The adenosine deaminase acting on RNA (ADAR) enzymes catalyse the hydrolytic deamination of adenosine (A) to inosine (I) in double stranded (ds) RNA. Mutations in ADAR1 underlie the autoimmune disorder Aicardi Goutiѐres syndrome (AGS). Patients with AGS display heightened levels of type I interferon (IFN) and IFN stimulated genes (ISGs). The first aim of my thesis was to determine whether the mutations found in the human ADAR1 gene affected RNA editing. I found that the ADAR1 mutants identified in the AGS patients have reduced editing activity. Interestingly, the mutations have a greater effect on the IFN-inducible cytoplasmic isoform, ADAR1p150 than on the constitutive ADAR1p110 isoform. These results imply that A-to-I editing plays a role in regulating the type I IFN response. The Adar1 null mouse dies by E12.5 with a type I IFN signature similar to that observed in the AGS patients. The second aim of my thesis was to characterize the immune signalling pathway aberrantly activated in the absence of Adar1. A colleague in our research group rescued the Adar1 null mouse to birth by blocking the cellular response to cytoplasmic dsRNA by generating a double mutant with the mitochondrial antiviral signalling adaptor, Mavs. In the Adar1-/-; Mavs-/- mutant I found that the aberrant immune response is rescued at E11.5. This indicates that MAVS is the downstream adaptor in the aberrant immune response that underlies the embryonic lethality in the Adar1-/- mouse. The third aim of my thesis was to determine if the lack of inosine modification within cellular RNA was triggering the aberrant immune response in the Adar1-/- mouse. To study this, Adar1-/-; p53 -/- mouse embryonic fibroblasts (MEFs) were generated. By reintroducing various ADAR isoforms into the Adar1-/-; p53 -/- MEFs I found that to rescue the aberrant immune response requires both catalytic activity and the location of an ADAR protein within the cytoplasm. Moreover, I demonstrated that transfecting inosine-containing dsRNA oligonucleotides into Adar1-/-; p53 -/- MEFs suppresses the aberrant immune response. Overall my results suggest that A-to-I editing by ADAR1 is an essential RNA modification that is required by the cell to distinguish between ‘self’ and ‘non-self’ RNA. Editing of cellular RNAs prevents an autoimmune response whereas editing of viral RNA may act to suppress a heightened antiviral immune response and prevent long-term damage to the cell.

Detection of evolutionarily conserved pathogen motifs by pattern recognition receptors (PRRs), particularly on dendritic cells (DCs), is crucial for adequate immune responses. Defects in DC function are known to be associated with inflammatory bowel disease (IBD). The endocannabinoid system (ECS) is the system through which exocannabinoids such as Δ⁹-tetrahydrocannabinol and cannabidiol signal. Regarding inflammation, cannabinoids generally exert anti-inflammatory effects, including on experimental colitis. However, most work has been performed in animal models and less is known about the function of this system in human immune cells, particularly DCs. Monoacylglycerol lipase (MGLL) is the key enzyme for hydrolysis of the endocannabinoid 2-arachidonoylglycerol, and a member of the serine hydrolase enzyme superfamily. This thesis defines the activity of serine hydrolase enzymes for the first time in human DCs upon stimulation by NOD2/TLR2 ligands using activity-based protein profiling (ABPP). MGLL is shown to be ubiquitously upregulated upon stimulation of DCs and in monocyte-derived macrophages. Through pharmacological inhibition studies, MGLL is demonstrated to regulate cellular and secreted lipids, not limited to endocannabinoids. However, overall DC function is independent of this enzyme suggesting that the effects of lipid modulation may be on bystander cells. Challenging the current literature, MGLL inhibition with a novel inhibitor worsens murine Citrobacter rodentium colitis. Finally, ABPP demonstrates a rich serine hydrolome in colonic tissue from human IBD with many enzymes previously undefined in this disease. Gene expression of ECS components suggests the enzymes ABHD12 and DAGLα/β may be potential markers of field change in IBD.

