Inflammasome mediated autoinflammatory disorders

NLRP3 intrinsic inflammasomopathies

Cryopyrin-associated periodic syndromes (CAPS) or cryopyrinopathies refer to three phenotypically distinct disorders caused by mutations in the NLRP3 gene[12,13,14], and are inherited in an autosomal dominant manner or secondary to de novo mutations[15]. The distinct CAPS phenotypes are not linked with specific mutational clustering in the NLRP3 gene[13], although the majority of mutations arise within the NACHT-domain encoding exon 3 locus[14,16,17]. Mutations result in gain of function with constitutive activation of the NLRP3 inflammasome and resultant excessive IL-1β production[18].

The spectrum of CAPS ranges from the relatively mild familial cold autoinflammatory syndrome (FCAS), to the intermediate Muckle-Wells syndrome (MWS), and to the severe neonatal onset multisystem inflammatory disorder (NOMID, also known as chronic infantile neurological cutaneous and articular syndrome or CINCA). Despite clinical heterogeneity, these conditions share overlapping features of inflammation, including fever, rash, conjunctivitis, and arthralgia. Common laboratory findings include leukocytosis with neutrophilia, and elevation in acute phase reactants such as C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR)[19]. The commonly reported rash is described as urticaria-like in appearance but is infrequently pruritic and oftentimes painful with biopsy findings of neutrophilic dermal infiltrates[20].

Treatment of CAPS has been difficult as non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and disease-modifying antirheumatic drugs (DMARDs) have not provided significant benefit[21,22]. However, patients have exhibited dramatic improvement with treatment targeting IL-1. Available IL-1 inhibitors include 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. Anakinra was the first studied IL-1 targeted therapy, and was FDA approved for use in rheumatoid arthritis in 2001. Given its short half-life of 4 to 6 hours, it is administered subcutaneously daily, with commonly reported injection-site reactions, and a few reports of increased infections[23]. This led to the development of longer-acting IL-1 blocking agents including rilonacept and canakinumab. Rilonacept was FDA approved for the treatment of CAPS in 2008. It has a half-life of greater than 8 days, is administered weekly, and is generally well tolerated, with some reports of minor injection-site reactions, and increased frequency of infections[24]. Canakinumab was FDA approved for the treatment of CAPS in 2009. It has the longest half-life of 28 to 30 days, requires administration only every 8 weeks, and is associated with minimal injection-site reactions, and some increased risk of infections. Use of canakinumab in CAPS resulted in prompt and persistent improvement in clinical and laboratory parameters of inflammation[25].

Familial Cold Autoinflammatory Syndrome

Familial cold autoinflammatory syndrome (FCAS) is the mildest form of CAPS, and was first described in 1940[26]. It is inherited in an autosomal dominant manner, and does not vary in severity with each generation. Age of presentation is usually less than 10 years of age, however, symptoms can present in adulthood. A unique feature of FCAS is that systemic symptoms are precipitated by generalized cold exposure, and can occur up to 8 hours following cold exposure. Attacks are characterized by rash, fever, arthralgia, and an estimated 80% of subjects develop conjunctivitis[27]. Other associated symptoms include headaches, nausea, fatigue, and sensorineural hearing loss[24]. Episodes typically resolve within 24 hours, but can persist for up to 72 hours. Complications of FCAS include reactive systemic amyloidosis, which occurs secondary to deposition of serum amyloid A (SAA), a hepatocyte-produced acute phase reactant. An estimated 2% of patients develop manifestations of renal amyloidosis. Given the principal role of the NLRP3 inflammasome and IL-1β production in disease pathogenesis, treatment with IL-1 inhibitors has been pursued. Treatment with anakinra has shown to decrease cold-induced attacks of fever, rash, fatigue, arthralgia, with a corresponding decrease in leukocytosis, and IL-6[28]. Use of rilonacept has also been shown to decrease cold-induced symptoms, and biochemical markers of inflammation[24].

