The last decade has been witness to a number of seminal discoveries in the field of innate immunity. The discovery that microbial molecules and endogenous danger signals can be detected by germ-line encoded receptors has changed the way we study the immune system. Indeed, the characterization of Toll in Drosophila as a sensor of microbial products in 1997 then led to the discovery of a family of Toll Like Receptors (TLRs) in mammals. TLRs are critical for the induction of inflammatory responses and the generation of a successful adaptive immune response. The array of ligands that these transmembrane proteins recognized mediates defense against bacteria, viruses, fungus and parasites, as well as, possibly, cancerous cells. In addition to this membrane-bound family of recognition proteins, two families of pattern recognition receptors have been recently shown to respond to microbial and chemical ligands within the cytosol. These represent the Nod Like Receptors (NLRs) and RIGI-like helicase receptor (RLH) families. Nod1 and Nod2 are members of the NLR family of proteins, which are responsible for the recognition of components derived from the bacterial cell wall, more precisely, moieties of peptidoglycan. As such, Nod1 and Nod2 are implicated in the recognition and the defense against bacterial pathogens. Importantly, the genes encoding these two proteins have also been linked to the etiology of several inflammatory disorders such as Crohn’s disease and asthma. In this thesis, we show that recognition of Nod1 and Nod2 ligands generates a rapid and transient inflammatory response in vivo. When co-injected with a model protein, Nod1 and Nod2 ligands exhibit adjuvant properties that lead to the generation of an antigen-specific Th2 type adaptive immune response. Surprisingly, recognition of the Nod1 ligand in non-hematopoietic cells is critical for the generation of this immune response. In contrast, TLRs classically tip the balance towards a Th1 response and interestingly, co-injection of TLR and Nod ligands synergize to generate a more potent immune response characterized by the generation of Th1, Th2 and Th17 T cell respones. To study the role of Nod1 and Nod2 in the context of a bacterial infection in vivo, we used an intestinal mouse pathogen, Salmonella enterica serovar Typhimurium. We were able to show that Nod1-deficient mice, but not Nod2-deficient mice, are more susceptible to the strain of this bacterium, which enters the host through the active pickup in the intestinal lumen by underlying myeloid cells. This sampling mechanism is mediated by a subset of dendritic cells that populate the intestinal lamina propria. Accordingly, the defect seen in Nod1-deficient mice localizes to the mucosal barrier where these dendritic cells appear to have an impaired response towards the bacteria. Taken together, these results increase our knowledge on the general role of Nod1 and Nod2 in immunity and might generate new avenues of research and potential therapeutic targets.

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 123-136).

Regulation of the immune system and of uterine tissue homeostasis, growth, and remodelling are deeply intertwined during pregnancy and are essential for successful reproduction. Recent findings showed that tissue-resident innate lymphoid cells (ILCs) are crucial regulators of both physiology and pathology of the tissues they populate. Uterine natural killer (uNK) cells are a subtype of ILCs known to regulate trophoblast invasion, uterine vascular adaptation to pregnancy, and foetal growth. We recently described additional types of ILCs in the uterus of women and mice. However, the role of these ILCs during reproduction is unknown. Among them, group 2 ILCs (ILC2s) have been previously characterised in other tissues, in which they modulate immune cells and tissue homeostasis by producing type-2 cytokines and growth factors (i.e. IL-4, IL-5, IL-13, and Amphiregulin). Based on these premises, I hypothesized that uterine ILC2s (uILC2s) regulate uterine immune homeostasis and thus contribute to successful reproduction. To test this, I first characterised the uILC subtypes present in humans and mice at various stages of the reproductive cycle. Secondly, I addressed the functional role of uILC2s during pregnancy by taking advantage of a uILC2 knockout mouse model. My results show that uterine ILC2s represent < 1% and < 0.1% of murine and human uterine leukocytes, respectively. However, as they can quickly produce large amounts of cytokines, uILCs are capable of potently affect both other immune cells and the surrounding tissue. Indeed, I found that compared to other tissue-resident ILC2s, uILC2s produce high levels of IL-5 and Areg even in the absence of any stimulation. On the contrary, non-uterine ILC2s mainly produce IL-13, which is lowly expressed by uILC2s. To further characterize the tissuespecific properties of uILC2s, I then performed RNAseq on uILC2s isolated from virgin, midgestation, and term murine uterus, and I compared their transcriptomes with those of ILC2s from lung, intestine, and bone marrow. Interestingly, uILC2s specifically express granzymes and genes typical of regulatory T cells. Therefore, uILC2s have tissue-specific properties