Browsing by Subject "Innate immunity"

In the 21st century, the world has already experienced two pandemics caused by respiratory RNA viruses, the 2009 H1N1 “swine-flu” pandemic, and the ongoing COVID-19 pandemic. The 2009 pandemic was, thankfully, mild in terms of casualties; however, it revealed certain failings in existing systems. As a circulating virus, we had approved, in-use antiviral treatments for influenza, but the 2009 H1N1 viruses were resistant to an entire class, adamantanes. Also, the requirement for annually updated influenza vaccines meant there were systems in place for large-scale vaccine production, yet reliance on eggs dramatically limited the ability to scale-up a new vaccine quickly. Finally, global vaccine inequities meant many countries remained relatively unvaccinated against the H1N1 virus, even as the pandemic was declared over in August 2010. The COVID-19 pandemic has been a very different story on some fronts, most notably the death toll, which exceeds 6 million globally. Also, the decade of progress in vaccine research since 2009 has been evident in the rapid development of many highly effective COVID-19 vaccines using different platforms, while vaccine distribution issues remain. Finally, an entire lack of approved coronavirus antivirals required swift action to identify existing drugs and treatments to alleviate disease. The research and clinical response to the COVID-19 pandemic took advantage of existing technologies, pharmaceuticals, and systems and adapted them to solve the present crisis. The problem of this dissertation is to extend these efforts by investing in adaptable research tools, methods, and subjects to prepare for potential future respiratory RNA virus pandemics.In Chapters 2 and 3, we generated virus-specific research tools that are rapidly adaptable to new viral strains. Chapter 2 describes a fluorescent reporter of coronavirus protease activity that may be used to screen antiviral drugs and is compatible with diverse coronavirus protease proteins. Chapter 3 discusses a novel method for generating influenza reporter viruses with wide research applications, notably this manner of introducing exogenous proteins requires minimal genome rearrangements increasing transferability to newly identified strains. These projects demonstrate that even research tools requiring updates with the emergence of new strains can be designed to prioritize rapid adaptability. In Chapters 4 and 5, we interrogate the innate immune responses that determine the outcomes of viral infections. Chapter 4 identifies ETV7 as a negative regulator of the antiviral type I interferon response using CRISPR activation screening. ETV7 was previously known to be induced by interferon, but its role during the response to viral infection remained undetermined. We found ETV7 limits transcription of interferon stimulated gene expression, influencing the antiviral state of a responding cell. Chapter 5 reviews the impact of influenza viral disease outside the site of infection, the respiratory tract, and established methods of studying these effects using animal models. Many of the circulating cytokines implicated in non-respiratory influenza disease from these models, IL-6, IFNs, and TNF-alpha, are known to play a role in influenza and COVID-19 patient disease severity. These investigations show that our understanding of how innate immunity is regulated, and dysregulated, continues to require updating even as the main pathway members and downstream effectors have been identified.

The key components of innate immune defense to pathogens are various migratory as well as tissue resident innate immune cells, however, their interactions with pathogens as well as their immune-orchestrating roles are often poorly understood. While immune cells encounter pathogens at barrier sites and mount the first line of defense, pathogens are well adapted to bypass, inactivate and even exploit the functions of these cells. Better understanding of the interactions between pathogens and innate immune cells can teach us how pathogens avoid or exploit immune cells and how to overcome these mechanisms of pathogenesis by therapeutic interventions. In this work, we examined two scenarios of pathogen invasion and sought to understand the complex ways of external targeting of innate immunocytes that can either benefit the pathogen or the host.

First, we studied the migratory innate immunocytes in draining lymph nodes upon entry of Yersinia pestis via the skin and identified how this plague-causing bacterium coopted host cell death pathways of infiltrated mononuclear phagocytes. By employing time-lapse microscopy and flow cytometry, we demonstrated that within the confines of infected lymph nodes, bacteria-triggered necroptotic cell death resulted in the release of intracellular bacteria into the extracellular environment and attracted neighboring phagocytic cells, promoting their infection by these recently released bacteria. This expansion of bacteria-bearing immune cells which eventually migrate to secondary lymph nodes, enables large numbers of Y. pestis to disseminate from one node to the next via the lymphatic system. We show this mechanism of dissemination being essential for the transition of plague from a bubonic to septicemic stage and demonstrate immunotherapeutic potential of necroptosis inhibitors.

