Intracellular innate immune receptors: Life inside the cell

Introduction

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.

Inflammasomes

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.

Figure 1.

Intracellular innate immune sensors and their downstream signaling pathways. This figure provides a graphical summary of the reviews in this collection and depicts the various cytosolic and nuclear pattern recognition receptors that are discussed, along with their key signaling pathways. AIM2, absent in melanoma 2; ASC, apoptosis-associated speck-like protein containing a CARD; CARD, caspase activation and recruitment domain; CASP, caspase; FADD, fas-associated death domain; GBPs, guanylate binding proteins; GSDMD, gasdermin D; MDA5, melanoma differentiation-associated protein 5; MLKL, mixed lineage kinase domain-like pseudokinase; IRF, interferon regulatory factor; NLRs, nucleotide-binding domain, leucine-rich repeat-containing (or NOD-like) receptors; pMLKL, phospho-MLKL; RIG-I, retinoic acid-inducible gene I; RIPK, receptor-interacting serine/threonine-protein kinase; TLRs, Toll-like receptors; ZBP1, Z-DNA-binding protein 1

NLRP1

NLRP1 (NLR family, pyrin domain-containing 1) was the first PRR to be identified to form an inflammasome¹, 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 mechanism². 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 disease². 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

NLRP3 has been the most well-studied inflammasome sensor. NLRP3 was first found to be activated under physiological conditions in response to bacterial components³, uric acid crystals⁴, 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 inflammasome⁶. It was later found that ZBP1 acts as the innate immune sensor to trigger NLRP3 activation during influenza A virus (IAV) infection⁷. My own review with Zheng provides a general overview of the NLRP3 inflammasome and its activation and then focuses on the ZBP1-NLRP3 inflammasome⁸. This inflammasome is known to form in response to ZBP1 sensing Z-nucleic acids, which occurs during IAV infection⁷. When the Zα2 domain, the portion of ZBP1 responsible for Z-nucleic acid sensing, is deleted, NLRP3 inflammasome activation in response to IAV is abolished⁹. 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 cleavage²³. 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-11²⁶. 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 beneficial²⁶. 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 infection²⁷. 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 Leishmania²⁸. 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 host²⁹. 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.

NLRC4

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 are controlled by IRF8³². The review by Kay et al. comprehensively describes the mechanisms of NAIP-NLRC4 inflammasome activation and details the cell type-specific consequences of this activation³³. 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 cancer³³. 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

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 signaling³⁶. 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

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 proteins⁴¹. Yersinia produces proteins that both activate and inhibit the pyrin inflammasome, and the authors cover the details of these pathways⁴¹. 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 models⁴². Also, pyrin is protective during colorectal cancer, as it drives IL-18 production to regulate intestinal barrier integrity⁴³. 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.

Inflammasomes in metabolic disease

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 diseases⁴⁴. Dysregulated cholesterol metabolism can activate the NLRP3 and AIM2 inflammasomes, and NLRP3 can sense a variety of metabolic DAMPs, including cholesterol crystals and oxidized mtDNA⁴⁴. The functions of lipid metabolism are diverse and complex, and understanding these functions has significant implications for diseases that are driven by the dysregulated metabolism, such as obesity, atherosclerosis, and diabetes.

Therapeutic modulation of inflammasome pathways

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 trials⁴⁵. 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 specific contexts. The goal of treatment should be to facilitate optimal inflammasome activation while preventing hyperactivation and systemic inflammation. Balancing these two outcomes is often difficult, and more work is needed to understand how to achieve this equilibrium in patients.

Other intracellular PRRs

Beyond the inflammasomes, there are also several non-inflammasome–forming intracellular PRRs that are crucial for innate immune responses. NLRC1 and NLRC2, also known as NOD1 and NOD2, were the first intracellular sensors of bacterial peptidoglycan to be discovered^48–50, and the details of their molecular pathways have been extensively studied since then. After sensing peptidoglycan, NLRC1 and NLRC2 activate inflammatory signaling through the NF-κB and MAPK signaling pathways as well as activating the production of type I IFNs. The review by Trindade and Chen examines our current understanding of how NLRC1 and NLRC2 sense microbes and cellular stress⁵¹. The authors also focus on their downstream signaling pathways and detail how these proteins play a role in adaptive immunity and regulate host responses⁵¹. NLRC1 and NLRC2 have also been implicated in a number of inflammatory and autoinflammatory diseases, including type 1 and 2 diabetes, atherosclerosis, sarcoidosis, and asthma. Despite the detailed understanding of their peptidoglycan sensing, the mechanisms underlying how NLRC1 and NLRC2 contribute to inflammatory disease pathogenesis remain to be fully elucidated.

In addition to cytosolic PRRs, nuclear sensors play a key role in detecting intracellular perturbations and maintaining homeostasis. The review by Lin and Cao discusses a number of nuclear sensors, including IFI16, hnRNPA2B1, and SAFA⁵². Nuclear sensors must walk a fine line to avoid aberrant activation in response to self-nucleic acids while still being ready to eliminate foreign threats. Lin and Cao cover the mechanisms employed by these sensors to ensure this important differentiation is maintained and describe how these sensors drive innate immune signaling⁵². Nuclear sensors can contribute to innate immunity by shuttling to the cytoplasm upon activation to initiate signaling cascades there, or they can perform functions directly in the nucleus and exert epigenetic control or regulate mRNA modifications⁵². Their direct nuclear action is a unique characteristic among the PRRs and may allow nuclear sensors to act more quickly than cytosolic sensors. The kinetics of effects mediated by nuclear sensors versus their cytosolic counterparts and how this impacts health and disease is an interesting topic for future study.

