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Objective: To describe the pregnancy course and outcome, and use of anakinra, a recombinant selective IL-1 receptor blocker, during pregnancy in patients with cryopyrin-associated periodic syndromes (CAPS), including familial cold auto-inflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and neonatal onset multi-system inflammatory disease (NOMID). Methods: Women currently enrolled in natural history protocols (NCT00059748, and/or NCT00069329 under IND) who have been pregnant were included. Subjects underwent a structured, standardized interview with regards to maternal health, pregnancy and fetal outcomes. Medical records were reviewed. Results: Nine women (four with FCAS, one with MWS and four with NOMID) reported one to four pregnancies, each resulting in a total of fifteen FCAS, three MWS, and six NOMID pregnancies. Six births from FCAS mothers and three births from NOMID mothers occurred while patients were receiving anakinra. If a woman became pregnant while taking anakinra, the pre-pregnancy anakinra dose was continued. Anakinra dose was increased during one twin pregnancy. No preterm births or serious complications of pregnancy were observed. One fetus of the twin pregnancy had renal agenesis and suffered fetal demise. Genetic testing showed the deceased twin carried the same NLRP3 c.785T>C, p.V262A mutation as the mother. The other twin is healthy and mutation negative. Conclusions: Anakinra was continued during pregnancy in women with CAPS and provided significant, persistent CAPS symptom relief while continuing to prevent the long-term sequelae of CAPS. Anakinra was well tolerated. Although a causal relation between anakinra and renal agenesis seems unlikely, further safety data are needed.

BACKGROUND The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation. METHODS We analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-β, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls. RESULTS We identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mononuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibroblasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients’ lymphocytes was reduced by JAK inhibitors. CONCLUSIONS STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disease caused by gain-of-function mutations in TMEM173. (Funded by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases; ClinicalTrials.gov number, NCT00059748.)

Cryopyrin-associated periodic syndromes (CAPS) represent a spectrum of CIAS1 gene-mediated autoinflammatory diseases characterized by recurrent systemic inflammation. The clinical spectrum of CAPS varies from mild to severe and includes the syndromes historically described as familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID). This article presents the largest cohort of patients with CAPS. The objective is to describe the pathogenesis, otolaryngologic, and audiologic manifestations of CAPS.Prospective (2003-2009).National Institutes of Health.Fifty-seven patients with a diagnosis of CAPS were identified (31 NOMID, 11 NOMID/MWS, 9 MWS, and 6 FCAS). Comprehensive data regarding clinical manifestations, audiologic phenotype, and fluid attenuation inversion recovery MRI (FLAIR-MRI) of the brain and inner ear were obtained.Complete audiologic data obtained on 70% of ears revealed conductive hearing loss in 4 (11%) NOMID ears and mixed hearing loss in 5 (13%) NOMID and 2 (14%) NOMID/MWS ears. Sensorineural hearing loss (SNHL), worse in higher frequencies, was the most common type of hearing loss and was present in 23 (61%) NOMID, 10 (71%) NOMID/MWS, and 4 (33%) MWS ears. All of the patients with FCAS had normal hearing except 2, who had SNHL from 4 to 8 kHz. On FLAIR-MRI sequence, cochlear enhancement was noted in 26 of 29 (90%) NOMID, 6 of 11 (55%) NOMID/MWS, 3 of 9 (33%) MWS, and 1 of 6 (17%) FCAS patients and was significantly associated with the presence of hearing loss. Maxillary sinus hypoplasia and mucosal thickening were found in 39% and 86% of the cohort, respectively.CIAS1 pathway–mediated CAPS is associated with unregulated autoinflammation mediated by interleukin-1 in the cochlea and hearing loss. Timely diagnosis is crucial to initiate early treatment with interleukin-1 receptor antagonists.

