Inflammasomes in the Pathophysiology of Autoinflammatory Syndromes

Abstract

Inflammasomes are a specialized group of intracellular sensors that are key components of the host innate immune system. Autoinflammatory diseases are disorders of the innate immune system that are characterized by recurrent inflammation and serious complications. Dysregulation of the inflammasome is associated with the onset and progression of several autoinflammatory and autoimmune diseases, including cryopyrin-associated periodic fever syndrome, familial Mediterranean fever, rheumatoid arthritis, and systemic lupus erythematosus. In this review, we discuss the involvement of various inflammasome components in the regulation of autoinflammatory disorders and describe the manifestations of these autoinflammatory diseases caused by inflammasome activation.

Summary Sentence

Review of how genetic alterations in inflammasome components instigate autoinflammatory disorders and the implications of inflammasome activation to the development and manifestations of autoinflammatory diseases.

1. INTRODUCTION

Inflammation is a protective response by the host to ensure the removal of detrimental stimuli and the development of a healing process to repair damaged tissue.[1] The innate immune system is the first line of defense against microbial and cellular insults. Germline-encoded pattern recognition receptors (PRRs) sense microbe-specific molecular signatures known as pathogen-associated molecular patterns (PAMPs) [2] and self-derived molecules from damaged cells known as damage-associated molecular patterns (DAMPs).[3, 4] These PRRs include members of the toll-like receptor,[5–7] nucleotide-binding oligomerization domain (NOD)-like receptor (NLR),[8, 9] retinoic acid-inducible gene-I–like receptor,[10] C-type lectin receptor,[11] and absent in melanoma 2 (AIM2)-like receptor families.[12, 13]

NLRs constitute a large family of intracellular PRRs, several of which are well characterized, such as NOD1, NOD2, and NLRP3 (Fig. 1A).[9, 14, 15] These NLRs regulate inflammation by inducing cytokines, chemokines, and antimicrobial genes. Upon activation, some NLRs form multiprotein complexes termed inflammasomes, whereas others mediate caspase-independent nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase signaling.[16] Inflammasome activation results in the oligomerization and activation of inflammatory caspases. Caspase-1 is the quintessential inflammatory caspase and is the primary enzyme responsible for the cleavage and activation of interleukin (IL)-1β and IL-18.[17] Upon activation, the inflammasome also promotes an inflammatory form of cell death termed pyroptosis, a process regulated by the formation of pores in the plasma membrane by the N-terminal domain of gasdermin D (GSDMD) (Fig. 1B).[18, 19] The current literature indicates that 5 PRRs assemble the inflammasome complex after sensing their respective stimuli: NLRP1, NLRP3, NLRC4, AIM2, and pyrin.[20, 21] Emerging evidence indicates that several other members of the NLR and PYHIN families, including NLRP2, NLRP6, NLRP7, NLRP12, and interferon (IFN)-γ–inducible protein 16 (IFI16), can also form inflammasomes, but there is little information available about their composition.[21]

Figure 1. Schematic representation of the basic structure of individual NLRs and their activation.

(A) Schematic representation of the domains of individual human NLRs based on the human NLRs. Human NLRs are sub-classified into 5 categories: NLRA, NLRB, NLRC, NLRP, and NLRX. All 22 human NLRs contain a central NACHT domain, and all except NLRP10 contain a C-terminal ligand-sensing LRR domain. The N-terminal domains ascribe functional properties to the NLRs; however, the functions of the N-terminal domain of NLRC3 and NLRC5 and the C-terminal FIIND domain in NLRP1 are still unclear. The MT domain targets NLRX1 to the mitochondria, but no sequence homology with the traditional mitochondrial targeting sequence has been reported.

(B) Among the NLRs, NLRP1, NLRP3, and NLRC4 participate in the formation of the inflammasome platform. AIM2 and pyrin, which do not belong to the NLR family, have also been shown to form part of a specific inflammasome. Inflammasome activation leads to caspase-1 activation that in turn cleaves its downstream effectors, the newly identified pyroptosis executioner gasdermin D and the pro-forms of the cytokines IL-1β and IL-18.

Because of its role in inflammation, inflammasome activity needs to be tightly regulated at multiple levels. Priming or “signal 1”, typically delivered via NF-κB activation by toll-like receptor ligands, stimulates NLRP3 and IL-1β transcription and prepares the cell for a vigorous response upon activation.[9] Regulation of the NLRP3 inflammasome by non-transcriptional priming and deubiquitination has also been reported to be an important intermediate step for priming.[22] Subcellular compartmentalization contributes to inflammasome regulation, and anti-inflammatory signals may block inflammasome assembly by preventing the co-localization of inflammasome components.[23] Other adaptor and signaling molecules, such as caspase-8, also participate in inflammasome regulation.[21] The ultimate assembly and activation of the inflammasome requires “signal 2” in the form of ligands specific for different inflammasome scaffolds or cellular metabolic changes. These can include signals as diverse as crystalline materials, oxidative stress, potassium efflux, and nucleic acids.[24–27]

The innate immune response is programmed for immediate action, often resulting in overwhelming and self-perpetuating inflammation that can be detrimental to the host. The term “autoinflammatory disease” was coined to distinguish a set of inflammatory autosomal dominant diseases from self‐directed autoimmune diseases.[28] Autoinflammatory syndromes are characterized by recurrent attacks of systemic inflammation with fevers and are distinguished from autoimmune diseases by the absence of autoantibody and autoantigen-specific T and B cells.[29, 30] Chronic inflammation presents a major threat for the development and progression of autoinflammatory diseases, and inflammasome activation is pivotal to instigate inflammatory responses in macrophages. The inflammasomes are key for resisting microbial infections; yet, mutations in inflammasome sensor-encoding genes are involved in several hereditary autoinflammatory syndromes.[30] In this review, we discuss the contribution of inflammasomes in inflammatory/autoimmune disorders. Insight into the mechanisms and functions of inflammasome activation may lead to the development of novel therapeutics for the treatment of autoinflammatory disorders.

