Cryopyrin-associated periodic syndromes
Cryopyrin-associated periodic syndromes (CAPS), which include familial cold autoinflammatory syndrome (FCAS), Muckle–Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID) are caused by autosomal dominant mutations in the NACHT domain of NLRP3 [
63‐
65,
98]. Additionally, myeloid-restricted somatic mosaicism and mutations in
NLRP12 and
NLRC4 may account for the inflammatory responses in CAPS patients negative for mutations in
NLRP3 [
66]. NLRP3 is believed to switch from a closed and inactive conformation to an open state in response to PAMP- or DAMP-induced cues;
NLRP3-activating mutations locked this protein in the active state, leading to the constitutive assembly of the inflammasome [
67]. Common features of CAPS include recurring fever episodes, urticaria, conjunctivitis, and joint pain whereas central nervous system complications and arthropathies characterized by bone deformities, bulky epiphyses, leg length discrepancy, and short stature are hallmarks of NOMID, the most severe manifestation of these disorders [
66,
68‐
70]. Consistent with the tumor-like features of bony outgrowths, induced pluripotent stem cells from NOMID patients are more proliferative and exhibit higher differentiation potential than normal cells [
71]. Epiphyseal abnormalities undoubtedly predispose NOMID patients to joint instability and subsequent development of osteoarthritis. It is worth noting that skeletal phenotyping is based on radiographic observations and limited histology that reveal heterogeneously calcified bone matrix, and severely disorganized and hypocellular growth plate [
70].
Murine models of CAPS in which wild-type
Nlrp3 alleles are replaced by murine or human alleles carrying mutations found in patients, reproduce several features of human disorders including early onset of systemic inflammation, skin and joint pathologies, and growth retardation. Cryopyrinopathies are in general more severe in rodents than in humans as mutant mice of all phenotypes (i.e., CAPS, MWS, and NOMID) exhibit short lifespan (3–4 weeks) [
72‐
74]. NOMID mice in which NLRP3 is activated globally exhibit normal patterning of skeletal elements but display hypocellular epiphyses due to massive chondrocyte death, and disorganized growth plate matrix protruding towards the bone marrow cavity [
73]. Unexpectedly, conditional activation of NLRP3 in myeloid cells but not in osteochondro-progenitors reproduces the abnormal cartilage features, suggesting that the phenotype is not chondrocyte autonomous [
75]. CAPS mice also exhibit severe low bone mass, a phenotype that correlates with a massive expansion of osteoclast precursors, exuberant osteoclastogenesis, and increased osteoclast activity [
41,
73,
75‐
77]. Thus, while the magnitude of bone resorption in CAPS patients is not known, this process is prominent and well characterized in mouse models.
Systemic inflammation and multiple organ pathologies, including bone abnormalities, are entirely prevented in NOMID mice lacking IL-1 receptor [
75]. However, persistent residual inflammation is reported in FCAS mice and MWS mice deficient in IL-1 and IL-18 signaling [
78,
79]. These findings align with clinical studies, which consistently show that epiphyseal lesions and outgrowths continue to expand for a significant number of NOMID patients on IL-1 biologics despite the resolution of disease-associated inflammatory symptoms [
70,
99‐
101]. Collectively, these observations suggest that IL-1β is not the primary driver of skeletal outcomes in CAPS, and that inflammasome-dependent responses other than IL-1β play a role in these disorders. This view is consistent with findings showing that levels of TNF-α remain elevated in certain CAPS patients on IL-1 blockers, and neutralization of TNF-α activity improves inflammatory endpoints in CAPS mice [
78]. This view is further supported by recent evidence indicating that the pathogenesis of NOMID in mice is prevented by (i) genetic ablation of GSDMD and (ii) a novel inhibitor of the interactions of p38α MAPK and MAPK-activated kinase 2, which inhibits not only IL-1β, but also IL-6 and TNF-α [
41,
77]. Thus, multiple responses, including pyroptosis, contribute to inflammasomopathies.