Next, we focused on mast cells, a resident innate immunocyte in the context of skin infection by Staphylococcus aureus. We showed that connective tissue mast cells promoted recruitment of neutrophils at the early stage and CD301b+ dendritic cells at the later stages of infection, which played critical roles in infection control and repair, respectively. We further demonstrated that exogenous activation of skin mast cells via a mast cell-specific G protein-coupled receptor controlled infection as well as enhanced mobilization of dendritic cells to draining lymph nodes in a mast-cell dependent manner and protected mice from re-infection. Therefore, selective activation of mast cells appears to orchestrate immunomodulation integrating both the innate and adaptive immune arms.

These studies reveal the yin and yang of innate immune cells in two very different infectious settings. They emphasize how different strategies to target these cells at the immune checkpoints can be beneficial for host-directed therapy against bacterial infections.

Concurrent with the global escalation of the AIDS pandemic, cryptococcal infections are increasing and are of significant medical importance. Although improvements in antifungal therapy have advanced the treatment of cryptococcosis, the mortality rate is approximately 12% in medically advanced countries, and approaches 50% in less developed regions. Additionally, C. neoformans can cause infection in seemingly healthy individuals, elevating its status as a primary human pathogen. Although numerous studies have examined virulence properties, less is understood regarding host immune factors in the lungs during early stages of fungal infection. In the present thesis studies, I examined the roles played by pulmonary surfactant proteins in response to C. neoformans in vitro and in vivo. We demonstrate that SP-D, but not SP-A, binds to the yeast and increases phagocytosis of poorly encapsulated yeast cells by macrophages, yet concomitantly protects the pathogenic microbes from macrophage-mediated defense mechanisms. Furthermore, we show that SP-D functions as risk factor in vivo by protecting the yeast cells against oxidant species and thus facilitating disease progression. The results of these studies provide a new paradigm on the role played by surfactant protein D during host responses to C. neoformans and, consequently, impart insight into potential future treatment strategies for cryptococcosis.

Dectin-1 is a C-type lectin receptor with diverse functions in orchestrating the innate immune response. Previous studies have primarily focused on the function of Dectin-1 in the setting of fungal infection. However, emerging evidence supports an important role for Dectin-1 in the context of autoimmunity and sterile inflammation. In this thesis, I investigated the function of Dectin-1 in central nervous system (CNS) autoimmunity. Specifically, I studied the role of Dectin-1 signaling in experimental autoimmune encephalomyelitis (EAE), a mouse model of Multiple Sclerosis (MS). Genetically modified mouse lines and immunophenotyping approaches were used to study Dectin-1 function in EAE. In addition, ex vivo studies using small molecule inhibitors and next-generation sequencing were used to perform in-depth characterization of Dectin-1 signaling mechanisms. Here I report that Dectin-1 limited EAE, while its downstream signaling molecule, Card9, promoted the disease. Myeloid cells mediated the pro-resolution function of Dectin-1 in EAE with enhanced gene expression of the neuroprotective molecule, Oncostatin M (Osm), through a Card9-independent pathway, mediated by the transcription factor NFAT. Furthermore, I found that the Osm receptor (OsmR) functioned specifically in astrocytes to reduce EAE severity. Notably, Dectin-1 did not respond to heat-killed Mycobacteria, an adjuvant to induce EAE. Instead, endogenous Dectin-1 ligands, including galectin-9, in the central nervous system (CNS) were involved to limit EAE. This thesis research reveals a mechanism of beneficial myeloid cell-astrocyte crosstalk regulated by a Dectin-1 pathway and identifies potential therapeutic targets for autoimmune neuroinflammation.

