Genes modulating innate immunity in mammals are generally considered the first line of defense with respect to invading pathogens and therefore it has become important to characterize naturally occurring genetic variation, and subsequently determine whether this variation is likely to be benign, beneficial, or detrimental to the host. Relevant to this study, the mammalian Toll-like receptor proteins (TLR), encoded by members of the TLR gene family, have the capacity to recognize a wide variety of pathogen ligands, and mutations within these genes have been shown to influence disease susceptibility or resistance within mammalian species. Two studies which sought to determine the frequency and distribution of naturally occurring genetic variation within the bovine and equine TLR genes revealed a large number of discrete point mutations, which were subsequently used to reconstruct haplotypes for each investigated gene across a large number of samples. Detailed analyses of haplotypes provided evidence for extensive haplotype sharing among specialized breeds, subspecies, and even divergent species. Classical and new tests of selection provided evidence for significant deviations from a strictly neutral model of molecular evolution for both cattle as well as equids, with some of the same TLR genes deviating from a strictly neutral model among divergent species. As a first step toward determining whether naturally occurring bovine TLR variation is likely to be benign, beneficial, or detrimental, we tested validated variation from bovine TLR genes capable of recognizing components of Mycobacteria for associations with Mycobacterium avium subspecies paratuberculosis (MAP) infection in dairy cattle, and found several SNPs that were nominally associated with disease status, thereby providing evidence for small-effect loci potentially influencing risk for differential susceptibility to Johne's disease.

<p>Sepsis is a major cause of morbidity and mortality in the United States, and Staphylococcus aureus (S. aureus) is the bacteria most commonly cultured from septic patients. In severe sepsis, the relationship between the systemic inflammatory response and the resulting mitochondrial and metabolic dysfunction is not fully understood, especially with respect to the mechanisms of mitochondrial damage resolution. The process of mitochondrial biogenesis, which leads to the restoration of metabolic and anti-oxidative functions in damaged or stressed cells and tissues, is pro-survival and is a critical protective response in sepsis. Mitochondrial biogenesis requires the coordinated expression of multiple regulatory proteins, including the PPARgamma-coactivator (PGC) family of proteins. Previous work in sepsis has focused on mitochondrial biogenesis in response to late signals of mitochondrial damage; however, for acute sepsis, we have hypothesized a direct and early link between the innate immune response and the transcriptional activation of mitochondrial biogenesis. Since the Toll-like receptors (TLRs) are a major part of the innate immune response, we hypothesized that they could activate mitochondrial biogenesis in bacterial sepsis. Earlier work showed that TLR4 (which responds to components of Gram-negative bacteria) was necessary for mitochondrial biogenesis induction in response to heat-killed E. coli challenge. For this work, the objective was to investigate whether signaling by TLR2 (which responds to components of Gram-positive bacteria) would activate mitochondrial biogenesis in response to S. aureus sepsis in mice. The sepsis model was initially characterized in wild-type (WT) mice by PCR analysis of hepatic RNA, in which the up-regulation of several regulatory proteins for mitochondrial biogenesis, including all three PGC family members, was observed. In contrast, in both TLR2-/- and TLR4-/- mice, the mitochondrial biogenesis response was deficient and delayed. In addition, PGC-1alpha and PGC-1beta were differentially regulated in WT, TLR2-/-, and TLR4-/- mice. To identify the mechanisms involved in this induction pattern, the known TLR signaling pathways were systematically probed for activation using several strains of genetic knockout mice. These data demonstrated that the differential regulation of the PGC family is independent of the MyD88 adapter protein and is caused in part by IRF7 signaling. IRF7 is a pro-inflammatory transcription factor that is normally involved in the interferon response; in this case, IRF7 was found to be necessary but not sufficient for PGC-1alpha/beta induction. In addition, a second level of regulation was identified in the microRNA mmu-mir-202-3p, which is inversely correlated with the expression of PGC-1alpha and PGC-1beta mRNA in WT, TLR2-/-, and TLR4-/- mice and was shown to functionally decrease PGC-1alpha mRNA. If these observations are confirmed in humans, IRF7 and mir-202-3p may be potential therapeutic targets for the up-regulation of PGC-1alpha/beta levels in the clinical setting of sepsis and impaired mitochondrial biogenesis.</p> / Dissertation

