The NLR family of innate immune and cell death sensors

SUMMARY

Nucleotide-binding oligomerization domain (NOD)-like receptors, also known as nucleotide-binding leucine-rich repeat receptors (NLRs), are a family of cytosolic pattern recognition receptors that detect a wide variety of pathogenic and sterile triggers. Activation of specific NLRs initiates pro- or anti-inflammatory signaling cascades, and the formation of inflammasomes - multi-protein complexes that induce caspase-1 activation to drive inflammatory cytokine maturation and lytic cell death, pyroptosis. Certain NLRs and inflammasomes act as integral components of larger cell death complexes – PANoptosomes - driving another form of lytic cell death, PANoptosis. Here, we review the current understanding of the evolution, structure and function of NLR subfamilies in health and disease. We discuss the concept of NLR networks and their roles in driving cell death and immunity. An improved mechanistic understanding of NLRs may provide therapeutic strategies applicable across infectious and inflammatory diseases and in cancer.

eTOC blurb

Innate immunity relies on the NLR family of cytosolic pattern recognition receptors to detect pathogenic and sterile insults. In this issue of Immunity, Kanneganti and colleagues review the evolution and molecular characteristics of NLRs and discuss the concept of NLR networks in the context of the roles of these proteins in innate immunity, cell death and disease.

Introduction

The innate immune system acts as the first line of defense against pathogens and homeostatic perturbations. To detect invading pathogens and sterile insults, the innate immune system uses pattern recognition receptors (PRRs),^1–3 germline-encoded host proteins that are either membrane-bound or cytosolic. The membrane-bound PRRs include Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), while cytosolic PRRs include nucleotide-binding oligomerization domain (NOD)-like receptors (also known as nucleotide-binding leucine-rich repeat receptors, NLRs), absent in melanoma 2 (AIM2)-like receptors (ALRs), and RIG-I–like receptors (RLRs), among others.⁴ Different classes of PRRs have evolved to recognize distinct microbial components, or pathogen-associated molecular patterns (PAMPs), to induce innate immune responses against invading pathogens.^5,6 PAMPs are highly conserved molecular structures that include lipids, proteins, and nucleic acids, such as lipopolysaccharides (LPS), lipoteichoic acid (LTA), and bacterial DNA,^5,6 molecules not found in host cells during homeostasis. In addition, PRRs can also recognize host-derived damage-associated molecular patterns (DAMPs) that are released as a result of infection or cellular damage.^7,8 The PRR sensing of PAMPs and DAMPs is critical for the activation of innate immune signaling pathways such as nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), and interferon (IFN) pathways.⁹ These signaling pathways lead to the production of proinflammatory cytokines and chemokines that are critical for clearing infections and damaged cells. However, overactivation of these pathways is associated with excess inflammation and inflammatory diseases. Therefore, PRR sensing and innate immune activation must be tightly controlled to maintain the balance between health and disease.

Among the PRR families, the NLR family is one of the largest. NLR proteins sense a diverse array of pathogenic and sterile triggers to induce distinct signaling mechanisms. For instance, NLR family members can activate multiple innate immune pathways, including NF-κB signaling and cytokine production, and they can induce innate immune cell death. Additionally, NLR functions are diverse and far-reaching; some NLRs are positive or negative regulators of inflammation, modulators of transcription to regulate major histocompatibility complex (MHC) class I and class II gene expression, or key reproductive molecules.^6,10 Furthermore, certain NLRs can form multi-protein inflammasome complexes that trigger proteolytic cleavage of caspase-1 to induce its downstream functions, including processing of IL-1β and IL-18 from their pro-forms to produce their active forms and cleaving the pore-forming molecule gasdermin D (GSDMD) to induce a form of lytic cell death known as pyroptosis.^11–18 Moreover, recent extensive genetic and biochemical evidence has shown that NLRs can have distinct roles in PANoptosis, a unique, lytic, inflammatory innate immune cell death pathway driven by caspases and receptor-interacting serine/threonine kinases (RIPKs) and regulated by PANoptosome complexes.^19–23 Given their diverse functions and importance across innate immune responses and in maintaining homeostasis, mutations in NLRs can lead to aberrant inflammation and inflammatory disease.^24,25 Therefore, NLRs are potential therapeutic targets, and many pharmaceutical companies have focused on identifying new drugs and repurposing existing chemical scaffolds to target these molecules.^26,27

In this review, we discuss our current understanding of NLR biology, including the evolution, domain organization, and architecture of NLRs, along with their functions, activation mechanisms, and roles in immune signaling and innate immune cell death. In addition, we discuss how NLRs can function as networks, and highlight their roles in driving inflammatory and infectious diseases. Given the diverse roles of NLRs in modulating inflammatory responses, leveraging our fundamental understanding of NLRs can provide critical insights to advance therapeutics for various infectious and inflammatory diseases, as well as cancers.

The broad and diverse NLR family

Foundational concepts in innate immune have come from studies in plants. The plant immune system uses two classes of specialized PRRs. One system recognizes PAMPs at the cell surface to induce pattern- or PAMP-triggered immunity (PTI); the other identifies specific pathogen effectors that are often meant to circumvent PTI and are delivered inside the cell, leading to effector-triggered immunity (ETI).^28–30 ETI is driven by resistance (R) genes in plants and generally results in hypersensitive response (HR)-mediated cell death.³¹ Pathogen avirulence (avr) genes, also known as effector-encoding genes, encode products that interact with R proteins; the presence or absence of these AVR and R proteins is responsible for susceptibility and resistance.^32,33 The majority of plant R genes are NLR-encoding genes, with the number of NLR genes varying widely between plant species.³⁴ This flexibility in number and arrangement suggests that these genes can be diversified rapidly to respond to distinct pathogenic threats encountered by different species.^35,36

Originally, the gene-for-gene hypothesis proposed that the host R and pathogen AVR proteins interact physically to induce innate immune activation.^37,38 However, plant genetics and functional genomics identified diverse NLR networks in host defense, providing evidence for a complex wiring of immune proteins and supporting a model of indirect pathogen detection that goes beyond the basic gene-for-gene hypothesis.^37–40 The evolutionary arms race between host and pathogen drives a continuous increase in NLR complexity, leading to the differentiation of functions among the molecules. The result is a range of NLRs where some can function as “singleton” NLRs, directly sensing the effector and driving downstream immune responses, while others have more specialized, divided functions to work as pairs or broader networks incorporating sensor NLRs and helper NLRs that facilitate the downstream signaling and cell death, and there is functional redundancy among many helper NLRs (Figure 1A).^41,42 There are multiple types of NLR connections in plants and their functions are conceptualized in various models. There is the guard model, where R proteins function as guards for the effector target and detect modifications introduced by the effector; e.g., the tomato R protein Prf detects the Pseudomonas syringae effectors, AvrPto and AvrPtoB, through their phosphorylation of the host protein Pto.⁴³ There is also the decoy model, where a host protein’s sole function is to act as a bait for an effector-mediated modification that can then be sensed by other host proteins, such as Xanthomonas campestris HopZ1a acetylation of ZED1 that is sensed by host ZAR1 to trigger its activation and an immune response.⁴⁴ The combinations of these diverse NLR networks drive an effective innate immune response for host defense. Plant NLR networks have been extensively reviewed elsewhere,^37,45 and parallels are beginning to be drawn for NLR networks in mammalian species.^22,46

Figure 1. Evolutionary dynamics of NLR genes in the tree of life.

