NAIP/NLRC4 inflammasome activation in MRP8+ cells is sufficient to cause systemic inflammatory disease.

Inflammasomes are cytosolic multiprotein complexes that initiate protective immunity in response to infection, and can also drive auto-inflammatory diseases, but the cell types and signalling pathways that cause these diseases remain poorly understood. Inflammasomes are broadly expressed in haematopoietic and non-haematopoietic cells and can trigger numerous downstream responses including production of IL-1β, IL-18, eicosanoids and pyroptotic cell death. Here we show a mouse model with endogenous NLRC4 inflammasome activation in Lysozyme2 + cells (monocytes, macrophages and neutrophils) in vivo exhibits a severe systemic inflammatory disease, reminiscent of human patients that carry mutant auto-active NLRC4 alleles. Interestingly, specific NLRC4 activation in Mrp8 + cells (primarily neutrophil lineage) is sufficient to cause severe inflammatory disease. Disease is ameliorated on an Asc -/- background, and can be suppressed by injections of anti-IL-1 receptor antibody. Our results provide insight into the mechanisms by which NLRC4 inflammasome activation mediates auto-inflammatory disease in vivo

Leukotrienes provide an NFAT-dependent signal that synergizes with IL-33 to activate ILC2s.

Group 2 innate lymphoid cells (ILC2s) and type 2 helper T cells (Th2 cells) are the primary source of interleukin 5 (IL-5) and IL-13 during type 2 (allergic) inflammation in the lung. In Th2 cells, T cell receptor (TCR) signaling activates the transcription factors nuclear factor of activated T cells (NFAT), nuclear factor κB (NF-κB), and activator protein 1 (AP-1) to induce type 2 cytokines. ILC2s lack a TCR and respond instead to locally produced cytokines such as IL-33. Although IL-33 induces AP-1 and NF-κB, NFAT signaling has not been described in ILC2s. In this study, we report a nonredundant NFAT-dependent role for lipid-derived leukotrienes (LTs) in the activation of lung ILC2s. Using cytokine reporter and LT-deficient mice, we find that complete disruption of LT signaling markedly diminishes ILC2 activation and downstream responses during type 2 inflammation. Type 2 responses are equivalently attenuated in IL-33- and LT-deficient mice, and optimal ILC2 activation reflects potent synergy between these pathways. These findings expand our understanding of ILC2 regulation and may have important implications for the treatment of airways disease

MicroRNA regulation of type 2 innate lymphoid cell homeostasis and function in allergic inflammation.

MicroRNAs (miRNAs) exert powerful effects on immunity through coordinate regulation of multiple target genes in a wide variety of cells. Type 2 innate lymphoid cells (ILC2s) are tissue sentinel mediators of allergic inflammation. We established the physiological requirements for miRNAs in ILC2 homeostasis and immune function and compared the global miRNA repertoire of resting and activated ILC2s and T helper type 2 (TH2) cells. After exposure to the natural allergen papain, mice selectively lacking the miR-17∼92 cluster in ILC2s displayed reduced lung inflammation. Moreover, miR-17∼92-deficient ILC2s exhibited defective growth and cytokine expression in response to IL-33 and thymic stromal lymphopoietin in vitro. The miR-17∼92 cluster member miR-19a promoted IL-13 and IL-5 production and inhibited expression of several targets, including SOCS1 and A20, signaling inhibitors that limit IL-13 and IL-5 production. These findings establish miRNAs as important regulators of ILC2 biology, reveal overlapping but nonidentical miRNA-regulated gene expression networks in ILC2s and TH2 cells, and reinforce the therapeutic potential of targeting miR-19 to alleviate pathogenic allergic responses

Bile acid–sensitive tuft cells regulate biliary neutrophil influx

Inflammation and dysfunction of the extrahepatic biliary tree are common causes of human pathology, including gallstones and cholangiocarcinoma. Despite this, we know little about the local regulation of biliary inflammation. Tuft cells, rare sensory epithelial cells, are particularly prevalent in the mucosa of the gallbladder and extrahepatic bile ducts. Here, we show that biliary tuft cells express a core genetic tuft cell program in addition to a tissue-specific gene signature and, in contrast to small intestinal tuft cells, decreased postnatally, coincident with maturation of bile acid production. Manipulation of enterohepatic bile acid recirculation revealed that tuft cell abundance is negatively regulated by bile acids, including in a model of obstructive cholestasis in which inflammatory infiltration of the biliary tree correlated with loss of tuft cells. Unexpectedly, tuft cell–deficient mice spontaneously displayed an increased gallbladder epithelial inflammatory gene signature accompanied by neutrophil infiltration that was modulated by the microbiome. We propose that biliary tuft cells function as bile acid–sensitive negative regulators of inflammation in biliary tissues and serve to limit inflammation under homeostatic conditions

