Host survival during plague, caused by the Gram-negative bacterium Yersinia pestis, is favored by a robust early innate immune response initiated by IL-1β and IL-18. Precursors of these cytokines are expressed downstream of TLR signaling and are then enzymatically processed into mature bioactive forms, typically by caspase-1 which is activated through a process dependent on multi-molecular structures called inflammasomes. Y. pestis evades immune detection in part by using a Type three secretion system (T3SS) to inject effector proteins (Yops) into host cells and suppress IL-1β and IL-18 production. We investigated the cooperation between two effectors, YopM and YopJ, in regulating inflammasome activation, and found that Y. pestis lacking both YopM and YopJ triggers robust caspase-1 activation and IL-1Β/IL-18 production in vitro. Furthermore, this strain is attenuated in a manner dependent upon caspase-1, IL-1β and IL-18 in vivo, yet neither effector appears essential for full virulence. We then demonstrate that YopM fails to inhibit NLRP3/NLRC4 mediated caspase-1 activation and is not a general caspase-1 inhibitor. Instead, YopM specifically prevents the activation of a Pyrin-dependent inflammasome by the Rho-GTPase inhibiting effector YopE. Mutations rendering Pyrin hyperactive are implicated in the autoinflammatory disease Familial Mediterranean Fever (FMF) in humans, and we discuss the potential significance of this disease in relation to plague. Altogether, the Y. pestis T3SS activates and inhibits several inflammasome pathways, and the fact that so many T3SS components are involved in manipulating IL-1β/IL-18 underscores the importance of these mechanisms in plague.

Yersinia pestis, the causative agent of plague, is estimated to have claimed the lives of 30-50% of the European population in five years. Although it can now be controlled through antibiotics, there are still lurking dangers of outbreaks from biowarfare and bioterrorism; therefore, ongoing research to further our understanding of its strong virulence factors is necessary for development of new vaccines. Many Gram-negative bacteria, including Y. pseudotuberculosis, the evolutionary ancestor of Y. pestis, produce a hexa-acylated lipid A/LPS which can strongly trigger innate immune responses via activation of Toll-like receptor 4 (TLR4)-MD2. In contrast, Y. pestis grown at 37ºC generates a tetra-acylated lipid A/LPS that poorly induces TLR4-mediated immune activation. We have reported that expression of E. coli lpxL in Y. pestis, which lacks a homologue of this gene, forces the biosynthesis of a hexa-acylated LPS, and that this single modification dramatically reduces virulence in wild type mice, but not in mice lacking a functional TLR4. This emphasizes that avoiding activation of innate immunity is important for Y. pestis virulence. It also provides a model in which survival is strongly dependent on innate immune defenses, presenting a unique opportunity for evaluating the relative importance of innate immunity in protection against bacterial infection. TLR signaling is critical for the sensing of pathogens, and one implication of TLR4 engagement is the induction of the pro-forms of the potent inflammatory cytokines IL-1β and IL-18. Therefore Y. pestis is able to suppress production of these which are generated through caspase-1-activating nucleotide-binding domain and leucine-rich repeat (NLR)-containing inflammasomes. For my thesis, I sought to elucidate the role of NLRs and IL-18/IL-1β during bubonic and pneumonic plague infection. Mice lacking IL-18 signaling led to increased susceptibility to wild type Y. pestis, and an attenuated strain producing a Y. pseudotuberculosis-like hexa-acylated lipid A. I found that the NLRP12, NLRP3 and NLRC4 inflammasomes were important protein complexes in maturing IL-18 and IL-1β during Y. pestis infection, and mice deficient in each of these NLRs were more susceptible to bacterial challenge. NLRC4 and NLRP12 also directed interferongamma production via induction of IL-18 against plague, and minimizing inflammasome activation may have been a central factor in evolution of the high virulence of Y. pestis. This is also the first study that elucidated a pro-inflammatory role for NLRP12 during bacterial infection.

L'inflammasome est complexe protéique intracellulaire de l'immunité innée permettant la reconnaissance de pathogènes intracellulaires. NLRC4, un Nod-like récepteur permettant l'activation de l'inflammasome est impliqué dans la reconnaissance du flagelle ainsi que du système de sécrétion de type 3 (SST3) de Pseudomonas aeruginosa, une bactérie majoritairement extracellulaire. Nous avons donc déterminer l'impact de la reconnaissance de P. aeruginosa par le NLRC4-inflammasome in vivo dans un modèle murin de pneumonie aiguë. De façon surprenante, l'activation du NLRC4-inflammasome par le SST3 de P. aeruginosa contribue à diminuer la survie de l'hôte en diminuant la clairance bactérienne pulmonaire et en augmentant la lésion pulmonaire induite. En effet, la perte de l'activation de l'inflammasome chez les souris NLRC4/- permet d'une part, une réponse précoce méfiée par l'IL-17A. Cette réponse dépendant de l'IL-17A conduit à une expression majeure de peptides antimicrobiens par l'épithélium pulmonaire et diminue la lésion pulmonaire en diminuant le recrutement des cellules immunitaires inflammatoires. L'administration d'IL-18 recombinante murine ou l'inhibition de cette voie par un anticorps anti-IL-17A inhibe cette réponse IL-17A dépendante. Ces résultats mettent en évidence un nouveau rôle du SST3 de P. aeruginosa, qui en plus de son effet cytotoxique et de la translocation d'exotoxines, permet d'activer l'inflammasome pour échapper à la réponse immunitaire innée de l'hôte en inhibant une voie IL-17 dépendante.