and are modulated during pregnancy. Furthermore, the ability of uILC2s to produce IL-5 and Areg suggests that they may be crucial in the regulation of uterine type-2 immunity. I then studied the phenotype of $Rora^{flox/flox}Il7ra^{cre/wt}$(ILC2KO) mouse models, as well as that of mice lacking the ILC2 activating cytokine IL-33 ($IL33^{cit/cit}$; IL33KO). I examined the immune microenvironment in both the myometrium and decidua in ILC2KO mice and found alterations in type-2 cytokines and myeloid cell homeostasis. In particular, in absence of ILC2s, IL-4 and IL-5 are dramatically reduced, IL-13 is absent, and decidual inflammatory cytokines IL1β and IL-6 are increased. Furthermore, uterine dendritic cells (uDC), uterine macrophages (uMac), and uterine neutrophils (uN) increase, while uterine eosinophils (uEo) are virtually absent. These results show that uILC2s regulate uterine type-2 immunity, suggesting that uILC2s could be important during pregnancy. Accordingly, I found that lack of uILC2s leads to insufficient spiral artery remodelling and restricted foetal growth. Type-2 cytokines and in particular IL-4 regulates alternative activation of Macrophages (Mac) and Dendritic Cells (DCs), which promote the development of an anti-inflammatory environment and facilitate tissue remodelling. I hypothesised that similar mechanisms occur in the uterus and that uILC2s have a central role in the polarisation of the immune response. To explore this, I studied in more detail the characteristics of uEo, uMac, and uDCs dissected from wild type and ILC2KO mice. I found a reduction in genes associated with alternative activation in uMac and uDCs in the uterus of pregnant ILC2KO mice. Additionally, I showed that uEo are the main producers of the IL-4. This demonstrates that uILC2s promote alternative activation of myeloid cell population by modulating the uterine immune microenvironment. I then assessed the role of uILC2s-dependent type-2 immunity in inflammatory pathology following a type-1 response to bacterial infection. When challenged with LPS, pregnant ILC2KO mice showed more pronounced foetal demise. Therefore, uILC2s regulate uterine type-2 immune homeostasis and this prevents inflammatory pathology. Collectively, my work advances our knowledge of the innate immune mechanisms that control physiological and pathological events during pregnancy. These findings have implications to the field of immunology of pregnancy and may lead to clinical progress in diagnosis and prevention of infection-induced abortion in human pregnancies.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Erika R. Geisbrecht / Maintenance of muscle tissue during development is greatly dependent upon the extracellular matrix (ECM) to stabilize, sense, and compensate for changes in the local environment. Muscle has a particularly high demand for a dynamic ECM to allow for contraction and to transmit forces necessary for generating movement. Inefficient contraction and/or detachment can lead to muscle tissue damage and the release of damage-associated molecular patterns (DAMPs), which overactivate immune responses and drive the progression of muscle diseases. Our lab uses the Drosophila muscle attachment site (MAS) as a model to characterize novel genes and mechanisms involved in muscle maintenance. Initially, we were focused on characterizing a novel ECM protein, Fondue (Fon), which had previously been shown as a critical mediator of ECM stability in the hemolymph clot. Mutations in fon and the knockdown of fon through RNAi causes body wall muscles to detach and also creates large gaps between muscle hemisegments. TEM analysis of fon mutant MASs revealed a loss of ECM integrity and important support features including disruption of cuticle and tendon architectures, a lack of muscle-tendon interdigitation, and a loss of electron-dense matrix accumulation. More interestingly, a sensitized background screen revealed a subset of coagulation proteins, fon, Tiggrin, and Lsp1γ, that were necessary for stabilizing muscle attachment sites. Further investigation into gene expression profiles of mutants experiencing hypercontraction-induced muscle tissue stress indicated a clear trend of innate immune activation, suggesting a broader connection between muscle development and innate immunity. In fon mutants with muscle detachment, we also observe abnormal melanin accumulation as melanotic tumors or along the larval MASs, activation of Toll signaling in the fat body, and constitutive expression of the antimicrobial peptide (AMP), drosomycin. In a fon-sensitized background assay, we identified genetic interactions between fon and Toll pathway members, including the NFκB inhibitor/IκB, cactus. At the local level, fon-mediated muscle detachment and muscle hypercontraction mutants, Mhc[superscript]S1 and Brkd[superscript]J29, cause JAK/STAT activation within muscle tissue. We propose a model where muscle tissue stress caused by disruptions to muscle homeostasis progresses muscle disease through overactivation of the innate immune system. Understanding the mechanisms by which these two biological processes are intertwined will advance our knowledge of how tissue stresses can be sensed and elicit multi-tissue responses.