2. INFLAMMASOME-MEDIATED AUTOINFLAMMATORY DISEASES

2.1. CRYOPYRIN-ASSOCIATED PERIODIC SYNDROMES (CAPS)

Since the initial association of NLRP3 with a rare autosomal dominant fever disorder known as familial cold autoinflammatory syndrome (FCAS) [31], 2 phenotypically similar diseases, Muckle-Wells syndrome and neonatal-onset multisystem inflammatory disease (NOMID), also known as chronic infantile neurological cutaneous and articular syndrome (CINCA) [32, 33] containing NLRP3 mutations have been identified. These 3 phenotypically distinct disorders caused by mutations in the NLRP3 gene are referred to as cryopyrinopathies or CAPS.[34] Patients with CAPS experience recurrent episodes of fever with localized neutrophilic inflammation in multiple tissues, a hive-like rash, joint pain and swelling, red eyes, and headaches. The spectrum of CAPS ranges from the relatively mild FCAS to the intermediate Muckle-Wells syndrome and to the severe NOMID (Table 1). [35] CAPS-associated NLRP3 mutations are inherited in an autosomal dominant manner or are secondary to de novo mutations.[36] These mutations result in a gain of function with constitutive activation of the NLRP3 inflammasome and a resultant excess in IL-1β production.[37] Monocytes from patients with CAPS show enhanced inflammasome activation compared with the cells from normal human controls, both in the presence or absence of a low-dose stimulation or mild hypothermia.[38–40] Results of several studies in mouse models suggest that inflammasome function increases in CAPS, with increase in caspase-1 activity, release of IL-1β, and various forms of cell death observed.[41–43] Remarkably, all NOMID-associated inflammatory symptoms are prevented upon ablation of GSDMD [44], suggesting that GSDMD-dependent actions are required for the pathogenesis of NOMID in mice.

Table 1.

Clinical features and pathophysiology of monogenic autoinflammatory diseases.

Disease	Responsible gene, protein	Inheritance pattern	Clinical features	Treatment
CAPS[38, 53]	NLRP3, NLRP3 (cryopyrin)	Autosomal dominant	Fever, rigors, urticaria-like rash, arthralgia/arthritis, headache, conjunctivitis FCAS: episodes lasting < 24 h; cold-induced Muckle-Wells syndrome: episodes lasting 1–2 d; progressive neurosensory hearing loss NOMID: chronic daily symptoms; bony overgrowth of knees, chronic aseptic meningitis, papilledema, uveitis, developmental delay, seizures	IL-1 blockade FCAS: anakinra, canakinumab, rilonacept Muckle-Wells syndrome: anakinra, canakinumab, rilonacept NOMID: anakinra, canakinumab
FMF[64, 72]	MEFV, PYRIN	Autosomal recessive	Episodes lasting 1–3 d, abdominal pain, chest pain, arthralgia/arthritis, myalgia	Colchicine, anakinra, canakinumab
NLRC4-associated disorders[66, 109]	NLRC4, NLRC4	Autosomal dominant	Variable; recurrent fever, diarrhea, vomiting, urticaria-like rash, arthritis; might have MAS-like symptoms	Anakinra, anti–IFN-γ mAb
PAPA[154, 155]	PSTPIP1, PSTPIP1	Autosomal dominant	Episodes induced by mild trauma or infection; fever, arthralgia/arthritis, severe cystic acne, recurrent ulcers, sterile skin abscesses	Corticosteroids, etanercept, anakinra; acne might require oral tetracycline or isotretinoin
HIDS or MKD[156]	MVK, mevalonate kinase	Autosomal recessive	Episodes lasting 7–10 d; fever, arthralgia, cervical lymphadenopathy, abdominal pain, maculopapular rash	NSAIDs, etanercept, anakinra
TRAPS[157]	TNFSFR1A, TNFSFR1A	Autosomal dominant	Episodes lasting days to weeks; fever, periorbital edema, arthritis/myalgia, abdominal pain, centrifugal rash	Corticosteroids, anakinra, canakinumab, etanercept, NSAIDs
DIRA[158, 159]	IL1RN, IL-1RA	Autosomal recessive	Chronic symptoms noted in neonatal period; failure to thrive, irritability, pustular rash, hepatosplenomegaly, oral ulcers, joint swelling, arthritis	Anakinra