Une reconnaissance appropriée des parasites est essentielle pour une réponse immunitaire efficace, assurant l'activation adéquate des mécanismes de défense immunitaire. Chez les vertébrés, il a été démontré que les gènes codant pour les récepteurs de l'immunité adaptative impliqués dans la reconnaissance des agents pathogènes sont soumis à une intense pression sélective. En revanche, beaucoup moins d’études se sont intéressées à la sélection agissant sur les récepteurs de l'immunité innée. Le but de cette thèse est de décrire la variabilité naturelle des gènes de l'immunité innée impliqués dans la détection des agents pathogènes chez les rongeurs et d’analyser les mécanismes responsables de leur évolution. Ce travail s’est focalisé principalement sur les rongeurs de la sous-famille des Murinae et de leur rôle potentiel en tant que réservoirs d’agents pathogènes dangereux pour l’Homme. Tout d´abord nous avons étudié la variabilité intraspécifique de cinq Toll-like récepteurs ciblant les bactéries (TLR1, TLR2, TLR4, TLR5 et TLR6) pour des lignées consanguines de souris domestiques issues d’une population sauvage de deux sous-espèces : Mus musculus domesticus (Mmd) et Mus musculus musculus (Mmm). Les souches consanguines constituent un outil adapté à l'étude de la variabilité des gènes immunitaires car elles confèrent une information sur les allèles présents dans les populations naturelles tout en bénéficiant de génotypes homozygotes. Les résultats les plus significatifs concernent la découverte d'un codon stop dans l'exon 2 du Tlr5 chez une lignée de Mmm et l’absence de variabilité du Tlr4 chez Mmd. A la suite de ces résultats, nous avons décidé de vérifier si l‘absence de polymorphisme du Tlr4 chez Mmd reflète une absence de variabilité dans les populations naturelles, ou si il s’agit plutôt d’un effet de l'échantillonnage ou des croisements ultérieurs. Nous avons donc séquencé le gène Tlr4 pour les deux sous-espèces provenant de la région du Paléarctique Occidentale (au total 39 Mmm et 62 Mmd) puis nous avons comparé ces résultats avec la variabilité génétique d’un gène mitochondrial (cytochrome b). Nous avons confirmé notre prédiction : la variabilité de Tlr4 chez Mmd est fortement réduite par rapport à Mmm, probablement à cause d’agents pathogènes ayant exercé une sélection purifiante chez Mmd durant la colonisation vers l’ouest. Cependant, l'influence de mécanismes évolutifs neutres, tel que la dérive consécutive à un goulot d’étranglement démographique, ne peut être exclue sur la base de nos données. La dernière partie a été consacrée à la comparaison interspécifique de deux récepteurs : TLR4 et TLR7. Ces deux TLRs se différencient à la fois par leur localisation et leur capacité de détection. TLR4 est un TLR extracellulaire reconnaissant principalement les ligands bactériens, essentiellement les lipopolysaccharides, tandis que TLR7 est localisé dans la cellule et détecte les virus à ARN simple brin. L‘objectif était de décrire la variabilité inter-spécifique de chaque récepteur et de révéler les mécanismes de sélection s’exerçant sur ces gènes au cours de leur évolution sur une échelle de temps plus importante. Nous avons analysé 23 espèces de Murinae provenant surtout d’Asie. Nos résultats suggèrent que la sélection purifiante est la force principale ayant agit sur l’évolution des deux TLRs. Cependant, nous avons également mis en évidence des épisodes de sélection diversifiante qui ont pu être à l’origine des variations intra-spécifiques de TLRs observée aujourd’hui chez les rongeurs. Des sites sous sélection positive sont principalement concentrés dans les domaines extracellulaires des deux récepteurs, domaines responsables de la reconnaissance des agents pathogènes. Enfin, la comparaison entre ces deux TLRs montre que le TLR7 est soumis à une sélection négative plus forte. Cette sélection peut s’expliquer en raison des interactions du TLR7 avec les acides nucléiques viraux. / Appropriate recognition of parasites is crucial for effective immune response, ensuring activation of adequate defence mechanisms. In vertebrates, it has frequently been demonstrated that genes encoding proteins involved in pathogen recognition by an adaptive immune system are often subject to intense selection pressures. On the contrary, much less information has been provided on the evolution of recognition mechanisms of innate immunity. The aim of this thesis is to describe the pattern of natural variation of innate immunity genes involved in pathogen recognition