Lipopolysaccharide stimulation test on cultured PBMCs assists the discrimination of cryopyrin-associated periodic syndrome from systemic juvenile idiopathic arthritis.

Systemic juvenile idiopathic arthritis (sJIA) and cryopyrin-associated periodic syndrome (CAPS) share many common manifestations. We aim to identify an applicable method to assist disease discrimination. Inflammatory cytokines were measured in the plasma of patients with CAPS, sJIA with persistent disease course and healthy controls. Supernatants collected from non-stimulated peripheral blood mononuclear cells (PBMCs) and those undergone inflammasome stimulation tests utilizing lipopolysaccharide (LPS) with and without adenosine triphosphate (ATP) were investigated. Inflammatory cytokines in patient plasma fail to differentiate sJIA from CAPS. PBMCs from sJIA secrets higher amount of IL-1ß and IL-18 while CAPS PBMCs produces more caspase-1 without stimulation. IL-1ß, IL-18, and caspase-1 were significantly elevated among CAPS PBMCs (all p < 0.05) upon LPS stimulation, but not when additional ATPs were provided. Levels of cytokines and PBMC responses to the stimulation assays were similar among all sJIA patients regardless of their history of macrophage activation syndrome. Unstimulated PBMC activities and the LPS inflammasome stimulation assay without exogenic ATPs can assist the differentiation of CAPS from sJIA with persistent disease course.

Progressive waves of IL-1ß release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.

IL-1ß is an essential cytokine, but its release needs to be strictly controlled to avoid severe inflammatory manifestations. Lacking a signal sequence, IL-1ß does not follow the endoplasmic reticulum-Golgi route. Several pathways have been proposed to mediate its release. One involves the translocation of pro-IL-1ß into intracellular vesicles of lysosomal origin that eventually fuse with the plasma membrane. Another exploits pores formed on the plasma membrane upon proteolytic cleavage of gasdermin D (GSDMD). Here we investigated how primary monocytes-the main source of IL-1ß in humans-control IL-1ß release in response to pro-inflammatory stimuli of increasing intensity and found that two different routes are induced depending on the strength of activation. Triggering of Toll-like receptor 4 (TLR4) by LPS induces slow IL-1ß release through LAMP2A+ vesicles. In contrast, the simultaneous stimulation of TLR2, TLR4 and TLR7/8 drives high levels of ROS, GSDMD cleavage and faster IL-1ß secretion. Drugs blocking ROS production prevent GSDMD cleavage supporting a role of oxidative stress in GSDMD-mediated secretion. Singly stimulated monocytes undergo apoptosis, whereas triple stimulation triggers pyroptosis, which might amplify inflammation. In both cases, however, IL-1ß secretion precedes cell death. Inhibition of caspases 4/5 prevents GSDMD cleavage and pore-mediated secretion, but not vesicular release. The two pathways also display other distinct pharmacologic sensitivities that reflect the underlying mechanisms. Remarkably, single TLR4 stimulation is sufficient to activate massive, GSDMD-mediated IL-1ß secretion in monocytes from patients affected by Cryopyrin Associated Periodic Syndrome (CAPS), an autoinflammatory disease linked to NLRP3 mutations. The exaggerated sensitivity to activation correlates with high basal ROS levels in CAPS monocytes. In conclusion, the vesicular pathway limits IL-1ß release upon low pathogen load while stronger stimulation or concomitant cell stress induce instead uncontrolled secretion via GSDMD leading to detrimental inflammatory manifestations.

Effects of cold sensitivity in the extremities on circulating adiponectin levels and metabolic syndrome in women.