The overall aim of this project was to determine the molecular mechanisms by which the flagellin gene from Salmonella enterica serovar Typhimurium (S. Typhimurium) activates the NAIP/NLRC4 inflammasome and its contribution to the host protective immune response against salmonellosis. Inflammasomes are multi-protein complexes formed in response to the activation of pattern recognition receptors (PRRs). The NOD-like receptor (NLR)-family of inflammasome complexes are formed from the cytosolic NLR receptors, ASC adaptor and caspase-1 in response to pathogen- associated molecules or danger-associated signals. The NAIP/NLRC4 inflammasome is activated by the S. enterica flagellar filament protein (FliC), the SPI-1 type III secretion system needle (PrgI) and inner rod proteins (PrgJ). Recognition of these bacterial ligands by the NAIP receptors allows oligomerisation with NLRC4 and subsequent recruitment of caspase-1. Caspase-1 mediates pyroptosis, while recruitment of ASC is also required for cleavage of pro-IL-1β and pro-IL-18 to their active forms by caspase-1. Differential recognition of the flagellar filament proteins (flagellin) by the NAIP/NLRC4 inflammasome forms an important part of my thesis. Here, I have looked at the molecular mechanisms and immunological consequences of the differential recognition of flagellin by the NAIP/NLRC4 inflammasome using S. Typhimurium SL1344 and the non-pathogenic E. coli strain K12-MG1655. An important part of my work was to try and determine which regions of fliC are required for NAIP/NLRC4 inflammasome activation and whether they can be mutated while preserving motility. To do this a panel of ten strains expressing chimeric fliC genes were created and characterised in macrophage infection experiments and bacterial motility assays. My results confirm the C-terminus of FliC is critical for both inflammasome activation and motility in agreement with published reports. To further investigate this differential recognition by the NAIP/NLRC4 inflammasome I modified S. Typhimurium strain of moderate virulence (M525P) to express flagellin from E. coli K12-MG1655. This strain (M525PΔfliC::fliCK12-MG1655CmR) retained motility and both in vitro and in vivo characterisation was carried out in macrophages and using a murine model of sublethal salmonellosis respectively. Activation of the NAIP/NLRC4 inflammasome was impaired in murine macrophages infected with M525PΔfliC::fliCK12-MG1655CmR when compared to those infected with M525P. Mice infected with M525PΔfliC::fliCK12-MG1655CmR had increased liver and spleen bacterial burdens compared to those infected with M525P, indicating that optimal NAIP/NLRC4 inflammasome activation is key for efficient control of microbial spread in vivo. The role of NAIP receptors in inflammasome formation was further investigated with the use of CRISPR/Cas9 to generate mutant murine macrophage cell lines. To investigate the consequence of gene deletions cell lines were designed to lack NAIP 1, 2, 5 and 6, while others were designed to express tagged NAIP proteins to elucidate the cellular localisation of the NAIP proteins during inflammasome formation by microscopy. Characterisation of these cell lines is ongoing, with extensive optimisation of the CRISPR/Cas9 technique undertaken during this study.

Antimicrobial peptides (AMPs) are innate host defense peptides that protect against pathogenic microbes by neutralizing toxins or via a direct killing mechanism. AMPs are classified based on their physical properties such as charge, structure, and binding motifs. Here we investigated the antimicrobial and immune-modulating effects of the Regenerating Islet-Derived Protein 3 (REG3) family and LL-37 REG3 peptides are C-type lectins and have been demonstrated to have antimicrobial activity against Gram-positive bacteria by binding to sugars on the peptidoglycan membrane of these bacteria. A similar strategy is also employed by the lectin Surfactant Protein-D which has been shown to bind and neutralize Influenza A Virus (IAV). REG3 peptides were shown to be expressed in the lungs of mice infected with IAV. We observed reduction of IAV infected cells when IAV was pre-incubated with an Escherichia coliexpressed recombinant version of human REG3A peptide. This peptide also modified interaction of IAV with primary human neutrophils. However, these effects were lost when using a mammalian cell expressed recombinant REG3A. A second member of the REG3 family, REG3G, showed minimal inhibition of IAV infection. While the mechanism remains unclear, LL-37 has demonstrated killing activity against a spectrum of microbes including IAV. Previous work from our group identified the core domain of LL-37 responsible for IAV neutralization. In addition, our group showed that LL-37 modulates interaction of IAV with neutrophils. Here we tested three modified versions of LL-37 that retain the overall size and charge of LL-37, but with modifications in the core domain reducing hydrophobicity. We observed that these mutants retain IAV killing activity across multiple strains. In addition, these mutants retain the modulation of IAV induced neutrophil responses. We also found that the compounds sodium butyrate and Entinostat, which can upregulate endogenous expression of LL-37, have variable effects in IAV infection. We believe these findings will aid in the development of LL-37 derivatives to expand the repertoire of antimicrobials.