in rodents and to analyze the mechanisms of their evolution. We used murine rodents (subfamily Murinae) as a principal model group because they are potential reservoirs of various pathogens dangerous to humans. First, we studied the intraspecific variability of five bacterial sensing Toll-like receptors (TLR1, TLR2, TLR4, TLR5, and TLR6) in inbred strains derived from two subspecies of the house mouse (M. m. musculus, hereafter abbreviated as Mmm and Mus musculus domesticus, Mmd). Wild-derived inbred strains are suitable tools for studying variation of immunity genes because they provide information about alleles that occur in natural populations, and at the same time they occur at homozygous state. The most significant results include the findings of a stop codon in exon 2 of the Tlr5 gene in one Mmm strain and no variability in Tlr4 of Mmd. Following these results we decided to check whether the absence of Tlr4 polymorphism in Mmd reflects the pattern found in natural populations, or whether it is a consequence of insufficient sampling or subsequent breeding. We therefore sequenced Tlr4 in both subspecies across a large part of the Western Palearctic region (in total 39 Mmm and 62 Mmd individuals), then we compared these results with variability on mitochondrial DNA (cytochrome b). The result confirmed our prediction that observed variability in Mmd is strongly reduced also in free-living populations (compared to Mmm), probably due to strong purifying selection by pathogens with which they met during the westward colonization. However, the influence of random evolutionary processes (e.g. drift during bottlenecks) cannot be excluded based on our data. At the intraspecific level, we could not find any sign of positive selection. The last part of my dissertation is devoted to interspecific comparison of two receptors, TLR4 and TLR7. These two TLRs differ in the exposure and the ligands detection. TLR4 is an extracellular receptor detecting mainly bacterial ligands (especially lipopolysaccharides), while TLR7 is located inside the cell and detects ssRNA viruses. The aim of this part of the thesis was to describe variability of both receptors at the interspecific level and to reveal selection forces acting on TLRs in longer evolutionary time scale. In total we analyzed 23 rodent species of the subfamily Murinae in Europe, Asia and Africa. Our results suggest that purifying selection has been a dominant force in evolution of the Tlr4 and Tlr7 genes, but we also demonstrated that episodic diversifying selection has shaped the present species-specific variation in rodent Tlrs. Sites under positive selection were concentrated mainly in the extracellular domain of both receptors, which is responsible for ligand binding. The comparison between two TLRs lead us to the conclusion that the intracellular TLR7 is under much stronger negative selection pressure, presumably due to its interaction with viral nucleic acids.

Estudos em nosso laboratório caracterizaram camundongos B10.A e A/J, respectivamente, como susceptíveis e resistentes à infecção pulmonar pelo fungo Paracoccidioides brasiliensis. A imunidade inata desempenha papel fundamental no controle inicial dos patógenos e na regulação da resposta imune adquirida. Como os macrófagos alveolares são as primeiras células do hospedeiro a interagir com o fungo, propusemo-nos a estudar a capacidade fungicida e secretora dos macrófagos alveolares de camundongos susceptíveis e resistentes ao P. brasiliensis para melhor compreender a PCM pulmonar. Camundongos B10.A e A/J normais (n: 10-15) foram submetidos a lavagem bronco-alveolar (LBA) e a suspensão celular obtida (2x105 células/poço) foi pré-ativada durante a noite com IFN-γ, IL-12 ou a combinação destas duas citocinas (50.000, 10.000 e 2.000 pg/rnL) e desafiados in vitro com leveduras viáveis de P. brasiliensis (relação fungo-macrófagos de 1:50). Após 72h de incubação a atividade fungicida dos macrófagos foi avaliada através da contagem do número de fungos viáveis pelo método de unidades formadoras de colônias (UFC). A produção de nitrito e de citocinas foi avaliada no sobrenadante de cultivo através da reação de Griess e ELISA, respectivamente. Os dados foram expressos como média ± EP e analisados pelo teste T -Student. Nossos resultados mostraram que os macrófagos de camundongos B 10.A pré-ativados com IFN-γ, IL-12 ou ambas as citocinas nas diferentes concentrações ensaiadas apresentaram elevada capacidade fungicida (51-97%) acompanhada de produção aumentada