A) NLR networks and their complexity/development over evolutionary time from multifunctional ancestors to specialized NLR singletons with dual roles as sensors and helpers, followed by receptor specialization to divide their functions, with distinct NLR proteins taking on sensor and helper roles, and finally functional diversification. Representation was inspired by previous descriptions of NLR networks.³⁷ B) Phylogenetic distribution of NLR genes per genome among selected organisms representing major phyla across three primary domains: bacteria, archaea, and eukarya (including algae, fungi, plants, and animals). The distribution is mapped on a simplified tree, and branch lengths are not drawn to scale. The scientific names of the organisms used in the tree are indicated here: (i) Bacteria: Steptomyces coelicolor (Steptomyces; Gram positive), Rickettsia conorii (Rickettsia; Gram negative), (ii) Archaea: Methanothrix soehngenii (methanogenic archaea), Prometheoarchaeum syntrophicum (asgard), (iii) Algae: Chromochloris zofingiensis (green algae), (iv) Fungi: Cenococcum geophilum (sac fungi; Ascomycota), Agaricus bisporus (mushroom; Basidiomycota), (v) Plants: Land plants: Physcomitrella patens (earthmoss; Bryophyta), Marchantia polymorpha (liverwort; Marchantiophyta), Vascular plants: Selaginella moellendorffii (spikemoss; Lycophyte), Ceratopteris richardii (fern; Monilophyte), Flowering plants: Malus domestica (apple; Eudicot angiosperm), Arabidopsis thaliana (Arabidopsis; Eudicot angiosperm), Solanum tuberosum (potato; Eudicot angiosperm), S. lycopersicum (tomato; Eudicot angiosperm), Hordeum vulgare (barley; Monocot angiosperm), Triticum aestivum (wheat; Monocot angiosperm), Oryza sativa (rice; Monocot angiosperm), Zea mays (maize; Monocot angiosperm), Non-flowering plants: Ginkgo biloba (ginko; Gymnosperm), (vi) Animals: Amphimedon queenslandica (sponge; Porifera), Acropora digitifera (coral; Cnidaria), Caenorhabditis elegans (worm; Nematoda), Hydra magnipapillata (fresh-water polyp; Mollusca), Drosophila melanogaster (fruit fly; Arthropoda), Capitella teleta (bristle worm; Annelida), Strongylocentrotus purpuratus (sea urchin; Echinodermata), Ciona intestinalis (sea squirt; Tunicata), Branchiostoma floridae (lancelet; Leptocardii), Mus musculus (mouse; Rodentia), Homo sapiens (human; Primates), Takifugu rubripes (Japanese puffer; Tetraodontiformes), Cyprinus carpio (carp; Cypriniformes) and Danio rerio (zebrafish; Cypriniformes). The number of NLR genes were extracted from multiple reports. Representation was inspired by previous tree representations;⁴⁰ tree designs were adapted from SVG SILH source (https://svgsilh.com) and modified using Biorender.

Mammalian NLRs were identified based on their molecular similarity to plant R genes, and they are also functionally similar, with key roles in innate immune and cell death responses.^47–52 NLR proteins are expressed across all kingdoms of life. While there are at least 23 NLRs in humans and 34 in mice, other species contain many more NLRs, such as the 266 found in zebrafish and the 246 found in sea urchins (Figure 1B); however, some species, such as Drosophila and Caenorhabditis, have no NLR proteins identified so far, suggesting they may use alternative receptors for intracellular pathogen detection.⁵³ There are proteins containing the NACHT domain, a hallmark of NLRs, in bacteria and these can provide immunity against phage infection.⁵⁴ This evolutionary conservation highlights the importance of NLRs for organismal survival. NLRs evolved through duplication, uneven cross over, and recombination events,⁵⁵ and likely experienced positive diversifying selection,⁵⁶ which can enhance the ability to sense various ligands and initiate different functional outcomes.

The modular structure of NLRs contributes to both their specificity and broad range of activities. The architecture of plant and mammal NLRs is similar, with the proteins containing three core domains: 1) an N-terminal variable domain for downstream signaling; 2) a central nucleotide-binding domain for oligomerization, the NACHT domain (often referred to as NBD/NOD or NBS for plants); and 3) a C-terminal leucine-rich repeat (LRR) domain. The NACHT domain is composed of four sub-domains, the nucleotide-binding domain (NBD), helical domain 1 (HD1), winged-helix domain (WHD) and helical domain 2 (HD2), which is necessary for ATPase activity. NACHT oligomerizes to form a scaffold for assembling signal transducing molecules to mount an immune response.⁵³ Cannonically,the LRR domain is thought to sense PAMPs and DAMPs in host cells to initiate innate immune response, although there is increasing evidence that not all mammalian NLRs are direct ligand-binding sensors.^{22,46,57–62} Among the mammalian NLRs, exceptions to the general N-terminal variable domain, central NACHT domain, and C-terminal LRR domain structure include NLRP1, which contains a CARD domain at its C-terminus, and NLRP10, which lacks an LRR domain at its C-terminus.

The major functions of NLRs are determined by their specific domains, and therefore NLRs are categorized based on N-terminal domain structure. In plant NLRs, there are TNL proteins containing a Toll-IL-1 receptor (TIR) domain and CNL proteins containing a coiled-coil (CC) domain.^10,63 In mammalian NLRs, these classifications include the NLRA (acidic transactivating domain [AD]-containing NLR protein), NLRB (baculovirus inhibitor of apoptosis protein repeat [BIR]-containing NLR protein), NLRC (caspase activation and recruitment domain [CARD]-containing NLR protein), NLRP (pyrin domain [PYD]-containing NLR protein), and NLRX (NLRs containing an “other” domain with no similarity to known NLR subfamily members; currently assigned as ‘X’ in the nomenclature) groups (Figure 2).^{10,53,64–67}

Figure 2. NLR architecture.

Domains of nucleotide-binding and oligomerization (NOD)-like receptors (NLRs), or nucleotide-binding domain (NBD) and leucine-rich repeat (LRR) proteins, are shown for human and mouse isoforms. Five different subgroups are classified in human and mouse NLR families based on N-terminal domains. These include NLRA (NLRs containing an acidic transactivation domain, AD); NLRB (NLRs containing a baculovirus inhibitor of apoptosis repeat, BIR), also known as NAIPs; NLRC (NLRs containing a caspase activation and recruitment domain, CARD); NLRP (NLRs containing a pyrin domain, PYD); and NLRX (NLRs containing a domain with no similarity to known NLR subfamily members; currently assigned as ‘X’ in the nomenclature). NLRC1 and NLRC2 are more commonly known as NOD1 and NOD2, respectively, and this terminology is reflected here. NLRP3 and NLRP12 are unique in having the full fish-specific NACHT-associated (FISNA) domain, while NLRP6 has a similar domain with only some of the FISNA characteristics. Domain boundaries were extracted from NCBI GenPept entries and are shown from human sequence, unless otherwise indicated for mouse. The domains are not shown to scale. Abbreviations: CIITA, MHC class II transactivator; FIIND, domain with function to find, also called the autoproteolytic domain; NAIP, neuronal apoptosis inhibitor protein; PST, proline/serine/threonine.

a Form-III and IV of CIITA lack the N-terminal CARD domain, and form-II shows low transcriptional activity.

b Naip3 exists as a pseudogene.

c Nlrp4d exists as a pseudogene.

The N-terminal effector domain facilitates signal transduction by initiating protein-protein interactions with other death domain-containing proteins, such as caspases and inhibitor of apoptosis (IAP) family members. The best characterized of these interactions is inflammasome formation. Upon sensing their PAMPs or DAMPs, specific cytosolic innate immune sensors, such as NLRP3, NLRP1, NLRC4, and the non-NLR proteins Pyrin or AIM2,^{11,12,17,68–74} undergo a conformational change that allows them to associate with the adaptor protein ASC through PYD or CARD homotypic interactions.^75–77 ASC contains both a PYD and CARD, which allows it to interact with these diverse sensors. The sensor-ASC oligomeric complex can then recruit caspase-1 through CARD-CARD interactions. This recruitment induces caspase-1 autoproteolytic cleavage and activation. The active caspase-1 subsequently cleaves its substrates, including the pore-forming molecule GSDMD to generate C-terminal and N-terminal fragments,⁷⁸ as well as pro–interleukin (IL)-1β and pro–IL-18 to release their mature forms⁷⁹. Translocation of the N-terminal GSDMD fragments to the plasma membrane then forms oligomeric pores, and NINJ1 is also recruited to facilitate plasma membrane rupture.⁸⁰ The pore formation and plasma membrane rupture lead to cell lysis through water influx and the release of cytokines, including IL-1β and IL-18, and other DAMPs.^{14,15,81–83} This form of inflammatory cell death is known as pyroptosis. Pyroptosis can also be driven by the activation of caspase-11 (mice) or caspase-4/5 (humans) in a process called non-canonical NLRP3 inflammasome activation, in which these inflammatory caspases induce GSDMD activation which then leads to ion efflux that triggers NLRP3 inflammasome activation and inflammatory cell death.^78,81–84

Collectively, mammalian NLR domains carry out diverse functions to facilitate protein-protein interactions, assemble multi-protein cell death complexes, induce innate immune signaling and cell death pathways, and regulate MHC class I and II gene expression,^6,53 among many other functions. Below we discuss the different NLR subfamilies and their functions. The expression pattern of NLRs, their canonical functions and diseases associated with mutations in relevant genes are summarized in Table 1.

