NLRP3 inflammasome inhibition is disrupted in a group of auto-inflammatory disease CAPS mutations.

Inflammasomes are positioned to rapidly escalate the intensity of inflammation by activating interleukin (IL)-1ß, IL-18 and cell death by pyroptosis. However, negative regulation of inflammasomes remains poorly understood, as is the signaling cascade that dampens inflammasome activity. We found that rapid NLRP3 inflammasome activation was directly inhibited by protein kinase A (PKA), which was induced by prostaglandin E2 (PGE2) signaling via the PGE2 receptor E-prostanoid 4 (EP4). PKA directly phosphorylated the cytoplasmic receptor NLRP3 and attenuated its ATPase function. We found that Ser295 in human NLRP3 was critical for rapid inhibition and PKA phosphorylation. Mutations in NLRP3-encoding residues adjacent to Ser295 have been linked to the inflammatory disease CAPS (cryopyrin-associated periodic syndromes). NLRP3-S295A phenocopied the human CAPS mutants. These data suggest that negative regulation at Ser295 is critical for restraining the NLRP3 inflammasome and identify a molecular basis for CAPS-associated NLRP3 mutations.

CANE, a Component of the NLRP3 Inflammasome, Promotes Inflammasome Activation.

The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3, also called cryopyrin) inflammasome is an intracellular innate immune complex, which consists of the pattern-recognition receptor NLRP3, the adaptor apoptosis-assciated speck-like protein containing a caspase recruitment domain, and procaspase-1. Aberrant activation of the NLRP3 inflammasome causes an autoinflammatory disease called cryopyrin-associated periodic syndrome (CAPS). CAPS is caused by gain-of-function mutations in the NLRP3-encoding gene CIAS1; however, the mechanism of CAPS pathogenesis has not been fully understood. Thus, unknown regulators of the NLRP3 inflammasome, which are associated with CAPS development, are being investigated. To identify novel components of the NLRP3 inflammasome, we performed a high-throughput screen using a human protein array, with NLRP3 as the bait. We identified a NLRP3-binding protein, which we called the cryopyrin-associated nano enhancer (CANE). We demonstrated that CANE increased IL-1ß secretion after NLRP3 inflammasome reconstitution in human embryonic kidney 293T cells and formed a "speck" in the cytosol, a hallmark of NLRP3 inflammasome activity. Reduced expression of endogenous CANE decreased IL-1ß secretion upon stimulation with the NLRP3 agonist nigericin. To investigate the role of CANE in vivo, we developed CANE-transgenic mice. The PBMCs and bone marrow-derived macrophages of CANE-transgenic mice exhibited increased IL-1ß secretion. Moreover, increased autoinflammatory neutrophil infiltration was observed in the s.c. tissue of CANE-transgenic versus wild-type mice; these phenotypes were consistent with those of CAPS model mice. These findings suggest that CANE, a component of the NLRP3 inflammasome, is a potential modulator of the inflammasome and a contributor to CAPS pathogenesis.

Isoliquiritigenin inhibits NLRP3 inflammasome activation with CAPS mutations by suppressing caspase-1 activation and mutated NLRP3 aggregation.

The nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome contributes to the development of inflammatory diseases. Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory disease caused by NLRP3 gene mutations that results in excessive IL-1ß production. We previously identified isoliquiritigenin (ILG), a component of Glycyrrhiza uralensis extracts, as a potent inhibitor of the NLRP3 inflammasome. Here, we aimed to investigate whether ILG inhibits the activation of NLRP3 inflammasome caused by NLRP3 gene mutations. We demonstrated that ILG significantly inhibited NLRP3 inflammasome-mediated lactate dehydrogenase (LDH) release and IL-1ß production in two CAPS model THP-1 cell lines, NLRP3-D303N and NLRP3-L353P, in a dose-dependent manner. Interestingly, the NLRP3 inhibitor MCC950 inhibited LDH release and IL-1ß production in NLRP3-D303N cells, but not in NLRP3-L353P cells. Western blotting and caspase-1 activity assays showed that ILG, as well as caspase inhibitors, including Z-VAD and YVAD, suppressed caspase-1 activation. Notably, ILG prevented cryo-sensitive foci formation of NLRP3 without affecting the levels of intracellular Ca2+. We concluded that ILG effectively prevents the constitutive activation of the inflammasome associated with NLRP3 gene mutations by inhibiting the aggregation of cryo-sensitive mutated NLRP3.