de NO, IL-12 e MCP-l e baixa síntese de IL-10 e GM-CSF. Por outro lado, somente o tratamento com a mais alta concentração de IFN-γ foi capaz de induzir atividade fungicida em macrófagos alveolares de animais A/J. A síntese de NO ocorreu sempre em baixos níveis e nos sobrenadantes dos co-cultivos observamos altos níveis de IL-10 e GM-CSF associados a baixas concentrações de IL-12 e MCP-l. A inibição da síntese de NO por aminoguanidina demonstrou que a atividade fungicida dos macrófagos de animais B10.A, mas não de A/J, era mediada por este composto tratamento dos macrófagos com anticorpo monoclonal anti-IL-10 não alterou a capacidade fungicida dos macrófagos de ambas as linhagens, entretanto induziu franca produção de NO que, para animais A/J, não se traduziu em atividade microbicida aparente. Por outro lado. a neutralização de TGF-β induziu alta capacidade fungicida das células de animais A/J níveis aumentados de NO e TNF-α aliados a níveis reduzidos de IL-10. O mesmo tratamento não alterou a já elevada capacidade fungicida dos macrófagos alveolares de animais B10.A mas aumentou a produção de NO, IL-12 e TNF-α. Em conclusão, os macrófagos alveolares de camundongos B10.A são facilmente ativáveis por 1FN-γ e IL-12, apresentam elevada capacidade fungicida, NO-dependente, sobre leveduras do P. brasiliensis associada com a síntese de níveis elevados de NO e IL-12. Por outro lado, os macrófagos de camundongos A/J são pobremente ativados por 1FN-γ e IL-12, produzem baixos níveis de NO, a sua capacidade fungicida, que parece ser NO-independente, pode ser aumentada pela neutralização do TGF-β endógeno, mas não pelo tratamento com anti-IL-10. / Previous studies in our laboratory characterized B10.A and A/J mice as susceptible and resistant strains to pulmonary Paracoccidioides brasiliensis infection. Innate immunity plays a fundamental role on the control of the initial growth of pathogens as well as in the acquired immunity that subsequently develops. As alveolar macrophages are the first host cells to interact with P. brasiliensis we decided to study the fungicidal and secretory ability of alveolar macrophages from resistant and susceptible mice to P. brasiliensis to better understand the pulmonary model of paracoccidioidomycosis. Normal B10.A and A/J mice (n=10-15) were submitted to bronchoalveolar lavage (BAL) and cell suspensions (2x105 cells/well) were pre-activated ovemight with IFN-γ, IL-12 or the combination of these two cytokines (50,000, 10,000 and 2,000 pg/rnL). After that, macrophages were in vitro challenged with P. brasiliensis yeasts (1:50 fungus: macrophage ratio) and 72h later fungicidal activity was determined by colony forming units counts (CFU). Nitrite and cytokines production were determined in culture supematants by Griess reaction and ELISA, respectively. Data were expressed as means ± SE and analyzed by Student\'s t test. Our results showed that B10.A macrophages pre-activated with the different assayed concentrations of IFNγ, IL-12 or both cytokines presented elevated fungicidal ability (51¬-97%) concomitant with the presence of high levels of NO, IL-12 and MCP-l and low amounts of IL-10 and GM-CSF. NO synthesis occurred in low levels but high concentrations of IL-10 and GM-CSF associated to low amounts of IL-12 and MCP-1 were detected in the co-cultures supematants. NO synthesis inhibition by aminoguanidine clearly showed that the fungicidal ability of B10.A but not of A/J macrophages was NO¬ dependent. Treatment with anti-IL-10 monoclonal antibodies did not alter the fungicidal ability of macrophages from both mouse strains but enhanced NO synthesis which, however, did not alter the absent microbicidal ability of A/J macrophages. On the contrary, anti-TGF-β treatment induced an increased fungicidal ability of A/J cells associated with enhanced levels of NO and TNF-α besides diminished amounts of IL-10. The same treatment did not alter the high fungicidal ability of B10.A alveolar macrophages but increased NO, IL-12 and TNF-α production. In conclusion, alveolar macrophages from susceptible mice are easily activated by IFNγ and IL-12, present a high and NO-dependent ability to kill P. brasiliensis yeasts and secrete elevated levels of NO and IL-12. In contrast, alveolar macrophages from resistant mice are poorly activated by IFNγ and IL¬12, secrete low amounts of NO and high of IL-10. Their fungicidal ability which appears to be NO-independent can be restored by neutralization of endogenous TGF-β but not by anti-IL-10 treatment.