Muckle-Wells Syndrome

Muckle-Wells syndrome (MWS) is of intermediate phenotype on the CAPS spectrum, and was initially reported in 1962[29]. Mutational analysis reports have found that approximately 25% of subjects with MWS are NLRP3 mutation-negative, however, share clinical similarity, suggesting mutations in other inflammasome components may be responsible[15]. Disease presentation is seen primarily in childhood, and symptoms can be continuous in nature. In contrast to FCAS, symptoms are not precipitated by cold exposure, and include fever, rash, conjunctivitis, episcleritis, optic disc edema, and arthritis[30]. Subjects frequently manifest progressive perceptive hearing loss, and reactive systemic amyloidosis is seen in up to 25% of patients[19]. Treatment with anakinra in MWS has been associated with improvement in inflammatory symptoms and decrease in amyloidosis induced nephrotic syndrome[22].

Neonatal Onset Multisystem Inflammatory Disorder

Neonatal onset multisystem inflammatory disorder (NOMID) is the most severe form of CAPS, and was originally described in 1981[31]. Approximately 50% of patients with NOMID are found to be NLRP3 mutation-negative, however, clinical presentation does not differ between the mutation-positive and negative groups[13]. NOMID presents during the neonatal period or early infancy, and symptoms of inflammation are typically chronic. Initial features include fever, lymphadenopathy, hepato-splenomegaly[32], and the characteristic rash is seen in about 75% of subjects at birth[33]. A majority of patients exhibit arthropathy, which is characterized by patellar and epiphyseal long bone ossification, and secondary osseous overgrowth. Skeletal deformity is a common cause of disability, and includes joint contractures, genu varus or valgus, leg length discrepancy, and short stature[34]. Most patients develop central nervous system (CNS) inflammation, with manifestations of aseptic leptomeningitis, increased intracranial pressure (ICP), ventriculomegaly, and cerebral atrophy[21]. Consequential neurological complications include seizures, cognitive impairment, optic nerve atrophy with progressive vision loss, and cochleitis with sensorineural hearing loss. The approximate mortality in untreated patients is estimated to be about 20%[35]. Treatment with anakinra has been shown to decrease symptoms of fever, rash, ophthalmologic symptoms, headache, and arthralgia. Therapy was also shown to decrease CNS inflammation, as evidenced by decrease in ICP, cerebrospinal fluid protein, enhancement on brain MRI, and improved hearing[21].

Complex or Acquired Inflammasomopathies

While the above diseases have been definitively identified as causally associated with the NLRP3 inflammasome through specific mutation identification in afflicted patients, other disorders have been suggested to be associated with inflammasome activation primarily through murine studies. Here we will discuss these findings and their applicability to human disease processes.

Crystalline arthropathies

Molecular insight into the pathogenesis of CAPS led to further investigation of the NLRP3 inflammasome. This resulted in the discovery that endogenous danger signals, including particulates implicated in crystalline arthritis, can activate the NLPR3 inflammasome. Acute gouty arthritis is induced by the deposition of monosodium urate (MSU) crystals in articular and periarticular tissues. Similarly, the inflammatory joint disorder pseudogout is caused by deposition of calcium pyrophosphate dihydrate (CPPD) crystals. The presence of crystals in the joint cavity promotes an acute inflammatory response, with ensuing tissue damage[36]. Insight into disease pathogenesis was provided when studies demonstrated that MSU and CPPD crystals could mediate NLRP3 inflammasome activation, and increased IL-1β secretion[37]. Trials of anakinra use conducted in select patients with gout and pseudogout arthritis refractory to conventional therapy have revealed clinical benefit[38,39].

Silicosis and Asbestosis

Silicosis is a pulmonary fibrotic disorder caused by inhalation of silica particles. Inhalational exposure to silica is seen in several occupational settings, including mining and construction work. Asbestosis is a similar fibrosing lung disorder, which occurs following inhalation of asbestos particles. Both disorders have a long latency period, which is inversely proportional to the level of particulate exposure. Alveolar macrophages have a prime role in disease pathogenesis. Macrophages trigger an inflammatory response following ingestion of crystal particles in the lungs, a process that eventually leads to fibrogenesis[40]. The NLRP3 inflammasome has been implicated in mediating these fibrosing lung disorders in studies that have indicated murine macrophages secrete IL-1β in response to silica and asbestos crystals in a NLRP3 dependent manner[41,42,43].