Antisense Oligonucleotide Therapy Decreases IL-1ß Expression and Prolongs Survival in Mutant Nlrp3 Mice.

Antisense oligonucleotides (ASOs) are a novel therapeutic strategy that targets a specific gene and suppresses its expression. The cryopyrin-associated periodic syndromes (CAPS) are a spectrum of autoinflammatory diseases characterized by systemic and tissue inflammation that is caused by heterozygous gain-of-function mutations in the nucleotide-binding and oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) gene. The aim of this study was to investigate the efficacy of an Nlrp3-specific ASO treatment in CAPS. An Nlrp3-specific ASO was designed and tested in murine cell lines and bone marrow-derived macrophages (BMDMs) from wild-type and CAPS mouse models. Nlrp3 knock-in mice were treated in vivo with Nlrp3-specific ASO, survival was monitored, and expression of organ-specific Nlrp3 and IL-1ß was measured. Nlrp3-specific ASO treatment of murine cell lines and BMDMs showed a significant downregulation of Nlrp3 and mature IL-1ß protein expression. Ex vivo treatment of Nlrp3 mutant mouse-derived BMDMs with Nlrp3-specific ASO demonstrated significantly reduced IL-1ß release. In vivo, Nlrp3-specific ASO treatment of Nlrp3 mutant mice prolonged survival, reduced systemic inflammation, and decreased tissue-specific expression of Nlrp3 and mature IL-1ß protein. The results of this study demonstrate that Nlrp3-specific ASO treatment downregulates Nlrp3 expression and IL-1ß release in CAPS models, suggesting ASO therapy as a potential treatment of CAPS and other NLRP3-mediated diseases.

Lipids in inflammasome activation and autoinflammatory disorders.

Autoinflammatory diseases (AIDs) are a group of rare monogenetic disorders characterized by recurrent episodes of fever and systemic inflammation. A major pathologic hallmark of AIDs is excessive inflammasome assembly and activation, often the result of gain-of-function mutations in genes encoding core inflammasome components, including pyrin and cryopyrin. Recent advances in lipidomics have revealed that dysregulated metabolism of lipids such as cholesterol and fatty acids, especially in innate immune cells, exerts complex effects on inflammasome activation and the pathogenesis of AIDs. In this review, we summarize and discuss the impact of lipids and their metabolism on inflammasome activation and the disease pathogenesis of the most common AIDs, including familial Mediterranean fever, cryopyrin-associated periodic syndromes, and mevalonate kinase deficiency. We postulate that lipids hold diagnostic value in AIDs and that dietary and pharmacologic intervention studies could represent a promising approach to attenuate inflammasome activation and AID progression.

A novel Nlrp3 knock-in mouse model with hyperactive inflammasome in development of lethal inflammation.

NOD-like receptor family, pyrin domain-containing 3 (NLRP3) is a central protein contributing to human inflammatory disorders, including cryopyrin-associated periodic syndrome and sepsis. However, the molecular mechanisms and functions of NLRP3 activation in various diseases remain unknown. Here, we generated gain-of-function knock-in mice associated with Muckle-Wells syndromes using the Cre-LoxP system allowing for the constitutive T346M mutation of NLRP3 to be globally expressed in all cells under the control of tamoxifen. The mice were treated with tamoxifen for 4 days before determining their genotype by PCR and sequence analysis. In vitro, we found that bone marrow-derived macrophage from homozygous T346M mutation mice displayed a robust ability to produce IL-1ß in response to lipopolysaccharide exposure. Moreover, ASC specks and oligomerization were observed in the homozygous mutant bone marrow-derived macrophages in the presence of lipopolysaccharides alone. Mechanistically, K+ and Ca2+ depletion and mitochondrial depolarization contribute to the hyperactivation of mutant NLRP3. In vivo, homozygous mice carrying the T346M mutation exhibit weight loss and mild inflammation in the resting state. In the lipopolysaccharide-mediated sepsis model, homozygous mutant mice exhibited higher mortality and increased serum circulating cytokine levels, accompanied by serious liver injury. Furthermore, an increase in myeloid cells in the spleen has been suggested to be a risk factor for inducing sepsis sensitivity. Altogether, we describe a cryopyrin-associated syndrome animal model with the T346M mutation of NLRP3 and suggest that the hyperactivated inflammasome aggregated by the mutant NLRP3 lowers the inflammatory response threshold both in vitro and in vivo.