The innate immune system is the first line of defence against infection. It is comprised of physicochemical barriers and a variety of cell types including macrophages and dendritic cells. Pathogens express specific pathogen associated molecular patterns (PAMP) which are recognised by pattern recognition receptors (PRR) on macrophages to initiate an innate immune response. Gram-negative bacteria such as Salmonella enterica serovar Typhimurium express a range of bacterial PAMPs recognised by Toll-like receptors (TLRs) including lipopolysaccharides (LPS) recognised by TLR-4 and lipoproteins by TLR-2. The activation of TLRs results in activation of nuclear factor κB (NF-κB) to drive transcription of mRNA coding for pro-inflammatory proteins such as tumor necrosis factor α (TNF-α) and pro-interleukin (IL) 1β. Myeloid cells also possess intracellular PRRs including the nucleotide-binding domain and leucine-rich repeat (NLR) family. NLR family CARD domain- containing protein 4 (NLRC4) and NLR family pyrin domain-containing protein 3 (NLRP3) are the main NLRs engaged in recognising S. Typhimurium infection, leading to formation of the inflammasome. The inflammasome is a macromolecular complex assembled in the cytoplasm, and usually contains a NLR, the structural protein apoptosis-associated speck-like protein containing a CARD (ASC) and effector enzymes such as cysteine-dependent aspartate-directed protease (caspase) -1 and caspase-8. This structure is responsible for processing the cytokines pro- IL-1β and pro-IL-18 to their mature form and is involved in triggering a pro-inflammatory process of cell death termed pyroptosis. The formation of the inflammasome therefore results in cell death and secretion of proinflammatory cytokines which play important roles in controlling infections. Inflammasome activity must be tightly coordinated, as its dysregulation is associated with a variety of auto-inflammatory and auto-immune diseases. The signalling events leading to inflammasome assembly are poorly understood and the molecules involved in fine-tuning its activity are only beginning to be discovered. The aim of this thesis was to discover new molecules involved in inflammasome activation and/or in keeping its activity in check. To achieve this goal, I performed S. Typhimurium infection assays in primary bone marrow derived macrophages (BMDM) derived from C57BL/6 mice wild type (WT) and compared the resulting cellular viability, intracellular bacteria counts and IL-1β production to that of BMDMs derived from C57BL/6 mice lacking proteins involved with, or suspected to be involved with, innate immune activity. Amongst the proteins I studied, caspase recruitment domain 9 (CARD9) inhibited inflammasome-mediated IL-1β production. Multiple independent genome-wide association studies link this protein to inflammatory pathologies such as Crohn's disease, but its role in canonical inflammasomes was largely unexplored. To investigate how CARD9 inhibits inflammasome-mediated IL-1β production I have conducted assays in WT and Card9-/- BMDMs, including stimulation of specific NLRs with their purified ligands, infection with bacterial strains deficient in NLRC4 activation, and infection assays in presence of pharmacological inhibitors. By employing these approaches, I observed that CARD9 has a negative role on NLRP3-dependent IL-1β production. Specifically, in response to activation of the NLRP3 by Salmonella infection, CARD9 negatively regulates pro-IL-1β transcription, and decreases IL-1β processing by inhibiting spleen tyrosine kinase (SYK)-mediated NLRP3 activation and represses caspase-8 activity in the inflammasome. CARD9 expression is suppressed in the course of S. Typhimurium infection which may act as a mechanism to increase IL-1β production during the infection. In conclusion, I have established a connection between CARD9 and IL-1β production by the canonical NLRP3 inflammasome and elucidated some of the mechanisms involved in this process. I have also found evidence that other proteins are likely to be involved in inflammasome regulation and the elucidation of their roles will be addressed in future studies.

O objetivo geral deste trabalho foi avaliar a influência do uso precoce de antimicrobianos na microbiota intestinal, resposta imune e ocorrência de diarreias em bezerras recém-nascidas. Foram selecionadas 26 bezerras Holandesas distribuídas de acordo com a aplicação (ATB+) ou não (ATB-) de tulatromicina (2,5 mg/Kg - SC) até 12h de vida. Capítulo 1 Este capítulo avaliou a influência do uso precoce de antimicrobiano na sanidade de bezerras holandesas recém-nascidas, por meio do exame clínico geral. As diarreias foram detectadas por sistema de escore e matéria seca (%) nas fezes, além do escore respiratório e palpação externa da região umbilical. O desempenho das bezerras foi determinado pela estimativa do ganho de peso. As bezerras ATB- apresentaram maior frequência de mucosas esbranquiçadas (P=0,066) e inflamações umbilicais no ATB- em D3-5 (P=0,063) e D13-15 (P= 0,066). A frequência de escore fecal 2 e 3 foi semelhante entre os grupos, observando-se pico no D13-15. Os valores médios para matéria seca (%) foram menores