Many mutations described in the NLRP3 gene are located in the nucleotide-binding domain (NBD) (Fig. 2A); yet, few mutations have been characterized in animal models, and several discordant effects in human and mouse models were found.[45–47] Some of the variants represent somatic gain-of-function mutants of NLRP3 restricted to myeloid cells.[48, 49] The development of knock-in mice with CAPS-associated mutations in the mouse homolog of NLRP3 have helped to investigate the pathophysiological mechanisms of these mutations. These models reproduce the autoinflammatory phenotypes found in human patients. However, the tissue-specific models showed increased mortality dependent on cellular location, especially in myeloid cells, which does not occur in humans.[38, 50] Knock-in mice globally expressing the D301N NLRP3 mutation, an orthologue of the D303N NLRP3 mutation found in human patients with NOMID, present with neutrophilia in the blood, inflammatory cell recruitment in knee joints, and high levels of inflammatory mediators in the serum with specific bone-remodeling abnormalities and osteolysis.[51] These features were also observed in myeloid cell-specific D301N expression, demonstrating that this is a gain-of-function mutation within osteoclasts and that it is associated with the degradation of poly (ADP-ribose) polymerase 1, an inhibitor of osteoclastogenesis.[51, 52]

Figure 2. Schematic outline of mutations in the domains of NLRP3 and PYRIN.

(A) Schematic representation of residue substitutions in the NLRP3 protein and reported point mutations. Mutations reported to be involved in Muckle-Wells syndrome and familial cold autoinflammatory syndrome (FCAS) are highlighted in red and blue, respectively.

(B) The domain organization of human PYRIN, indicating the four major domains and residue substitutions. Mutations reported to be involved in familial Mediterranean fever (FMF) are highlighted in red.

Recent data suggest there is a role for IL-18 in CAPS pathogenesis. Similar to IL-1β, IL-18 is also cleaved by caspase-1 and is often secreted at higher levels in CAPS-affected human monocytes and mouse hematopoietic cells.[53] Deletion of IL-18R, IL-1R, or IL-1β on a CAPS mutation background in mice has demonstrated that IL-18 signaling is vital to the initial development of inflammation in murine CAPS, whereas IL-1β leads to later systemic inflammation [53]. Interestingly, when CAPS-mutant mice were bred onto an Il1r^–/–Il18^–/– background, the mice exhibited reduced inflammatory activity, although the inflammatory activity was still significant.[53] Based on the known role of NLRP3 mutations in these inflammatory activities, it can be speculated that murine CAPS is mediated by NLRP3 inflammasome-dependent factors but independent of the biological activity of IL-1β and IL-18.

Treatment of CAPS with non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and disease-modifying antirheumatic drugs has not provided any significant benefits for patients.[54, 55] However, dramatic improvements have resulted from using IL-1–targeted treatments, including anakinra, a recombinant human IL-1 receptor antagonist (IL-1RA); rilonacept, a recombinant fusion protein consisting of portions of the IL-1 receptor and IgG1 Fc region; and canakinumab, a humanized anti–IL-1β monoclonal antibody.[54, 55] Unlike anakinra and rilonacept, canakinumab has a long half-life of 28–30 days, requires administration once every 8 weeks, and is associated with minimal injection-site reactions but some increased risk of infections. Use of canakinumab in CAPS resulted in prompt and persistent improvement in the clinical and laboratory parameters of inflammation.[54, 56, 57]

2.2. FAMILIAL MEDITERRANEAN FEVER (FMF)

FMF is the most common autosomal recessive, autoinflammatory disorder; it is highly prevalent in individuals from the eastern Mediterranean region.[58, 59] It is characterized by episodic fever and neutrophil-mediated inflammation of serosal tissues (Table 1). MEFV has been described as the gene responsible for FMF.[60, 61] MEFV encodes for pyrin, a protein of 781 amino acids with an N-terminal pyrin domain, a central boxed-box and coiled-coil domain, and a C-terminal PRY/SPRY (B30.2) domain.[62] Pyrin is primarily expressed in myeloid cells including neutrophils, eosinophils, monocytes, dendritic cells, and synovial fibroblasts and forms an inflammasome complex in response to disturbances in cytoplasmic homeostasis.[63, 64]

More than 300 MEFV gene variants have been described (Fig. 2B) [65], but only 14 occur frequently in FMF (E148Q, E167D, T267I, P369S, F479L, I591T, M680I, I692del, M694I, M694V, K695R, V726A, A744S, and R761H). Interestingly, in contrast to the gain-of-function mutations in NLRP3, NLRC4, or NLRP1 observed in CAPS and in NLRC4-and NLRP1-associated inflammasomopathies [31, 66–68], FMF-associated MEFV mutations do not lead to constitutive pyrin inflammasome activation and require a specific stimulus, such as low doses of Clostridium difficile toxin B.[69–71] Most pathogenic MEFV mutations in humans occur in exon 10, which encodes the B30.2 domain. Strikingly, this domain is absent in the murine ortholog.[64]

To study the effect of these mutations, a collection of FMF knock-in (FMF-KI) mouse strains were generated that express a chimeric pyrin protein harboring the human B30.2 domain with mutations observed in patients with FMF.[64] One such FMF-KI strain is Mefv^V726A/V726A, which expresses the murine pyrin spliced to the B30.2 domain with a valine-to-alanine substitution at amino acid position 726. These mice develop an autoinflammatory disorder characterized by systemic wasting and neutrophilia that is caused by GSDMD-mediated IL-1β release in response to aberrant pyrin inflammasome activation independent of IL-1α and caspase-8.[64, 72, 73] Recent study results highlight the central role of tumor necrosis factor (TNF)/TNF receptor (TNFR) signaling in regulating the pyrin inflammasome and the distinct features of pyrin inflammasomopathy.[74] Intriguingly, colchicine therapy is primarily effective as a prophylactic treatment for FMF attacks. It is recommended in all patients regardless of the frequency and intensity of attacks [75]; however, the mechanism of action is still unclear. Because colchicine has been shown to prevent TNF-induced toxicity in vivo,[76] it can be speculated that blocking chronic TNF signaling could be more effective in limiting inflammation in FMF.