Equally potent: Nlrp3 mutation in macrophages or neutrophils is sufficient to drive autoinflammation.

Gain-of-function mutation in NLRP3 is associated with a spectrum of autoinflammatory disorders including familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and neonatal onset multisystem inflammatory disease, collectively known as cryopyrin-associated periodic syndrome (CAPS). However, the cell types mediating the pathogenesis of CAPS are not completely understood. Two studies in EMBO Reports now demonstrate that gain-of-function Nlrp3 mutation in either macrophages or neutrophils alone is sufficient to trigger systemic autoinflammation and lethality in mice.

Nlrp3 inflammasome activation in macrophages suffices for inducing autoinflammation in mice.

Cryopyrin-associated periodic syndromes (CAPS) are a spectrum of autoinflammatory disorders caused by gain-of-function NLRP3 mutant proteins that form hyperactive inflammasomes leading to overproduction of the pro-inflammatory cytokines IL-1ß and IL-18. Expressing the murine gain-of-function Nlrp3A350V mutant selectively in neutrophils recapitulates several autoinflammatory features of human CAPS, but the potential contribution of macrophage inflammasome hyperactivation to CAPS development is poorly defined. Here, we show that expressing Nlrp3A350V in macrophages is sufficient for driving severe multi-organ autoinflammation leading to perinatal lethality in mice. In addition, we show that macrophages contribute to autoinflammation also in adult mice, as depleting macrophages in mice ubiquitously expressing Nlrp3A350V significantly diminishes splenic and hepatic IL-1ß production. Interestingly, inflammation induced by macrophage-selective Nlrp3A350V expression does not provoke an influx of mature neutrophils, while neutrophil influx is still occurring in macrophage-depleted mice with body-wide Nlrp3A350V expression. These observations identify macrophages as important cellular drivers of CAPS in mice and support a cooperative cellular model of CAPS development in which macrophages and neutrophils act independently of each other in propagating severe autoinflammation.

Optical coherence tomography assessment of disease activity in cryopyrin-associated periodic syndrome.

BACKGROUND AND PURPOSE: Cryopyrin-associated periodic syndrome is a rare autoinflammatory disease caused by gain-of-function mutations or variants in the NLRP3 gene. Clinically, patients suffer from a broad spectrum of both systemic and neurological symptoms. The aim of this study was to determine whether systemic inflammation demonstrated by serum amyloid A (SAA) elevation is associated with neuroinflammation assessed by optical coherence tomography (OCT). METHODS: Thirty eyes of 15 patients with NLRP3 low penetrance mutations (PwNLRP3) and 20 eyes of 10 age- and sex-matched healthy controls were examined by spectral-domain OCT as part of routine clinical care. All retinal layers and clinical features were evaluated. RESULTS: At baseline no significant retinal neuroaxonal inflammation or degeneration was observed in all measured retinal layers amongst PwNLRP3 compared with healthy controls. In a pooled analysis of all individual OCT time points a significant difference regarding the macular retinal nerve fibre layer was detected. Increased levels of SAA showed a positive association with averaged combined outer plexiform layer and outer nuclear layer volumes (ρ < 0.0001, r2 = 0.35). CONCLUSION: In cryopyrin-associated periodic syndrome increased combined outer plexiform layer and outer nuclear layer volumes are mirrored by SAA increase, an acute phase reactant indicating systemic inflammation. Our findings identify OCT as a candidate biomarker to monitor subclinical neuroinflammation and to assess disease activity in PwNLRP3.

Identifying high-risk neurological phenotypes in adult-onset classic monogenic autoinflammatory diseases: when should neurologists consider testing?