no ATB+ no D7-9 (P=0,063) e D27-30 (P=0,084). O agente etiológico das diarreias foi o Cryptosporidium e/ou rotavírus. O ganho de peso diário (GPD) foi igual entre grupos, observando-se intensa redução no GPD em D13-15. Capítulo 2 O objetivo deste capítulo foi avaliar a influência do uso precoce de antimicrobianos na colonização bacteriana intestinal, por meio da detecção de material genético dos principais grupos bacterianos por meio do PCR em tempo real. Não foram observadas diferenças entre os grupos para bactérias totais e C. perfringens. As bezerras ATB- apresentaram maior quantidade de Bifidobacterium, nas fezes no D3-5 (P=0,002) e D7-9 (P=0,018). A população de Lactobacillus também apresentou tendência (P=0,097) para maior número de cópias de DNA no grupo ATB-. Ao contrário, a Escherichia coli apresentou tendência para maiores valores no grupo ATB+ no D20-23 (P=0,052) e D27-30 (P=0,072). Capítulo 3 O objetivo deste capítulo foi avaliar a influência do uso precoce de antimicrobianos no perfil inflamatório de bezerras holandesas recém-nascidas por meio de provas hematológicas, imunológicas, bioquímicas e eletroforéticas. Observou-se tendência para menores valores de hemoglobina (P=0,071), CHCM (P=0,057) e ferro sérico (P=0,051) no D3-5 no ATB-. Neste momento, a frequência de anemias também foi maior no ATB- (P=0,016). O número de neutrófilos (P=0,051) e a produção espontânea de EROs foi maior no ATB-, enquanto o número de linfócitos foi maior no ATB+ (P=0,061) no D7-9. Estes dados são condizentes com as análises imunes, no qual a proporção de populações de mononucleares 1 e 2 expressando o CD62L+ apresentou tendência para maior valor no ATB+ em D3-5 (P=0,098) e D7-9 (P=0,083). Após as diarreias (D13-15), os granulócitos (P=0,043) e mononucleares (P=0,089) das bezerras ATB+ apresentaram maior intensidade de fluorescência para expressão CD62L no D20-23. O grupo ATB+ também apresentou maiores teores de beta-globulina (P=0,057), gama-globulina (P=0,069) e haptoglobina (P=0,032) no D13-15. O uso precoce de tulatromicina influenciou na microbiota intestinal, imunidade inata e ocorrência de doenças no período neonatal. / The general goal of this research was to evaluate the influence of early antimicrobial use in the gut microbiota, immune response and occurrence of diarrhea in neonate heifers. It was screened 26 neonate Holstein heifers distributed according to the application (ATB+) or not (ATB-) of tulathromycin (2.5mg/Kg - SC) from birth until 12 hours of life. Chapter 1 This chapter evaluated the influence of early use of antimicrobial in the health of neonate Holstein heifer by general clinical examination. Diarrheas were detected using score system and fecal dry matter (%), besides of respiratory score and external palpation of navel region. The performance of calves was evaluated by daily weight gain. Heifers ATB- had high frequency of paleness mucous (P=0.066) and umbilical inflammation in ATB- group on D3-5 (P=0.063) and D13-15 (P= 0.066). The frequency of fecal score 2 and 3 was similar between groups, observing peak of diarrhea on D13-15. The mean values of fecal dry matter were low in ATB+ group on D7-9 (P=0.063) and D27-30 (P=0.084). The main etiologic agent of diarrheas was Cryptosporidium and/or rotavírus. The daily weight gain (DWG) was similar between groups, observing intense decrease of DWG on D13-15. Chapter 2 The aim of this chapter was to evaluate the influence of early use of antimicrobial in the microbial gut colonization by detection of genetic bacterial material using real time PCR. It was not found difference between groups for total bacteria and C. perfringens. The calves from ATB- group had high quantity of Bifidobacterium in the fecal material on D3-5 (P=0.002) and D7-9 (P=0.018). The Lactobacillus population had a tendency (P=0.097) to have high number of DNA copies in the ATB- group. On the other hand, Escherichia coli had tendency to high values in ATB+ group on D20-23 (P=0.052) and D27-30 (P=0.072). Chapter 3 The aim of this chapter was to evaluate the influence of early use of antimicrobial in the inflammatory profile of neonate Holstein heifers by hematological, immunological and biochemical tests, besides electrophoresis. It was possible to observe lower values of hemoglobin (P=0.071), CHCM (P=0.057) and serum iron (P=0.051) in ATB- group on D3-5. In this moment, the frequency of anemia also was high in ATB- group (P=0.016). The number of neutrophils (P=0.051) and spontaneous ROSs was high in ATB- group, while the number of lymphocytes was high in ATB+ group (P=0.061) on D7-9. These data were according to immune analysis, where the proportion of mononuclear population 1 and 2 expressing CD62L+ had a tendency for high values in ATB+ group on D3-5 (P=0.098) and D7-9 (P=0.083). After diarrhea (D13-15), granulocytes (P=0.043) and mononuclear cells (P=0.089) showed high fluorescence intensity of CD62L in ATB+ heifers on D20-23. ATB+ group also had high rate of beta globulin (P=0.057), gamma globulin (P=0.069) and haptoglobin (P=0.032) on D13-15. The early use of tulathromycin had influence on gut microbiota, innate immunity and occurrence of disease in the neonatal period.