Table 1:

NLR expression, canonical functions and diseases associated with mutations in NLR genes

Human NLR	Mouse NLR	Primary expression	Canonical function	Disease associations
CIITA	Ciita	B cells, macrophages, and dendritic cells	Regulation of MHC class II expression	Bare lymphocyte syndrome;47 primary mediastinal B cell lymphoma and classical Hodgkin lymphoma;326 susceptibility to rheumatoid arthritis, multiple sclerosis, and myocardial infarction;327 Behcet’s Disease328
NAIP	Naip1, 2, 4, 5, 6, 7	Intestinal epithelial cells, monocytes, macrophages	Detection of T3SS and flagellin; association with NLRC4 for inflammasome activation	Spinal muscular atrophies101
NOD1 (NLRC1)	Nod1 (Nlrc1)	Intestinal epithelial cells	Detection of peptidoglycan ligand (Tri-DAP); regulation of NF-κB and MAPK activation	Asthma;339 atopic eczema;340 inflammatory bowel disease;329 low-grade duodenal eosinophilia in Helicobacter pylori-related dyspepsia;341 Behcet’s Disease328
NOD2 (NLRC2)	Nod2 (Nlrc2)	Monocytes, intestinal epithelial cells, and keratinocytes	Detection of MDP; regulation of NF-κB and MAPK activation	Crohn’s disease;103,104 Blau syndrome;342,343 Yao syndrome;344 atopic dermatitis;345 susceptibility to leprosy346
NLRC3	Nlrc3	T cells	Negative regulation of NF-κB, IFN responses, and mTOR signaling pathways	Unknown
NLRC4	Nlrc4	Intestinal epithelial cells, monocytes, macrophages	Inflammasome formation	Susceptibility to bacterial infection, multiple sclerosis, and autoinflammation with infantile enterocolitis;127,128 FCAS;129 ulcerative colitis347
NLRC5 (CITA)	Nlrc5 (Cita)	Hematopoietic cells	Regulation of MHC class I expression	Chronic periodontitis;139 pulmonary aspergillosis140
NLRP1	Nlrp1a, b, c	Epithelial cells and keratinocytes	Inflammasome formation	Vitiligo;144,145 multiple self-healing palmoplantar carcinoma and familial keratosis lichenoides chronica;142 arthritis and dyskeratosis;146 recurrent respiratory papillomatosis;148 Alzheimer’s disease;348 Celiac disease;349 Addison disease, type 1 diabetes;350 autoimmune thyroid disorders;351 systemic lupus erythematosus;352 systemic sclerosis;145 giant cell arteritis;353 congenital toxoplasmosis;354 rheumatoid arthritis;355 chronic obstructive pulmonary disease147
NLRP2	Nlrp2	Oocytes and ovaries	Negative regulation of NF-κB and IFN responses; embryogenesis	Beckwith–Wiedemann syndrome;247 female infertility332
NLRP3	Nlrp3	Monocytes, macrophages, and dendritic cells	Inflammasome formation	Susceptibility to infection; CAPS (FCAS1, MWS,25 and NOMID356), autosomal dominant deafness;357 keratoendotheliitis fugax hereditarian;358 myelodysplastic syndrome;359 cancers and metabolic disorders (obesity,292,293 diabetes,294,295 atherosclerosis,296 cardiovascular disease,297 gout,69,298 others);172 neurodegenerative diseases (Alzheimer’s and Parkinson’s);299,300 and many others
NLRP4	Nlrp4a, b, c, e, f, g	Placenta, oocytes, testes, spleen, pancreas, liver, lung, kidney, and thymus,	Autophagy inhibition and type I IFN regulation	Exacerbation of asthma in smokers331
NLRP5	Nlrp5	Oocytes and ovaries	Embryogenesis	Female infertility248,332
NLRP6	Nlrp6	Hepatocytes and intestinal epithelial cells	Negative regulation of NF-κB; inflammasome formation	Rheumatoid arthritis360
NLRP7	-	Oocytes and ovaries	Detection of lipopeptide; inflammasome formation	Recurrent hydatidiform moles249
NLRP8	-	Oocytes and ovaries	Unknown	Unknown
NLRP9	Nlrp9a, b, c	Oocytes and ovaries	Reproduction Mouse Nlrp9b: Detection of dsRNA; inflammasome formation	Unknown
NLRP10	Nlrp10	Heart, skeletal muscle, brain, and skin tissues	Unknown	Atopic dermatitis330
NLRP11	-	Monocytes, B cells, testes, ovaries, lungs	Negative regulation of NF-κB and IFN response; association with NLRP3 for inflammasome activation	Unknown
NLRP12	Nlrp12	Neutrophils and eosinophils	Negative regulation of NF-κB and IFN responses; PANoptosome formation	FCAS2;361 atopic dermatitis;362 polymyalgia rheumatica;363 adult onset autoinflammation with gastrointestinal and neurological symptoms325
NLRP13	-	Ovary, gallbladder, and colon tissues; platelets	Unknown	Unknown
NLRP14	Nlrp14	Oocytes and ovaries	Spermatogenesis	Spermatogenic failure246
NLRX1	Nlrx1	Nearly ubiquitous tissue expression; highest in esophagus and skin	Antiviral response; type I IFN inhibition; ROS regulation; autophagy	Multiple sclerosis333

Abbreviations: CAPS, cryopyrin-associated periodic syndrome; CITA, MHC class I transactivator; CIITA, MHC class II transactivator; dsRNA, double-standard ribonucleic acid; FCAS, familial cold autoinflammatory syndrome; IFN, interferon; MWS, Muckle-Wells syndrome; MAPK, mitogen-activated protein kinase; MDP, muramyl dipeptide; mTOR, mammalian target of rapamycin; MHC, major histocompatibility complex; NLR, nucleotide-binding domain (NBD) leucine-rich repeat (LRR)-containing protein; NLRC, NBD-, LRR- and caspase activation and recruitment domain (CARD)-containing protein; NLRP, NBD-, LRR- and pyrin domain (PYD)-containing protein; NLRX1, NLR family member X1; NOD, nucleotide-binding oligomerization domain-containing protein; NOMID, neonatal-onset multisystem inflammatory disease; NAIP, neuronal apoptosis inhibitory protein; NF-κB, nuclear factor kappa-light chain enhancer of activated B; ROS, reactive oxygen species; T3SS, type III secretion system; tri-DAP, L-Ala-γ-D-Glu-mDAP.

NLRA subfamily

CIITA (MHC class II transcription activator) is a founding member of the NLR family in mammals, and it is the only member of the NLRA subfamily (Figure 2). CIITA is constitutively expressed in antigen presenting cells (APCs) such as B cells, macrophages, and dendritic cells.⁸⁵ However, CIITA expression can also be induced by IFN-γ treatment in THP-1 cells.⁸⁶ Four isoforms of CIITA are known, and they are designated I to IV based on their promoter number in their upstream gene sequence. Form-I contains an N-terminal CARD, while forms-III and IV lack this CARD, and form-II is not highly expressed and, therefore, remains difficult to understand functionally.⁸⁷ All CIITA isoforms contain AD and proline/serine/threonine (PST) domains, which are not found in other NLRs. CIITA acts as a master transcriptional regulator for the expression of MHC class II genes, providing a link between innate and adaptive immune responses.^47,88 Currently, there is no evidence that CIITA directly binds to DNA, but its N-terminal AD allows transcription coregulators to bind and regulate gene expression. Furthermore, CIITA is recruited and physically associated with MHC class II promoters and forms an MHC class II enhanceosome.⁸⁸ Dysregulated expression of MHC class II genes is observed in Bare lymphocyte syndrome (BLS), an inherited condition of immune dysfunction.⁴⁷ CIITA shuttles between the nucleus and cytoplasm to perform its functions, and defects in its nuclear translocation can lead to a form of type II BLS.⁸⁹ In addition, pathogens evolved to evade immune defenses by inhibiting the expression of CIITA and MHC class II genes. For example, human immunodeficiency virus (HIV) inhibits CIITA through its transcriptional transactivator (Tat), reducing MHC class II expression.⁹⁰ Moreover, the expression of CIITA and MHC class II genes is inhibited in response to infections with chlamydia,⁹¹ human cytomegalovirus,⁹² Toxoplasma gondii,⁹³ Mycobacterium bovis Bacillus Calmette-Guerin,⁹⁴ and Epstein-Barr virus.⁹⁵ The targeting of CIITA by multiple pathogens highlights its critical role in linking innate and adaptive immune responses and providing host defense.