In addition to the mutations that cause FMF, 2 distinct, dominant MEFV mutations that cause a different autoinflammatory syndrome termed pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND) have been identified (pS242R and pE244K).[77, 78] These mutations abolish or reduce protein kinase N-1/2–mediated phosphorylation of pyrin on serine 242 and impair the binding of 14–3-3 proteins, leading to constitutive activation of the pyrin inflammasome. Interestingly, PAAND-associated mutations do not map to the B30.2 domain, where most pathogenic FMF-associated mutations are found. Furthermore, FMF-associated MEFV mutations do not seem to affect pyrin phosphorylation levels or 14–3-3 binding. [70, 79, 80]

Although both autoinflammatory syndromes described above are caused by direct MEFV mutations, there are other monogenic syndromes that indirectly affect pyrin inflammasome activation. For example, PAPA syndrome is defined by symptoms of sterile, purulent arthritis; pyoderma gangrenosum; and cystic acne, as summarized by its acronym (Table 1) [54]. PAPA syndrome is a dominantly inherited disease associated with missense mutations in CD2-binding protein 1, more commonly described by its murine ortholog Pstpip1 which encodes the proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP-1).[81] PSTPIP-1 interacts with pyrin and promotes its ability to interact with ASC and to trigger its oligomerization. In PAPA syndrome, PSTPIP1 mutations decrease the ability of PSTPIP-1 to interact with proline-, glutamic acid-, serine-and threonine-rich phosphatase, leading to hyperphosphorylated PSTPIP-1 variants that have increased avidity for pyrin [82]. Although there is clear evidence of inflammasome deregulation in PAPA syndrome [83], the syndrome’s pathophysiology is complex and probably involves functions of PSTPIP-1 other than its regulation of the pyrin inflammasome.[84] These studies demonstrate that pyrin, although known for its association with FMF, can also modulate other autoinflammatory skin disorders.

2.3. RHEUMATOID ARTHRITIS (RA)

RA is a chronic inflammatory/autoimmune disease that carries a substantial burden for both the individual and society.[85] This disease is characterized by persistent synovial inflammation and causes multi-joint destruction and cartilage degradation with limited treatment options leading to poor patient outcomes.[86–88] Since RA is the inflammatory disease with the highest worldwide incidence of 0.5% to 1%,[89] the role of inflammasomes in RA has been widely studied.

The gene expression of NLRP3 inflammasome components, including apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), full-length NLRP3, short-length NLRP3, and caspase-1, is increased in peripheral blood mononuclear cells (PBMCs) of RA patients compared with those of healthy controls, and there is some evidence of an association between single-nucleotide polymorphisms (SNPs) at the NLRP3 locus and susceptibility for RA.[90, 91] Another study suggested that polymorphism in the NLRP1 gene is associated with susceptibility to RA in the Han Chinese population. Individuals with an NLRP1 polymorphism associated with RA risk had significantly higher NLRP1 mRNA levels than those with the non-risk genotype.[92] A subsequent study showed that inhibiting mRNA expression of P2X4, a purinergic receptor, suppresses joint inflammation and damage in collagen-induced arthritic (CIA) mice and affects synovial cells of patients with RA by suppressing the activation of the NLRP1 inflammasome and effectively reducing the serum level of IL-1β.[93] Collectively, these studies clearly indicate that inflammasomes and their polymorphisms are strongly associated with RA pathogenesis.

Recent studies also indicate that different methods of arthritis induction in animal models may have differential effects on immune cells and effector molecules. These differential effects could lead to different experimental outcomes.[94, 95] The CIA model showed that Nlrp3^–/– and Caspase1^–/– mice were susceptible to arthritis, whereas Asc^–/– mice were protected.[94] However, arthritis induced by the K/BxN transgenic mice serum transfer model containing autoantibodies recognizing glucose-6-phosphate isomerase (GPI) [96] did not affect immune cells in Caspase1^–/– mice.[95] Animal model studies focused on RA have shown that induction of chronic arthritis (characterized by minimal numbers of neutrophils) in Caspase1^–/– mice led to reduced joint inflammation and less cartilage damage. Alternatively, in a model of acute (neutrophil-dominated) arthritis, Caspase1^–/– mice developed joint inflammation similar to that in wild-type control mice.[95] These findings suggest that caspase-1 inhibitors may be of therapeutic value only in inflammatory conditions in which limited numbers of neutrophils are present.