Activation of the cysteine protease caspase-1 and the subsequent processing and secretion of the pro-inflammatory cytokines IL-1Β and IL-18 is central to the inflammatory response as well as the induction of adaptive immune responses. Caspase-1 is activated as a part of a high-molecular weight multi-protein complex termed the inflammasome. The NLRP3 inflammasome is by far the best studied of these complexes, and it is the most promiscuous in terms of activating signals. The diversity of NLRP3 activating signals makes it likely that NLRP3 does not recognize each agonist directly, rather it detects a molecule that is generated, revealed, or altered by cellular stress. Recent studies have indicated that mitochondrial dysfunction is crucial for NLRP3 inflammasome activation, yet the activating ligand has not yet been identified. Appropriate and timely activation of this inflammatory pathway is required for host immunity to a variety of pathogens, however dysregulated activation leads to autoinflammation and potentially autoimmunity. Hence it is important to identify mechanisms for inflammasome activation and regulation. Therefore, this dissertation has focused on investigating the mechanisms for activation and regulation of the NLRP3 inflammasome, and the biological consequences of these changes. We show that the mitochondrial lipid cardiolipin is required for NLRP3 inflammasome activation. We have also identifying a novel mechanism by which inflammasome activation is regulated. Data presented in this dissertation shows that IgG immune complexes effectively suppress inflammasome activation and the subsequent processing and secretion of IL-1Α and IL-1Β. Furthermore we show that immunization with IgG immune complexes suppresses both Th2 and Th17 immune responses. Together these data provide novel insights into the activating and regulatory pathways of both the innate and adaptive immune systems.

Equine herpesvirus-1 (EHV-1) is one of the most important and prevalent viral pathogens of horses causing a major threat to the equine industry throughout most of the world. EHV-1 primarily causes respiratory disease but viral spread to distant organs enables the development of more severe sequelae; abortion and neurologic disease. In order to produce disease, EHV-1 has to overcome the innate barrier of the type-I interferon (IFN) system in host cells. However, the underlying mechanisms employed by EHV-1 to circumvent the type-I IFN response in host cells are not well understood. In this project study, using molecular techniques, we explored how EHV-1 is able to escape the type-I IFN response in host cells during infection. We also investigated whether EHV-4, a closely related but less pathogenic virus, has similar effects on type-I IFN as a clue to understanding how widespread IFN suppressive function is found among equine alphaherpesviruses. Our data showed that inhibition of the type-I IFN response in host cells is not a function of neuropathogenicity of EHV-1 strains. However, a reduced type-I IFN response correlated with pathogenicity as EHV-4, unlike EHV-1, was unable to down-regulate the type-I IFN response in equine endothelial cells (EECs). Investigation of the mechanisms employed by EHV-1 to suppress type-I IFN revealed that the virus sequentially prevented outside-in signaling events that lead to type-I IFN production. Specifically, EHV-1 blocked the expression of Toll-like receptors (TLR) 3 and TLR4 at 6 hours post-infection (hpi) and 12 hpi. EHV-1 also prevented the transcription of IRF7 and IRF9 at different time-points during infection. The virus also perturbed the JAK-STAT signaling pathway by negatively regulating the cellular levels of TYK2 and phosphorylation-mediated activation of STAT2 molecules. Immunofluorescence data revealed that during infection, EHV-1 was able to sequester STAT2 molecules from nuclear translocation. This may be a limiting step preventing the formation of interferon- stimulated gene factor 3 (ISGF3) whose nuclear translocation is required to transactivate interferon-stimulated genes (ISGs) including IRF7. Further investigation showed that unlike EHV-1, EHV-4 only interfered with phosphorylation-mediated activated STAT1 and STAT2 molecules at 3 and 6 hpi. EHV-4 was unable to block TLR3/4 and IRF7/9 mRNA expression at any time-point. Intriguingly, while viral late gene of EHV-1 mediates inhibition of STAT phosphorylation, our data showed that for EHV-4, a virus late gene did not mediate the inhibition of STAT phosphorylation. The findings from this study help illuminate how EHV-1 strategically interferes with limiting steps required for type-I IFN response in host cells to promote pathology. Our data also strengthen the hypothesis that the ability to shut off host factors required for type-I IFN production might be directly related to the degree of pathogenicity of the EHV subtypes.