Endogenous retroviruses (ERVs) are ancient deactivated viral elements that have integrated into the human genome over the course of millennia of evolution. ERVs are normally kept transcriptionally repressed, but recent evidence has demonstrated that ERV transcription can be upregulated in response to a variety of stimuli and pharmacological treatments. Furthermore, ERV transcription in the form of double-stranded RNA (dsRNA) was shown to upregulate a host of immune signaling pathways, with implications for the future of cancer therapy. In this study, we utilize the transcriptional corepressor KRAB-Associated Protein 1 (KAP1), a chromatin modulator responsible for suppressing ERV transcription sites, as a tool to investigate ERV expression in cancer therapy.

We demonstrate that radiotherapy, historically believed to mediate tumor cell repression through direct cell killing, can also upregulate ERV transcription, which stimulates downstream interferon production and interferon-stimulated genes through the MDA5/MAVS innate antiviral immunity pathway, a separate pathway from cGAS/STING signaling. KAP1 depletion enhances the effect of radiation, as ERV and interferon transcription is significantly upregulated. Additionally, KAP1 depletion enhances the effect of radiation-induced anti-tumor response in vivo in two separate tumor models. Our findings indicate a novel and understudied pathway of radiotherapy-induced tumor control.

We also demonstrate that KAP1 depletion is sufficient to provoke interferon signaling and immune effects. KAP1 depletion upregulates ERV transcription, driving a similar effect to that seen with irradiation. Furthermore, KAP1 depletion is sufficient to inhibit tumor growth of B16F10 tumors in in vivo studies, but this effect is dependent on an intact host immune system. Tumor growth inhibition in vivo is driven by increased recruitment of immune cells to the tumor microenvironment, along with upregulated of interferon-stimulated genes. Patient data support our findings, as mRNA analysis of TCGA patient cohorts reveals that Trim28 expression is negatively associated with survival and immune recruitment. Taken as a whole, our work indicates KAP1 as a crucial modulator of ERVs, with significant consequences for cancer therapy.

Urinary tract infections (UTIs) are the second most common type of infection identified in the clinical setting and disproportionately afflict women. UTIs most frequently manifest in the form of infection of the lower urinary tract, involving the bladder. Uropathogens, particularly uropathogenic E. coli, progressively colonize the urethra and ascend to the bladder, where they initiate cystitis. In some cases, infection further ascends through the ureters and reaches the kidneys, where it causes pyelonephritis. Infection of both the upper and lower urinary tract can have serious ramifications for the host, and this is in large part due not to infection itself but to host-directed responses to bacterial insults.

In this thesis, I will describe and discuss two distinct aspects of UTIs. In the first study, in vivo work in a mouse model of urinary tract infection revealed a novel role for mast cells, which are tissue-resident granulated innate immune cells, in directing the detachment and death of epithelial cells during cystitis, facilitating the clearance of bacteria from the bladder. An ex vivo porcine bladder infection model suggested a specific role for mast cell granules and the proteases contained therein, which was corroborated with in vitro experiments utlizing isolated mast cell granules and human epithelial cells to demonstrate granule-induced exfoliation and cell death. From this work, it is clear that mast cells play a highly targeted role in modulating urothelial integrity during bladder infection by mediating host-directed epithelial loss.

In the second study described in this dissertation, the synergistic roles of both pyelonephritis and vesico-ureteric reflux (VUR), a congenital urinary tract defect that results in the improper backflow of urine from the bladder to the kidney, in the development of reflux nephropathy, a fibrotic host response characterized by renal scar formation, were elucidated in a series of in vivo experiments. Specifically, the C3H mouse, which is naturally susceptible to VUR, was utilized to characterize the dynamics of kidney infection and the onset of reflux nephropathy. Renal scarring was dependent on the presence of sustained kidney infection and the accompanying inflammatory response due to VUR, while neither transient infection nor reflux alone were sufficient to provoke nephropathy. Thus, the development of reflux nephropathy is dependent upon the confluence of both infection and VUR.

This body of work reveals the double-edged sword of the host inflammatory response to urinary tract infection. In the bladder, mast cell activation and degranulation leads to granule-induced epithelial exfoliation and consequently a reduction in the bacterial burden in the bladder. However, the sustained inflammatory response that accompanies pyelonephritis in vesico-ureteric reflux-affected individuals results in significant damage to the kidney without any accompanying reduction in infection. These findings highlight the dueling roles of the host inflammatory response to infection in the upper and lower urinary tract and strongly suggest that differential clinical approaches to cystitis and pyelonephritis are necessary to promote an effective mast cell in the bladder in the former and facilitate the clearance of renal infection while mitigating tissue damage in the latter.