BACKGROUND: In adipose tissues, adipokine levels, including adiponectin and leptin, are involved in insulin sensitivity and are reciprocally induced by cold temperature stress. Thermogenic response in the extremities (hands and feet) against cold stress can be negatively related to fat mass accumulation, particularly in the abdomen. However, the relationship between the sensation of cold in the extremities and circulating levels of adipokines is not fully understood. Here, we investigated whether adipokine levels are associated with cold hypersensitivity in the hands and feet (CHHF), independent of body mass, and whether the CHHF is related to metabolic syndrome (MS). METHODS: Associations of the CHHF with serum levels of adipokines and MS risk were evaluated in 1021 Koreans (372 men and 649 women), using a linear regression model while controlling for thermogenic factors and a logistic regression model, respectively. RESULTS: The adiponectin levels were positively associated with the CHHF, particularly in women, irrespective of thermogenic factors, including body mass index (ß = 1.23 µg/mL, 95% confidence interval [1.04-1.45]). Logistic regression analysis for MS risk via the CHHF showed that there was a significant inverse association in women (odds ratio = 0.449, 95% confidence interval [0.273-0.737]). CONCLUSIONS: In summary, our founding indicated that the CHHF could induce increased levels of circulating adiponectin and in turn reduce the MS risk in women. Despite complaints of feeling cold, these women could be at lower risk of cardiovascular disease.

Diagnostic criteria for cryopyrin-associated periodic syndrome (CAPS).

Cryopyrin-associated periodic syndrome (CAPS) is a rare, heterogeneous disease entity associated with NLRP3 gene mutations and increased interleukin-1 (IL-1) secretion. Early diagnosis and rapid initiation of IL-1 inhibition prevent organ damage. The aim of the study was to develop and validate diagnostic criteria for CAPS. An innovative process was followed including interdisciplinary team building, item generation: review of CAPS registries, systematic literature review, expert surveys, consensus conferences for item refinement, item reduction and weighting using 1000Minds decision software. Resulting CAPS criteria were tested in large cohorts of CAPS cases and controls using correspondence analysis. Diagnostic models were explored using sensitivity analyses. The international team included 16 experts. Systematic literature and registry review identified 33 CAPS-typical items; the consensus conferences reduced these to 14. 1000Minds exercises ranked variables based on importance for the diagnosis. Correspondence analysis determined variables consistently associated with the diagnosis of CAPS using 284 cases and 837 controls. Seven variables were significantly associated with CAPS (p<0.001). The best diagnosis model included: Raised inflammatory markers (C-reactive protein/serum amyloid A) plus ≥two of six CAPS-typical symptoms: urticaria-like rash, cold-triggered episodes, sensorineural hearing loss, musculoskeletal symptoms, chronic aseptic meningitis and skeletal abnormalities. Sensitivity was 81%, specificity 94%. It performed well for all CAPS subtypes and regardless of NLRP3 mutation. The novel approach integrated traditional methods of evidence synthesis with expert consensus, web-based decision tools and innovative statistical methods and may serve as model for other rare diseases. These criteria will enable a rapid diagnosis for children and adults with CAPS.

Serum amyloid protein A concentration in cryopyrin-associated periodic syndromes patients treated with interleukin-1 beta antagonist.

OBJECTIVES: Cryopyrin-associated periodic syndromes (CAPS) are a group of chronic, relapsing autoinflammatory disorders which may be complicated by systemic AA amyloidosis. The aim of our study was to evaluate serum amyloid protein A (SAA) level in CAPS patients treated with Interleukin-1beta (IL-1ß) antagonist and to correlate its level with treatment response. METHODS: All patients of CAPS Italian Register treated with IL-1ß inhibitor were enrolled. SAA levels before starting therapy, and at last visit were evaluated. Patients were then divided in complete responders and partial responders. RESULTS: Twenty-five patients were enrolled. SAA level before starting therapy was increased (median 118.5 mg/L, IQR 96.4-252.8; normal value <6.4 mg/L), while at last visit SAA was significantly reduced (median 4.3 mg/L, IQR 2.3-12.7) (p<0.001). However 12 patients still presented SAA levels beyond normal range, 10/25 patients (40%) showed a complete response to treatment. Conversely, 15 patients presented only a partial response, of which 12 for increased SAA value and 3 for increased CRP value. Patients with partial response had SAA values significantly higher than patients with complete response (median 12.6 mg/L; IQR 8.3-20.0 vs. 2.7 mg/L; IQR 1.6-4.1, p<0.001). CONCLUSIONS: Our results confirm the long term efficacy of anti IL-1ß treatment in CAPS and the decrease of SAA levels; however 48% of patients still presented SAA elevation despite treatment. The real risk of these patients in developing amyloidosis is not clear but the persistent increase of SAA needs a close follow-up.