BACKGROUND Autoinflammatory diseases manifest inflammation without evidence of infection, high-titer autoantibodies, or autoreactive T cells. We report a disorder caused by mutations of IL1RN, which encodes the interleukin-1-receptor antagonist, with prominent involvement of skin and bone. METHODS We studied nine children from six families who had neonatal onset of sterile multifocal osteomyelitis, periostitis, and pustulosis. Response to empirical treatment with the recombinant interleukin-1-receptor antagonist anakinra in the first patient prompted us to test for the presence of mutations and changes in proteins and their function in interleukin-1-pathway genes including IL1RN. RESULTS We identified homozygous mutations of IL1RN in nine affected children, from one family from Newfoundland, Canada, three families from the Netherlands, and one consanguineous family from Lebanon. A nonconsanguineous patient from Puerto Rico was homozygous for a genomic deletion that includes IL1RN and five other interleukin-1-family members. At least three of the mutations are founder mutations; heterozygous carriers were asymptomatic, with no cytokine abnormalities in vitro. The IL1RN mutations resulted in a truncated protein that is not secreted, thereby rendering cells hyperresponsive to interleukin-1 beta stimulation. Patients treated with anakinra responded rapidly. CONCLUSIONS We propose the term deficiency of the interleukin-1-receptor antagonist, or DIRA, to denote this autosomal recessive autoinflammatory disease caused by mutations affecting IL1RN. The absence of interleukin-1-receptor antagonist allows unopposed action of interleukin-1, resulting in life-threatening systemic inflammation with skin and bone involvement. (ClinicalTrials.gov number, NCT00059748.)

Objective: To describe the pregnancy course and outcome, and use of anakinra, a recombinant selective IL-1 receptor blocker, during pregnancy in patients with cryopyrin-associated periodic syndromes (CAPS), including familial cold auto-inflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and neonatal onset multi-system inflammatory disease (NOMID). Methods: Women currently enrolled in natural history protocols (NCT00059748, and/or NCT00069329 under IND) who have been pregnant were included. Subjects underwent a structured, standardized interview with regards to maternal health, pregnancy and fetal outcomes. Medical records were reviewed. Results: Nine women (four with FCAS, one with MWS and four with NOMID) reported one to four pregnancies, each resulting in a total of fifteen FCAS, three MWS, and six NOMID pregnancies. Six births from FCAS mothers and three births from NOMID mothers occurred while patients were receiving anakinra. If a woman became pregnant while taking anakinra, the pre-pregnancy anakinra dose was continued. Anakinra dose was increased during one twin pregnancy. No preterm births or serious complications of pregnancy were observed. One fetus of the twin pregnancy had renal agenesis and suffered fetal demise. Genetic testing showed the deceased twin carried the same NLRP3 c.785TC, p.V262A mutation as the mother. The other twin is healthy and mutation negative. Conclusions: Anakinra was continued during pregnancy in women with CAPS and provided significant, persistent CAPS symptom relief while continuing to prevent the long-term sequelae of CAPS. Anakinra was well tolerated. Although a causal relation between anakinra and renal agenesis seems unlikely, further safety data are needed.

Background/Purpose: Neonatal-onset multisystem inflammatory disease (NOMID) is an autoinflammatory disease characterized by fever, chronic urticarial rash, CNS manifestations, elevated acute phase reactants, and arthropathy. About 50% of patients with NOMID have de novo missense mutations in NLRP3/CIAS1, leading to constitutive inflammasome activation and IL-1 production in monocytes, and caspase-1 mediated persistence of osteoblast progenitor cells that are also found in other fibroblastoid tumors ([1]). Poorly differentiated chondrocytes are seen with abnormal enchondral bone formation without inflammatory cells in NOMID arthropathy with bony overgrowth and premature fusion of physis. While blocking IL-1 with anakinra reduces organ-specific (central nervous system, inner ear, eye) and systemic inflammation (fever, rash, acute phase reactants), it has little effect on preexisting bone lesions. Methods: NOMID individuals with at least 5 months of follow up data (clinical, laboratory, and radiographic) on IL-1 blocking treatment were included for review. Results: 18 (36.7%) of the NOMID patients (n = 49; 21 male/28 female; age at study entry: 9.4 yo, 0.8–46.3 yo; duration of follow-up: 4.6 yrs, 0.4–9.5 yrs) had bony involvement, most commonly the knee (16/49 = 32.7%, 12/49 = 24.5% with patellar involvement). 11/49 (22.4%) had multi-joint involvement, typically asymmetric, including 4 elbows, 5 wrists, 6 ankles, 3 shoulders, and 2 hips. Ongoing abnormal enchondral bone formation continues until physeal fusion, which often occurs prematurely at involved sites, despite anaanakinra treatment. Lesions eventually ossify normally but may cause bone deformities. 3 patients had patellar subluxations (1 managed surgically) and 2 had patella resections. Leg length discrepancy (8/49 = 16.3%) and bowed leg deformities (9/49 = 18.3%) present due to premature, asymmetric fusion of physis required osteomy and/or stapling in 3. 2 developed long twisted fibula to accommodate shortened tibia, 1 surgically managed with stapling. One individual developed hip subluxation due to a longer left leg, which resulted in left hip avascular necrosis. 2 individuals had pseudofractures with development of horizontal linear defects noted in long-bones consistent with residual changes from abnormal enchondral ossification rather than true fracture. 1 individual developed severe contractures with bilateral knee arthropathy and subsequently had bilateral total knee replacement. Conclusion: 1. Unique bony findings in NOMID are due to persistence of immature osteoblast precursors with abnormal enchondral bone growth which continues until physeal fusion despite anakinra treatment, often with premature fusion at affected sites, with eventual normal ossification. 2. Surgical intervention can be safely considered for those with significant pain/disability from NOMID arthropathy. 3. Consideration of horizontal linear pseudofractures in long bones and risk of hip avascular necrosis is important in those with NOMID ([1]).