NLRB subfamily

NLRB, also known as the NAIP subfamily, contains one member in humans, but seven members in mice, with Naip3 identified as a pseudogene (Figure 2). Expression of mouse Naip1, Naip2, Naip5, and Naip6 has been observed most abundantly in intestinal epithelial cells.⁹⁶ These proteins feature three N-terminal BIR domains, which are members of the zinc-finger domain family. BIR domains are frequently found in IAP family members, where they mediate the inhibition of apoptotic caspases. However, NAIPs are distinct from other BIR-containing proteins, as they have a unique ability to detect bacterial type III secretion system (T3SS) components⁹⁷. NAIPs themselves do not form an inflammasome, but they induce NLRC4 inflammasome activation and inflammatory cell death,^{58–60,98,99} which will be discussed in detail in subsequent sections.

In humans, the single NAIP protein can sense flagellin and bacterial T3SS ligands, such as rod and needle proteins.^58,59,98,99 However, in mice, different NAIPs have distinct sensing capabilities, and their expression is regulated by IFN regulatory factor 8 (IRF8).¹⁰⁰ NAIP5 and NAIP6 sense bacterial flagellin.^59,60 Moreover, NAIP5 directly interacts with flagellin and promotes the physical NAIP5-NLRC4 association.^59,60 In contrast, NAIP2 detects the T3SS rod protein,^59,60 while NAIP1 detects the needle protein.⁵⁸ Mutations in the NAIP locus have been linked to spinal muscular atrophies (SMA).¹⁰¹

NLRC subfamily

Both humans and mice have five NLRC subfamily members (Figure 2). NLRC1 and NLRC2 are also known as NOD1 and NOD2, respectively. NOD1 and NLRC4 have similar domain arrangements, including an N-terminal CARD, while NOD2 has two tandem N-terminal CARDs.^10,53,66,67 NLRC3 has a less-defined N-terminal CARD, which is generally considered a putative CARD and has structural similarities to both CARD and PYD domains. NLRC5 has an atypical CARD with distinct structural features and variations in its α-helices.¹⁰²

NOD1 and NOD2 were the first mammalian NLRs characterized, and they play crucial roles in innate immune responses. Mutations in NOD2 are associated with Crohn’s disease (CD) and chronic inflammatory bowel diseases.^103,104 NOD1 expression has been most notably reported in intestinal epithelial cells,¹⁰⁵ whereas NOD2 expression has been found in monocytes,¹⁰⁶ intestinal epithelial cells^107,108 and keratinocytes.¹⁰⁹ NOD1 and NOD2 are critical for the activation of NF-κB signaling in response to bacterial peptidoglycan fragments from different Gram-positive and Gram-negative bacteria.^110–112 NOD1 directly interacts with the bacterial peptidoglycan ligand L-Ala-γ-D-Glu-mDAP (Tri-DAP),¹¹³ while NOD2 interacts with muramyl dipeptide (MDP).¹¹⁴ Additionally, DHHC5-mediated S-palmitoylation of NOD1 and NOD2 is necessary for an effective immune response against bacterial peptidoglycans.¹¹⁵

Upon recognizing their respective PAMPs, NOD1 and NOD2 undergo oligomerization and CARD-mediated recruitment of RIPK2.¹¹⁶ This recruitment activates NF-κB and MAPK signaling pathways to produce inflammatory cytokines and chemokines.¹¹⁶ NOD1 can also mediate the activation of apoptosis through RIPK2.¹¹⁷ These functions are critical to host defense against bacterial infection. However, the physiological roles of the CARD of NOD1 and NOD2 interacting with other CARD-containing proteins, such as the inflammasome adaptor ASC, and inducing inflammatory cell death require further study.

NLRC3 negatively regulates NF-κB and IFN signaling in response to multiple triggers, including LPS,¹¹⁸ lymphocytic choriomeningitis virus infection, experimental autoimmune encephalomyelitis,¹¹⁹ cytosolic DNA, cyclic di-GMP, DNA viruses,¹²⁰ and Mycobacterium tuberculosis infection.¹²¹ Animals deficient in NLRC3 prolong activation of CD4⁺ T cell responses,¹¹⁹ and the relative expression of NLRC3 is generally high in T cells.¹²² Moreover, NLRC3 suppresses the activation of mTOR signaling pathways and inhibits cellular proliferation, providing protection against colorectal cancer.¹²³ While the biology of NLRC3 remains relatively poorly understood, these current evidences suggest that NLRC3 negatively regulates NF-κB signaling and IFN responses to maintain homeostasis.

NLRC4 (also known as Ipaf, or ICE protease-activating factor) responds to Gram negative bacterial pathogens and was the first NLR found to associate with pro–caspase-1.^16–18 Cytosolic bacterial flagellin induces the formation of the NLRC4 inflammasome, resulting in caspase-1 activation, the secretion of the mature form of IL-1β, and pyroptosis.^70,71 Furthermore, NLRC4 is not a direct sensor; instead, NAIPs act as the upstream innate immune sensor proteins for NLRC4 inflammasome activation.^{58–60,98,99} While NLRC4 can directly interact with caspase-1 through CARD-CARD interactions to induce caspase-1 activation,¹²⁴ NLRC4 can also interact through the inflammasome adaptor molecule ASC, which contains a PYD and a CARD. The CARD of ASC is critical for optimal caspase-1 recruitment to the inflammasome complex, allowing its activation and proteolytic cleavage of pro–IL-1β and pro–IL-18.^46,125,126 Gain of function mutations in NLRC4 induce the constitutive release of inflammatory cytokines and dysregulation in inflammation, which can drive the autoinflammatory disease macrophage activation syndrome (MAS), though other conditions can also induce MAS.^127–130

The final member of the NLRC subfamily is NLRC5, although it also has several similarities across subfamilies with the NLRA protein CIITA. NLRC5 is highly expressed in hematopoietic cells such CD4⁺ T cells, CD8⁺ T cells, CD19⁺ B cells, natural killer (NK) cells, and natural killer T (NKT) cells (Table 1).¹³¹ Similar to CIITA, NLRC5 can translocate between the nucleus and cytoplasm, and it is also a transcriptional regulator, though it regulates MHC class I expression and is therefore referred to as MHC class I transactivator (CITA).^132–135 Loss of MHC class I is a common immune evasion strategy used by various cancers; therefore, reduced expression of NLRC5 is linked to impaired CD8⁺ T-cell activation and poor patient prognosis in different cancer types.¹³⁶ Beyond its roles as a transcriptional regulator, NLRC5 remains enigmatic, with both pro- and anti-inflammatory roles observed. NLRC5 negatively regulates NF-κB by interacting with IKKα and IKKβ and blocking their phosphorylation. It can also inhibit type I IFN responses by interacting with RIG-I and MDA5.¹³⁷ In contrast, NLRC5 has been associated with NLRP3 inflammasome activation to induce caspase-1 activation and IL-1β and IL-18 maturation in response to bacterial infections, PAMPs, and DAMPs.¹³⁸ Polymorphisms in NLRC5 can affect susceptibility to chronic periodontitis and pulmonary aspergillosis,^139,140 highlighting its key connections to disease. The multifaceted roles of NLRC5 are continuing to be investigated.

NLRP subfamily

Humans have 14 NLRPs, while mice have 19, including multiple isoforms for NLRP1, NLRP4, and NLRP9 (Figure 2). Most members of this subfamily share the same architecture, with an N-terminal PYD, a central NACHT, and a C-terminal LRR, but there are a few exceptions. For instance, the NLRP1 proteins contain a function to find domain (FIIND; also called the autoproteolytic domain), and the human NLRP1 and mouse NLRP1a and b proteins contain a C-terminal CARD. Mouse NLRP1a, b, and c proteins lack the N-terminal PYD,¹⁴¹ and NLRP1c also lacks the characteristic C-terminal CARD. Additionally, NLRP3 and NLRP12 have an additional domain close to the NACHT domain, known as the FISNA (fish-specific NACHT associated) domain. FISNA regulates the conformational states and oligomerization of NLRP3. While NLRP6 also has a domain that constitutes certain FISNA characteristics, including the initial polybasic motif and nucleotide-interacting residues, it does not fully conserve the FISNA domain.

Within the NLRP subfamily, several proteins are known to be canonical inflammasome sensors, while others have both inflammasome-dependent and inflammasome-independent functions. We will first discuss the canonical inflammasome sensors.