The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response.

Assembly of the NLRP3 inflammasome activates caspase-1 and mediates the processing and release of the leaderless cytokine IL-1ß and thereby serves a central role in the inflammatory response and in diverse human diseases. Here we found that upon activation of caspase-1, oligomeric NLRP3 inflammasome particles were released from macrophages. Recombinant oligomeric protein particles composed of the adaptor ASC or the p.D303N mutant form of NLRP3 associated with cryopyrin-associated periodic syndromes (CAPS) stimulated further activation of caspase-1 extracellularly, as well as intracellularly after phagocytosis by surrounding macrophages. We found oligomeric ASC particles in the serum of patients with active CAPS but not in that of patients with other inherited autoinflammatory diseases. Our findings support a model whereby the NLRP3 inflammasome, acting as an extracellular oligomeric complex, amplifies the inflammatory response.

Anakinra for cryopyrin-associated periodic syndrome.

Cryopyrin-associated periodic syndrome (CAPS) is a very rare auto-inflammatory syndrome, which has recently served as a pure model of IL-1ß-driven diseases. CAPS is caused by mutations into the NLRP3 gene that encodes crypoyrin, which serves as a receptor of the innate immunity that senses danger signals and pathogens. Constitutive activation of cryopyrin in CAPS leads to an excessive secretion of IL-1ß. CAPS patients experience symptoms of systemic inflammation, intense fatigue and have poor quality of life. In the most severe forms, they may develop serious organ damage such as visual and hearing impairment, neurological deterioration and renal insufficiency. Anti-IL-1 drugs are effective in treating symptoms of almost all CAPS patients and have radically transformed their lives. We describe the history of the 'revival' of CAPS patients through anti-IL-1 treatments with a special focus on anakinra, the first drug used in cohorts with variable disease severity and number of patients.

A taste of periodic fever syndromes.

Periodic fevers are acquired or inherited disorders of innate immunity, which were first described in the 1940s. The patients are typically young at onset and have regularly recurring fevers for a few days to a few weeks with systemic inflammatory symptoms that are interrupted by symptom-free periods. There is a variety of clinical manifestations including gastrointestinal complaints, myalgias, arthralgias, and rash. A differential diagnosis in these patients may include recurrent infections, other inflammatory disorders, and neoplastic disease. This clinical review focuses on a sample of autoinflammatory disorders including familial Mediterranean fever, tumor necrosis factor receptor 1-associated periodic syndrome, hyperimmunoglobulinemia D syndrome, the cryopyrin-associated periodic syndrome, and periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome. We review the basics, pertinent clinical and laboratory features, and management of each entity.

Safety and efficacy of canakinumab in Japanese patients with phenotypes of cryopyrin-associated periodic syndrome as established in the first open-label, phase-3 pivotal study (24-week results).