Cells detect and respond to environmental changes using intracellular networks, and defects in the wiring of these networks contribute to diseases. For example, Toll-like receptors (TLRs) sense microbial molecules and trigger pathways critical for host defense. Genetic defects in components of the TLR and other pathogen-sensing pathways have been linked to human diseases. Hence, rational targeting of these pathways should help to manipulate immune responses associated with infections, autoimmunity, or vaccines. A fundamental challenge is to dissect the intracellular networks mobilized by pathogen-sensing pathways. Here we present approaches to dissect the TLR network of innate immune dendritic cells (DCs), focusing on two regulatory layers: signaling and transcription. First, we present a strategy to systematically perturb candidate regulators and monitor cellular transcriptional responses. We apply this approach to derive regulatory networks that control the transcriptional response to TLR engagement by microbial molecules. Our approach revealed the regulatory functions of 125 transcription factors (TFs), chromatin modifiers, and RNA binding proteins, which enabled the construction of a network model consisting of 24 core regulators and 76 “fine-tuners” that help explain how TLR pathways achieve specificity. Second, we report the systematic discovery of signaling components in TLR responses. By combining transcriptional profiling, genetic and small molecule perturbations, and phosphoproteomics, we uncover 35 signaling regulators, including 16 known members of the TLR signaling pathways. In particular, we find that Polo-like kinases (Plk) 2 and 4 are essential components of antiviral pathways in vitro and in vivo and activate a signaling branch involving a dozen proteins, among which is Tnfaip2, a gene associated with autoimmune diseases but whose role was unknown. Lastly, we expand these approaches to integrate functional and physical interactions linking the ‘signaling-to-transcription’ TLR network. By combining our perturbation-based approach with measurements of physical interactions, including phosphorylation, protein complexes, and TF binding to DNA, we uncover 30 signaling regulators mechanistically linked to 19 downstream TFs. The integration of these datasets into a model reveals the organization of the TLR response. Overall, these studies illustrate the power of combining systematic measurements and perturbations to elucidate complex intracellular circuits and discover potential therapeutic targets.

The innate immune system is of central importance to the early containment of infection. When receptors of innate immunity recognize molecular patterns on pathogens, they initiate an immediate immune response by inducing the expression of cytokines and other host defense genes. Altered expression or function of the receptors, the molecules that mediate the signal transduction cascade, or the cytokines themselves can predispose individuals to infectious or autoimmune diseases. Here we used genomic approaches to uncover novel components underlying the innate immune response to cytosolic DNA and to characterize variation in the innate immune responses of human dendritic cells to bacterial and viral ligands. In order to identify novel genes involved in the cytosolic DNA sensing pathway, we first identified candidate proteins that interact with known signaling molecules or with dsDNA in the cytoplasm. We then knocked down 809 proteomic, genomic, or domain-based candidates in a high-throughput siRNA screen and measured cytokine production after DNA stimulation. We identified ABCF1 as a critical protein that associates with DNA and the known DNA-sensing components, HMGB2 and IFI16. We also found that CDC37 regulates stability of the signaling molecule, TBK1, and that chemical inhibition of CDC37 as well as of several other pathway regulators (HSP90, PPP6C, PTPN1, and TBK1) potently modulates the innate immune response to DNA and to retroviral infection. These proteins represent potential therapeutics targets for infectious and autoimmune diseases that are associated with the cytosolic DNA response. We also developed a high-throughput functional assay to assess variation in responses of human monocyte-derived dendritic cellsto LPS (receptor: TLR4) or influenza (receptors: RIG-I and TLR3), with the goal to ultimately map genetic variants that influence expression levels of pathogen-responsive genes. We compared the variation in expression between the dendritic cells of 30 different individuals, and within paired samples from 9 of these individuals collected several months later. We found genes that have significant inter- vs. intra-individual ariation in response to the stimuli, suggesting that there is a substantial genetic component underlying variation in these responses. Such variants may help to explain differences between individuals’ risk for infectious, autoimmune, or other inflammatory diseases.

It is not clear whether genes of the innate immune system of vertebrates are subject to the same selective pressures as genes of the adaptive immune system, despite the fact that innate immunity genes lie directly at the interface between host and pathogens. The lack of consensus about the incidence, type, and strength of selection acting on vertebrate innate immunity genes motivated this study. The goal of this work was to elucidate the general principles of innate immune receptor evolution within and between species. A phylogenetic analysis of the Toll-like receptor 5 (TLR5) in primates showed an excess of nonsynonymous substitutions at certain codons, a pattern that is consistent with recurrent positive selection. The putative sites under selection often displayed radical substitutions, independent parallel changes, and were located in functionally important regions of the protein. In contrast with this interspecific pattern, population genetic analysis of this gene in humans and chimpanzees did not provide conclusive evidence of recent selection. The frequency and distribution of a TLR5 null mutation in human populations further suggested that TLR5 function might be partially redundant in the human immune system (Appendix A). Comparable analyses of the remaining nine human TLRs produced similar results and further pointed to a biologically meaningful difference in the pattern of molecular evolution between TLRs specialized in the recognition of viral nucleic acids and the other TLRs (Appendix B). The general picture that emerges from these studies challenges the conventional idea that pattern recognition receptors are subject to an extreme degree of functional constraint dictated by the recognition of molecules that are essential for microbial fitness. Instead, TLRs display patterns of substitution between species that reflect an old history of positive selection in primates. A common theme, however, is that only a restricted proportion of sites is under positive selection, indicating an equally important role for purifying selection as a conservative force in the evolution of this gene family. A comparative analysis of evolutionary rates at fifteen loci involved in innate, intrinsic and adaptive immunity, and mating systems revealed that more promiscuous species are on average under stronger selection at defense genes (Appendix C). Although the effect is weak, this suggests that sexual promiscuity plays some role in the evolution of immune loci by affecting the risk of contracting infectious diseases.