BACKGROUND: Monogenic autoinflammatory disorders result in a diverse range of neurological symptoms in adults, often leading to diagnostic delays. Despite the significance of early detection for effective treatment, the neurological manifestations of these disorders remain inadequately recognized. METHODS: We conducted a systematic review searching Pubmed, Embase and Scopus for case reports and case series related to neurological manifestations in adult-onset monogenic autoinflammatory diseases. Selection criteria focused on the four most relevant adult-onset autoinflammatory diseases-deficiency of deaminase 2 (DADA2), tumor necrosis factor receptor associated periodic fever syndrome (TRAPS), cryopyrin associated periodic fever syndrome (CAPS), and familial mediterranean fever (FMF). We extracted clinical, laboratory and radiological features to propose the most common neurological phenotypes. RESULTS: From 276 records, 28 articles were included. The median patient age was 38, with neurological symptoms appearing after a median disease duration of 5 years. Headaches, cranial nerve dysfunction, seizures, and focal neurological deficits were prevalent. Predominant phenotypes included stroke for DADA2 patients, demyelinating lesions and meningitis for FMF, and meningitis for CAPS. TRAPS had insufficient data for adequate phenotype characterization. CONCLUSION: Neurologists should be proactive in diagnosing monogenic autoinflammatory diseases in young adults showcasing clinical and laboratory indications of inflammation, especially when symptoms align with recurrent or chronic meningitis, small vessel disease strokes, and demyelinating lesions.

Pathogenic variants in the NLRP3 LRR domain at position 861 are responsible for a boost-dependent atypical CAPS phenotype.

BACKGROUND: Cryopyrin-associated periodic syndrome (CAPS) is associated with NLRP3 pathogenic variants, mostly located in the NACHT (neuronal apoptosis inhibitor protein, MHC class 2 transcription activator, incompatibility locus protein from Podospora anserina, telomerase-associated protein) domain. Cold-induced urticarial rash is among the main clinical features. However, this study identified a series of 14 patients with pathogenic variants of the Y861 residue (p.Tyr861) of the LRR domain of NLRP3 and minimal prevalence of cold-induced urticarial rash. OBJECTIVES: This study aimed to address a possible genotype/phenotype correlation for patients with CAPS and to investigate at the cellular levels the impact of the Y861C substitution (p.Tyr861Cys) on NLRP3 activation. METHODS: Clinical features of 14 patients with CAPS and heterozygous substitution at position 861 in the LRR domain of NLRP3 were compared to clinical features of 48 patients with CAPS and pathogenic variants outside the LRR domain of NLRP3. IL-1ß secretion by PBMCs and purified monocytes from patients and healthy donors was evaluated following LPS and monosodium urate crystal stimulation. RESULTS: Patients with substitution at position 861 of NLRP3 demonstrated a higher prevalence of sensorineural hearing loss while being less prone to skin urticarial. In contrast to patients with classical CAPS, cells from patients with a pathogenic variant at position 861 required an activation signal to secrete IL-1ß but produced more IL-1ß during the early and late phase of secretion than cells from healthy donors. CONCLUSIONS: Pathogenic variants of Y861 of NLRP3 drive a boost-dependent oversecretion of IL-1ß associated with an atypical CAPS phenotype.

Complex regulation of alarmins S100A8/A9 and secretion via gasdermin D pores exacerbates autoinflammation in familial Mediterranean fever.

BACKGROUND: Familial Mediterranean fever (FMF), caused by mutations in the pyrin-encoding MEFV gene, is characterized by uncontrolled caspase-1 activation and IL-1ß secretion. A similar mechanism drives inflammation in cryopyrin-associated periodic fever syndrome (CAPS) caused by mutations in NLRP3. CAPS and FMF, however, result in largely different clinical manifestations, pointing to additional, autoinflammatory pathways involved in FMF. Another hallmark of FMF is extraordinarily high expression of S100A8 and S100A9. These alarmins are ligands of Toll-like receptor 4 and amplifiers of inflammation. However, the relevance of this inflammatory pathway for the pathogenesis of FMF is unknown. OBJECTIVE: This study investigated whether mutations in pyrin result in specific secretion of S100A8/A9 alarmins through gasdermin D pores' amplifying FMF pathology. METHODS: S100A8/A9 levels in FMF patients were quantified by enzyme-linked immunosorbent assay. In vitro models with knockout cell lines and specific protein inhibitors were used to unravel the S100A8/A9 secretion mechanism. The impact of S100A8/A9 to the pathophysiology of FMF was analyzed with FMF (MEFVV726A/V726A) and S100A9-/- mouse models. Pyrin-S100A8/A9 interaction was investigated by coimmunoprecipitation, immunofluorescence, and enzyme-linked immunosorbent assay studies. RESULTS: The S100A8/A9 complexes directly interacted with pyrin. Knocking out pyrin, caspase-1, or gasdermin D inhibited the secretion of these S100 alarmins. Inflammatory S100A8/A9 dimers were inactivated by tetramer formation. Blocking this inactivation by targeted S100A9 deletion in a murine FMF model demonstrated the relevance of this novel autoinflammatory pathway in FMF. CONCLUSION: This is the first proof that members of the S100 alarmin family are released in a pyrin/caspase-1/gasdermin D-dependent pathway and directly drive autoinflammation in vivo.