Distinct microbial communities colonize diverse vertebrate mucosal surfaces including the intestinal tract. These microorganisms, termed the intestinal microbiota, impact many aspects of host physiology including metabolism, behavior, and immune development. A majority of gut associated microbes are genetically intractable and thus the mechanisms that mediate their colonization and influence on the host remain undefined. To define genetic mechanisms of bacterial colonization and their subsequent impact on host innate immune development and function, we utilized a gnotobiotic zebrafish model. Using germ free zebrafish, devoid of microbiota, compared to zebrafish colonized with either large isogenic mutant pools or complex microbial communities, we identify bacterial mechanisms of colonization (Chapter 2) and mechanisms of microbial control of host innate immune function (Chapter 3).

In Chapter 1, I introduce the intestinal microbiota and experimental strategies to interrogate their influence on host immunity. I highlight mechanisms by which animals recognize microbiota derived signals and products. Further I detail the cellular responses that lead to phenotypic alterations of host epithelial and innate immune cells following microbiota colonization. In Chapter 2 we use gnotobiotic zebrafish to investigate the mechanisms of intestinal colonization of a gut bacterial commensal, Exiguobacterium acetylicum. We performed parallel in vitro and in vivo competitions of large pools of E. acetylicum mutants. These experiments identified several mutations that are differentially enriched specifically in vivo. We also elucidated the ability of different E. acetylicum strains to colonize the zebrafish intestine utilizing high resolution live imaging of labeled strains. Our data indicate that traits, such as motility, are not always the major drivers of successful colonization.

The intestinal microbiota influences the development and function of myeloid lineages such as neutrophils, but the underlying molecular mechanisms are unresolved. In Chapter 3, we identified the immune effector Serum amyloid A (Saa) as one of the most highly induced transcripts in digestive tissues following microbiota colonization in gnotobiotic zebrafish. Saa is a conserved secreted protein produced in the intestine and liver with enigmatic in vivo functions. We engineered saa mutant zebrafish to test requirements for Saa on innate immunity in vivo. Zebrafish mutant for saa displayed impaired neutrophil responses to wounding but augmented clearance of pathogenic bacteria. Saa’s effects on neutrophils further depend on microbiota colonization, suggesting this protein mediates the microbiota’s effects on host innate immunity. To test tissue-specific roles of Saa on neutrophil function, we over-expressed saa in the intestine or liver and found that sufficient to partially complement neutrophil phenotypes observed in saa mutants. These results indicate Saa produced by the intestine in response to microbiota serves as a systemic signal to neutrophils to restrict aberrant activation, decreasing inflammatory tone and bacterial killing potential while simultaneously enhancing their ability to migrate to wounds. These findings identify a host molecular mechanism that promotes innate immune tolerance to the commensal microbiota. In Chapter 4, I offer perspectives as to how Saa may function at the crossroads between host metabolism and immunity. I posit that Saa, and other microbiota-induced host factors from IECs, are the molecular underpinnings of animal-microbiota symbiosis, orchestrating systemic alterations in host metabolism and immune function.

Although monophosphoryl lipid A was recently approved by the Food and Drug Administration, more vaccine adjuvants are needed to meet the demand for vaccines against new, emerging, and re-emerging diseases. Additionally, characterizing the mechanisms of action of potent vaccine adjuvants is important for moving toward more rational vaccine design based on the careful selection of antigens and adjuvants to stimulate only the desired immune responses. Two experimental vaccine adjuvants, compound 48/80 (C48/80) and IL-1, were evaluated in these studies. The safety and efficacy of the mast cell activator C48/80 was evaluated when used as an adjuvant delivered intradermally (ID) with recombinant anthrax protective antigen (rPA) in comparison with two well-known adjuvants. Mice were vaccinated in the ear pinnae with rPA or rPA + C48/80, CpG oligodeoxynucleotides (CpG), or cholera toxin (CT). All adjuvants induced similar increases in serum anti-rPA IgG and lethal toxin-neutralizing antibodies. C48/80 induced balanced cytokine production (Th1/Th2/Th17) by antigen-restimulated splenocytes, minimal injection site inflammation, and no antigen-specific IgE. Our data demonstrate that C48/80 is a safe and effective adjuvant, when used by the intradermal route, to induce protective antibody and balanced Th1/Th2/Th17 responses. Histological analysis demonstrated that vaccination with C48/80 reduced the number of resident mast cells and induced an injection-site neutrophil influx within 24 hours. Nonetheless, rPA + C48/80 significantly increased antigen-specific IgG titers in mast cell-deficient mice compared to antigen alone, suggesting that C48/80 has mast cell-dependent and mast cell-independent mechanisms of action.