NLRP1 was the first NLR reported to assemble the inflammasome complex in a cell-free system, leading to the coining of the term ‘inflammasome’.⁶⁸ NLRP1 is expressed most highly in epithelial cells and keratinocytes, and mutations in NLRP1 are associated with inflammatory disorders in the skin due to spontaneous inflammasome activation and IL-1β secretion.^142–148 Due to NLRP1’s unique domain architecture, it undergoes distinct regulation. The NLRP1 FIIND undergoes constitutive autoproteolysis, resulting in two NLRP1 fragments that continue to interact, causing autoinhibition.^149,150 This inactive state is maintained by dipeptidyl peptidases 8 and 9 (DPP8/9), and inhibiting them by treatment with Val-boroPro can block the autoinhibitory cleavage.^151–153 Subsequent proteasomal degradation of the N-terminal fragment can then release the C-terminal region containing the CARD needed to form an active inflammasome complex and trigger caspase-1 activation.^154–156 In mice, NLRP1b can be activated by Bacillus anthracis lethal toxin-induced degradation or endogenous proteasomal degradation following exposure to the ubiquitin ligase Shigella flexneri protein IpaH7.8, leaving the C-terminal free to form an inflammasome complex.^{141,154,157,158} In addition to bacterial toxins, viral proteases can also degrade the N-terminus of NLRP1 to induce inflammasome activation.^159,160 Furthermore, the addition of artificial cleavage sites in the N-terminal is sufficient to induce NLRP1 inflammasome activation,¹⁶¹ suggesting that degradation of the N-terminus is a common mechanism to induce NLRP1 inflammasome activation.

However, there are alternative mechanisms for NLRP1 activation. NLRP1 can directly bind to viral dsRNA during Semliki Forest virus infection through its LRR domain, triggering NLRP1-dependent inflammasome activation.¹⁶² Moreover, ultraviolet B- and toxin-induced ribotoxic stress responses (RSR) activate human NLRP1 through the kinases ZAKα and p38.^163,164 Exotoxin A, a ribotoxin released by the Pseudomonas aeruginosa type 2 secretion system (T2SS) during chronic infection, and diphtheria toxin also induce RSR-dependent NLRP1 inflammasome activation.^165,166 Alternatively, protein folding stress and reductive stress potentiate NLRP1 degradation and induce inflammasome activation,^167,168 and oxidized thioredoxin-1 binds to the NACHT-LRR region to inhibit the NLRP1 inflammasome.¹⁶⁹ Furthermore, nigericin triggers potassium efflux in primary human skin, nasal, and corneal epithelial cells to induce NLRP1 inflammasome activation.¹⁷⁰ These studies collectively suggest that NLRP1 responds to various cellular stress events to induce inflammasome activation.

NLRP3 is the most well-studied member of the NLR family, with over 17,000 articles published, and it has garnered significant attention from pharmaceutical companies aiming to target it. This high level of interest is due to the association of NLRP3 with several diseases. NLRP3 is expressed in splenic neutrophils, macrophages, monocytes, and dendritic cells.¹⁷¹ Mutations in NLRP3 drive cryopyrin-associated periodic syndrome (CAPS), a rare hereditary inflammatory disorder encompassing a continuum of diseases including familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID), also known as chronic infantile neurological, cutaneous and articular (CINCA) syndrome.^24,25 Furthermore, NLRP3 inflammasome activation and release of proinflammatory cytokines occur in other inflammation-associated diseases such as cancer, atherosclerosis, diabetes, obesity, and many more.¹⁷²

NLRP3 responds to a diverse array of PAMPs, DAMPs, and homeostatic alterations to induce inflammasome activation, cytokine release and cell death, which is likely why it has been associated with so many diseases.^11,12,69 However, a single, unifying mechanism of activation for NLRP3 remains unclear. Canonically, two steps are required to induce the fully functional NLRP3 inflammasome: priming and activation. The priming step generally involves TLR-dependent recognition of PAMPs or DAMPs to induce NF-κB activation and increase the expression of NLRP3, caspase-1, and pro–IL-1β.^173–175 Post-translational modifications also regulate the priming step, and TLR-independent priming can also occur under TNF priming conditions and during Streptococcus pyogenes infection.^10,176,177 The subsequent activation step involves oligomerization of NLRP3 through homotypic interactions between NACHT domains, allowing the adaptor protein ASC to associate with NLRP3 and then recruit caspase-1 to the complex.^75,76 Other regulatory mechanisms occur in context-dependent manners. For example, post-translational modifications can regulate NLRP3 activation, with phosphorylation¹⁷⁸ and deubiquitination^179,180 promoting activation, ubiquitination¹⁸¹ and sumoylation¹⁸² suppressing activation, and palmitoylation having both activating and suppressing effects^183,184. Potassium efflux,^185,186 reactive oxygen species (ROS), oxidized mitochondria,^187–191 and disruption of the trans-Golgi network¹⁹² also provide regulation in various contexts.

In addition to the cellular events associated with NLRP3 activation, specific proteins also provide regulation. Caspase-8 regulates NLRP3 inflammasome activation in response to different pathogens,¹⁹³ and the key stress granule protein DDX3X is also required for NLRP3 inflammasome activation.¹⁹⁴ Furthermore, NIMA-related kinase (NEK7) oligomerization with NLRP3 is critical for inflammasome activation,^195–198 although signaling through IKKβ can circumvent the requirement for NEK7 in human cells.¹⁹⁹

Given the variety of cellular events and diverse ligands that can activate NLRP3, it is likely that it does not directly bind to any of these ligands. Instead, NLRP3 may function as part of innate immune sensing networks, similar to the NLR networks seen in plants.⁴¹ Growing evidence supports these connections. NLRP3 interacts with other NLR and non-NLR innate immune sensors in multiple complexes. For example, in response to influenza A virus (IAV) infection, the innate immune sensor Z-DNA binding protein 1 (ZBP1) detects viral nucleic acids and recruits the NLRP3 inflammasome to form a larger cell death-inducing complex called the ZBP1-PANoptosome.^23,57,200 Additionally, NLRP3 can interact with the NLRC4 inflammasome in response to Salmonella infection,⁴⁶ and the NLRP3 inflammasome can be recruited with NLRP12 to the NLRP12-PANoptosome in response to heme and PAMPs.²² While early studies of NLRP3 often used individual PAMPs or DAMPs as triggers, using whole pathogens and more physiologically relevant conditions in these recent studies sheds new light on the ability of NLRs to form networks to induce innate immune cell death.

In addition to NLRP1 and NLRP3, which are well-known for their inflammasome formation, other NLRPs can also form inflammasomes in context-dependent manners while also maintaining key inflammasome-independent functions. NLRP6 (also known as PYPAF5)²⁰¹ is most highly expressed in hepatocytes and intestinal epithelial cells.²⁰² It induces secretion of the inflammasome-dependent cytokine IL-18, maintains the integrity of the epithelial barrier in the dextran sodium sulfate (DSS)-induced colitis model,^203–205 and suppresses inflammation and carcinogenesis by regulating tissue repair in the colon.²⁰⁴ NLRP6 may regulate the gut microbiome to carry out these functions,^203,206 but this remains controversial.^207,208 NLRP6 also performs inflammasome-dependent functions during infection. It senses bacterial LTA for non-canonical inflammasome activation through caspase-11, without cleavage of GSDMD.²⁰⁹ During murine coronavirus mouse hepatitis virus (MHV) infection, NLRP6 binds to dsRNA and undergoes liquid–liquid phase separation dependent on its disordered poly-lysine sequence (K350-354) to drive inflammasome formation and innate immune signaling.²⁰² In addition to its inflammasome-dependent functions, NLRP6 also modulates inflammatory signaling. It negatively regulates inflammation during bacterial infections by reducing the production of NF-κB- and MAPK-dependent cytokines and chemokines.²¹⁰ NLRP6 also controls intestinal viral infections through DHX15-NLRP6 sensing of RNA and the subsequent regulation of type I and type III IFN signaling.²¹¹ NLRP6 can also attenuate non-alcoholic fatty liver disease,²¹² alcoholic hepatitis,²¹³ and hepatocellular carcinoma²¹⁴ in mice by negatively regulating NF-κB and TLR4 signaling, thereby reducing the production of inflammatory cytokines.

NLRP9 exists as a single isoform in humans but as three isoforms (NLRP9a, b, and c) in mice. It is most well-known for its roles in reproduction, as it is highly expressed in the reproductive system. NLRP9 also induces inflammasome formation, although the specific physiological relevance remains unclear. In mice, NLRP9b associates with the RNA helicase DHX9 in response to rotavirus dsRNA during infection, allowing NLRP9b to form an inflammasome in the intestine that is important for clearing the virus.²¹⁵ NLRP9b also contributes to acute lung injury in a mouse model,²¹⁶ but it is unclear whether this is due to its inflammasome-dependent or independent functions. While human NLRP9 can also interact with ASC in response to rotavirus RNA,²¹⁵ the NLRP9 PYD crystal structure suggests that it does not form oligomers,^217,218 which prevents it from forming a fully functional inflammasome. Therefore, the role of the NLRP9 inflammasome in humans requires further investigation.