Suicidal Cells and Lipid Storms: Mechanisms and Consequences of Cytokine–independent Inflammasome Activation

To protect against infectious disease, the innate immune system must mount a rapid response that distinguishes between self and non-self, and between pathogen and harmless commensal. Invasion of the host cell cytosol, either directly or through the injection of effector proteins, is a uniquely pathogen-associated event. Therefore, the innate immune system includes numerous cytosolic sensors to detect foreign molecules or the disruption of intracellular homeostasis. Among these sensors is the NBD–LRR family of proteins, which oligomerize into large complexes called “inflammasomes”, and serve as a molecular platform for the activation of the protease CASPASE–1. The outcome of innate sensing in the cytosol is determined by the downstream effector functions of each pathway. In most cases, this occurs through the transcriptional induction of effector proteins, such as inflammatory and chemotactic cytokines. In contrast, inflammasome effector functions are carried out by CASPASE–1 in the absence of de novo transcription or translation, making inflammasome activation one of the most rapid innate signaling responses. Experiments in mice deficient for CASPASE–1 or other inflammasome components, have revealed the importance of inflammasome activation in restricting bacterial pathogens in vivo. Bacterial restriction is achieved by the effector functions of CASPASE–1, the best characterized of which are the processing and secretion of the inflammatory cytokines IL–1β and IL–18, and the induction of a lytic cell death called pyroptosis. The relative contribution of CASPASE–1 effector functions to immunity varies depending on the disease model; however, in several cases inflammasome-dependent protection is completely retained in IL–1β/IL–18–/– mice, suggesting cytokine-independent inflammasome functions. In this dissertation I explore the molecular mechanisms that regulate cytokine-independent inflammasome activity, and describe a novel CASPASE–1 effector function in vivo. The dissertation begins with an overview of inflammasome activation and function, highlighting some of the important areas for future research. Next, I describe a full-genome siRNA screen we undertook to identify novel proteins required for inflammasome signaling, and in particular for pyroptosis. Although the screen led to the unbiased identification of known inflammasome components, all novel candidate genes failed to validate after further analysis. Perhaps this negative result indicates a functional redundancy among the CASPASE–1 substrates required for pyroptosis. In chapter 3, we examine how autoproteolysis determines the effector functions of CASPASE–1. We show that a non-cleavable allele of CASPASE–1 retains the ability to initiate pyroptosis, but fails to process IL-1β/IL–18. This finding explains the previous observation that in the absence of the inflammasome adaptor protein ASC, inflammasome activation leads to cell death without release of cytokines. Furthermore, we show that pyroptosis and cytokine processing can be initiated from spatially and structurally distinct inflammasome complexes, suggesting a mechanism for tuning the outcome of CASPASE–1 activation. In chapter 4, we identify a novel CASPASE–1 effector function using an in vivo model of inflammasome activation. We show that systemic cytosolic delivery of flagellin, a potent inflammasome agonist, leads to massive vascular fluid loss and can kill mice in less than 30 minutes. This unexpected response is dependent on CASPASE–1, but independent of IL–1β and IL–18. Instead, CASPASE–1 activation leads to a cellular calcium influx that triggers the biosynthesis of eicosanoids — a family of inflammatory lipids that includes the prostaglandins and leukotrienes. Mice deficient in COX–1, an enzyme required for synthesis of prostaglandins, are resistant to the rapid pathology caused by inflammasome activation. Our findings therefore link the rapid sensing capacity of inflammasomes to the potent activity of eicosanoids. Activated within minutes, this pathway represents one of the most rapid cellular innate immune responses described to date, and suggests a model for the initiation of inflammation at the site of infection. In the 5th and final chapter, I discuss this model and other emerging themes in inflammasome research, highlighting several mouse models I have developed to uncover additional inflammasome functions in vivo. Although to date the emphasis has been on IL–1β and IL–18, the findings reported in this dissertation highlight how much there is to learn about the cytokine–independent functions of inflammasomes