IL-1alpha and beta have been shown to have strong mucosal adjuvant activities, but little is known about their mechanism of action. Bone marrow chimeric mice were intranasally vaccinated with Bacillus anthracis lethal factor (LF) with or without 4 µg IL-1alpha or a control adjuvant (cholera toxin) to determine if IL-1R1 expression on stromal cells or hematopoietic cells was sufficient for the maximal adjuvant activity of nasally delivered IL-1alpha. IL-1alpha was not active in IL-1R1-deficient (Il1r1-/-) mice given Il1r1-/- bone marrow, demonstrating that the adjuvant activity of IL-1 was due to the presence of IL-1R1 and not contaminants. Cytokine and chemokine responses induced by vaccination with IL-1alpha were predominantly derived from the stromal cell compartment and included G-CSF, IL-6, IL-13, MCP-1, and KC. Nasal vaccination of Il1r1-/- mice given wild-type bone marrow (WT-->KO) and WT-->WT mice with LF + IL-1alpha induced maximal adaptive immune responses, while vaccination of wild-type mice given Il1r1-/- bone marrow (KO-->WT) mice resulted in significantly decreased production of LF-specific serum IgG, IgG subclasses, lethal toxin-neutralizing antibodies, and mucosal IgA compared to WT-->KO and WT-->WT mice (p < 0.05). Our results suggest that IL-1R1 expression in the hematopoietic compartment is sufficient for the maximal induction of antigen-specific adaptive immunity after nasal vaccination adjuvanted with IL-1alpha and that while stromal cells are required for maximal adjuvant-induced cytokine production, the adjuvant-induced stromal cell cytokine responses are not required for effective induction of adaptive immunity.

The innate immune system is the front line of host defense against microbial infections, but its rapid and uncontrolled activation elicits microbicidal mechanisms that have deleterious effects. Increasing evidence indicates that the metazoan nervous system, which responds to stimuli originating from both the internal and the external environment, functions as a modulatory apparatus that controls not only microbial killing pathways but also cellular homeostatic mechanisms. Exploiting simple organism Caenorhabditis elegans, we performed whole-animal screen and identified antibiotic colistin and dopamine antagonists as immune activators that target conserved immune pathways. The goal of this work is to investigate the role of dopamine signaling and underlying neuronal circuits in regulating the C. elegans innate immune response.

Through genetic and pharmacological studies, we identified a D1-like dopamine receptor, DOP-4, which suppresses the conserved PMK-1/p38 immune pathway in C. elegans. We also demonstrated that the manipulation of a dopaminergic neural circuit can alter the immune response upon pathogen infection. Previous studies showed that an octopamine receptor, OCTR-1, functions in chemosensory neurons to inhibit innate immunity. We found that OCTR-1-expressing neurons, ASH, and interneurons, AIA, are involved in controlling the resistance to pathogen infection. This work provides direct evidence that a neuronal network exists in C. elegans to orchestrate defenses against pathogen invasion.