Similar to NLRP6, NLRP12 (also known as Monarch-1 or PYPAF7) is also a negative regulator of inflammation and an inflammasome sensor. NLRP12 negatively regulates NF-κB signaling,^219,220 and it can suppress RIG-I activation via TRIM25-mediated ubiquitination.²²¹ In vivo, NLRP12 suppresses inflammation during Salmonella infection,²²² reduces colonic inflammation and colitis-associated colorectal cancer in mouse models by inhibiting NF-κB and ERK signaling,^223–225 and prevents atypical neuroinflammatory symptoms.²²⁶ Moreover, NLRP12 regulates caspase-1 activation and IL-1β and IL-18 secretion in response to Yersinia pestis²²⁷ and Plasmodium chabaudi²²⁸ in an inflammasome-dependent manner. NLRP12 is also the innate immune sensor that drives inflammatory cell death, IL-1β and IL-18 secretion, and pathogenesis in response to heme plus PAMPs by forming the NLRP12-PANoptosome.²² Additionally, mutations in NLRP12 are associated with familial cold autoinflammatory syndrome type 2 (FCAS2),²²⁹ highlighting the multifaceted roles of NLRP12 as an innate immune sensor in health and disease.

Beyond the NLRPs that can form inflammasomes, little is known about the sensing and activation mechanisms of other NLRPs. NLRP4 is widely expressed in the placenta, oocytes, testes, spleen, pancreas, liver, lung, kidney, and thymus,^230–232 and it can negatively regulate NF-κB activation²³¹ and type I IFN signaling in response to dsRNA or DNA.²³² NLRP10 is the only mammalian NLR protein that lacks an LRR domain at its C-terminal, though the functional implications of this deficiency remain unclear. NLRP10 is highly expressed in heart, skeletal muscle, brain, skin tissues, and distal colonic intestinal epithelial cells (IECs),^233,234 and it induces an anti-inflammatory response during Leishmania major infection.²³⁵ The phospholipase C activator, 3m3-FBS, triggers mitochondrial destabilization to induce NLRP10 inflammasome activation and cytokine secretion,^236,237 suggesting NLRP10 may contribute to mitochondria-induced inflammatory conditions. NLRP11 is highly expressed in monocytes, B cells, testes, ovaries, and lungs,^238–241 and it negatively regulates NF-κB activation, MAPK signaling, and proinflammatory cytokine production,²³⁸ as well as type I IFN signaling in response to RNA virus infection.²⁴² Moreover, NLRP11 interacts with NLRP3 to promote NLRP3 inflammasome assembly and activation,²⁴³ further suggesting the NLR network concept from plants is conserved in mammals. In addition, a cluster of NLRP proteins is critical for embryogenesis, fertilization, and reproduction. NLRP2, NLRP4, NLRP5, NLRP7, NLRP8, NLRP9, NLRP11, NLRP13, and NLRP14 are all involved in reproduction, and these proteins are highly expressed in oocytes and ovaries.^240,244,245 Mutations in these NLRP proteins are associated with infertility,^246–251 emphasizing their importance in reproduction.

NLRX subfamily

NLRX1 is the sole protein in the NLRX subfamily, and it contains a mitochondrial targeting sequence in its N-terminal region, a feature absent in all other NLRs.^252,253 NLRX1 localizes to the mitochondrial outer membrane, and it can interact with the CARD of mitochondrial antiviral signaling adaptor (MAVS).²⁵² NLRX1 regulates the production of mitochondrial ROS.²⁵³ Moreover, NLRX1 negatively regulates type I IFN production and NF-κB signaling in response to viral infection;^252,254,255 however, it can also regulate early antiviral responses by post-transcriptionally controlling IRF1 expression.²⁵⁶ Moreover, NLRX1 regulates autophagy and mitophagy in response to Listeria monocytogenes and vesicular stomatitis virus infections.^257,258

NLR networks bridging innate immunity and cell death

The multidomain structure of NLRs makes them multifaceted proteins capable of diverse protein-protein interactions and the formation of NLR networks that can mechanistically connect pathogen, PAMP, DAMP, and homeostatic alteration sensing with innate immune cell death. In plants, this concept has been well-studied, and there are diverse NLR networks critical in host defense that have evolved in parallel with pathogens to counteract immune evasion strategies and effector functions. While some NLRs function as “singleton” NLRs to directly sense pathogen molecules and induce the immune response, others form NLR pairs or broader networks with separate sensor and helper NLRs to form functionally redundant connections between pathogen sensing and immune activation.^41,42 In this system, sensor NLR activation induces confirmational changes that can propagate to other NLRs. This immune activation and signaling often leads to a form of programmed cell death called HR in plants.³¹ Parallels with these plant NLR networks and cell death activation also exist in mammalian species,^22,46 though the specific mechanisms of action are expected to differ.²⁵⁹ The most direct illustration of the plant NLR network concept in mammals is the NAIP-NLRC4 connection. In response to pathogen effectors, NAIPs serve as the sensor NLR, and then NLRC4 acts as the helper NLR to induce inflammasome formation and downstream cell death activation.^{58–60,98,99} In humans, where there is only one NAIP, this suggests an NLR pair is formed. In contrast, in mice, NAIP1, NAIP2, and NAIP5/6 have distinct sensing capabilities,^58–60, suggesting that they form an NLR network with NLRC4 to induce pyroptosis, where multiple sensor NLRs can interact with the same helper NLR (Figure 1).

Other NLRs have also been implicated in network formation to drive innate immune cell death. Given the diverse array of triggers that activate NLRP3, NLRP3 is likely a key helper NLR in conjunction with multiple sensors. For example, NLRP3 acts as a helper NLR to induce inflammasome activation and IL-1β and IL-18 release as part of an NLRP12-mediated NLR network that drives cell death in response to heme and PAMPs.²² NLRP3 also serves as a helper NLR in conjunction with non-NLR sensors, as seen in the context of an innate immune sensor for IAV infection, ZBP1. Activation of ZBP1 recruits the NLRP3 inflammasome to induce cytokine release and cell death.^23,57,200 Continued investigations into these and other NLR networks will advance our understanding of NLR networks and their roles in inflammation and disease in mammalian systems.

Beyond the traditional NLR network constructs, there is also extensive molecular crosstalk and sharing of components between pyroptosis and other cell death pathways.^{57,193,260–271} For example, NLRC4 and NLRP3 inflammasome activation can induce caspase-1–mediated cleavage and activation of the apoptotic caspase-7²⁶⁰ or the apoptotic substrate PARP1.²⁶¹ Inflammasomes can also act upstream of apoptosis through GSDMD-mediated formation of mitochondrial pores to induce cytochrome c release²⁷² and caspase-1–mediated activation of the BCL-2 family protein BID in the absence of GSDMD.²⁷³ Additionally, functional redundancies occur between cell death components downstream of NLR engagement, with ASC associating with caspase-8 when caspase-1 is absent to induce cell death.^274,275 Furthermore, caspase-8 regulates both canonical and non-canonical inflammasomes,¹⁹³ and it can be recruited to the NLRP3 or NLRC4 inflammasome complexes in context-dependent manners.^125,193,276 Caspase-8 also activates pyroptotic effectors in response to transforming growth factor-β-activated kinase 1 (TAK1) inhibition.²⁶⁴ Additional crosstalk has also been extensively reviewed previously.^277,278

As a result of the extensive crosstalk and functional redundancies, exhaustive genetic and biochemical analyses found that NLRs and inflammasomes act as integral components of PANoptosomes, multi-protein complexes that regulate a unique innate immune, lytic and inflammatory cell death pathway driven by caspases and RIPKs called PANoptosis. To date, four PANoptosome complexes have been identified, with NLRs and NLR networks as central components of several of these (Figure 3): 1) the ZBP1-PANoptosome, containing ZBP1 along with NLRP3, ASC, caspase-1, caspase-8, RIPK3, among others; 2) the RIPK1-PANoptosome, also containing NLRP3, ASC, caspase-1, caspase-8, RIPK3; 3) the NLRP12-PANoptosome, containing NLRP12, with NLRP3 as an optional component, along with ASC, caspase-8, and RIPK3; and 4) the AIM2-PANoptosome.^{22,57,279,280} PANoptosomes form in response to PAMPs, DAMPs, cytokines, and homeostatic alterations that induce PRR activation and the subsequent recruitment of the complex molecules. PANoptosome molecules putatively come together through homotypic domain interactions involving PYD, CARD, DED, and RHIM domains, though some heterotypic interactions, such as that between the ASC PYD and caspase-8 DED are also likely.^281,282 These complexes induce the activation of multiple caspases, including caspase-1, −3, −7, −8, as well as RIPK1 and RIPK3, and the downstream executioners GSDMD, GSDME, and MLKL, among other molecules. Within the PANoptosome, the inflammasome drives IL-1β and IL-18 cytokine maturation but not cell death. This is illustrated in the NLRP12-PANoptosome, where the NLRP3 inflammasome drives caspase-1 activation and cytokine release, but only has a minor role in the execution of cell death.²² This further supports the NLR network concept in mammals, wherein distinct NLRs are responsible for different components of the innate immune response, and they come together to form a multifaceted signaling complex that drives inflammatory cell death along with cytokine and DAMP release, contributing to a broader immune response. This forms the basis for innate immune cell death, which has been implicated across the disease spectrum as a key host defense mechanism during infection but also as a driver of cytokine storms and inflammation that can induce a myriad of diseases.^{19,21,22,262–264,280,283–291}

Figure 3. NLRs in inflammasome and PANoptosomes.