Sterile particles underlie the pathogenesis of numerous inflammatory diseases. These diseases can often become chronic and debilitating. Moreover, they are common, and include silicosis (silica), asbestosis (asbestos), gout (monosodium urate), atherosclerosis (cholesterol crystals), and Alzeihmer’s disease (amyloid Aβ). Central to the pathology of these diseases is a repeating cycle of particle-induced cell death and inflammation. Macrophages are the key cellular mediators thought to drive this process, as they are especially sensitive to particle-induced cell death and they are also the dominant producers of the cytokine responsible for much of this inflammation, IL-1β. In response to cytokines or microbial cues, IL-1β is synthesized in an inactive form (pro-IL-1β) and requires an additional signal to be secreted as an active cytokine. Although a multimolecular complex, called the NLRP3 inflammasome, controls the activation/secretion of IL-1β (and has been thought to also control cell death) in response to particles in vitro, the in vivo inflammatory response to particles occurs independently of inflammasomes. Therefore, I sought to better understand the mechanisms governing IL-1β production and cell death in response to particles, focusing specifically on the role of lysosomal cathepsin proteases. Inhibitor studies have suggested that one of these proteases, cathepsin B, plays a role in promoting inflammasome activation subsequent to particle-induced lysosomal damage, however genetic models of cathepsin B deficiency have argued otherwise. Through the use of inhibitors, state-of-the-art biochemical tools, and multi-cathepsin-deficient genetic models, I found that multiple redundant cathepsins promote pro-IL-1β synthesis as well as particle-induced NLRP3 activation and cell death. Importantly, I also found that particle-induced cell death does not depend on inflammasomes, suggesting that this may be why inflammasomes do not contribute to particle-induced inflammation in vivo. Therefore, my observations suggest that cathepsins may be multifaceted therapeutic targets involved in the two key pathological aspects of particle-induced inflammatory disease, IL-1β production and cell death.

The innate immune system is an evolutionarily conserved primary defense system against microbial infections. One of the central components of innate immunity are the pattern recognition receptors which sense infection by detecting various conserved molecular patterns of pathogens and trigger a variety of signaling pathways. In this dissertation, the signaling pathways of several classes of these receptors were dissected. In chapters II and III, the role of two NOD-like receptors, NLRP3 and NLRC4 were investigated in the context of infection with the fungal pathogen, C. albicans. C. albicans is an opportunistic pathogen that causes diseases mainly in immunocompromised humans and innate immunity is critical to control the infection. In chapters II and III, we demonstrate that a multiprotein-inflammasome complex formed by the NLR protein, NLRP3 and its associated partners, ASC and caspase-1 are critical for triggering the production of mature cytokine IL-1β in response to C. albicans. NLRC4, another inflammasome forming NLR that is activated by intracellular bacterial pathogens, was not required for this process in macrophages. Thus, our data indicates that NLRP3 inflammasome responds to fungal infections in addition to its known stimuli such as bacterial and viral infections, toxic, crystalline and metabolic signals. Interestingly, this NLRP3 dependent inflammasome response was maintained even when the pathogen is not viable, and is either formalin fixed or heat-killed (HK). Hence, in chapter III, we examined β-glucans, a structural cell wall component, as the potential immunostimulatory component of C. albicans and dissected the inflammasome responses to β -glucans. We observed that NLRP3-ASC-caspase-1 inflammasome was critical for commercially obtained particulate β-glucans similar to the case of C. albicans. β-glucan sensing C-lectin receptor dectin-1 and the complement receptor CR3 mediated inflammasome activation, IL-1β production in response to the glucan particles. Interestingly, CR3 which recognizes glucans as well as complement opsonized pathogens was strongly required for HK C. albicans induced IL-1β, and partially required for that of live C. albicans, while dectin-1 was not required. Consistent with the receptor studies, blocking of β -glucan receptors by pre-incubating cells with nonstimulatory, soluble glucans led to decreased IL-1β production in response to HK C. albicanswith no effect on IL-1β in response to the live fungus. Dectin-1, CR3 and β-glucan sensing also triggered a moderate dendritic cell death response to β-glucans and HK C. albicans. Live C. albicans induced cell death requires phagocytosis but not the inflammasome, β-glucan sensing, dectin-1 or CR3. The Drosophila caspase-8 like molecule DREDD plays an essential, nonapoptotic role in the Drosophila NF-κB pathway called the ‘IMD’ pathway. Owing to the remarkable evolutionary conservation between Drosophila and mammalian innate immune NF-κB pathways, we explored the potential role of caspase-8 in inflammasomes and in TLR signaling. Using casp8-/- Rip3-/- macrophages and dendritic cells, we observed that caspase-8, specifically augments β-glucan and HK C. albicans induced IL-1β as well as cell death in a caspase-1 independent manner, but not that of live C. albicans, in chapter III. We also found that caspase-8 differentially regulates TLR4 and TLR3 induced cytokine production (chapter IV). Caspase-8 specifically promotes TLR4 induced production of cytokines such as TNF, IL-1β in response to LPS and E. coli. On the other hand, caspase-8 negatively regulates TRIF induced IFNβ production in TLR4 and TLR3 signaling in response to LPS and dsRNA. Caspase-8 executed a similar mode of regulation of the cytokine RANTES in MEFs, in part, by collaborating with RIP3. Strikingly, caspase-8 deficiency alone triggers higher macrophage death and IL-1β production in response to TLR ligands, due to the presence of RIP3. Thus, in addition to its conventional roles in apoptosis, caspase-8 modulates TLR4 and TLR3 induced cytokine production and prevents RIP3 mediated hyper inflammation in response to TLR signals. Together, our findings provide valuable information on fungal pattern recognition and inflammasome pathways and define the contribution of β-glucan sensing to C. albicans induced inflammasome responses. In addition, we demonstrate how caspase-8 adds a layer of specificity to inflammasome as well as TLR signaling. Overall, these results also shed light on the cross talk between death signaling components and innate immune pathways to mount a specific and potentially effective innate immune response against microbial pathogens.