Myeloid-cell-specific deletion of the RA susceptibility gene Tnfaip3/A20 in mice triggers a spontaneous erosive polyarthritis resembling RA in patients. This destructive polyarthritis was TNF independent but relies on the NLRP3 inflammasome and IL-1R signaling. Deletion of NLRP3 or caspase-1/11 or IL-1R significantly rescues arthritis in myeloid-cell-specific A20^–/– mice [97], indicating that myeloid cell-specific A20^–/– mice can be used as a suitable pre-clinical model to validate any experimental therapies for RA targeting inflammasomes and/or IL-1 signalling. Further studies demonstrated enhanced activity of the NLRP3 inflammasome in peripheral blood cells of patients with active RA.[98] These patients expressed higher basal intracellular levels of NLRP3, ASC, active caspase-1, and pro–IL-1β [98], suggesting that targeting NLRP3 or downstream caspases may be of benefit in suppressing IL-1β production in RA.

The efficacy of anakinra treatment in RA has been evaluated in several controlled studies.[99] Although no direct comparison is available between the efficacy of IL-1 blockade and the overwhelming number of competing biologic agents, including TNFα blockers, based upon indirect comparisons, anakinra seem to be modestly efficacious biologic therapy for RA.[100] Similar to anakinra, the anti-IL-1β monoclonal antibody canakinumab led to reduced disease severity in patients with RA, including those who were unresponsive to anti-TNFα therapies[101]; however, unlike anakinra, long-term preservation of joint function with canakinumab remains unstudied. Taken together, these data suggest that inflammasome activation and IL-1R signaling are risk factors for RA pathogenesis, and their selective inhibition reduces RA symptoms and progression.

3. OTHER INFLAMMASOME-RELATED AUTOINFLAMMATORY DISORDERS

3.1. AIM2 in autoinflammation

AIM2 had not been linked to inherited autoinflammatory diseases, and no gain‐of function mutations have been identified within the Aim2 gene. This could be because mutations in the oligomerization domains of NLRP3 and pyrin are associated with autoinflammatory disorders, whereas AIM2 does not possess known oligomerization domains, which may be surrogated by its multivalent ligand dsDNA.[102] Mutations in the receptors that diminish DNA binding may result in reduced oligomerization and signaling and a compromised immune response to microbial infections, whereas mutations that enhance DNA binding or destabilize the autoinhibited state may be linked to autoimmune disorders.[102]

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates innate immune responses through production of the second messenger cGAMP, which activates the adaptor molecule STING. Aberrant activation of the cGAS pathway by self DNA can also lead to autoimmune and inflammatory disease. In a mouse model of polyarthritis‐like disease caused by DNase II deficiency, both the cGAS‐STING pathway and the AIM2 inflammasome pathway contribute to autoinflammation caused by the increased release of self DNA within the cytosol.[103, 104] The inability to degrade self DNA also contributes to the pathogenesis of autoimmune polyarthritis. Lacking the lysosomal endonuclease DNase II (DnaseII^−/−) is embryonically lethal in mice due to the impaired ability of macrophages to degrade self DNA.[105] Genomic deletion of type I IFN receptor (IFNAR) rescues DnaseII^−/− mice from embryonic lethality [106]; however, the mice lacking both IFNAR and DNase II (DnaseII^−/−Ifnar^−/−) eventually develop polyarthritis.[107] Intriguingly, genomic deletion of AIM2 prevents inflammasome activation, inflammatory cytokine production, macrophage infiltration in the joint, and arthritis development in DnaseII^−/−Ifnar^−/− mice.[103, 104] Genomic deletion of STING also protects DnaseII^−/−Ifnar^−/− mice from joint inflammation,[104] indicating that multiple DNA sensors might contribute to the inappropriate DNA recognition driving clinical manifestation. In psoriatic lesions, abundant cytosolic DNA in keratinocytes acts as a DAMP that can elicit the AIM2 inflammasome, leading to IL‐1β maturation and the autoinflammation signs observed in patients with psoriasis.[108] Overall, aberrant AIM2 activation by self DNA is a key driver of inflammatory and autoimmune diseases.

3.2. NLRC4 in autoinflammation

Gain-of-function mutations in the NLRC4 gene are associated with early-onset autoinflammation with enterocolitis or recurrent macrophage activation syndrome (MAS), depending on the mutation.[67, 109] Patients with such alterations are characterized by mutations in the NBD region of NLRC4 and benefit from recombinant human IL-18–binding protein therapy.[110] The autoinflammatory-associated NLRC4 mutation H443P can constitutively activate caspase-8 and induce apoptosis by interacting with the suppressor of Gal 1 component of the 26S proteasome and with ubiquitinated cellular proteins.[111] Additionally, a macrophage-intrinsic defect can drive the MAS phenotype in the absence of a primary cytotoxic defect, thus providing a new paradigm for the pathogenesis of MAS.[67]