Background/Purpose: Macrophage Activation Syndrome (MAS) is a life-threatening systemic inflammatory disorder of unknown etiology. While MAS has no known genetic basis, clinical similarity with a genetic disorder of impaired cytotoxicity known as primary Hemophagocytic Lymphohistiocytosis (HLH) has suggested shared pathogenesis. In contrast, other investigations have suggested innate immune dysregulation drives MAS. Methods: We performed detailed clinical, genetic, and immunologic evaluation of a patient with early onset, recurrent MAS-like disease including whole exome sequencing and analyses of serum cytokines, whole blood transcription, and stimulated monocyte and macrophage responses. We subsequently tested the effects of a de novo mutation in transfection experiments with a monocytic cell line. Results: We identified a 7 year-old female with recurrent episodes of fever, splenomegaly, transaminitis, pancytopenia, occasional evanescent rash, and massive hyperferritinemia since 6 months of age. Her Natural Killer cell function was normal, and she had no mutations in genes associated with HLH or hereditary periodic fever syndromes. Whole exome sequencing identified a de novo missense mutation in a highly conserved area in the nucleotide-binding region of the inflammasome component NLRC4. Interleukin (IL)-1b, Interferon a2, and particularly IL-18 were elevated in the patient's peripheral blood even during clinical quiescence. The patient's monocytes produced more of a number of inflammatory cytokines in response to stimulation than controls, while monocyte-derived macrophages specifically over-produced IL-1b and IL-18. Transfection of THP1 monocytes with a plasmid bearing a mutant NLRC4 construct resulted in increased cell death and IL-1b production versus a wild-type plasmid. The patient showed short-term clinical improvement and ability to wean steroids with recombinant IL-1 receptor antagonist (IL-1ra) therapy. Conclusion: While NLRP3 inflammasome hyperactivity has been implicated in the pathogenesis of a variety of autoinflammatory conditions, our data suggest that a mutation in the ADP binding domain of NLRC4 can result in MAS-like disease. This mutation may destabilize NLRC4's auto-inhibited conformation by altering ADP binding ([1]), resulting in enhanced/spontaneous activation and pathogenic production of IL-1b and IL-18. NLRC4 hyperactivity represents a novel mechanism of MAS/HLH pathogenesis.