The innate immune system provides an essential first line of defense against infection. Innate immune cells detect pathogens through several classes of Pattern Recognition Receptors (PRR) allowing rapid response to a broad spectrum of infectious agents. Activated receptors initiate signaling cascades that lead to the production of cytokines, chemokines and type I interferons all of which are vital for controlling pathogen load and coordinating the adaptive immune response. Detection of nucleic acids by the innate immune system has emerged as a mechanism by which infection is recognized. Recognition of DNA is complex, influenced by sequence, structure, covalent modification and subcellular localization. Interestingly certain synthetic oligodeoxynucleotides comprised of the TTAGGG motif inhibit proinflammatory responses in a variety of disease models. These suppressive oligodeoxynucleotides (sup ODN) have been shown to directly block TLR9 signaling as well as prevent STAT1 and STAT4 phosphorylation. Recently AIM2 has been shown to engage ASC and assemble an inflammasome complex leading to the caspase-1-dependent maturation of IL-1β and IL-18. The AIM2 inflammasome is activated in response to cytosolic dsDNA and plays an important role in controlling replication of murine cytomegalovirus (MCMV). In the second chapter of this thesis, a novel role for the sup ODN A151 in inhibiting cytosolic nucleic acid sensing pathways is described. Treatment of dendritic cells and macrophages with the A151 abrogated type I IFN, TNF-α and ISG induction in response to cytosolic dsDNA. A151 also reduced INF-β and TNF-α induction in BMDC and BMDM responding to the herpesviruses HSV-1 and MCMV but had no effect on the responses to LPS or Sendai virus. In addition, A151 abrogated caspase-1-dependent IL-1β and IL-18 maturation in dendritic cells stimulated with dsDNA and MCMV. Although inhibition of interferon-inducing pathways and inflammasome assembly was dependent on backbone composition, sequence differentially affected these pathways. While A151 more potently suppressed the AIM2 inflammasome, a related construct C151, proved to be a more potent inhibitor of interferon induction. A151 suppressed inflammasome signaling by binding to AIM2 and competing with immune-stimulatory DNA. The interaction of A151 and AIM2 prevented recruitment of the adapter ASC and assembly of the macromolecular inflammasome complex. Collectively, these findings reveal a new route by which suppressive ODNs modulate the immune system and unveil novel applications for suppressive ODNs in the treatment of infectious and autoimmune diseases. The innate immune response to HSV-1 infection is critical for controlling early viral replication and coordinating the adaptive immune response. The cytokines IL-1β and IL-18 are important effector molecules in the innate response to HSV-1 in vivo. However, the PRRs responsible for the production and maturation of these cytokines have not been fully defined. In the third chapter of this thesis, The TLR2-MyD88 pathway is shown to be essential for the induction of pro-IL-1β transcription in dendritic cells and macrophages responding to HSV-1. The HSV-1 immediate-early protein ICP0 has previously been shown to block TLR2 responses and in keeping with this finding, ICP0 blocked pro-IL-1β expression. Following translation, pro-IL-1β exists as an inactive precursor that must be proteolytically cleaved by a multiprotein complex known as the inflammasome to yield its active form. Inflammasomes are composed of cytoplasmic receptors such as NLRP3 or AIM2, the adapter molecule ASC, and pro-caspase-1. In the present study we found that the NLRP3 inflammasome is important for maturation of IL-1β in macrophages and dendritic cells responding to HSV-1. In contrast the related NLRP12 protein controls IL-1β production in neutrophils. These data indicate that sensing of HSV-1 by TLR2 drives pro-IL-1β transcription and infection activates the inflammasome to mature this cytokine. Moreover, these studies reveal cell type-specific roles for NLRP3 and NLRP12 in inflammasome assembly.