Activating mutations in NLRC4 cause a new autoinflammatory disease presenting as a periodic fever syndrome with MAS;, distinct from CAPS and partially responsive to IL-1 inhibition.[67] The persistence of high serum IL-18 levels and macrophage hyper-responsiveness even after the initiation of IL-1–blocking treatment suggests a role for IL-18 in MAS and warrants an exploration of IL-18 as a therapeutic target. Physical manifestations in patients with NLRC4 mutations include rash and splenomegaly; laboratory findings include increased levels of inflammatory markers, including ferritin, increased levels of lactate dehydrogenase, and pancytopenia.[66] Leukocytes from some of these patients also displayed increased production of IL-1β, IL-18, or both, with increased caspase-1 cleavage and the appearance of ASC specks, consistent with inflammasome activation.[66, 67, 109] A mouse with a similar mutation observed in one of the patient families had dermatitis and arthritis that was worsened when exposed to cold. The mutation in NLRC4 increased its oligomerization and resulted in hyperactivation of caspase-1. Bone marrow-derived cells from these mice also produce increased levels of IL-1β and neutrophilic inflammation that is dependent on IL-17.[66] Thus, NLRC4 has finally provided a link between autoinflammation and the systemic inflammatory response syndrome (SIRS) via a hyperinflammatory SIRS subtype known as MAS.

Collectively, mutations in genes encoding inflammasome sensor proteins, such as NLRP1, NLRP3, NLRC4, or pyrin, contribute to the development and progression of a number of autoinflammatory disorders due to uncontrolled activation of caspase-1 and the aberrant release of inflammation-inducing cytokines (Fig. 3).

Figure 3. Inflammasomes and their association with autoinflammatory syndromes.

NLRP1, NLRP3, NLRC4, and pyrin form canonical inflammasomes with ASC and caspase-1 that process pro–IL-1β and pro–IL-18 into their active forms, IL-1β and IL-18. Boxes with lightening bolts denote proteins that have been reported to be mutated in the respective diseases. Gain-of-function mutations in these proteins lead to constitutively active inflammasomes. Diseases associated with corresponding mutations are: NLRP1, multiple self-healing palmoplantar carcinoma (MSPC) and familial keratosis lichenoides chronica (FKLC) [153]; NLRP3, cryopyrin-associated periodic syndrome (CAPS) and neonatal onset multisystem inflammatory disease (NOMID); NLRC4, macrophage activation syndrome (MAS) and systemic inflammatory response syndrome (SIRS); pyrin, familial Mediterranian fever (FMF) and pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). Mutations in mevalonate kinase (MVK) and proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) associated with mevalonate kinase deficiency (MKD) and/or hyper IgD syndrome (HIDS) and purulent arthritis, pyoderma gangrenosum, and cystic acne (PAPA), respectively, affect the activity of the pyrin inflammasome through various mechanisms and lead to increased IL-1β secretion. Deficiency in the IL-1 receptor antagonist (IL-1RA), which is associated with deficiency of IL-1 receptor antagonist (DIRA), leads to unchecked IL-1 signaling. Activated caspase-1 can also cleave gasdermin D (GSDMD), and its released N-terminal domain (N-term) forms cytotoxic pores in the plasma membrane, thus inducing pyroptosis, an inflammatory form of cell death.

4. INFLAMMASOME-MEDIATED AUTOIMMUNE DISEASES

4.1. SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)

SLE is a systemic autoimmune disorder characterized by increased type I IFN production and immune complex deposition within the tissues, leading to varied inflammatory responses and often devastating organ involvement.[112] Dysregulation of both the innate and adaptive immune responses is central to SLE development. Recent evidence from human and murine studies indicates that inflammasomes are central contributors to the development of lupus nephritis and the associated organ damage.[113]

The NLRP3 and AIM2 inflammasome have received a significant amount of attention as contributors to SLE in murine models and have been actively explored. Upregulation of inflammasome components in lupus nephritis biopsies has been reported,[114] suggesting a priming effect for inflammasome activation. Most studies focused on the upregulation of inflammasome components were conducted using human monocytes and associated with neutrophil extracellular trap (NET) formation. NETs are extracellular structures composed of chromatin, with specific proteins and enzymes from the neutrophilic granules critical for host defense [115] that play a key role in SLE development.[116] NETs can activate the NLRP3 inflammasome in macrophages derived from patients with SLE, resulting in release of active IL-1β and IL-18. This activation of the NLRP3 inflammasome utilizes P2X7 receptor-mediated potassium efflux.[117] Importantly, IL-18 can induce NETosis, a form of cell death, suggesting that inflammasome-activated IL-18 stimulates a perpetual cycle of NET formation and inflammasome activation.[117] This mechanism yields an intriguing possibility suggesting a feed-forward inflammatory loop that could potentially lead to disease flares and/or organ damage.

A hallmark of SLE is the production of autoantibodies that recognize nuclear antigens. Circulating double-stranded DNAs (dsDNAs) and anti-dsDNA antibodies are commonly found in the sera of patients with SLE.[118, 119] These immune complexes can activate the NLRP3 inflammasome, and SLE-derived macrophages are hyper-responsive to innate immune stimuli, leading to enhanced activation of the inflammasome and production of inflammatory cytokines.[120]