Macrophage activation syndrome
The NLRC4 inflammasome senses bacterial type III and IV secretion systems and flagellin via NLR family apoptosis inhibitory proteins (NAIPs). As noted above,
NLRC4-activating mutations are found in certain FCAS patients. Moreover, recent evidence implicates the NLRC4 inflammasome in the pathogenesis of sterile inflammatory disorders as patients with
NLRC4 gain-of-function mutations develop cytopenia, high ferritin levels, hemophagocytosis, and splenomegaly. These symptoms are associated with excessive levels of IL-18 and IL-1β, and recurring fever flares, a phenotype that is reminiscent of macrophage activation syndrome (MAS) [
12,
80]. MAS is a frequent complication of systemic juvenile idiopathic arthritis (sJIA), a disease that interferes with healthy skeletal development and bone mass acquisition [
102]. Although IL-1β levels are in general lower in MAS compared with CAPS, IL-1 biologics are efficacious in the treatment of sJIA [
103,
104]. Consistent with the role of mutated NLRC4 in the development of joint pathologies, transgenic mice expressing constitutively active NLRC4 produce high levels of IL-1β and IL-18, and develop arthritis [
81]. The NLRC4 inflammasome is also activated by nucleotide-derived metabolites (e.g., adenine and N-4-acetylcytidine) [
6] and fatty acids (e.g., lysophosphatidylcholine and palmitate) [
89] and is upregulated by bone-derived DAMPs during osteoclastogenesis [
90]. An interplay between the NLRP3 and NLRC4 inflammasomes has been noted in these models of sterile inflammation as well as in response to
Salmonella infection [
105,
106].
Familial Mediterranean fever
MEFV encodes pyrin, which activates caspase-1 through ASC upon sensing post-translationally modified Rho GTPase [
107]. These modifications include phosphorylation, ADP-ribosylation, and glycosylation and occur upon cell exposure to
Clostridium toxins and in conditions of mevalonate kinase deficiency or proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) gain-of-function [
82,
108‐
110]. Hyper-activation of the pyrin inflammasome by
MEFV-activating mutations causes Familial Mediterranean fever (FMF), a disease that is characterized by high levels of IL-1β, IL-6, IL-8, and IL-12, recurring fever episodes, arthritis, and low bone mass [
83,
111]. FMF is the most prevalent monogenic autoinflammatory disorder; it affects over 100,000 persons worldwide and causes sporadic and chronic symptoms [
98]. FMF mice develop severe systemic inflammation and exhibit massive cartilage and bone erosion [
112]. These mice do not display systemic inflammatory symptoms upon deletion of GSDMD, IL-1 receptor, or ASC [
112,
113].
Arthritis
Inflammasomes are activated in several autoimmune diseases, including rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Components of the NLRP3 inflammasomes and various cytokines including TNF-α, IL-1β, IL-6, IL-7, and IL-17 are highly expressed in RA [
84,
114]. Specific interactions among these cytokines and other inflammatory mediators may drive systemic and focal osteolysis in arthritis [
115]. Systemic arthritis and bone loss induced by TNF over-expression are abolished upon ablation of IL-1β signaling despite the presence of synovial inflammation, suggesting that the effects of TNF-α on bone are mediated by IL-1β [
116]. These findings positioning IL-1β downstream of TNF-α are in line with the observation that TNF-α-induced RANKL production by murine stromal cells is dependent on IL-1β [
85,
117]. As noted above, the upregulation of TNF-α by IL-1β is also well known. Components of the inflammasomes, including NLRP3, ASC, and caspase-1 are upregulated in AS, a disease that is also associated with high levels of IL-1β, TNF-α, IL-6, IL-23, and IL-17 [
118]. Bone manifestations in AS include excessive focal bone formation in joints whereas pronounced trabecular bone loss occurs in vertebral bodies [
119].
