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Open Access Publications from the University of California

Scholarly Works (14 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2012)

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.

    Cover page: Suicidal Cells and Lipid Storms: Mechanisms and Consequences of Cytokine–independent Inflammasome Activation
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2014)
    Innate type 2 immune cells are activated in response to helminths, allergens, and certain types of proteases and particulates. Recently, innate type 2 immune pathways have also been implicated in protective host responses to homeostatic perturbations, such as metabolic dysfunction, atherosclerosis, and tissue injury. In this context, innate type 2 cytokines stimulate local tissues, recruit eosinophils, and alternatively activate macrophages to restore homeostasis. As the major source of innate interleukin (IL)-5 and IL-13, group 2 innate lymphoid cells are positioned to initiate and maintain homeostatic type 2 responses. The absence of exogenous stimuli in these processes implicates endogenous pathways in the activation of type 2 immunity and suggests an alternative evolutionary trajectory for type 2 immunity, apart from its role in response to helminths and allergens.
      Cover page: I-L-C-2 it: type 2 immunity and group 2 innate lymphoid cells in homeostasis
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2017)
      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.
        Cover page: NAIP/NLRC4 inflammasome activation in MRP8+ cells is sufficient to cause systemic inflammatory disease
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2016)
        Parasitic helminths and allergens induce a type 2 immune response leading to profound changes in tissue physiology, including hyperplasia of mucus-secreting goblet cells and smooth muscle hypercontractility. This response, known as 'weep and sweep', requires interleukin (IL)-13 production by tissue-resident group 2 innate lymphoid cells (ILC2s) and recruited type 2 helper T cells (TH2 cells). Experiments in mice and humans have demonstrated requirements for the epithelial cytokines IL-33, thymic stromal lymphopoietin (TSLP) and IL-25 in the activation of ILC2s, but the sources and regulation of these signals remain poorly defined. In the small intestine, the epithelium consists of at least five distinct cellular lineages, including the tuft cell, whose function is unclear. Here we show that tuft cells constitutively express IL-25 to sustain ILC2 homeostasis in the resting lamina propria in mice. After helminth infection, tuft-cell-derived IL-25 further activates ILC2s to secrete IL-13, which acts on epithelial crypt progenitors to promote differentiation of tuft and goblet cells, leading to increased frequencies of both. Tuft cells, ILC2s and epithelial progenitors therefore comprise a response circuit that mediates epithelial remodelling associated with type 2 immunity in the small intestine, and perhaps at other mucosal barriers populated by these cells.
          Cover page: Tuft-cell-derived IL-25 regulates an intestinal ILC2–epithelial response circuit
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2017)
          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.
            Cover page: Leukotrienes provide an NFAT-dependent signal that synergizes with IL-33 to activate ILC2s
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2018)
            The small intestinal tuft cell-ILC2 circuit mediates epithelial responses to intestinal helminths and protists by tuft cell chemosensory-like sensing and IL-25-mediated activation of lamina propria ILC2s. Small intestine ILC2s constitutively express the IL-25 receptor, which is negatively regulated by A20 (Tnfaip3). A20 deficiency in ILC2s spontaneously triggers the circuit and, unexpectedly, promotes adaptive small-intestinal lengthening and remodeling. Circuit activation occurs upon weaning and is enabled by dietary polysaccharides that render mice permissive for Tritrichomonas colonization, resulting in luminal accumulation of acetate and succinate, metabolites of the protist hydrogenosome. Tuft cells express GPR91, the succinate receptor, and dietary succinate, but not acetate, activates ILC2s via a tuft-, TRPM5-, and IL-25-dependent pathway. Also induced by parasitic helminths, circuit activation and small intestinal remodeling impairs infestation by new helminths, consistent with the phenomenon of concomitant immunity. We describe a metabolic sensing circuit that may have evolved to facilitate mutualistic responses to luminal pathosymbionts.
              Cover page: A Metabolite-Triggered Tuft Cell-ILC2 Circuit Drives Small Intestinal Remodeling
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2017)
              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.
                Cover page: MicroRNA regulation of type 2 innate lymphoid cell homeostasis and function in allergic inflammation
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2013)
                Eosinophils are specialized myeloid cells associated with allergy and helminth infections. Blood eosinophils demonstrate circadian cycling, as described over 80 years ago, and are abundant in the healthy gastrointestinal tract. Although a cytokine, interleukin (IL)-5, and chemokines such as eotaxins mediate eosinophil development and survival, and tissue recruitment, respectively, the processes underlying the basal regulation of these signals remain unknown. Here we show that serum IL-5 levels are maintained by long-lived type 2 innate lymphoid cells (ILC2) resident in peripheral tissues. ILC2 cells secrete IL-5 constitutively and are induced to co-express IL-13 during type 2 inflammation, resulting in localized eotaxin production and eosinophil accumulation. In the small intestine where eosinophils and eotaxin are constitutive, ILC2 cells co-express IL-5 and IL-13; this co-expression is enhanced after caloric intake. The circadian synchronizer vasoactive intestinal peptide also stimulates ILC2 cells through the VPAC2 receptor to release IL-5, linking eosinophil levels with metabolic cycling. Tissue ILC2 cells regulate basal eosinophilopoiesis and tissue eosinophil accumulation through constitutive and stimulated cytokine expression, and this dissociated regulation can be tuned by nutrient intake and central circadian rhythms.
                  Cover page: Type 2 innate lymphoid cells control eosinophil homeostasis
                  • Article
                  • Peer Reviewed
                  UC San Francisco Previously Published Works (2022)
                  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.
                    Cover page: Bile acid–sensitive tuft cells regulate biliary neutrophil influx
                    • Article
                    • Peer Reviewed
                    UC San Francisco Previously Published Works (2022)
                    While the lung bears significant regenerative capacity, severe viral pneumonia can chronically impair lung function by triggering dysplastic remodeling. The connection between these enduring changes and chronic disease remains poorly understood. We recently described the emergence of tuft cells within Krt5+ dysplastic regions after influenza injury. Using bulk and single-cell transcriptomics, we characterized and delineated multiple distinct tuft cell populations that arise following influenza clearance. Distinct from intestinal tuft cells which rely on Type 2 immune signals for their expansion, neither IL-25 nor IL-4ra signaling are required to drive tuft cell development in dysplastic/injured lungs. In addition, tuft cell expansion occurred independently of type I or type III interferon signaling. Furthermore, tuft cells were also observed upon bleomycin injury, suggesting that their development may be a general response to severe lung injury. While intestinal tuft cells promote growth and differentiation of surrounding epithelial cells, in the lungs of tuft cell deficient mice, Krt5+ dysplasia still occurs, goblet cell production is unchanged, and there remains no appreciable contribution of Krt5+ cells into more regionally appropriate alveolar Type 2 cells. Together, these findings highlight unexpected differences in signals necessary for murine lung tuft cell amplification and establish a framework for future elucidation of tuft cell functions in pulmonary health and disease.
                      Cover page: Injury-induced pulmonary tuft cells are heterogenous, arise independent of key Type 2 cytokines, and are dispensable for dysplastic repair
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