Intracellular innate immune receptors: Life inside the cell

Abstract

The innate immune system is the critical first line of defense against infectious and sterile insults. A cell’s ability to sense these insults relies on a series of germline-encoded receptors, generally referred to as pattern recognition receptors (PRRs). PRRs are responsible for recognizing unique molecular patterns from microbes known as pathogen-associated molecular patterns (PAMPs) and endogenous molecules released from damaged and dying cells known as damage-associated molecular patterns (DAMPs). There are several different PRRs found throughout the cell, including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), nucleotide-binding domain, leucine-rich repeat-containing (or NOD-like) receptors (NLRs), absent in melanoma 2 (AIM2), IFI16, pyrin, Z-DNA-binding protein 1 (ZBP1), retinoic acid-inducible gene I (RIG-I), MDA5, and many more. PRRs can be found on the membrane, in the cytosol, and in the nucleus. Some PRRs can induce the formation of a multiprotein complex called the inflammasome that leads to the processing and release of the proinflammatory cytokines IL-1β and IL-18 and cell death in the form of pyroptosis. Within the NLR family of PRRs, there are some proteins that form an inflammasome, such as NLRP1, NLRP3, and NLRC4, and some that do not, such as NLRC1 and NLRC2 (NOD1 and NOD2). AIM2 and pyrin are also well-established as sensors that form an inflammasome. Whether they form inflammasomes or not, PRRs are each important for sensing their respective ligands and initiating signaling pathways that drive gene expression, protein production, cytokine and chemokine release, and cell death while also shaping the adaptive immune response, dictating the overall fitness of the immune system. Within the cell, membrane-bound PRRs are responsible for sensing external insults, while intracellular cytosolic and nuclear PRRs are essential for detecting intracellular pathogens or alterations in cellular homeostasis. In this issue of Immunological Reviews, we explore the intracellular innate immune receptors, characterizing their sensing and signaling pathways and detailing their diverse roles in health and disease. We also describe the therapeutic implications of modulating these pathways. Downstream of PRR sensing of PAMPs and DAMPs, several signaling cascades are initiated (Figure 1). These pathways lead to proinflammatory cytokine and chemokine secretion and cell death, among other outcomes. Among the intracellular PRRs, some have the ability to form a multiprotein complex known as the inflammasome. NLRP1, NLRP3, NLRC4, AIM2, and pyrin are well-characterized to form inflammasomes, while several other receptors have also been recognized to form an inflammasome in context-dependent manners. The inflammasome is typically composed of a sensor, the adaptor protein ASC, and the effector protein caspase-1. Inflammasome assembly provides the platform for autocatalytic cleavage and activation of caspase-1. Activated caspase-1 can then go on to cleave pro–IL-1β and pro–IL-18 into their active forms and gasdermin D to release its N-terminus. The gasdermin D N-terminus forms pores in the membrane to execute an inflammatory form of cell death known as pyroptosis and allows the release of IL-1β and IL-18, along with other cellular contents. NLRP1 (NLR family, pyrin domain-containing 1) was the first PRR to be identified to form an inflammasome,1 although much about its biology has remained unclear since that discovery in 2002. NLRP1 is unique among the NLRs in that it contains a C-terminal function-to-find (FIIND) domain that undergoes autoproteolysis before inflammasome activation. This produces two fragments that remain associated, preventing inflammasome formation until an additional stimulus is received. Recently, it has been shown that NLRP1 is activated in response to the Bacillus anthracis lethal toxin and Toxoplasma gondii, among other stimuli. NLRP1 activation requires the stimulus to induce proteasome-mediated degradation of its N-terminus, freeing the C-terminal region to form the inflammasome. The review by Taabazuing et al discusses NLRP1 and the related molecule CARD8, which shares the FIIND domain and this proteasome-mediated activation mechanism.2 The authors describe the mechanistic details of inflammasome activation driven by these molecules and also explore how mutations in these sensors can contribute to diseases, such as vitiligo, Addison’s disease, and celiac disease.2 The full details of NLRP1 activation remain to be characterized, though, as both direct and indirect activators of NLRP1 have been identified with no clear link between their mechanisms. NLRP3 has been the most well-studied inflammasome sensor. NLRP3 was first found to be activated under physiological conditions in response to bacterial components,3 uric acid crystals,4 and LPS and ATP5 in 2006. Since then, it has been found that NLRP3 can be activated in response to several PAMPs from infectious agents, including bacteria, viruses, fungi, and parasites, and a number of DAMPs, such as cholesterol crystals. While the number of NLRP3-activating stimuli continues to grow, suggesting that NLRP3 is a global sensor of PAMPs and DAMPs, a unifying mechanism to describe these activation processes is yet to be identified. Nonetheless, the field has made great strides in delineating many of the molecular details of the NLRP3 pathway. NLRP3 was first found to be activated by viral products in 2006, shortly after the initial descriptions of the NLRP3 inflammasome.6 It was later found that ZBP1 acts as the innate immune sensor to trigger NLRP3 activation during influenza A virus (IAV) infection.7 My own review with Zheng provides a general overview of the NLRP3 inflammasome and its activation and then focuses on the ZBP1-NLRP3 inflammasome.8 This inflammasome is known to form