Various inflammasomes are assembled in mouse models of arthritis. For example, NLRP3 and AIM-2 are both upregulated in the synovium of IL-10-deficient mice exposed to antigen-induced arthritis, and osteoclast differentiation from bone marrow cells isolated from these mutant mice is blunted by the inhibitors of NLRP3 and AIM-2 inflammasomes [
120]. Moreover, arthritis induced by DNase II deficiency, which is associated with accrual of self-DNA, is attenuated by AIM2 ablation [
86,
87]. The complexity of inflammasome functions is underscored by the observations that NLRP3, NLRP1, NLRC4, and caspase-1, but not ASC are dispensable for collagen-induced and antigen-induced arthritis [
88,
121]; yet NLRP3 is a key player in joint destruction in a mouse model of A20 deficiency, in which NLRC4 and AIM2 are expendable [
122]. Thus, the role of the inflammasomes in experimental arthritis is mouse model-dependent.
Osteomyelitis
Defective innate immune defense mechanisms including the inflammasomes underlie the pathogenesis of periodontitis and osteomyelitis commonly caused by
Porphyromonas gingivalis and
Staphylococcus aureus, respectively.
P. gingivalis–derived PAMPs such as LPS are potent activators of priming signals, which through TLR4 signaling drive the expression of several components of the inflammasomes including IL-1β, NLRP3, AIM2, and caspase-11 [
57,
58]. Accordingly, the massive alveolar bone destruction caused by
P. gingivalis is attenuated upon loss of NLRP3 [
59].
S. aureus bacterial products include peptidoglycans, hemolysins, bacterial lipoproteins, and Panton-Valentine leucocidin stimulate the inflammasomes through TLR2-mediated activation of NF-kB [
123]. Moreover, some of these factors promote osteoclastogenesis [
124]. The osteoblasts also express the NLRP3 inflammasome [
60] though to lower levels compared with myeloid cells [
73] and contribute to the pathogenesis of periodontitis and osteomyelitis [
61,
125‐
127].
Autoinflammatory reactions of unknown etiology causes confined chronic non-bacterial osteomyelitis (CNO) or systemic chronic recurrent multifocal osteomyelitis (CRMO). Components of the NLRP3 inflammasome are expressed in osteoclasts in bone specimens from CRMO patients [
62]. Mice carrying an inactivating -mutation in the proline-serine-threonine phosphatase-interacting protein 2 gene (
Pstpip2) develop a phenotype reminiscent of CRMO, which is associated with over-production of IL-1β, enhanced osteoclastogenesis and bone resorption, responses that depend on IL-1 receptor and IL-1β, but not IL-1α, and are driven by neutrophils [
21,
22]. Neutrophils in CRMO mice over-produce IL-1β via redundant actions of caspase-8 and the NLRP3 inflammasome [
23].
Inflammasomes have been linked to age- and menopause-related osteoporosis [
6,
90,
91]. Estrogen profoundly affects the skeleton through various mechanisms including immunomodulation, suppressive effects on the expression of TNF-α and IL-1β, induction of osteoclast apoptosis through ERα, suppression of osteoclast differentiation, inhibition of RANKL production by osteoblasts, T and B cells, and stimulation of osteoprotegerin (OPG) expression [
31,
128,
129]. Consistent with increased levels of pro-inflammatory cytokines in conditions of estrogen deficiency, blockade of TNF-α or IL-1β in post-menopausal patients leads to a decrease in the levels of bone resorption markers [
130]. Accordingly, inhibition of TNF-α or deletion of NLRP3 protects against ovariectomy-induced bone loss in mice [
90,
131].
Aging is associated with low-grade chronic inflammation. This process is referred to as inflammaging and is associated with increased levels of circulating IL-18, IL-1 receptor antagonist, and IL-6 [
132]. Inflammasome gene modules including NLRC4 and IL-1β are upregulated in older people compared with younger individuals; persistent expression of these genes correlates with the occurrence of age-related complications, including chronic production of inflammatory cytokines, metabolic dysfunction, and oxidative stress [
6]. The NLRP3 inflammasome also modulates age-related inflammation in peripheral tissues and age-related bone loss in mice, though the underlying mechanisms are unknown [
91].