Bovine mastitis remains one of the costliest diseases affecting the dairy industry. Individual susceptibility to mastitis and severity of infection varies between animals and can only be partially explained by genetics. As such, understanding how genetic predisposition coordinately interacts with epigenetic modifications and environmental exposures is necessary to bridge the gap in missing heritability. The role of DNA methylation in regulating the response to bacterial lipopolysaccharide (LPS) was first determined by performing reduced representation bisulfite sequencing on fibroblasts isolated from heifers at 5- and 16-months of age that exhibit an age-dependent up-regulation in LPS-responsiveness. More than 14,000 differentially methylated sites were identified between the two sets of cultures with a trend towards decreased methylation with age. Young cultures were also hyper-methylated in gene promoters regulated by NF-κB and exhibited lower expression in genes that regulate the innate immune response, suggesting that methylation contributes to gene regulation in fibroblast innate response. Previously, TLR4 expression was shown to differ in the age-dependent fibroblast model, however, it was not known if variation in TLR4 expression would affect mastitis severity. Therefore, fibroblasts were isolated from sixty lactating, adult Holstein cows and their expression of TLR4, along with LPS-induced production of IL-8 and IL-6, was used to rank the animals from high to low. Six high responders and six low responders were then experimentally infected in one mammary gland with E. coli. Overall, severity of mastitis was quite variable, with a few notable differences between high and low responders. High responding animals had an earlier increase in somatic cell count and febrile response that coincided with more efficient bacterial clearance. However, tissue damage and milk production did not differ between the two groups, indicating that while rapid up-regulation of the innate response addresses bacterial clearance, subsequent down-regulation is required to alleviate damage within the mammary gland. Finally, one-week old bull calves were subjected to treatment with either saline or LPS to determine if neonatal exposure to endotoxin would make calves less responsive to a second LPS challenge at 32-days of age. The initial treatment showed a large effect of LPS as measured by higher plasma IL-6 and TNF-α concentrations in calves treated with LPS over saline. Subsequent treatment of all 10 calves with LPS showed a very similar response between the two treatment groups and significant inter-animal variability in clinical response. Fibroblasts and monocyte-derived-macrophages (MDMs) were also isolated following initial treatment to determine if any changes occurred at the cellular level as a result of LPS exposure. Fibroblasts isolated from calves at 20-days of age had a very low response to LPS that did not differ between the early life treatments. MDMs isolated from calves at 28-days of age were more responsive to LPS, but again no differences were detected between the early life treatments. In summary, our results suggest that DNA methylation likely plays a role in the cellular response to LPS and may partially contribute to differences between animals in severity of E. coli mastitis, however, the appropriate in vitro phenotype to detect susceptible animals still needs to be characterized before epigenetic biomarkers can be identified, and perhaps modified by environmental interventions.

Le système immunitaire inné est un mécanisme de défense commun à tous les métazoaires. Son activation peut être délétère lorsqu'elle est incontrôlée. L'étude des mécanismes qui sous-tendent cet équilibre entre l'activation ou non de la réponse immunitaire innée est à la base de mes travaux de thèse. La similarité entre les voies moléculaires - comme la voie NF-KB - relayant la réponse immunitaire innée chez les insectes et les mammifères fait de la drosophile un excellent modèle pour explorer la réponse immune. Après une stimulation immunitaire, l'arrêt des voies moléculaires de l'immunité est nécessaire pour éviter le développement de maladies auto-immunes ou du cancer. Mon premier projet s'est attaché à comprendre un mode de régulation original dépendant du temps, dans une des voies NF-KB de la drosophile. Mon deuxième projet··concerne l'activation de la réponse immunitaire. Une· protéine nucléaire contrôle l'implication de machinerie épigénétique dans le contrôle de l'expression d'une des voies NF-KB de la drosophile. Le tout permet de mieux saisir la dynamique de régulation de la réponse innée. / The innate immune system is a defense mechanism common to all metazoans. lts activation can be deleterious when it is uncontrolled. The study of the mechanisms underlying this balance between the activation or not of the innate immune response is the basis of my thesis work. The similarity of the molecular pathways - such as the NF-KB pathway - relaying the innate immune response in insects and mammals makes Drosophila an excellent model for exploring the immune response.After immune stimulation, stopping the molecular pathways of immunity is necessary to prevent the development of autoimmune diseases or cancer. My first project focused on understanding a time-dependent mode of regulation in one of Drosophila's NF-KB pathways. My second project concerns the activation of the immune response. A nuclear protein contrai the involvement of epigenetic machinery in controlling the expression of one of Drosophila's NF-KB pathways. Ali this makes it possible to better grasp the dynamics of regulation of the innate response.