Diabetes Mellitus

Diabetes mellitus (DM) is a metabolic disorder denoted by hyperglycemia, and is associated with vascular complications, including cardiovascular disease, nephropathy, and neuropathy. Type 1 DM occurs secondary to insulin deficiency in the setting of autoimmune destruction of the insulin-producing pancreatic β-cells. Type II DM arises due to peripheral insulin resistance, and a relative impairment in insulin production. The innate immune system, and the NLRP3 inflammasome in specific, have been implicated in the development of Type II DM. Studies have determined that elevated glucose concentrations can stimulate IL-1β production from human pancreatic β-cells, which subsequently mediates β-cell dysfunction and cell death. Consequent deterioration of β-cell function leads to worsened hyperglycemia, thereby propagating the inflammatory cycle[44]. Given the proposed role of IL-1 in disease modulation, anakinra treatment has been used in patients with Type II DM, and has been shown to improve β-cell secretory function and glycemic control[45]. Further investigation of glyburide, a sulfonylurea agent used for treatment of Type II DM, ascertained that its mechanism of action was dependent on the NLRP3 inflammasome. This study revealed that glyburide inhibits NLRP3 inflammasome activation by various stimuli, thereby preventing secretion of IL-1β[46]. A mechanistic link between NLRP3 and a protein previously linked to insulin resistance, thioredoxin (TRX)-interacting protein (TXNIP) has been postulated as well[47].

NLRP3 extrinsic inflammasopathies

The term extrinsic inflammasopathies has been used to denote IL-1β mediated inflammatory disorders provoked by defects in regulatory components extrinsic to the inflammasome[48]. These include several autoinflammatory conditions including Familial Mediterranean Fever (FMF), Pyogenic arthritis with pyoderma gangrenosum and acne (PAPA) syndrome, Hyperimmunoglobulinemia D with periodic fever syndrome (HIDS) or Mevalonate kinase deficiency (MKD), and Schnitzler’s syndrome (SS).

FMF is characterized by periodic fever, peritonitis, pleuritis, arthritis, and rash, with disease complication including systemic amyloidosis[49]. High carrier frequencies have been found in populations of the Mediterranean basin and the Middle East. MEFV is the gene implicated in FMF, and encodes pyrin[50] a protein that can modulate the inflammasome by associating with ASC[51]. However, the role of pyrin in IL-1β activation remains controversial, with some studies suggesting that it activates an inflammasome complex[52], while others suggesting that it inhibits IL-1β production[53]. The treatment of choice for FMF remains colchicine, however, in colchicine-refractory cases, IL-1 inhibitor therapy has been used successfully[48].

PAPA syndrome is defined by symptoms of sterile, purulent arthritis, pyoderma gangrenosum, and cystic acne, as summarized by its acronym[54]. Inheritance is autosomal dominant, with mutations detected in the CD2-binding protein 1 (CD2BP1), which is more commonly described by its murine ortholog, proline serine threonine phosphatase-interacting protein 1 (PSTPIP1)[55]. PSTPIP1 interacts with pyrin, and mutations in PSTPIP1 have been shown to augment this binding, thereby enhancing IL-β production[56]. Anakinra has been used in PAPA syndrome, and has been associated with improvement in arthritis and pyoderma gangrenosum[57,58].

HIDS is identified by features of recurrent fever, lymphadenopathy, abdominal pain, arthralgia, and a constitutively elevated serum immunoglobulin D (IgD) level[59]. HIDS is inherited in an autosomal recessive manner, and is caused by mutations in MVK, which encodes mevalonate kinase (MK)[59,60]. This mutation has been shown to disrupt the MK pathway, with resultant activation of the GTPase Rac1, phosphoinositide 3-kinase (PI3K), and protein kinase B (PKB). This process mediates caspase-1 activation, and enhanced IL-1β secretion[61]. Trials of anakinra in HIDS have also been performed, and have indicated beneficial clinical outcomes[62,63].

SS is characterized by an urticaria-like rash and monoclonal IgM or IgG gammopathy with associated fever, arthralgia, arthritis, lymphadenopathy, hepatomegaly, splenomegaly, or elevated acute phase reactants[64]. Disease pathogenesis is currently under investigation, however, given that treatment with IL-1 inhibitor therapy has shown clinical benefit, IL-1 is thought to have a principal role in disease modulation[65,66].