The onset of immune response against microbial stimuli activates induction of many anti- inflammatory genes and ISGs for effective clearance of the pathogen. This response includes transcriptional activation of several non-coding transcripts such as miRNAs and long non-coding RNAs (lncRNAs). LncRNAs constitutes the largest class of non-coding genome and are arbitrarily described as transcripts greater than 200 base pairs. Similar to protein coding mRNAs, lncRNAs are RNA polymerase II transcripts and undergo mRNA processing such as capping, splicing and polyadenylation. In recent years, high throughput sequencing has enabled an in-depth exploration of the human genome and subsequent discovery of lncRNAs. Several studies have highlighted the crucial role of lncRNAs in many biological processes including as regulators of gene expression as well as molecular effectors of host-pathogen driven immune responses. To date, majority of lncRNAs have been studied in murine models with limited understanding in human cells. In order to elucidate the role of lncRNAs in human immune cell regulation, the goal of this thesis is to identify and characterize novel lncRNAs critical to host-pathogen innate immune responses. RNA sequencing in LPS, IAV and HSV stimulated cells revealed lncRNA LUCAT1 as most differentially regulated lncRNA. CRISPR-cas9 and shRNA mediated depletion of LUCAT1 showed enhanced IFN-I genes signature, which was suppressed upon overexpression of LUCAT1. Additionally, LPS stimulated hDCs showed enrichment of LUCAT1 in the nucleus and its association with the chromatin markers. Further, LUCAT1 depletion contributed to enhanced occupancy of transcriptional coactivators at the promoters of IFN-I genes. Global identification of RNA associated proteins revealed LUCAT1 association with STAT1 in the nucleus thus emphasizing its role in transcriptional regulation of Type I IFN genes in inflammatory responses. This thesis furthers the understanding about the molecular factors affecting immune regulation and describes the novel role of LUCAT1 as an attenuator of immune cell response to pathogens.

The immune system rapidly mounts an innate immune response to invading pathogens that is accompanied by antigen-presentation, to promote the development of the adaptive immune response. These responses orchestrate through signal transduction by PRRs that recognize PAMPs, which results in the expression of various cytokines and mediators to promote pathogen control. Herein, we investigated the role of the type I interferon (IFN)- and the p38MAPK- pathways in response to infection with Salmonella Typhimurium (ST). We delved into the mechanisms through which IFNAR1-signaling results in host susceptibility against ST and show that while STAT2 and IRF9 promote susceptibility against ST, this is antagonized by STAT1. Our results indicate that IFNAR1-signaling induces IL-10 production through the ISGF3 complex, which indeed inhibits the production of IL-1β (via NLRP3 and caspase-1) resulting in a state of resistance against ST. Furthermore, our work elucidates that MK2, which is a p38MAPK substrate promotes host resistance, which is contradictory to type I IFNs despite the fact that MK2 regulates cytokine expression in a similar pattern to IRF9. We demonstrate that MK2 inhibits inflammasome signaling via NLRP3, caspase-1 and caspase11. We also reveal a role for MK2 in regulating IL-1β production via distinct signaling pathways including inhibition of MSK1/2 besides activation of the autophagic machinery; which also contribute to the enhanced inflammasome activation seen in Mk2- deficient cells. Thus, our observations illuminate the fact that the type I IFN pathway and the p38MAPK pathway are only dependent on each other to a certain extent in modulating the innate immune response to Salmonella infection, thereby bringing about varied outcomes in the infected host.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17815号 / 医博第3813号 / 新制||医||999(附属図書館) / 30630 / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 野田 亮, 教授 藤田 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Salmonella enterica serovar Typhimurium (Salmonella) is a Gram-negative bacterial pathogen capable of causing both gastroenteritis and bacteraemia in human hosts. During infection, Salmonella invokes a complex network of virulence factors, regulatory systems, and metabolic pathways to promote immune evasion, sometimes demanding antibiotic treatment for resolution. Unfortunately, antibiotic resistance has reached critical levels in this and other pathogens, necessitating the discovery of new anti-infective targets and treatment options. Herein, we have sought to exploit the dynamic interactions between Salmonella and the host immune system to identify new, conditionally active anti-Salmonella therapies. In chapter 2, we aim to identify chemical compounds that are selectively antimicrobial against intracellular Salmonella, and discover that the anxiolytic drug metergoline inhibits Salmonella survival in cultured macrophages and systemically infected mice. In chapter 3, we screen for anti-virulence compounds that target regulatory signaling in Salmonella, and characterize the inhibitory activity of methyl-3,4-dephostatin, which perturbs SsrA/B and PmrB/A signaling and enhances sensitivity to colistin in vitro and in vivo. In chapter 4, we identify several host-directed compounds that modulate macrophage immunity and investigate their ability to attenuate a multidrug resistant Salmonella infection. Together, the work presented in this thesis demonstrates the potential for drug screening in infection-relevant conditions to identify new anti-infectives with non-traditional targets. / Thesis / Doctor of Philosophy (PhD)