NLRP3 leucine-rich repeats control induced and spontaneous inflammasome activation in cryopyrin-associated periodic syndrome.

BACKGROUND: The cryopyrin-associated periodic syndromes (CAPS) comprise a group of rare autoinflammatory diseases caused by gain-of-function mutations in the NLRP3 gene. NLRP3 contains a leucine-rich repeats (LRR) domain with a highly conserved exonic organization that is subjected to extensive alternative splicing. Aberrant NLRP3 inflammasome assembly in CAPS causes chronic inflammation; however, the mechanisms regulating inflammasome function remain unclear. OBJECTIVE: We aimed to elucidate the mechanisms regulating NLRP3-mediated autoinflammation in human disease, characterizing the role of LRR in inflammasome activation. METHODS: We analyzed sequence read archive data to characterize the pattern of NLRP3 splicing in human monocytes and investigated the role of each LRR-coding exon in inflammasome regulation in genetically modified U937 cells representing CAPS and healthy conditions. RESULTS: We detected a range of NLRP3 splice variants in human primary cells and monocytic cell lines, including 2 yet-undescribed splice variants. We observe that lipopolysaccharides affect the abundance of certain splice variants, suggesting that they may regulate NLRP3 activation by affecting alternative splicing. We showed that exons 4, 5, 7, and 9 are essential for inflammasome function, both in the context of wild-type NLRP3 activation by the agonist molecule nigericin and in a model of CAPS-mediated NLRP3 inflammasome assembly. Moreover, the SGT1-NLRP3 interaction is decreased in nonfunctional variants, suggesting that alternative splicing may regulate the recruitment of proteins that facilitate inflammasome assembly. CONCLUSION: These findings demonstrate the contribution of the LRR domain in inflammasome function and suggest that navigating LRR exon usage within NLRP3 is sufficient to dampen inflammasome assembly in CAPS.

Cold-induced loss of interaction with HSC70 triggers inflammasome activity of familial cold autoinflammatory syndrome-causing mutants of NLRP3.

NLRP3 is a cytoplasmic receptor protein, which initiates caspase-1 mediated inflammatory immune response upon detection of invading pathogen or a wide array of internal distress signals. Several gain-of function mutations of NLRP3 cause hereditary disorder of cold-induced hyper-inflammation known as familial cold autoinflammatory syndrome-1 (FCAS1). Although, caspase-1 activation and downstream interleukin-1ß/interleukin-18 maturation are common effectors in pathophysiology of this disorder, molecular mechanisms of how exposure to subnormal temperature triggers mutant NLRP3-inflammsome activity is not understood. Here, we show that endogenous NLRP3 is in complex with HSC70 (HSPA8), and this interaction is reduced upon exposure to cold. FCAS-causing NLRP3-L353P and NLRP3-R260W mutants show enhanced interaction with HSC70. Upon exposure to subnormal temperature, NLRP3-L353P and NLRP3-R260W show enhanced inflammasome formation, increased caspase-1 activation and reduced interaction with HSC70. Knockdown of HSC70 results in increased inflammasome formation by L353P and R260W mutants of NLRP3. Our results suggest that interaction with HSC70 suppresses inflammasome formation by FCAS-causing NLRP3 mutants at physiological temperature, and loss of this inhibitory association at subnormal temperature causes aggravated inflammasome formation and caspase-1 activation leading to interleukin-1ß maturation. These results provide evidence for HSC70 being a cold-sensor and a temperature-dependent regulator of inflammatory signaling by FCAS-causing NLRP3 mutants.

Diagnostic and therapeutic algorithms for monogenic autoinflammatory diseases presenting with recurrent fevers among adults.