NLRP1 inflammasome

A second inflammasome implicated in human disease pathogenesis is the NLRP1 inflammasome. This is composed of NLRP1 (also known as NALP1, DEFCAP, NAC, CARD7), ASC, caspase-1 and caspase-5, and similar to the NLRP3 inflammasome, assembly results in the activation of caspase-1 with the subsequent processing and secretion of IL-1β. Vitiligo is a depigmenting skin disorder due to the progressive autoimmune loss of melanocytes, and is associated with increased frequency of other autoimmune diseases, including autoimmune thyroid disease, latent autoimmune diabetes, rheumatoid arthritis, psoriasis, pernicious anemia, systemic lupus erythematosus, and Addison’s disease. NLRP1 has been identified as one of the candidate genes mediating the vitiligo-autoimmune susceptibility [67]. It is unclear at this point how NLRP1 may be dysregulated in vitiligo-associated autoimmunity; to date, only anthrax lethal toxin (LeTx) produced by Bacillus anthracis has been shown to activate the NLRP1 inflammasome[7].

NOD2

The nucleotide oligomerization domain 2 (NOD2) is another member of the NLR family. It is encoded by the CARD 15 (also known as NOD2), and is composed of two amino-terminus CARD domains, a central nucleotide-binding domain (NBD), and carboxy-terminus LRR. NOD2 recognizes bacterial peptidoglycan-derived muramyl dipeptide (MDP), and activates nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase signaling pathways. Defects in CARD 15 have been linked to both Crohn’s disease (CD) and Blau syndrome (BS)[7].

CD is an inflammatory bowel disorder characterized by transmural inflammation of the intestine. It commonly affects the ileum and colon, but can involve any part of the gastrointestinal (GI) tract. Disease manifestations include abdominal pain, diarrhea, weight loss, and less commonly hematochezia, with complications including intestinal granuloma formation, strictures, and fistulas. Etiology of CD is complex, and is attributed to a combination of genetic and environmental variables. CARD15/NOD2 is one of the susceptibility genes identified in CD[68,69]. A majority of the CD-associated CARD15/NOD2 polymorphisms have been detected in the LRR region[70]. This gene variant is estimated to account for about 20% of the genetic predisposition to CD[71], and is associated with a stricturing phenotype[70]. Theories on disease pathogenesis include impaired recognition and clearance of intestinal bacteria due to loss of function CARD15/NOD2 mutations or loss of tolerance against commensal microbes[72].

BS is an autosomal dominant disorder of early onset, characterized by rash, uveitis, and granulomatous arthritis. Complications of arthritis include camptodactyly and large synovial cysts[73]. CARD15/NOD2 mutations have also been implicated in BS, however, unlike in CD, mutations have been localized to the NBD region[74].

Psoriasis is an inflammatory skin disorder characterized by hyperproliferation and can be associated with psoriatic arthritis (PsA). PsA is a seronegative spondyloarthropathy manifesting with symptoms of arthritis, dactylitis, and enthesitis. Etiopathogenesis of PsA is attributed to an interplay between genetic and environmental factors. CARD15/NOD2 had been identified as a candidate gene in PsA in a study conducted in a Newfoundland population[75]. However, subsequent studies in American, German, and Italian populations failed to reproduce this association[76,77,78].

NLRP12

NLRP12 is an NLR encoded by NLRP12 (also known as NALP12, Monarch-1, or PYPAF7), and functions as a negative regulator of NF-κB activation[79]. Mutations in NLRP12 have been associated with Guadeloupe variant periodic fever syndrome, an autosomal dominant inherited autoinflammatory syndrome. Mutations result in loss of function and subsequent decreased NLRP12 mediated inhibition of NK-κB signaling. Guadeloupe variant periodic fever syndrome presents usually during the first year of life, and attacks are triggered by cold-exposure, similar to FCAS. Symptoms reported include fever, urticaria-like rash, lymphadenopathy, arthralgias, myalgias, and complications include sensorineural hearing loss[80].