The ability of the NLRP3 inflammasome to detect a wide variety of endogenous DAMPs is likely an important driver in the development of age- and metabolic-related pathologies. These DAMPs include crystalline cholesterol, extracellular ATP, purine and pyrimidine metabolites, and debris from damaged tissues [
6,
133]. For example, metabolites from the purine and pyrimidine pathways stimulate the NLRP3 and NLRC4 inflammasomes in THP-1 cells, activate human platelets and neutrophils in cultures, and promote hypertension and inflammation in mice [
6]. The NLRP3 inflammasome may also be involved in hyper-multinucleation of murine osteoclasts caused by purinergic receptor P2X5 signaling [
134]. We have shown that bone matrix degradation products regulate the NLRP3 and NLRC4 inflammasomes in cells of the osteoclast lineage [
90]. Accordingly,
Nlrp3 null mice are protected from bone loss induced by ovariectomy, sustained exposure to parathyroid hormone or RANKL. Treatment of mice with zoledronic acid inhibits inflammasome activation, thus reinforcing the view that endogenous DAMPs are released from the bone matrix during bone resorption, causing autocrine and paracrine effects on osteoclastogenesis [
90].
Wear debris-induced osteolysis
Wear particles from articulating prosthetic joint surfaces such as those from cobalt-chromium-molybdenum (CoCrMo) implants induce inflammatory responses that cause aseptic loosening as a result of uncontrolled osteolysis [
135]. The osteolytic process is associated with the formation at the implant-bone interface of a cellular membrane enriched in cells of the monocyte-macrophage lineage [
136]. Activation of the NF-kB pathway through TLR2 signaling by prosthetic debris promote not only the expression of pro-inflammatory cytokines such as TNF-α, but also priming signals for the NLRP3 and AIM2 inflammasomes. Macrophages can also phagocytose these particles; cellular accumulation of these non-degradable materials enhances the production of reactive oxygen species and the rupture of the phagosomes, which releases cathepsins in the cytoplasm, events that activate the inflammasomes [
92]. The size and shape of CoCrMo alloys affect the amplitude of the inflammatory responses [
93]. Thus, wear debris can provide both priming and assembly signals that lead to aberrant inflammasome activation. Consistent with the role of the NLRP3 inflammasome in bone damage induced by prosthetic particles, bone resorption induced by polymethylmethacrylate (PMMA) particles is reduced in the absence of caspase-1 [
3,
145]. Thus, both the metal and plastic components of the prostheses activate the inflammasomes.
Crystal-induced arthropathies
Endogenous crystalline particles are involved in the pathogenesis of arthropathies. For example, gouty arthritis is caused by precipitation of monosodium urate (MSU) crystals [
94], calcium pyrophosphate deposition disease (CPDD) is driven by calcium pyrophosphate dihydrate (CPPD) crystals [
94], and degenerative arthropathies such as osteoarthritis and Milwaukee shoulder are the result of abnormal accumulation of basic calcium phosphate (BCP) crystals [
137]. Shared mechanisms among these diseases include phagocytosis of crystals by myeloid cells, an event that activates several inflammatory pathways including the inflammasomes, chondrocyte apoptosis, and matrix calcification [
138‐
140]. Bone erosions in gout stems from continuous recruitment of macrophages to tophi [
95]; sporadic CPDD is characterized by the presence of CPPD crystals in articular cartilage whereas patients with CPDD familial patterns have low bone mineral density though the extent to which bone resorption is impacted is not known [
141]. BCP crystals, particularly hydroxyapatite crystals, promote osteoclast formation in vitro in NLRP3 inflammasome-dependent manner [
90,
96,
142]. However, the actions of BCP on NLRP3 inflammasome-mediated skeletal pathology are controversial. Lack of components of the NLRP3 inflammasome prevents the development of neutrophil inflammation in the air-pouch model of synovitis and decreased pathology in the Ank-deficient model of arthritis [
97]. By contrast, inflammatory responses induced by the injection of BCP crystals into the knees or intra-peritoneally are independent of the NLRP3 inflammasome [
143,
144]. Alternative processing of IL-1β by caspase-8 and pyroptosis may account for these discrepant observations.