Macrophages represent long lived immune cells that are remarkably resistant to apoptosis, which allows them to perform in highly stressful environments. Apoptosis resistance is a characteristic that develops during the differentiation process from monocytes to macrophages. However, the signaling pathways that mediate the development of macrophage antiapoptotic phenotype during differentiation remain mostly unknown. Because of their decreased susceptibility to cell death, macrophages are also key viral reservoirs during HIV infection. My research aims to understand the molecular mechanisms and signaling pathways that mediate cell survival during and after monocyte to macrophage differentiation and the involvement of the main families of antiapoptotic proteins, IAPs (inhibitors of apoptosis) and Bcl2 in this process. HIV accessory protein Vpr was used as an apoptotic stimulus, due to its death inducing abilities in other cell types. My results show that survival of macrophages is distinctively regulated during and after differentiation. I have identified a signaling pathway consisting of PI3K/Akt activation of NFκB that is important in survival of differentiating macrophages by specifically sustaining antiapoptotic Bcl-xL expression. However, once differentiated, Mcl-1, but not Bcl-xL is dependent on PI3K/Akt activation. Moreover, differentiated macrophages are resistant to the effect of HIV-Vpr, which is highly apoptotic for monocytes. In contrast, resistance to HIV-Vpr induced apoptosis of human macrophages is specifically mediated by antiapoptotic IAP proteins, with no involvement of the Bcl2 family, which maintains macrophage viability in the absence of any apoptotic stimuli. In addition to their antiapoptotic properties, IAPs are also important regulators of macrophage function. By using chemical compounds (SMAC mimetics) that target IAPs for degradation, I have shown that IAPs positively modulate LPS-induced IL10, IL-27 and MIG (monokine induced by IFNγ) production in human macrophages, by promoting TRAF2, JNK and p38 signaling and NFκB activation. In addition, IAPs also contribute to LPS-induction of CD80/CD86 costimulatory molecules. Overall, my results suggest that both IAPs and Bcl2 families contribute to survival of human macrophages and that IAPs are also involved in innate immune responses. Unraveling the mechanisms that control macrophage survival and function in various settings would provide therapeutic strategies aimed at eliminating cells when their survival is no longer beneficial for the host, as in the case of HIV infection or autoimmune diseases.

S100A8 is a highly-expressed calcium-binding protein in neutrophils and activated macrophages, and has proposed roles in myeloid cell differentiation and host defense. Functions of S100A8 are not fully understood, partly because of difficulties in generating S100A8 knockout mice. Attempts to silence S100A8 gene expression in activated macrophages and fibroblasts using RNA interference (RNAi) technology were unsuccessful. Despite establishing validated small interfering RNA (siRNA) systems, enzymaticallysynthesized siRNA targeted to S100A8 suppressed mRNA levels by only 40% in fibroblasts activated with FGF-2+heparin, whereas chemically-synthesized siRNAs suppressed S100A8 driven by an S100A8-expression vector by ~75% in fibroblasts. Suppression of the gene in activated macrophages/fibroblasts was low, and some enzymatically-synthesized siRNAs to S100A8, and unrelated siRNA to GAPDH, induced/enhanced S100A8 expression in macrophages. This indicated that S100A8 may be upregulated by type-1 interferon (IFN). IFN-β enhanced expression, but did not directly induce S100A8. Poly (I:C), a synthetic dsRNA, directly induced S100A8 through IL-10 and IFN-dependent pathways. Induction by dsRNA was dependent on RNA-dependent protein kinase (PKR), but not cyclooxygenase-2, suggesting divergent pathways in LPS- and dsRNA-induced responses. New mechanisms of S100A8 gene regulation are presented, that suggest functions in anti-viral defense. S100A8 expression was confirmed in lungs from influenza virus-infected mice and from a patient with severe acute respiratory syndrome (SARS). Multiple pathways via mitochondria mediated S100A8 induction in LPS-activated macrophages; Generation of reactive oxygen species via the mitochondrial electron transport chain and de novo synthesis of ATP may be involved. This pathway also regulated IL-10 production, possibly via PKR. Extracellular ATP and its metabolites enhanced S100A8 induction. Results support involvement of cell stress, such as transfection, in S100A8 expression. A breast tumor cell line (MCF-7) in which the S100A8 gene was silenced, was established using micro RNA technology; S100A8 induction by oncostatin M was reduced by >90% in stably-transfected cells. This did not alter MCF-7 growth. The new approach to investigate the role of S100A8 in a human tumor cell line may assist in exploring its functions and lead to new studies concerning its role in cancer.