The results of murine lupus model studies further suggest a role for the inflammasome in lupus pathogenesis. The pristane-induced lupus mouse model pointed to an association between the hyperactivation of the NLRP3 inflammasome and severe lupus-like symptoms. In mice with the Nlrp3^R258W gain-of-function mutation, a more severe lupus-like syndrome was developed, and these mice exhibited significantly higher mortality upon pristane challenge as compared with wild-type mice.[121] Interestingly, the phenotype observed in this mouse strain was largely due to the myeloid cell-specific Nlrp3^R258W mutation, as depletion of this mutation from myeloid cells rescued mice from experimental lupus.[121] Inhibiting the P2X7 receptor, an activator of NLRP3 inflammasome assembly, also benefits mice with lupus nephritis.[122] Additionally, caspase-1 deficiency provided protection from autoantibody generation and lupus nephritis in the pristane-induced mouse model of lupus. Other investigations have reported that in a mild model of spontaneous lupus-like autoimmunity (C57BL/6-lpr/lpr mice), NLRP3 or ASC deficiency increased dendritic cell and macrophage activation, the expression of numerous proinflammatory mediators, necrosis of lymphocytes, and the expansion of most T-cell and B-cell subsets with no defect in plasma cells and autoantibody production. This unexpected immunosuppressive effect of NLRP3 and ASC could be attributed to their role in transforming growth factor (TGF)-β receptor signaling [123], as NLRP3 or ASC deficiency significantly suppressed the expression of numerous TGF-β target genes in C57BL/6-lpr/lpr mice.[123] Taken together, these studies demonstrate a critical role for NLRP3 in the development of SLE, although an inflammasome-independent function from NLRP3 or ASC likely accounts for the more severe disease; these findings suggest that modulating the inflammasome signal may help to control the inflammatory damage in SLE.

Overexpression of inflammasome scaffolds such as IFI16 and AIM2 in leukocytes from patients with SLE has been reported.[124] Immune complexes containing DNA or RNA antigens can prime the inflammasome for activation, subsequently triggering assembly of NLRP3 inflammasome complexes.[125, 126] The activated complement component, C3a, promotes adenosine triphosphate secretion, thus serving as a trigger for inflammasome activation.[127] The complement component C1q is a risk factor for SLE development,[128] and its deficiency suppresses NLRP3 expression in human macrophages.[129] Interestingly, C1q-mediated phagocytosis of apoptotic lymphocytes inhibited NLRP3 inflammasome activity,[129] suggesting that activation of NLRP3 inflammasomes due to lack of C1q plays a role in SLE pathogenesis. Anakinra treatment proved beneficial in a very small, non-placebo–controlled trial of patients with lupus and refractory arthritis.[130]

The inflammasome scaffold AIM2 has also been studied for its role in SLE pathogenesis. Repeated injections of apoptotic DNA in Balb/c mice results in a lupus-like syndrome, and the disease progression in this model correlates well with the elevated levels of AIM2 expression in different organs. Furthermore, these mice have a diminished lupus-like phenotype in the absence of AIM2.[131] Others, however, have demonstrated that inhibiting AIM2 may promote lupus pathogenesis. In mice, another IFN-inducible p200 family member, p202, is upregulated in many lupus-prone strains [132, 133] and negatively regulates AIM2 inflammasome activation, consequently decreasing cell death and promoting prolonged type I IFN production in response to cytosolic DNA.[134] Inhibiting AIM2 results in p202 upregulation.[135] Importantly, in T and B cells, type I IFN exposure resulted in AIM2 suppression and p202 upregulation.[136] This could lead to a repeating cycle of type I IFN production. Thus, inhibiting AIM2 may have both positive and negative effects on lupus pathogenesis, necessitating careful assessment of this strategy for disease management.

7. IL-1–RELATED BUT INFLAMMASOME-INDEPENDENT AUTOINFLAMMATORY DISORDERS

A spontaneous mutation in Shank-associated RH domain-interacting protein (Sharpin) causes chronic proliferative dermatitis (cpdm) in C57BL/6 mice.[137, 138] Sharpin^cpdm mice have a single base pair deletion in exon 1, resulting in early termination of the transcript and the complete absence of Sharpin protein mimicking Sharpin-deficient mice.[138] These mice are runted and develop spontaneous systemic inflammation with apparent inflammation of the skin as early as 3–4 weeks after birth.[138] Nlrp3, Caspase1, Caspase11, Il1r, and Il1b deficiency all delay the onset of dermatitis in Sharpin^cpdm mice.[139, 140] Sharpin^cpdm inflammasome component-deficient mice still develop a full spectrum of disease, suggesting that the IL-1 signaling pathway may play only a minor role in disease progression. Another recent study has determined important roles for RIPK-1, TNF, and myeloid differentiation primary response protein 88 (MyD88) signaling in the complete rescue of skin inflammation in Sharpin^cpdm mice.[141–143] Together, these studies show that Sharpin is a crucial regulator of both apoptotic and necroptotic cell death pathways in epidermal cells. Moreover, these findings have been further strengthened by the demonstration that Sharpin^cpdm mice lacking TNFR1 specifically in epidermal cells (Sharpin^cpdmTnfr1^fl/flK14^Cre) are completely protected from disease.[144] Although mutations in Sharpin have not been identified in human patients so far, Sharpin^cpdm mice have been very helpful in understanding the basic biology of how several immune components are involved in the progression of an autoinflammatory skin disease resembling dermatitis.