Cell death

One of the major outcomes of intracellular PRR signaling in response to pathogens is cell death. In addition to the pyroptotic cell death that occurs downstream of inflammasome activation described above, several other forms of cell death can be initiated in response to intracellular pathogens. Extensive crosstalk has been demonstrated between many cell death pathways, and this crosstalk led to the establishment of the concept of PANoptosis to describe the coregulation of pyroptosis, apoptosis, and necroptosis^7,10–19. PANoptosis has been implicated in infectious and autoinflammatory diseases, cancer, and beyond^{7,10,12,13,15,17–19}. The review from Demarco et al. covers the diverse yet converging cell death pathways, with an emphasis on the interplay between pathogen cell death evasion and host defense mechanisms⁵³. The authors provide a comprehensive overview of the mechanisms of cell death signaling, including the processes of pyroptosis, apoptosis, necroptosis, and NETosis, and discuss the physiological importance of the cell type-specific activation of these pathways⁵³. Cell death is one of the critical outcomes of innate immune signaling, as it can directly eliminate infected or damaged cells. But excessive cell death can lead to tissue damage and organ dysfunction. Therefore, it is important to tightly regulate these processes to prevent aberrant cell death that would lead to morbidity and mortality.

Intracellular receptors and disease modulation

The extensive array of intracellular innate immune sensors and their downstream pathways work together to protect the body against pathogenic insults and promote homeostasis. However, activation of innate immune pathways to counteract infection can have unintended consequences on the host’s susceptibility to a secondary infection. In the review by Rippee-Brooks et al., the authors explore this scenario by outlining how immune responses to viral infections impact subsequent responses to secondary viral, bacterial, parasitic, or fungal infections⁵⁴. These coinfection studies are particularly important due to the fact that coinfections frequently result in inflammation, immunopathology, and more severe outcomes than either infection alone. Understanding the pathways involved is important for dampening this inflammation and counteracting morbidity and mortality.

In addition to pathogenic microbes, the body is constantly exposed to commensals that make up the microbiome. To avoid chronic inflammation, the interaction between the innate immune system and the microbiome must be tightly controlled. Liwinski et al. discuss how the cytosolic innate immune receptors are involved in shaping host-commensal interactions, with an emphasis on the physiological outcomes of these interactions⁵⁵. The authors also discuss the challenges that exist in designing and executing experiments aimed at dissecting the microbiome-PRR relationship⁵⁵. This is an important area of future research with many unanswered questions remaining.

While the importance of the innate immune system in responding to microorganisms is well established, it is increasingly apparent that cytosolic receptors and their signaling pathways can also be involved in a wide variety of other disease processes. The contribution from Ennerfelt and Lukens focuses on the role of innate immunity in the development of a neurological disorder, Alzheimer’s disease⁵⁶. The authors discuss how the immune system has been implicated in the initiation and spread of inflammation due to the role of microglia in these processes⁵⁶. Improved understanding of how the innate immune system drives Alzheimer’s will likely identify new and exciting avenues for treating these patients. Additionally, due to the known role of chronic inflammation in the development of many chronic diseases, Alzheimer’s represents just one of the countless chronic diseases where the contribution of cytosolic innate immune receptors is likely to play a role in pathogenesis. These contributions to chronic disease are just beginning to be understood and are a key topic for future research to identify novel therapeutic targets.

Because the roles of intracellular innate immune sensors are critical and diverse, genetic mutations altering their function can cause severe immunological defects. Based on our growing understanding of these mutations and the effects they have on signaling pathways, significant progress is being made to improve the quality of life for patients with these genetic diseases. In the review by Van der Made et al., the authors discuss these immunodeficiencies and the mutations responsible, focusing on the rational basis for drug therapy and emphasizing the new hope being found in the management of these diseases as our knowledge of innate immunity grows⁵⁷. The use of personalized medicine, with custom-tailored treatment plans informed by genetics and patient-specific molecular information, has a high likelihood of success for these patients.

Summary

This selection of reviews covers a number of topics under the umbrella of intracellular innate immune receptors, highlighting the diversity of the molecules that act as sensors and emphasizing the importance of these pathways in preventing the unchecked replication and spread of pathogens. However, a central theme of these articles is the negative consequences that can result from aberrant activation of these pathways. Controlling the rampant inflammation and cell death while still allowing these pathways to clear pathogens effectively is a constant balancing act within the cell. Our growing understanding of these pathways provides more opportunities for us to modulate them for our benefit, which has already shown success with some therapeutic interventions. I am excited to see these avenues of research continue to grow in the future. I would like to personally thank each of the contributors who have generously taken the time to write the articles for this issue of Immunological Reviews, and I would also like to acknowledge all my colleagues who have contributed to the growth of this field over the years. I hope you will join me in my enthusiasm for the future of this exciting research area.