Nucleotide-binding and oligomerization (NOD)-like receptors (NLRs) can act as key sensors in multi-protein cell death complexes, including inflammasomes and PANoptosomes, in response to diverse pathogenic and sterile triggers. A) The result of inflammasome complex formation is caspase-1 (CASP1) autoproteolysis and activation, leading to its cleavage of gasdermin D (GSDMD) to induce membrane pore formation and pro–IL-1β and pro–IL-18 to release their mature forms (IL-1β and IL-18). Non-NLR proteins, including AIM2 and Pyrin, can also induce canonical inflammasome formation. B) The result of PANoptosome complex formation is the activation of multiple caspases and receptor-interacting protein kinases (RIPKs) to drive inflammatory cell death, PANoptosis. Abbreviations: ASC, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain; CASP6, caspase-6; CASP8, caspase-8; DAMPs, damage-associated molecular patterns; DPP8/9, dipeptidyl peptidases 8 and 9; HSV1, herpes simplex virus 1; IAV, influenza A virus; IFN, interferon; LPS, lipopolysaccharide; NAIPs, NLR family of apoptosis inhibitory proteins; NEI, nuclear export inhibitor; PAMPs, pathogen-associated molecular patterns; TAK1i, transforming growth factor-β-activated kinase 1 inhibitor; ZBP1, Z-DNA binding protein 1. Figure prepared using Biorender.

NLRs in disease

NLR activation can be beneficial to the host by reducing the pathogen load, eliminating cancer cells, and promoting homeostasis. However, the enhanced activation of NLRs can lead to cytokine storms, aberrant inflammation, cell death, tissue damage, and mortality (Table 1). Therefore, there is broad interest in targeting NLRs for therapeutic purposes. In this section, we will briefly discuss the beneficial and detrimental roles of the most well-studied NLRs.

NLRP3 inflammasome activation and inflammatory cytokine release have diverse roles in acute and chronic inflammatory diseases, infection, and cancer, and NLRP3 is associated with metabolic disorders such as obesity,^292,293 diabetes,^294,295 atherosclerosis,²⁹⁶ cardiovascular disease,²⁹⁷ gout,^69,298 and many others, as well as neurodegenerative diseases, such as Alzheimer’s and Parkinson’s^299,300. Therefore, maintaining homeostatic balance in inflammation is critical for host fitness. As the roles of NLRP3 in disease have been extensively reviewed elsewhere,^26,27,172 we will focus our discussion on a few seminal examples here.

Gain of function mutations in NLRP3 can induce constitutive IL-1β release and drive a rare hereditary inflammatory disorder called CAPS, which encompasses a continuum of conditions including FCAS, MWS, and NOMID that manifest with periodic fevers, rashes, joint pain, and other inflammatory symptoms.^24,25 In addition to autoinflammatory disorders, NLRP3 also contributes to host defense responses against infections. NLRP3 is critical for survival in response to pathogenic IAV infection in murine models.^301,302 Additionally, NLRP3, along with NLRP1, limits T. gondii proliferation and induces IL-18 signaling to mediate host resistance to acute toxoplasmosis.³⁰³

Moreover, NLRP3 induces colon inflammation in vivo, but whether this is protective or pathogenic remains unclear. Mice deficient in NLRP3, or the other NLRP3 inflammasome components ASC or caspase-1, have higher mortality than wildtype (WT) mice in response to DSS-induced colitis. This increased mortality in NLRP3-deficient mice is attributed to a loss of epithelial integrity, resulting in systemic dispersion of commensal bacteria, leukocyte infiltration, and increased chemokine production in the colon.³⁰⁴ While similar results have been observed in some cases,³⁰⁵ NLRP3-deficient mice can also have reduced proinflammatory cytokine levels in colonic tissue and protection from DSS-induced colitis.³⁰⁶ These contradictory findings may be due to differences in the microbiome between animal facilities or differences in genetic backgrounds or experimental techniques. Additional work is needed to fully understand the role of the NLRP3 inflammasome in the maintenance of intestinal homeostasis and colitis in humans.

NLRP3 is also implicated in cancers. For instance, NLRP3 activation in dendritic cells induces IL-1β-dependent adaptive immune responses against tumors. Moreover, anticancer chemotherapy is ineffective against tumors in NLRP3-deficient animals, providing a link between the innate and adaptive immune systems through NLRP3.³⁰⁷ In contrast, NLPR3 suppresses NK cell-mediated control of carcinogenesis and metastases in a B16F10 experimental lung metastases model.³⁰⁸ Overall, NLRP3 has diverse functions in both driving and preventing disease in several cancers and infectious and inflammatory diseases. Given its multifaceted roles in disease, there is significant interest in therapeutic targeting of NLRP3. Glyburide, a sulfonylurea-containing compound, was initially identified as an NLRP3 inhibitor,³⁰⁹ though it is now known to be non-specific. Based on the chemical structure of glyburide, more specific NLRP3 inhibitors have been developed. The best characterized, specific NLRP3 inhibitor, MCC950,³¹⁰ is effective in treating inflammatory diseases in murine models, although clinical trials in humans identified potential liver toxicity,²⁶ and additional work will be needed to determine the clinical path forward for MCC950. In addition to MCC950, several other NLRP3 inhibitors are in clinical and preclinical development, including both MCC950 derivatives and unique scaffolds.²⁷ Furthermore, several compounds that target molecules in the NLRP3 inflammasome pathway, including canakinumab, rilonacept, and anakinra, which inhibit IL-1β signaling by binding the cytokine itself or IL-1R, are FDA approved to reduce inflammation in a variety of inflammatory diseases.²⁷ While these successes highlight the potential clinical efficacy for targeting the NLRP3 inflammasome pathways, no specific NLRP3 inhibitor has received FDA approval for clinical use to date.

Beyond NLRP3, several other NLRs are also implicated in disease. Gain of function mutations in NLRC4 can trigger constitutive activation of caspase-1 along with increased production of IL-1β and IL-18.¹²⁷ Specifically, mutation in the HD1 domain causes constitutive inflammasome activation in syndromes such as neonatal-onset enterocolitis, periodic fever, and fatal or near-fatal episodes of autoinflammation.¹²⁸ Additionally, a gain of function mutation in the NLRC4 LRR domain can induce an increase in ASC complex formation and caspase-1–mediated IL-1β and IL-18 production, which is associated with recurrent fever, skin erythema, and inflammatory arthritis symptoms.^311,312

NLRC4 is also critical for the innate immune response to drive host defense during bacterial infections. In response to the foodborne bacterium Salmonella, NLRC4-deficient mice are more susceptible to S. Typhimurium infection and have increased bacterial loads in the cecum, liver, and spleen compared with WT mice.^{46,96,126,313–316} Similarly, gut epithelial cell-specific deletion of Naip1-6 in mice leads to an increased pathogen load after Salmonella infection.⁹⁶ NLRC4 also drives host defense against another enteric bacterial pathogen, Citrobacter rodentium,³¹⁷ as well as non-enteric bacteria, such as Legionella species^318–321 and P. aeruginosa.¹⁸ Furthermore, NLRC4 is implicated in inflammation and cancers. Compared with WT mice, NLRC4-deficient mice are more susceptible to DSS-induced colitis,³²² and they have increased tumor formation, reduced cell death in tumors, and increased proliferation of colonic epithelial cells during the early stage of disease in a colitis-associated colorectal cancer model.³²³

NLRP6-deficient mice are highly resistant to infection with the bacterial pathogens L. monocytogenes, S. Typhimurium, and Escherichia coli.^209,210 In response to encephalomyocarditis virus, NLRP6 is required to activate antiviral immune responses and reduce the viral load in a murine model.²¹¹ NLRP6 also induces host defense in response to MHV infection.²⁰² In a murine colitis model, NLRP6 resolves inflammation and repairs damaged epithelial cells.³²⁴ Consistently, NLRP6 also suppresses inflammation and carcinogenesis by regulating tissue repair,²⁰⁴ indicating that NLRP6 is critical in maintaining the integrity of the epithelial barrier and intestinal homeostasis.