OBJECTIVES: Cryopyrin-associated periodic syndrome (CAPS), a rare hereditary auto-inflammatory disease, is associated with mutations in the NLRP3 gene resulting in elevated interleukin-1ß (IL-1 ß) release. CAPS generally occurs in early childhood with most patients presenting with periodic fever, skin rash, osteoarthropathy, aseptic meningitis, sensorineural hearing loss and optic neuritis. Canakinumab, a fully human anti-IL-1ß monoclonal antibody which binds selectively to IL-1ß, has demonstrated good efficacy with CAPS. This is the first study to evaluate the safety and efficacy of canakinumab in Japanese patients with CAPS. METHODS: In this open-label study, 19 Japanese CAPS patients aged ≥2 years received canakinumab either 150 mg s.c. or 2 mg/kg for patients with a body weight ≤ 40 kg every 8 weeks for 24 weeks. The primary objective was to assess the proportion of patients who were free of relapse at week 24. RESULTS: A complete response was achieved in 18 (94.7%) patients with some requiring a dose and/or a frequency adjustment to attain full clinical response. The majority of patients (14/18; 77.8%) were in remission, i.e. free of relapse at week 24. Auto-inflammatory disease activity as assessed by physician's global assessment declined from baseline to end of the study (score of absent in 10.5% at baseline versus 31.6% at end of the study). Two patients had serious adverse events (SAEs), which resolved with standard treatment. One patient reported a mild injection-site reaction. No malignancies or deaths were reported during the study. CONCLUSIONS: Canakinumab 150 mg s.c. every 8 weeks was well-tolerated, highly efficacious and offered a convenient dosing regimen for treating Japanese patients with CAPS.

Sustained remission of symptoms and improved health-related quality of life in patients with cryopyrin-associated periodic syndrome treated with canakinumab: results of a double-blind placebo-controlled randomized withdrawal study.

INTRODUCTION: To assess the effect of canakinumab, a fully human anti-interleukin-1ß antibody, on symptoms and health-related quality of life (HRQoL) in patients with cryopyrin-associated periodic syndrome (CAPS). METHODS: In this 48-week, phase 3 study, patients with CAPS received canakinumab 150 mg subcutaneously at 8-week intervals. All patients (n = 35) received canakinumab during weeks 1 through 8; weeks 9 through 24 constituted a double-blind placebo-controlled withdrawal phase, and weeks 24 through 48 constituted an open-label phase in which all patients received canakinumab. Patient and physician assessments of symptoms, levels of inflammatory markers, and HRQoL were performed. RESULTS: Rapid symptom remission was achieved, with 89% of patients having no or minimal disease activity on day 8. Responses were sustained in patients receiving 8-weekly canakinumab. Responses were lost during the placebo-controlled phase in the placebo group and were regained on resuming canakinumab therapy in the open-label phase. Clinical responses were accompanied by decreases in serum levels of C-reactive protein, serum amyloid A protein, and interleukin-6. HRQoL scores at baseline were considerably below those of the general population. Improvements in all 36-item Short-Form Health Survey (SF-36) domain scores were evident by day 8. Scores approached or exceeded those of the general U.S. population by week 8 and remained stable during canakinumab therapy. Improvements in bodily pain and role-physical were particularly marked, increasing by more than 25 points from baseline to week 8. Therapy was generally well tolerated. CONCLUSIONS: Canakinumab, 150 mg, 8-weekly, induced rapid and sustained remission of symptoms in patients with CAPS, accompanied by substantial improvements in HRQoL. TRIAL REGISTRATION: Clintrials.gov NCT00465985.

MRP8 and MRP14, phagocyte-specific danger signals, are sensitive biomarkers of disease activity in cryopyrin-associated periodic syndromes.