Hepatitis C virus (HCV) evasion of the host immune system is largely mediated by the actions of the HCV NS3-NS4A protease complex, which consists of the serine protease and RNA helicase NS3 and its membrane targeting co-factor NS4A. NS3-NS4A has multiple functions in the HCV life cycle, with roles in both HCV replication and regulation of innate immune signaling. To regulate innate immune signaling, NS3-NS4A inactivates multiple signaling proteins, including MAVS, an adaptor protein in the RIG-I antiviral signaling pathway, and Riplet, an E3 ubiquitin ligase that activates RIG-I. Inactivation of these host proteins results in an inhibition of downstream signaling through the transcription factor IRF3 and inhibition of the subsequent induction of IFN-β. What directs the multiple functions of NS3-NS4A throughout the HCV life cycle is largely undetermined. Here, we identify a tyrosine residue within the transmembrane domain of NS4A that uncouples the various function of NS3-NS4A.

First, to uncouple the roles of NS3-NS4A in replication and immune evasion, I focused on the NS4A transmembrane domain and generated an NS4A mutant (Y16F) in a full-length HCV infectious clone, a subgenomic replicon, and an over-expression construct. I then assessed viral replication of HCV wild-type (WT) and Y16F viruses by measuring replication of a subgenomic HCV replicon in two related liver hepatoma cell lines: Huh7, which have functional RIG-I signaling, and Huh-7.5 cells, which lack functional RIG-I signaling. The HCV Y16F virus replicated to similar levels as WT HCV in Huh-7.5 cells. However, in Huh7 cells, replication of HCV Y16F was decreased compared to the HCV WT. I used CRISPR-Cas9 gene editing to delete proteins in the RIG-I pathway, including RIG-I, MAVS, and IRF3, in Huh7 cells, infected these cells with HCV WT or Y16F viruses, and then measured virus replication. I found that Y16F viral replication was not restored to the levels of WT in Huh7-RIG-I KO cells or the Huh7-MAVS KO cells, but it was restored to the levels of WT in the Huh7-IRF3 KO cells. I also found that the HCV NS3-NS4A Y16F mutation reduced the ability of over-expressed NS3-NS4A to block IRF3 activation, as measured through nuclear translocation via immunofluorescence microscopy. Further the NS3-NS4A Y16F mutation also had a reduced ability to block the induction of interferon-stimulated genes during both HCV replication and infection. This reveals that HCV NS4A Y16 can regulate a RIG-I-independent, yet IRF3-dependent, signaling pathway that limits viral replication.

Second, to further characterize this RIG-I-independent, IRF3-dependent signaling pathway, I examined the interactions of HCV NS3-NS4A with two of its known host substrates, MAVS and Riplet. To test whether the Y16F mutation prevented NS3-NS4A cleavage of MAVS, I performed a MAVS cleavage assay during both overexpression of NS3-NS4A and MAVS and also during infection with HCV WT and Y16F viruses. NS3-NS4A Y16F was able to cleave MAVS just like WT during both conditions. Next, I found that over-expression of NS3-NS4A WT changed Riplet intracellular localization and that NS4A interacted with Riplet. However, the NS4A Y16F mutation prevented NS4A-Riplet interactions in both of these contexts. Interestingly, I found that Huh-7.5 cells express lower levels of Riplet protein and mRNA compared to Huh7 cells. When full-length Riplet was added exogenously to Huh-7.5 cells, HCV Y16F virus replication was reduced compared to WT. However, when a Riplet construct missing the RING domain, which is essential for Riplet signaling, was added exogenously to Huh-7.5, both WT and Y16F viruses now replicated similarly. Taken together, these data identify NS3-NS4A Y16 as important for regulating a previously uncharacterized Riplet-mediated signaling pathway that limits HCV infection.

Mast cells (MCs) are increasingly recognized as powerful sentinel cells responsible for modulating the early immune responses to a wide range of infectious agents. This protective role is attributable in part to their preponderance at the host-environment interface and their innate capacity to rapidly release modulators of immune cell trafficking which promotes the early recruitment of pathogen-clearing immune cells from the blood. However, host-adapted pathogens had been a critical threat to human for a long time because they have evolved mechanisms directed at overcoming protective immunity.

In this work, we outline Salmonella enterica serovar Typhimurium has evolved a novel mechanism to inactivate peripheral MCs resulting in limited neutrophil responses at infection sites in early stage of infection. Because of the delay in bacterial clearance at the point of entry, Salmonella are able to multiply and rapidly disseminate to distal sites. Suppression of local MCs' degranulation restricted outflow of vascular contents into infection sites, thus facilitating bacterial spread.