A spontaneous mutation in the protein tyrosine phosphatase non-receptor type 6 (Ptpn6) gene encoding the Src homology region 2 domain-containing phosphatase 1 (SHP-1) protein causes skin lesions in mice that are heavily infiltrated with neutrophils.[145] These mice harbor a Y208N missense mutation in the Ptpn6 gene (Ptpn6^spin), and their lesions closely resemble those of neutrophilic dermatoses in humans.[146] Gene analyses from patients with neutrophilic dermatoses have suggested that there are PTPN6 splice variants or heterozygous mutations in affected individuals.[147] Compared to SHP-1–deficient mice, Ptpn6^spin mice have 70% less SHP-1 phosphatase activity and present with a more mild phenotype and no disease-associated mortality.[148] Most Ptpn6^spin mice develop spontaneous footpad swelling at 8–16 weeks of age with signs of neutrophilia, intra-epidermal pustules, and cutaneous tissue damage, all of which are characteristics of neutrophilic dermatoses.[148, 149] Interestingly, Ptpn6^spin disease symptoms can be completely rescued with Il1r and MyD88 deficiency.[148] Recent results from our group have shown that this disease progresses independent of inflammasome regulators. Crossing Ptpn6^spin mice with Nlrp3^–/–, Asc^–/–, or Caspase1^–/– mice did not protect them from the footpad inflammation.[149] Interestingly, disease induction in Ptpn6^spin mice is mediated by RIPK1 and IL-1α but not by IL-1β.[149] Furthermore, we have shown the involvement of TAK1, ASK1/2, SYK, and CARD9 signaling pathways in mediating the footpad inflammation in Ptpn6^spin mice.[150–152] Because pro–IL-1α is fully biologically active and constitutively expressed in a wide variety of cell types, understanding IL-1α cytokine biology, which remains significantly obscure, will shed more light on the pathogenesis of this inflammatory disorder and could provide much-needed targeted therapy for these rare diseases.

8. CONCLUDING REMARKS

During the past two decades, remarkable advancements have greatly increased our understanding of innate immunity, inflammasomes, and their related disorders through a growing number of murine models and advances in the patient-centered research. Despite these impressive strides, the clinical applications of these findings are still in their infancy. The genetic characterization of autoinflammatory disorders with respect to inflammasomes has substantially increased our understanding of NLR biology and has highlighted the inflammasome’s ability to contribute to many inflammatory diseases without major provocation of the adaptive immune system. This recognition coupled with insight into the central role of IL-1β in mediating inflammation in these disorders has led to markedly improved patient care through the successful therapeutic use of IL-1 inhibition. Further characterization of the high-resolution structure of inflammasome proteins will improve our understanding of the mechanisms governing inflammasome regulation. As this knowledge continues to evolve, additional targets and therapies to regulate inflammasome activity during disease will be discovered. Future research should also focus on whether inhibition of inflammasome components may serve as a viable target for therapeutic development in SLE and other autoinflammatory/autoimmune diseases. Challenges for the future include understanding the clinical significance of low-penetrance variants, the genetics and pathophysiology of different autoinflammatory diseases, and the long-term safety and efficacy of anti–IL-1 and anti–IL-18 therapies.

Abbreviations:

AIM2

absent in melanoma 2

ASC

apoptosis-associated speck-like protein containing a caspase recruitment domain

ATD

acidic transactivation domain

BIR

Baculoviral inhibition of apoptosis protein repeat domain

CAPS

cryopyrin-associated periodic syndromes

CARD

caspase association and recruitment domain

cGAMP

cyclic GMP-AMP

cGAS

cyclic GMP-AMP synthase

cpdm

chronic proliferative dermatitis

DAMP

damage-associated molecular pattern

DIRA

deficiency of IL-1 receptor antagonist

FCAS

familial cold autoinflammatory syndrome

FIIND

function to find domain

FMF

familial Mediterranean fever

GSDMD

gasdermin D

HIDS

hyper IgD syndrome

IFI16

interferon-γ–inducible protein 16

IFN

interferon

IFNAR

type I interferon receptor

IL

interleukin

IL-1RA

interleukin 1 receptor antagonist

KI

knock-in

LRR

leucine-rich repeats

MAS

macrophage activation syndrome

MKD

mevalonate kinase deficiency

MyD88

myeloid differentiation primary response protein 88

NBD

nucleotide-binding domain

NET

neutrophil extracellular trap

NF-κB

nuclear factor kappa B

NLR

nucleotide-binding oligomerization domain-like receptor

NOD

nucleotide-binding oligomerization domain

NOMID

neonatal-onset multisystem inflammatory disease

NSAIDs

non-steroidal anti-inflammatory drugs

PAAND

pyrin-associated autoinflammation with neutrophilic dermatosis

PAMP

pathogen-associated molecular pattern

PAPA

purulent arthritis, pyoderma gangrenosum, and cystic acne

PRR

pattern recognition receptor

PSTPIP-1

proline-serine-threonine phosphatase-interacting protein 1

PTPN6

protein tyrosine phosphatase non-receptor type 6

PYD

pyrin domain

RA

rheumatoid arthritis

Sharpin

Shank-associated RH domain-interacting protein

SHP-1

Src homology region 2 domain-containing phosphatase 1

SIRS

systemic inflammatory response syndrome

SLE

systemic lupus erythematosus

TGF-β

transforming growth factor-β

TNF

tumor necrosis factor

TNFR

tumor necrosis factor receptor

TNFSFR1A

tumor necrosis family receptor superfamily 1A

TRAPS

tumor necrosis factor receptor-associated periodic syndrome