Autoinflammatory diseases (AIDs) are defined as disorders of innate immunity. They were initially defined in contrast to autoimmune diseases because of the lack of involvement of the adaptive immune system and circulating autoantibodies. The four monogenic AIDs first described are called the 'historical' AIDs and include FMF (associated with MEFV mutations), cryopyrinopathies (associated with NLRP3 mutations), TNF receptor-associated periodic syndrome (associated with TNFRSF1A mutations) and mevalonate kinase deficiency (MKD; associated with MVK mutations). In the last 10 years, >50 new monogenic AIDs have been discovered due to genetic advances. The most important discovery for adult patients is VEXAS syndrome associated with somatic UBA1 mutations leading to an AID affecting mostly elderly men. Diagnosis of monogenic AIDs is based on personal and family history and detailed analysis of symptoms associated with febrile attacks in the context of elevated peripheral inflammatory markers. This review proposes a practical approach for the diagnosis of the main monogenic AIDs among adult patients.

Subdural hemorrhage, macrocephaly, rash, and developmental delay in an infant: A pathogenic variant in NLRP3 causes CINCA/NOMID.

Subdural hemorrhages (SDHs) in children are most often observed in abusive head trauma (AHT), a distinct form of traumatic brain injury, but they may occur in other conditions as well, typically with clear signs and symptoms of an alternative diagnosis. We present a case of an infant whose SDH initially raised the question of AHT, but multidisciplinary evaluation identified multiple abnormalities, including rash, macrocephaly, growth failure, and elevated inflammatory markers, which were all atypical for trauma. These, along with significant cerebral atrophy, ventriculomegaly, and an absence of other injuries, raised concerns for a genetic disorder, prompting genetic consultation. Clinical trio exome sequencing identified a de novo likely pathogenic variant in NLRP3, which is associated with chronic infantile neurological, cutaneous, and articular (CINCA) syndrome, also known as neonatal-onset multisystem inflammatory disease (NOMID). He was successfully treated with interleukin-1 blockade, highlighting the importance of prompt treatment in CINCA/NOMID patients. This case also illustrates how atraumatic cases of SDH can be readily distinguished from AHT with multidisciplinary collaboration and careful consideration of the clinical history and exam findings.

Socio-professional impact and quality of life of cryopyrin-associated periodic syndromes in 54 patients in adulthood.

OBJECTIVES: Cryopyrin-associated periodic syndromes (CAPS) belongs to the group of hereditary recurrent fever disorders characterised by interleukin1ß-mediated systemic inflammation. Specific treatment by IL-1 targeting drugs has significantly modified the disease evolution. We aimed to evaluate the socio-professional impact of CAPS in the long term and the influence of genetic variants in the phenotype. METHODS: We made a multicentre, observational and descriptive study and collected retrospective data from childhood to adulthood, and until the last year of follow-up. We assessed the quality of life (QoL) of the patients by phone interviews. We also used the SF36 questionnaire including 8 domains: physical function, physical role, body pain, general health, vitality, social function, emotional role and mental health. A high score means a better QoL. RESULTS: Fifty-four patients were evaluated (14 familial cold autoinflammatory syndrome, 27 Muckle-Wells syndrome, 7 chronic infantileneurological cutaneous and articular syndrome. The study showed improvement in symptoms in adulthood and good QoL in all domains apart from school (87%) and work (61%) absenteeism. The MWS group is intermediate in terms of symptoms but seems to describe a better QoL compared to the other groups. The genetic variant alone does not determine the expression of the disease. CONCLUSIONS: Our study shows that CAPS patients have an improvement of symptoms in adulthood and a satisfactory QoL for most of them. Anti-IL1 treatment is the main factor linked to this improvement and therefore early initiation should be encouraged.

NLRP12-associated autoinflammatory disease: much more than the FCAS phenotype.