We discover MC suppression is mediated by the Salmonella Protein Tyrosine Phosphatase (SptP), which shares structural homology with Yersinia YopH. Interestingly, SptP, not only shares homology with phosphatases found in MCs, they are also homologous to YopH an effector protein expressed by plague causing Yersinia pestis. We show that YopH had MC suppressing abilities as SptP suggesting that this activity is shared among some of the more virulent bacterial pathogens. The functionally relevant domain in SptP is its enzymatic site and that it works by dephosphorylating the vesicle fusion protein N-ethylmalemide-sensitive factor (NSF) and by blocking phosphorylation of Syk, which is located in downstream and upstream of tyrosine phosphorylation signaling pathway in MCs.

Without SptP, orally challenged S. Typhimurium failed to suppress MC degranulation and exhibited limited colonization of the mesenteric lymph nodes. Administration of SptP to sites of Escherichia coli infection markedly enhanced its virulence. Thus, SptP-mediated inactivation of local MCs is a powerful mechanism utilized by S. Typhimurium to impede early innate immunity. This finding provides a logical explanation for why previous attempts by others to demonstrate a protective role for MCs against Salmonella infections have resulted in equivocal results.

Taken together, this work highlights an overlooked virulence mechanism possessed by certain host adapted pathogens to avoid the host's innate immune system. Additionally, this innate immune-quelling property of SptP may hold future promise in tempering harmful inflammatory disorders in the body of an immune competent host.

The Immunity-Related GTPases (IRGs) are a family of dynamin-like proteins found in vertebrates that play critical roles in cell-autonomous resistance to bacteria and protozoa. The IRGs are divided into two subfamilies, with the GMS IRGs exerting a regulatory function over the GKS IRGs, affecting GKS IRG expression, localization, and ultimately function. The profound loss of host resistance seen in mice lacking the GMS protein Irgm1 suggests that GMS IRGs may additionally have broader functions beyond the regulation of GKS IRGs, though the nature of these functions remains poorly understood. In this dissertation, we address the regulatory functions of GMS IRGs in mouse cells.

We first addressed regulation of GKS IRGs (Irga6 and Irgb6) by GMS IRGs (Irgm1 and Irgm3). We found that in both fibroblasts and macrophages lacking these GMS IRGs, that the GKS IRGs relocalized to form punctate structures that were ubiquitin-, p62-, and LC3-positive. A biochemical analysis indicated that the GKS IRGs were directly ubiquitinated through K63 linkages. Collectively, these results suggested that GMS IRGs regulate aggregation of GKS IRGs and their transfer to autophagosomes through one of at least two possible mechanisms -- by the direct association of GMS IRGs with GKS IRGs to block their aggregation that subsequently leads to autophagic removal, and/or by directly promoting autophagic removal of spontaneously forming GKS aggregates. The latter hypothesis was addressed using a series of complementary assays, which ultimately showed that absence of Irgm1 has no effect on the maturation of autophagosomes in fibroblasts, and only a very small and statistically insignificant effect in macrophages. Thus, we conclude that the major mechanism through which GMS IRGs regulate GKS IRGs is by directly inhibiting their aggregation, rather than through general effects on autophagic initiation or maturation of GKS IRG-containing autophagosomes.

We also addressed the possibility of broad regulatory functions of GMS IRGs beyond the regulation of GKS IRGs by examining whether GMS IRGs can affect another family of dynamin-like GTPases, the guanylate-binding proteins (GBPs). Despite no previous evidence of interactions between these two protein families, we found that the absence of GMS IRGs had striking effects on the localization of the murine Gbp2, leading it to colocalize with GKS IRG aggregates formed as a consequence of GMS IRG deficiency. We further demonstrated that unlike the GKS IRGs, Gbp2 was not tagged with K63-linked ubiquitin chains, which might have targeted it for specific macroautophagy, implying that Gbp2 is not aggregating in the absence of Irgm1. We then showed both that Gbp2 forms puncta in the presence of generic protein aggregates, and that guanylate-binding proteins including Gbp2 promote the degradation of GKS IRG protein aggregates. These findings suggest that GMS IRGs do not exert direct control over GBPs, but rather that GBPs are involved in the macroautophagic degradation of protein aggregates as a primary function, and are thus influenced indirectly by GMS IRGs.