OBJECTIVES: To assess the sensitivity of the phagocyte-specific molecules myeloid-related protein (MRP) 8 and MRP14 (calprotectin) for monitoring disease activity during anti-interleukin (IL)-1 therapies in patients with cryopyrin-associated periodic syndromes (CAPS), including familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and chronic infantile neurological, cutaneous and articular (CINCA) syndrome. METHODS: A total of 39 patients with CAPS, including 5 FCAS, 16 MWS and 18 CINCA syndrome, received anti-IL-1 therapy. All patients with CINCA and 12 with MWS were treated with IL-1Ra (anakinra), 14 patients with MWS with a monoclonal anti-IL-1ß antibody (canakinumab) and patients with FCAS received IL-1 Trap (rilonacept). During serial clinical visits serum amyloid A, C-reactive protein, erythrocyte sedimentation rate and MRP8/14 serum levels were analysed. RESULTS: Untreated patients with CAPS had significantly elevated MRP8/14 values. In response to treatment there was a significant reduction of MRP8/14 levels in CINCA (2,830 (range 690 - 8,480) ng/ml to 670 ng/ml, p < 0.001) and MWS patients (anakinra-treated: 4,390 (1790 - 9780) ng/ml to 1,315 ng/ml (p = 0.003); canakinumab-treated: 3,000 (500 - 13060) ng/ml to 630 ng/ml (p=0.001)). However, in many patients with CAPS, MRP8/14 levels were still elevated compared with healthy individuals, reflecting residual disease activity. However, canakinumab-treated patients with CAPS showed normalised MRP8/14 levels, suggesting control of phagocyte activation. CONCLUSIONS: Monitoring of cellular systems involved in inflammatory cascades of the innate immunity was successfully applied to the IL-1-driven CAPS diseases. This is the first study illustrating different states of subclinical disease activity in all types of CAPS depending on the type of anti-IL-1 therapy. MRP8/14 is a sensitive biomarker for monitoring disease activity, status of inflammation and response to IL-1 blockade in patients with CAPS.

Role of IL-1 beta in the development of human T(H)17 cells: lesson from NLPR3 mutated patients.

BACKGROUND: T helper 17 cells (T(H)-17) represent a lineage of effector T cells critical in host defence and autoimmunity. In both mouse and human IL-1ß has been indicated as a key cytokine for the commitment to T(H)-17 cells. Cryopyrin-associated periodic syndromes (CAPS) are a group of inflammatory diseases associated with mutations of the NLRP3 gene encoding the inflammasome component cryopyrin. In this work we asked whether the deregulated secretion of IL-1ß secondary to mutations characterizing these patients could affect the IL-23/IL-17 axis. METHODOLOGY/PRINCIPAL FINDINGS: A total of 11 CAPS, 26 systemic onset juvenile idiopathic arthritis (SoJIA) patients and 20 healthy controls were analyzed. Serum levels of IL-17 and IL-6 serum were assessed by ELISA assay. Frequency of T(H)17 cells was quantified upon staphylococcus enterotoxin B (SEB) stimulation. Secretion of IL-1ß, IL-23 and IL-6 by monocyte derived dendritic cells (MoDCs), were quantified by ELISA assay. A total of 8 CAPS and 11 SoJIA patients were also analysed before and after treatment with IL-1ß blockade. Untreated CAPS patients showed significantly increased IL-17 serum levels as well as a higher frequency of T(H)17 compared to control subjects. On the contrary, SoJIA patients displayed a frequency of T(H)17 similar to normal donors, but were found to have significantly increased serum level of IL-6 when compared to CAPS patients or healthy donors. Remarkably, decreased IL-17 serum levels and T(H)17 frequency were observed in CAPS patients following in vivo IL-1ß blockade. On the same line, MoDCs from CAPS patients exhibited enhanced secretion of IL-1ß and IL-23 upon TLRs stimulation, with a reduction after anti-IL-1 treatment. CONCLUSION/SIGNIFICANCE: These findings further support the central role of IL-1ß in the differentiation of T(H)17 in human inflammatory conditions.

Risk factors for severe Muckle-Wells syndrome.

OBJECTIVE: Muckle-Wells syndrome (MWS) is an inherited autoinflammatory disease resulting in excessive interleukin-1 release. It is unknown whether demographic, clinical, or laboratory characteristics at the time of diagnosis may identify patients who are at high risk for severe disease activity. This study was undertaken to analyze clinical and laboratory features of MWS, compare genetically defined subcohorts, and identify risk factors for severe MWS. METHODS: A multicenter cohort study of consecutive MWS patients was performed. Parameters assessed included clinical features, MWS Disease Activity Score (MWS-DAS), inflammation markers, and cytokine levels. E311K mutation-positive patients were compared with E311K mutation-negative patients. Putative risk factors for severe MWS (defined as an MWS-DAS score of ≥10) were assessed in univariate analyses, and significant predictors were entered into a multivariate model. RESULTS: Thirty-two patients (15 male and 17 female) were studied. The most frequent organ manifestations were musculoskeletal symptoms and eye and skin disorders. Renal disease and hearing loss were seen in >50% of the patients. Genetically defined subcohorts had distinct phenotypes. Severe disease activity was documented in 19 patients (59%). Predictors of severe MWS identified at the time of diagnosis were female sex, hearing loss, musculoskeletal disease, increased erythrocyte sedimentation rate, and low hemoglobin level. Female sex and hearing loss remained significant after adjustment for age in a multivariate model (relative risk 1.8 and 2.6, respectively). CONCLUSION: MWS patients at high risk for severe disease can be identified at the time of diagnosis. Female patients presenting with hearing loss have the highest likelihood of manifesting severe MWS and should be considered a high-risk group.