in response to ZBP1 sensing Z-nucleic acids, which occurs during IAV infection.7 When the Zα2 domain, the portion of ZBP1 responsible for Z-nucleic acid sensing, is deleted, NLRP3 inflammasome activation in response to IAV is abolished.9 In our review, we detail the role of the ZBP1-NLRP3 inflammasome in inflammatory cell death and describe the concept of PANoptosis, a form of inflammatory cell death that involves the extensive crosstalk and coregulation between pyroptosis, apoptosis, and necroptosis.7, 10-21 In addition to forming the ZBP1-NLRP3 inflammasome, ZBP1 is involved in the formation of the ZBP1 PANoptosome to initiate PANoptosis.10, 19 The ZBP1-NLRP3 inflammasome, ZBP1 PANoptosome, and downstream process of PANoptosis are critical for host defense during IAV infection.7, 9, 19, 22 This protective role may also extend to other infections or stimuli, although additional studies are required to elucidate these functions. In addition to the canonical mechanism of NLRP3 inflammasome activation, an alternative mode, known as “noncanonical activation,” can be driven by the molecule caspase-11 in mice and caspase-4/5 in humans. Caspase-11 is activated in response to lipopolysaccharide (LPS). When caspase-11 senses cytoplasmic LPS, it undergoes autoproteolytic cleavage.23 The activated caspase-11 can then cleave gasdermin D, initiating pyroptosis and altering the ion levels in the cell to trigger NLRP3 inflammasome activation.24, 25 Abu Khweek and Amer provide a comprehensive discussion of both the pyroptotic and non-pyroptotic functions and caspase-11, including the regulatory mechanisms that exist to control its activation and an extensive characterization of ligands in addition to LPS that activate caspase-11.26 Furthermore, the authors describe the role of caspase-11/4/5 in allergy, where evidence suggests they are detrimental, and asthma, where they appear to be beneficial.26 While recent studies have begun to characterize the mechanisms of caspase-11 signaling, much work remains to be done to fully understand caspase-11, including identification of additional triggers and interacting proteins to fully establish its effector functions. As we have seen with IAV infection, it is well established that NLRP3 activation is crucial for killing infectious pathogens and removing infected or damaged cells. However, excessive activation of the NLRP3 inflammasome can induce pathological inflammation and have significant negative effects. The review by Harrington and Gurung highlights these divergent roles for NLRP3 inflammasome activation specifically in the context of Leishmania infection.27 The authors discuss the conflicting literature that reports both protective and pathogenic roles for the NLRP3 inflammasome during leishmaniasis and analyze the existing data to reconcile these divergent outcomes. On one hand, NLRP3 has been shown to be important for restricting the multiplication and spread of Leishmania.28 However, other studies have shown that the inflammasome is not required for clearance of the parasites, and IL-18 cytokine release downstream of NLRP3 inflammasome activation propagates a Th2 response and IL-4 secretion, which is detrimental to the host.29 These different outcomes have likely been the result of variations in experimental conditions, with different strains of mice and species of Leishmania being used. The lack of clarity regarding the role of NLRP3 in Leishmaniasis requires additional study. But more generally, the tug-of-war between pathogen clearance and excess inflammation and immunopathology caused by NLRP3 inflammasome activation is indicative of a broader immunological theme and reflects the fine line the immune system must walk to ensure optimal responses are achieved. Due to this inflammation, mutations linked to aberrant NLRP3 activation are associated with a range of inflammatory diseases, including cryopyrin-associated periodic syndromes (CAPS). Improved understanding of the NLRP3 molecular pathways will be important for restoring balance in the immune system and preventing excess inflammation. A third NLR protein that is established in inflammasome formation is NLRC4, although its regulation is not well characterized compared with that of NLRP3. NLRC4 is unique in that it requires another member of the NLR family, NAIP proteins, for its activation. The NAIPs provide ligand specificity and recognize flagellin and proteins of the type III secretion system of many Gram-negative bacteria.30, 31 After sensing their respective ligand, activated NAIPs bind NLRC4 to initiate inflammasome assembly. The expression of both NLRC4 and NAIPs is controlled by IRF8.32 The review by Kay et al comprehensively describes the mechanisms of NAIP-NLRC4 inflammasome activation and details the cell type-specific consequences of this activation.33 As this inflammasome has also been implicated in a wide array of diseases, the authors cover the extensive literature describing at times contradictory roles of these molecules in disease, particularly cancer.33 Even within the same tumor model, inconsistent results have been observed, highlighting the complexity of studying these innate immune pathways. Due to differences between mice and humans, particularly regarding the repertoire of NAIP proteins (mice have 7, while humans have only 1), more work needs to be done to understand how NLRC4 and NAIPs sense and activate in humans. This may shed light on some of the contradictory data and provide therapeutic targets for patients with NLRC4 and NAIP mutations and autoinflammatory diseases or cancer. AIM2 is a nucleic acid sensor that can respond to a wide variety of pathogens, including bacteria, viruses, and fungi. In response to infection, the transcription factor IRF1 upregulates the expression of guanylate binding proteins (GBPs) and IRGB10, and localization of these proteins to bacterial cell membranes ruptures