In total, our experiments contribute to the understanding of regulatory interactions among GMS IRGs, GKS IRGs, and GBPs. These results will be important in establishing the mechanisms through which these important families of proteins influence eradication of bacterial and protozoan pathogens through key innate immune mechanisms.

Vaccinia virus (VV) is the most thoroughly studied member of the Poxviridae family and the vaccine used to achieve the only successful eradication of a human disease. Over the years, it has proven itself as a useful tool for the study of antiviral immunity, vaccine development, and potentially cancer immunotherapy. VV is capable of eliciting a robust immune response; however the mechanisms by which VV accomplishes this task remain unknown. The overall goal of this thesis project is to determine how VV activates the innate immune system, and how this activation contributes to viral clearance in vivo. We determined that VV or VV-DNA activated the TLR8-MyD88 pathway in plasmacytoid dendritic cells (pDC), resulting in the production of type I interferons (IFN). We also demonstrated that TLR8-mediated production of type I IFN by pDC was crucial to efficient VV control and clearance in vivo. Moreover, we identified the polyA- and polyT-rich sequences in VV-DNA was the possible motif recognize by TLR8. Type I IFN, known for ability to establish the "antiviral state", are also critical mediators of NK cell activation. In the setting of VV infection, we demonstrated that direct action of type I IFN on NK cells, but not accessory cells such as DC, was necessary for NK cell activation in vivo. We further demonstrated that type I IFN-dependent activation of NK cells was required for optimal VV clearance in vivo. Given the importance of NK cells in anti-VV innate immunity, we next examined what role the TLR2-MyD88 pathway, critical for activation of cDC, played in the activation of NK cells. NK cells from TLR2-/- or MyD88-/- mice displayed a reduction in activation and cytolytic function, and this defect was independent of pro-inflammatory cytokine signaling. We were able to demonstrate that direct TLR2 signaling on NK cells was required for their optimal activation and function in response to VV infection. Moreover, we were able to demonstrate that TLR2-MyD88 signaling resulted in the activation of the PI3K-ERK pathway, which was necessary for NK cell cytotoxicity. In addition, we identified the NKG2D pathway as critical for efficient NK cell activation and function in response to VV infection, independent of the TLR2 pathway. Both the NKG2D and TLR2 pathways were crucial for optimal VV clearance and control in vivo. Collectively, this project illuminates the roles and mechanisms of the innate immune system in the control of VV in vivo.

An estimated twenty-five percent of the fifty-seven million annual deaths worldwide can be directly attributed to infectious disease. Mammals contain both adaptive and innate immune systems to deal with invading pathogens. The genetic model organism Caenorhabditis elegans lacks an adaptive immune system, which makes it a powerful model organism to study the innate immune system without the added complexity of an adaptive immune system. Multiple human pathogens can cause lethal infections in C. elegans and several C. elegans innate immune pathways have been identified that are conserved with mammals and protect the nematode from infection. The goal of this work was to identify novel bacterial virulence factors and innate immune defenses against pathogens by using the genetic model organism C. elegans. We established C. elegans as a model for Yersinia pestis infection and used this model to identify novel bacterial virulence factors that were also important for virulence in a mammalian model of infection. Previous studies demonstrated that C. elegans can identify bacterial pathogens using sensory neurons and activate an avoidance response that requires components of G-protein signaling pathways. We screened forty C. elegans strains containing mutations in chemosensory G-protein coupled receptors for altered survival on pathogen and identified npr-1 to be required for full C. elegans defense against pathogens. We found that activation of the NPR-1 nervous circuit enhances host susceptibility to microbial infection while inhibition of the circuit boosts innate immunity. This data provides the first evidence that innate immunity in C. elegans is directly linked to the nervous system and establishes the nematode as a novel system to study neuroimmunology. From this work, we have identified Y. pestis virulence-related genes and C. elegans innate immune effector genes required for innate immunity to human bacterial pathogens.