Thèse (M.Sc.)--Université Laval, 2007. / Bibliogr.: f. [76]-90. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thèse (M.Sc.)--Université Laval, 2008. / Titre de l'écran-titre (visionné le 25 mars 2009). Bibliogr.

Toll-like receptor (TLR)-4 is a pattern recognition receptor (PRR) that recognises the pathogen-associated molecular pattern (PAMP) lipopolysaccharide (LPS) produced by Gram-negative bacteria. LPS binds to Myeloid differentiation 2 (MD-2)/TLR-4 heterodimers, driving their dimerisation and inducing a conformational change of the intracellular TLR-4 toll/interleukin-1 receptor (TIR) domains. The adaptor protein Myeloid differentiation primary response gene 88 (MyD88)-adaptor-like (Mal)/TIR domain-containing adaptor protein (TIRAP) then binds to the TIR domains of TLR-4 and acts as a bridge for MyD88 which goes on to form the myddosome, a large protein complex of six to eight MyD88 molecules and four Interleukin-1 receptor- associated kinase (IRAK) 4 and four IRAK1/2 molecules. This triggers a signalling cascade which results in nuclear factor (NF)-κB transcription factor activation and production of pro-inflammatory effector molecules such as the cytokine Tumour Necrosis Factor (TNF)-α. Upon activation TLR-4 is also endocytosed where it interacts with a second set of adaptor proteins TIR-domain-containing adaptor- inducing interferon (IFN)-β (TRIF)-related adaptor molecule (TRAM) and TRIF to initiate the type I IFN response. How TLR-4 dimerisation results in the formation of the oligomeric myddosome is not fully understood, but it is possible that the stoichiometry of Mal/TIRAP may be important in the formation of this protein complex. The aim of my thesis was to determine the stoichiometry of Mal/TIRAP at the plasma membrane of immortalised bone marrow derived macrophages (iBMDMs) and whether this stoichiometry changes upon stimulation with different TLR-4 ligands. To investigate Mal/TIRAP stoichiometry I first developed a viral transduction experimental cell model to visualise fluorescently labelled Mal/TIRAP. Mal/TIRAP-/- iBMDMs were lentivirally transduced with a Mal/TIRAPHALO construct. The halotag was fluorescently labelled then the cells were stimulated with TLR-4 ligands, such as LPS, fixed at different time points, then imaged. Total internal reflection fluorescence (TIRF) microscopy was used to image the plasma membrane and photobleaching experiments performed to determine Mal/TIRAP stoichiometry. I developed a computer-based analysis pipeline to analyse the resulting photobleaching data. Under resting conditions, Mal/TIRAP is present at the plasma membrane in clusters of approximately ten Mal/TIRAP molecules per cluster. After five minutes of stimulation with 10 ng/ml LPS Mal/TIRAP redistributes into cluster sizes of approximately six, twelve and much larger. After ten and fifteen minutes stimulation with 10 ng/ml LPS the clusters return to the resting size of approximately ten Mal/TIRAP molecules per cluster with a few much larger clusters remaining present. This confirms the rapid time frame within which TLR-4 signalling occurs at the plasma membrane and is consistent with myddosome stoichiometry of six MyD88 molecules or proposed super myddosomes of twelve MyD88 molecules. The computer-based analysis pipeline developed can be used to analyse any protein of interest at the plasma membrane. Protein ligands have also been found to activate TLR-4; for example allergens, such as Fel d 1 and Der p 2, as well as endogenous damage associated molecular patterns (DAMPs), such as extracellular matrix (ECM) proteins, for example fragments of fibronectin and tenascin-C. The mechanism by which these proteins interact with TLR-4 and induce signalling is unclear. Proteins from the ECM (fragments FNIII1c, FNIII13-14, FNIII9-E and FNIII9-E-14 from fibronectin and the fibrinogen-like globe (FBG) domain of tenascin-C) were tested using a transient transfection assay in HEK293 cells and shown to activate TLR-4. In conclusion, I have developed new tools and methodology to investigate how TLR-4 signals in response to LPS and DAMPs in living cells. Whether DAMP- activated TLR-4 forms similar signalling complexes to those induced by LPS will form part of a future study.