Background/Purpose: Neonatal onset multisystem inflammatory disease (NOMID) is a rare autoinflammatory disease caused by NLRP3 mutations that lead to constitutive NLRP3 inflammasome activation and IL-1β release. Patients present with neutrophilic urticaria, chronic aseptic meningitis, and papilledema. IL-1β inhibitors improve the inflammatory manifestations, normalize acute phase reactants, decrease CSF WBC and protein, and proinflammatory cytokine levels. We found that cytokine levels decrease with anakinra treatment, but do not normalize during clinical remission. The higher IL-6 and IP-10 levels in CSF than in the blood suggest local production in the brain/CSF. Our objective is to examine whether CSF cytokines, particularly IL-6 and IP-10, correlate with CSF subpopulations of immune cells and CSF characteristics. Methods: Immunophenotyping and cytokine data were available on 2 baseline and 16 anakinra treated patients, 9/16, fulfilled clinical remission criteria (ESR ≤25 mm/hr, CRP ≤0.5 mg/dl, CSF WBC ≤5 cells/μl and protein ≤40 mg/dl) and control patients (n = 7). Cytokines levels (IL-1β, IL-6, IL-10, IP-10, IL-12 (p70) and (p40), IFN-γ, TNF-α, and IL-18) were correlated to CSF WBC, protein, albumin quotient (AQ), and opening pressure at baseline, post-treatment, and clinical remission. Correlation was assessed with Pearson and Spearman tests. Results: At baseline, monocytes, granulocyte, and T cells were higher in NOMID patients versus controls. Post-treatment monocytes and granulocyte counts decreased but remained higher even in clinical remission, relative to control. Only IL-6 and IP-10 correlated with monocytes (r = .66, p = .051; r = .86, p = .002) respectively. A correlation of IP-10 but not IL-6 with granulocytes was weaker than with monocytes (r = .66, p = .058). IL-6, IP-10, and IL-18 correlated with CSF protein, AQ, and WBC (except IL-18). At baseline, IL-6 and IL-18 significantly correlated with CSF protein (r = .68, p = .027; r = .80, p = .0065) and AQ (r = .82, p = .011; r = .78, p = .029) respectively. At post-treatment, IL-18 significantly correlated and IL-6 trend towards significance with CSF protein (r = .53, p = .022; r = .40, p = .096) and AQ (r = .83, p = .0002; r = .47, p = .087) respectively. Conclusion: 1) Inflammatory cells significantly decrease in NOMID patients, especially monocytes which remain elevated even in patients in clinical remission, suggesting residual inflammation. 2) The correlation of monocytes with IL-6 and IP-10 suggests their contribution to IL-6 and IP-10 production, which may contribute to the recruitment of other inflammatory cells including granulocytes. 3) IL-6, IP-10, and IL-18 may be a marker for blood brain barrier dysfunction in NOMID and their use in long term monitoring should be explored. 4) These results are consistent with the notion that NOMID is caused by dysfunction in innate immune cells, particularly monocytes.

Background/purpose Auto-inflammatory diseases constitute a group of disorders that manifest systemic inflammation in the absence of infection, auto-antibodies or auto-reactive T cells. A specific genetic mutation is identified in some of them, such as in neonatal onset multisystem inflammatory disease (NOMID) or chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) syndrome, while in others such as Chronic Recurrent Multifocal Osteomyelitis (CRMO), and Adult onset Still9s Disease (AOSD) a specific cause is yet to be elucidated. The rapid response to targeted cytokine blocking therapies suggests that these disorders are mediated by specific cytokines. Herein, the authors investigate whether each disease is characterised by a specific cytokine signature. Method Serum samples, on visits when patients had clinically active disease, were collected and assayed by Luminex for the presence of 43 inflammatory cytokines at 9 timepoints. Disease groups (mutation positive and negative NOMID; CANDLE syndrome; CRMO; adult and pediatric Still9s disease; rheumatoid arthritis, including juvenile idiopathic arthritis were compared to normal controls whose assays were run at the same time. Ratios of patient values divided by the average of corresponding set of healthy control values were statistically tested for whether they significantly differed from 1 and also among the various disease groups. Result A distinct cytokine profile is found in different auto-inflammatory diseases. Specifically, AOSD is characterised by markedly increased levels of Interleukin-18 (p=0.005, mean=3.068) and IL-1a (p=0.032, mean=1.5687) compared to controls. NOMID mutation positive patients had increased levels of IL-1a (p=0.00078, mean=1.7819), IL-6 (p=0.001, mean=1.5376) and IL-18 (p=0.001, mean=1.4924) compared to controls and NOMID mutation negative patients. Il-18 levels in AOSD patients were actually significantly higher than in patients with mutation positive and negative NOMID. CANDLE patients have highly increased IFNg inducing protein 10 (IP-10) levels (p=0.00053, mean=3.9872) compared to controls and the other autoinflammatory diseases. CRMO patients had low levels of IL-8 (p=0.005, mean=0.1697), GM-CSF (p=0.00684, mean=0.0969) and MCP-1 (p2=0.002, mean=0.0687) compared to controls and other diseases. These differences in cytokine profiles disappeared when patients were on effective therapies. Conclusion During active disease specific cytokine profiles may allow us to detect dysregulated cytokine pathways that discriminate between clinically different autoinflammatory syndromes. A comprehensive approach of cytokine profiling may be useful to develop a therapeutic plan. Further studies are needed to determine it this approach can be used to monitor therapy and help in the definition of inflammatory disease.