Two adult siblings with atypical cryopyrin-associated periodic syndrome due to a novel M299V mutation in NLRP3.

OBJECTIVE: The NALP3 inflammasome is a multiprotein complex that triggers caspase 1-mediated interleukin-1beta (IL-1beta) release. Mutations in the gene encoding NALP3 (NLRP3) underlie the cryopyrin-associated periodic syndrome (CAPS). The aim of this study was to report a novel NLRP3 mutation in 2 siblings of Swedish descent in whom symptoms first presented in adulthood. METHODS: Mutation analysis of NLRP3 was performed on DNA from patients with CAPS and 100 control subjects. For assessment of caspase 1 and IL-1beta, blood was collected from patients and age- and sex-matched healthy control subjects. Genetic constructs containing mutant or wild-type NLRP3 were transduced into THP-1 cells, followed by assessment of IL-1beta levels in cell supernatant. RESULTS: Both siblings carried a novel M299V mutation in NLRP3, which was not present in the control population. The samples obtained from the patients displayed increased caspase 1 activity and elevated IL-1beta levels at basal conditions as compared with healthy control subjects. THP-1 cells expressing mutated M299V revealed almost 10-fold higher IL-1beta production compared with the wild-type construct. CONCLUSION: M299V is an activating mutation in NLRP3 resulting in elevated spontaneous caspase 1 activity and IL-1beta levels. The classic CAPS phenotype was lacking in these adult siblings. Whereas one sibling displayed a milder phenotype that has so far responded satisfactorily to oral nonsteroidal antiinflammatory drugs in combination with low-dose corticosteroids, the inflammatory symptoms in the sibling with the more severe case responded well to IL-1beta blockade. Understanding the pathogenic mechanism underlying such disorders can be helpful for the physician. Our study reinforces the importance of genetic testing and laboratory investigations in combination with careful phenotypic evaluation for the diagnosis of such patients.

Cerebrospinal fluid neopterin and cryopyrin-associated periodic syndrome.

Cryopyrin-associated periodic syndrome is a category of autoinflammatory disorders caused by mutations of the NLRP3 gene, with chronic infantile neurologic cutaneous and articular syndrome being the severest clinical phenotype. Various pterins have been reported as mediating immunologic functions in the central nervous system, but to date studies of pterin cerebrospinal fluid (CSF) values and cryopyrin-associated periodic syndrome have been lacking. A 2-year-old child was affected with a severe atypical form of cryopyrin-associated periodic syndrome, suspected based on the analysis of neopterin in CSF. He initially presented isolated neurologic manifestations mimicking a neuroregressive disorder. Blood and CSF analyses did not present any routine inflammatory markers, but CSF neopterin was elevated. Later, the patient developed arthritis and recurrent episodes of fever, and the cryopyrin-associated periodic syndrome diagnosis was confirmed by genetic studies. Neopterin was the most altered indicator over the time. Child neurologists should be on the alert when unexplained neurologic signs appear, giving consideration to the possibility of inflammatory or immune-mediated diseases. The present case demonstrates the clinical utility of measurement of CSF neopterin levels in screening for these immune-mediated diseases, especially when neurologic symptoms are associated with normal results on routine CSF tests.