Mutations in NLRP12 cause FCAS2, a condition associated with episodes of fever, skin rash, and joint pain, often in response to cold temperatures; excess activation of NLRP12 and inflammatory signaling drive the symptoms.²²⁹ NLRP12 mutations are also associated with diverse autoinflammatory symptoms in adult patients, most often leading to gastrointestinal and neurological symptoms.³²⁵ Despite its role in autoinflammatory conditions, NLRP12 is also critical to induce innate immune responses and host defense during infection. NLRP12-deficient mice are highly susceptible to Y. pestis infection,²²⁷ and both the NLRP12 and NLRP3 inflammasomes drive caspase-1 activation and host defense in response to P. chabaudi infection.²²⁸ Additionally, NLRP12 has functions similar to NLRP6 to regulate the integrity of the epithelial barrier and intestinal homeostasis to prevent colonic inflammation and colitis-associated colorectal cancer in mouse models.^223–225 NLRP12 also drives PANoptosis, inflammation, and pathology in response to heme plus PAMPs or cytokines in hemolytic and inflammatory disease models,²² implicating a role for NLRP12 in hemolytic diseases, such as malaria or sickle cell disease, as well as in inflammatory and infectious conditions, where hemolysis is a common side effect.

In addition to the above-mentioned NLRs, which are more widely associated with disease, polymorphisms and mutations in other NLRs are also associated with autoinflammatory diseases and cancers. In the NLRA family, mutations in CIITA can cause dysregulation of MHC class II protein expression, which is associated with disorders such as BLS;⁴⁷ primary mediastinal B cell lymphoma and classical Hodgkin lymphoma;³²⁶ susceptibility to rheumatoid arthritis, multiple sclerosis, and myocardial infarction;³²⁷ and Behcet’s disease.³²⁸ In the NLRB family, a mutated form of NAIP can contribute to type I SMA, a genetic disorder caused by a deletion of the survival motor neuron;¹⁰¹ this association suggests a potential link between aberrant neuronal cell death and neuronal loss in response to NAIP mutations. In the NLRC family, NOD1 and NOD2 mutations are associated with inflammatory bowel disease³²⁹ and Crohn’s disease.^103,104 Additionally, polymorphisms in NLRC5 are associated with chronic periodontitis¹³⁹ and pulmonary aspergillosis.¹⁴⁰ In the NLRP family, dysregulation of NLRP1 activation can lead to skin inflammatory diseases such as vitiligo,^144,145 multiple self-healing palmoplantar carcinoma and familial keratosis lichenoides chronica,¹⁴² as well as arthritis and dyskeratosis.¹⁴⁶ Other skin conditions are liked to NLRP10, and atopic dermatitis is associated with a missense variant in NLRP10 in a Japanese population.³³⁰ Beyond skin inflammation, NLRPs are also linked to other autoinflammatory conditions, with single-nucleotide polymorphisms in NLRP4 associated with asthma exacerbation.³³¹ Additionally, frameshift mutations in NLRP2 contribute to the growth disorder Beckwith–Wiedemann syndrome,²⁴⁷ and mutations in NLRP2 and NLRP5 can lead to human early embryonic arrest and female infertility.³³² Other NLRPs are also associated with reproductive disorders, with mutations in NLRP7 linked with abnormal pregnancies²⁴⁹ and mutations in NLRP14 associated with spermatogenic failure.²⁴⁶ Finally, in the NLRX family, mutations in NLRX1 have been observed in patients with multiple sclerosis.³³³

Taken together, mutations in NLR proteins are associated with a broad spectrum of diseases, including susceptibility to infection, inflammatory diseases, metabolic diseases, neurodegenerative diseases, fertility-related disorders, and cancers. Continued research on NLRs will pave the way to identify therapeutic strategies to mitigate inflammation in these diverse conditions.

Concluding remarks

NLR family members play critical roles in regulating health and disease. Many detect pathogens, PAMPs, DAMPs, and homeostatic disruptions to drive robust innate immune responses and form cell death-inducing complexes. Furthermore, NLRs can regulate the expression of MHC class I and II genes for antigen presentation, creating a critical link to activate adaptive immunity. Several NLRs contribute to reproductive biology and development, and others negatively regulate the activation of NF-κB and IFN signaling to maintain homeostasis and prevent aberrant inflammation. Collectively, NLR proteins have broad and diverse pro- and anti-inflammatory functions. Therefore, controlled activation of NLR proteins and their downstream pathways is essential for the host innate immune response to prevent infections and cancer development, and aberrant NLR signaling can have deleterious consequences.

The evolutionary concept of NLR networks has been well-established in plants, and is increasingly evident in mammalian systems, especially in the context of NLRP3 as a helper NLR. NLRP3 associates with various NLR proteins in different cell types and conditions. For instance, the NLRP3 inflammasome can associate with NLRP11 in human macrophages,²⁴³ NLRC5 in primary human monocytes,¹³⁸ and NLRC4 in microglia and astrocytes³³⁴ and murine macrophages⁴⁶ in context-dependent manners. NLRP12 can also form a complex that contains NLRP3, and this NLRP12-PANoptosome complex drives innate immune cell death and the release of cytokines and DAMPs.²² Beyond NLRP3, the association of NAIPs with NLRC4 is critical for inflammasome activation in response Gram negative bacteria,^59,60 and provides another example of a mammalian NLR network. Interactions of multiple NLR proteins as part of an NLR network are required to fully induce innate immune responses and innate immune cell death in many contexts. Additional work to understand these NLR networks and potential functional redundancies between NLRs will be critical to identify strategies to target these proteins and their connections in disease processes. Moreover, the recent determination of cryo-EM structures for NLRP3 and NLRC4,^{198,335–338} among others, provides opportunities to investigate the mechanisms of NLR functions. To date, structural studies have significantly contributed to our understanding of high-order structures, including inflammasome and PANoptosome formation. Understanding the structural aspects of NLRs can guide new drug design strategies to block complex formation and alleviate inflammation and inflammatory disorders, and continued efforts in this area are needed for clinical translation.

Despite increasing molecular and structural understanding of NLRs and their signaling pathways, how NLRs interact with their ligands to induce innate immune responses remains poorly understood. In the context of NLRP3, the diversity of triggering ligands and events makes direct sensing unlikely, and it is possible that NLRP3 responds to a specific homeostatic alteration, such as potassium, chloride, or calcium ion fluxes or organellar changes through lysosomal disruption or trans-Golgi network disassembly.¹⁷² This also further supports the idea that NLRP3 serves as a helper NLR as part of NLR networks with multiple NLR and non-NLR sensors. Furthermore, the specific activating ligands or triggers for other NLRs remain unknown. Additional work is needed to understand the molecular mechanisms and signaling events that activate both sensor and helper NLRs to broaden the concept of NLR networks in mammals.

Overall, the NLR family of innate immune molecules is a multifaceted group of proteins with key roles in maintaining homeostasis, as well as in managing infectious and inflammatory diseases and cancer. Ongoing research continuously identifies new and crucial functions for these proteins and pathways. While drugs targeting NLR family members, especially NLRP3, have entered clinical trials, there has been limited success to date, and further work to identify small molecule activators and inhibitors is ongoing. Recognizing the activators and inhibitors of NLR proteins, especially in clinical settings, opens new avenues for therapeutic targets in various diseases. For example, identifying the specific ligands that activate a particular NLR can suggest disease contexts in which its activation is detrimental to inform therapeutic inhibition strategies. In parallel, determining specific NLR activators can also suggest clinical situations in which providing the necessary ligand to selectively activate the downstream pathways would be beneficial, such as to activate innate immune cell death during cancer to clear tumor cells. As our ability to selectively deliver therapeutics to specific tissues or organs improves, understanding how the NLRs drive host defense versus pathology in different organs and cell types is of paramount importance. Defining NLR functions in disease will also allow us to leverage NLRs as biomarkers for diagnosis and treatment selection to advance personalized medicine. These active areas of research on NLRs shape our fundamental understanding of innate immunity and innate immune cell death and hold promising potential for therapeutic strategies across the disease spectrum.