-macrophage colony stimulating factor (rhGM-CSF). Neutrophils from foals were purified and incubated for 2 h and 4 h with media or virulent R. equi. Total RNA was extracted from both adult and foal neutrophils immediately after purification to measure baseline expression levels (0 h), and immediately after each of the prescribed incubation times. For each sample, 1 µg of total RNA was reverse-transcribed and analyzed for differential gene expression using real-time PCR. After 2 h and 4 h incubation with virulent or avirulent R. equi, neutrophils from adult horses expressed significantly (P< 0.05) greater TNFα, IL-12p40, IL-6, IL-8, and IL-23p19 mRNA relative to expression by unstimulated neutrophils, but not IFNγ or IL-12p35 mRNA. Furthermore, virulent R. equi induced significantly greater IL-23p19 mRNA expression than avirulent R. equi. Stimulation with rhGM-CSF of adult equine neutrophils failed to induce significant changes in cytokine expression. In foal neutrophils, stimulation with virulent R. equi induced significantly greater expression of IFNγ, TNFα, IL-6, IL-8, IL-12p40, and IL-12p35 mRNA relative to expression by unstimulated neutrophils. Furthermore, there were significant effects of age on expression of IL-6, IL-8 and IL-12p40 mRNA. Neutrophil mRNA expression of IL-6 and IL-8 in newborn foals was significantly greater than expression at 2-, 4-, and 8-weeks of age. There was no significant difference between unstimulated and R. equi-stimulated neutrophils from newborn and 2-week-old foals in expression of IL-12p40; however, expression of IL-12p40 by R. equi-stimulated neutrophils from 4- and 8-week-old foals was significantly greater than expression by unstimulated neutrophils. These results demonstrate that R. equi-stimulated neutrophils are a source of many pro-inflammatory cytokines, and that the magnitude of this expression with respect to IL-6, IL-8, and IL-12p40 mRNA expression was influenced by age. Collectively, the data presented indicate a non-phagocytic role for neutrophils that may influence the type of adaptive immune response to R. equi.

Many pathogen infections in nature are accompanied by injury and subsequent coagulation. Despite the contribution of hemolymph coagulation to wound sealing, little is known about its immune function. Based on the molecular knowledge of Drosophila innate immunity, this thesis investigated the immune function of clot both in vitro and in vivo, the immune relevant genes involved in a natural infection model, involving entomopathgenic nematodes (EPN) and the factors leading to crystal cell activation. Transglutaminase (TG) and its substrate Fondue (Fon) have been identified as bona fide clot components in Drosophila larvae. By knocking down TG or Fon via RNAi, we observed an increased susceptibility to EPN in larvae. In addition, this increased susceptibility was associated with an impaired ability of hemolymph clots to entrap bacteria. Immunostaining revealed that both clot components (Fon and TG) were able to target microbial surfaces. All these data suggest an immune function for the Drosophila hemolymph clot. Strikingly, similar results were obtained when we ran parallel experiments with human FXIIIa, an ortholog of Drosophila TG, indicating a functional conservation. We also found evidence for the regulation on both clot and immunity by eicosanoids in Drosophila larvae. The combination of EPN infection with the Drosophila model system allowed us to discover an immune function for TEP3 and Glutactin. However the molecular mechanism underlying the involvement of these two proteins in this particular host-pathogen interaction remains to be elucidated. Prophenoloxidase, the proform of enzyme involved in hardening the clot matrix, has been shown to be released by rupture of crystal cells. This cell rupture is dependent on activation of the JNK pathway, Rho GTPases and Eiger. Our work further identified the cytoskeletal component, Moesin, and the cytoskeletal regulator Rac2 as mediators of cell rupture. Despite the possible role of caspases in crystal cell activation, such cell rupture was turned out to be different from apoptosis. The implication of Rab5 in this process indicated that proper endocytosis is required for cell activation and subsequent melanization. Our findings furthered not only our understanding of the release of proPO via cell rupture but also our knowledge on different paths of immune cell activation. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: In press. Paper 4: Manuscript.<strong></strong></p>