OBJECTIVES: NLRP12-associated autoinflammatory disease (NLRP12-AID) is a rarely seen periodic fever syndrome also known as familial cold autoinflammatory syndrome-2 (FCAS2), caused by autosomal dominant inherited mutations in the NLRP12 gene. We aimed to present our clinical experience constituting one of the largest paediatric NLRP12-AID cohort. METHODS: The patients with preliminary diagnosis of systemic autoinflammatory disease (SAID) other than familial Mediterranean fever (FMF) and PFAPA syndrome were evaluated with the next-generation-sequence (NGS) genetic-panel analysis between January-2016 and January-2022. Among children carrying NLRP12-variant, patients with recurrent episodes of autoinflammatory disease manifestations were diagnosed with NLRP12-AID. Demographic, clinical and laboratory data, treatments and outcomes of patients were presented. RESULTS: Seventeen patients were diagnosed with NLRP12-AID. The mean age at diagnosis was 114.7±69.5 months. The most frequently seen clinical manifestations were respectively; fever (100%), arthritis/arthralgia (58.8%), rash (52.9%), abdominal pain (52.9%), diarrhoea (41.2%), myalgia/fatigue (53.2%) and, conjunctivitis (11.7%). Clinical manifestations were triggered by cold exposure in three patients (17.6%). Seven patients had pathogenic, one had likely pathogenic, seven had VUS, and two had novel heterozygous variants. The most common defined variant in the NLRP12 gene was R352C. Complete response was achieved in 5 patients and partial response was in 6 with colchicine treatment. Attacks were prevented with anti-IL-1 treatments in 6 patients unresponsive to colchicine. CONCLUSIONS: In conclusion, the disease can cause effects on various tissues, especially the musculoskeletal and gastrointestinal systems, apart from FCAS symptoms. We think that a patient who can be defined as syndrome of undifferentiated recurrent fever should also be evaluated genetically in terms of NLRP12 previously.

Similarities and differences in autoinflammatory diseases with urticarial rash, cryopyrin-associated periodic syndrome and Schnitzler syndrome.

Cryopyrin-associated periodic syndromes (CAPS) and Schnitzler syndrome (SchS) are autoinflammatory diseases that present with urticaria-like rashes. CAPS is characterized by periodic or persistent systemic inflammation caused by the dysfunction of the NLRP3 gene. With the advent of IL-1-targeted therapies, the prognosis of CAPS has improved remarkably. SchS is considered an acquired form of autoinflammatory syndrome. Patients with SchS are adults of relatively older age. The pathogenesis of SchS remains unknown and is not associated with the NLRP3 gene. Previously, the p.L265P mutation in the MYD88 gene, which is frequently detected in Waldenström macroglobulinemia (WM) with IgM gammopathy, was identified in several cases of SchS. However, because persistent fever and fatigue are symptoms of WM that require therapeutic intervention, it is a challenge to determine whether these patients truly had SchS or whether advanced WM was misidentified as SchS. There are no established treatments for SchS. The treatment algorithm proposed with the diagnostic criteria is to use colchicine as first-line treatment, and systemic administration of steroids is not recommended due to concerns about side effects. In difficult-to-treat cases, treatment targeting IL-1 is recommended. If targeted IL-1 treatment does not improve symptoms, the diagnosis should be reconsidered. We hope that the efficacy of IL-1 therapy in clinical practice will serve as a stepping stone to elucidate the pathogenesis of SchS, focusing on its similarities and differences from CAPS.

In-vitro NLRP3 functional test assists the diagnosis of cryopyrin-associated periodic syndrome (CAPS) patients: A Brazilian cooperation.

OBJECTIVE: To report our five-years experience on the use of NLRP3 inflammasome functional assays in the differential diagnosis of Brazilian patients with a clinical suspicion of CAPS. PATIENTS AND METHODS: The study included 9 patients belonging to 2 families (I, II) and 7 unrelated patients with a clinical suspicion of AID according to Eurofever/PRINTO classification, recruited between 2017 and 2022. The control group for the NLRP3 functional assay consisted of 10 healthy donors and for the CBA cytokines measurement of 19 healthy controls. Patients underwent clinical evaluation, genetic and functional analysis. RESULTS: All members of the family I received the diagnosis of Muckle-Wells Syndrome (MWS), carried the NLRP3 Thr348Met variant and resulted positive for the functional assay. The 2 patients of the family II resulted negative for the mutational screening but positive for the functional assay compatible with a MWS clinical phenotype. In 2 unrelated patients with NLRP3 mutations, including a novel mutation (Gly309Val, Asp303His), a positive functional test confirmed the clinical diagnosis of NOMID. 3 unrelated MWS and 1 FCAS patients resulted negative to the genetic screening and positive for the functional test. One patient with a FCAS-like phenotype harbored the NLRP12 His304Tyr variant confirming the diagnosis of FCAS2. CONCLUSION: The NLRP3 inflammasome functional assay can assist the clinical diagnosis of CAPS even in patients with unknown genetic defects.