the bacteria and releases ligands for AIM2 and the noncanonical NLRP3 inflammasome to sense.34, 35 Beyond its roles in sensing pathogens, AIM2 has also been shown to sense self-DNA and be involved in inflammatory disease, autoimmunity, and cancer. The review by Kumari et al covers the most recent advances in our understanding of the functions and mechanisms of AIM2 inflammasome activation and intracellular signaling.36 The authors also focus on the crosstalk between AIM2 and other DNA sensing pathways, which is important for host-pathogen interactions. While the role of AIM2 in pathogenic infection is fairly well characterized, its functions in cancers, particularly those associated with viral infections, are less clear. Improved understanding of how AIM2 could be used as an anti-cancer sensor will be important for chemotherapeutic applications and cancer prevention. Pyrin is unique in that it does not sense pathogens directly but instead responds to inactivation of RhoA. Under homeostatic conditions, pyrin is phosphorylated by RhoA-activated kinases, and this phosphorylation allows the inhibitory protein 14-3-3 to bind.37, 38 However, several bacteria, such as Clostridium difficile and Yersinia species, produce proteins that inactivate Rho, which allows pyrin to dissociate from 14-3-3 to form the inflammasome.39, 40 The review by Malik and Bliska discusses the mechanistic details of pyrin inflammasome formation and regulation, particularly in the context of Yersinia effector proteins.41 Yersinia produces proteins that both activate and inhibit the pyrin inflammasome, and the authors cover the details of these pathways.41 Additionally, pyrin has been linked to several diseases. Mutations in pyrin are associated with the autoinflammatory disease familial Mediterranean fever (FMF), which is driven by IL-1β and caspase-1 downstream of pyrin inflammasome activation in mouse models.42 Also, pyrin is protective during colorectal cancer, as it drives IL-18 production to regulate intestinal barrier integrity.43 The diverse roles of pyrin in disease are just beginning to be elucidated, and more work needs to be done to fully understand its impact. Beyond the protective and pathogenic functions of inflammasome sensors in infectious and inflammatory diseases and cancers discussed above, these sensors can also play key roles in metabolic diseases. Anand discusses the role of inflammasome sensors, particularly NLRP3 and AIM2, in detecting perturbations in metabolic pathways, focusing on how lipids can be involved in inflammasome regulation and contribute to diseases.44 Dysregulated cholesterol metabolism can activate the NLRP3 and AIM2 inflammasomes, and NLRP3 can sense a variety of metabolic DAMPs, including cholesterol crystals and oxidized mtDNA.44 The functions of lipid metabolism are diverse and complex, and understanding these functions has significant implications for diseases that are driven by dysregulated metabolism, such as obesity, atherosclerosis, and diabetes. Due to the diverse roles of inflammasome sensors and their downstream signaling pathways in infectious, inflammatory, and metabolic diseases and cancers, molecules in these pathways have been enticing targets for therapeutic modulation. Chauhan et al discuss several of the molecules that have been targeted clinically and others that are being considered in preclinical trials.45 Targets of interest feature downstream molecules shared between the inflammasome pathways, including IL-1β, IL-18, caspase-1, and gasdermin D, and the inflammasome sensors themselves, such as NLRP3. Historically, IL-1β has been one of the most extensively targeted molecules, with several IL-1–targeted therapies obtaining FDA approval. Canakinumab, an anti–IL-1β antibody, has been approved for the treatment of CAPS and was recently evaluated in the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS). Here, it was found that, in addition to reducing the risk of cardiovascular events and mortality, canakinumab reduces the incidence of lung cancer.46, 47 Findings such as this reinforce the systemic importance of appropriately regulating inflammasome activation and downstream signaling in health and disease. Therapeutic modulation often has the goal of inhibiting inflammasomes to regain control of dysregulated pathways and reduce inflammatory signaling. This inhibition can provide benefits for patients with a variety of infectious, autoimmune, and metabolic diseases and cancers. However, it is important to remember that inflammasomes have protective roles in many infections and even in cancer, so developing treatments that can activate inflammasomes will also be important for these The goal of treatment be to optimal inflammasome activation while preventing and systemic inflammation. these two outcomes is often and more work is to understand how to this in Beyond the inflammasomes, there are also several intracellular PRRs that are crucial for innate immune NLRC1 and also known as and the first intracellular sensors of bacterial to be and the details of their molecular pathways have been extensively since After sensing NLRC1 and NLRC2 activate inflammatory signaling the and signaling pathways as well as the production of type I The review by and our understanding of how NLRC1 and NLRC2 sense microbes and cellular The authors also focus on their downstream signaling pathways and detail how these proteins play a role in adaptive and regulate host NLRC1 and NLRC2 have also been implicated in a number of inflammatory and autoinflammatory diseases, including type 1 and 2 atherosclerosis, and the understanding of their sensing, the mechanisms how NLRC1 and NLRC2 contribute to inflammatory disease remain to be fully In addition to cytosolic PRRs, nuclear sensors play a key role in detecting intracellular perturbations and homeostasis. 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