Transcriptional licensing is required for Pyrin inflammasome activation in human macrophages and bypassed by mutations causing familial Mediterranean fever.

Pyrin is a cytosolic immune sensor that nucleates an inflammasome in response to inhibition of RhoA by bacterial virulence factors, triggering the release of inflammatory cytokines, including IL-1ß. Gain-of-function mutations in the MEFV gene encoding Pyrin cause autoinflammatory disorders, such as familial Mediterranean fever (FMF) and Pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). To precisely define the role of Pyrin in pathogen detection in human immune cells, we compared initiation and regulation of the Pyrin inflammasome response in monocyte-derived macrophages (hMDM). Unlike human monocytes and murine macrophages, we determined that hMDM failed to activate Pyrin in response to known Pyrin activators Clostridioides difficile (C. difficile) toxins A or B (TcdA or TcdB), as well as the bile acid analogue BAA-473. The Pyrin inflammasome response was enabled in hMDM by prolonged priming with either LPS or type I or II interferons and required an increase in Pyrin expression. Notably, FMF mutations lifted the requirement for prolonged priming for Pyrin activation in hMDM, enabling Pyrin activation in the absence of additional inflammatory signals. Unexpectedly, in the absence of a Pyrin response, we found that TcdB activated the NLRP3 inflammasome in hMDM. These data demonstrate that regulation of Pyrin activation in hMDM diverges from monocytes and highlights its dysregulation in FMF.

Steroid hormone catabolites activate the pyrin inflammasome through a non-canonical mechanism.

The pyrin inflammasome acts as a guard of RhoA GTPases and is central to immune defenses against RhoA-manipulating pathogens. Pyrin activation proceeds in two steps. Yet, the second step is still poorly understood. Using cells constitutively activated for the pyrin step 1, a chemical screen identifies etiocholanolone and pregnanolone, two catabolites of testosterone and progesterone, acting at low concentrations as specific step 2 activators. High concentrations of these metabolites fully and rapidly activate pyrin, in a human specific, B30.2 domain-dependent manner and without inhibiting RhoA. Mutations in MEFV, encoding pyrin, cause two distinct autoinflammatory diseases pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND) and familial Mediterranean fever (FMF). Monocytes from PAAND patients, and to a lower extent from FMF patients, display increased responses to these metabolites. This study identifies an unconventional pyrin activation mechanism, indicates that endogenous steroid catabolites can drive autoinflammation, through the pyrin inflammasome, and explains the "steroid fever" described in the late 1950s upon steroid injection in humans.

Pyrin Inflammasome Activation Abrogates Interleukin-1 Receptor Antagonist, Suggesting a New Mechanism Underlying Familial Mediterranean Fever Pathogenesis.

OBJECTIVE: Aberrant pyrin inflammasome activity triggers familial Mediterranean fever (FMF) pathogenesis, but the exact mechanism remains elusive and an obstacle to efficient treatment. We undertook this study to identify pyrin inflammasome-specific mechanisms to improve FMF treatment and diagnostics in the future. METHODS: Pyrin-specific protein secretion was assessed by proteome analysis in U937-derived macrophages, and specific findings were confirmed in pyrin inflammasome-activated monocytes from healthy blood donors and patients with FMF, stratified according to MEFV genotype categories corresponding to a suspected increase in FMF disease severity. RESULTS: Proteome data revealed a differential secretion pattern of interleukin-1 receptor antagonist (IL-1Ra) from pyrin- and NLRP3-activated U937-derived macrophages, which was verified by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. Moreover, pyrin activation significantly reduced IL1RN messenger RNA expression (P < 0.001) and IL-1Ra secretion (P < 0.01) in healthy donor and FMF monocytes, respectively. Independent of MEFV genotype, unstimulated FMF monocytes from colchicine-treated patients secreted lower amounts of IL-1Ra compared to healthy donors (P < 0.05) and displayed decreased ratios of IL-1Ra:IL-1ß (P < 0.05), suggesting a reduced antiinflammatory capacity. CONCLUSION: Our data show an inherent lack of IL-1Ra expression specific to pyrin inflammasome activation, suggesting a new mechanism underlying FMF pathogenesis. The reduced IL-1Ra levels in FMF monocytes suggest a diminished antiinflammatory capacity that potentially leaves FMF patients sensitive to proinflammatory stimuli, regardless of receiving colchicine therapy. Thus, considering the potential clinical consequence of reduced monocyte IL-1Ra secretion in FMF patients, we suggest further investigation into IL-1Ra dynamics and its potential implications for FMF treatment in the future.

Soluble TREM-1 Levels in Familial Mediterranean Fever Related AA-Amyloidosis.

Objectives: Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) is a monocyte and neutrophil receptor functioning in innate immunity. TREM-1 activity has been studied in various autoimmune diseases such as RA and SLE but there is no data in autoinflammatory pathologies. We studied soluble TREM-1 (sTREM-1) activity in Familial Mediterranean Fever (FMF) cases to evaluate the clinical role of TREM-1 in amyloidosis. Methods: The study includes 62 patients with FMF (42 with amyloidosis) who are regular attendees of a tertiary center for autoinflammatory diseases. For control purposes, 5 patients with AA amyloidosis secondary to other inflammatory diseases, and 20 healthy individuals were also included. Soluble TREM-1 levels were measured using enzyme-linked immunosorbent assay (ELISA). All FMF patients were in an attack-free period during the collection of the blood samples.Results: Soluble TREM-1 levels were found to be significantly higher in the FMF amyloidosis group compared to FMF without amyloidosis group and healthy controls (p = .001 and 0.002). Nevertheless, this difference between sTREM-1 levels was not found among FMF amyloidosis and other AA amyloidosis groups (p = .447) as well as between only FMF patients and healthy controls (p = .532). Soluble TREM-1 levels were found in correlation with creatinine and CRP in the FMF patient group regardless of their amyloidosis diagnosis (r = 0.314, p = .013; r = 0.846, p < .001).Conclusion: TREM-1 seems to be related to renal function rather than disease activity in FMF. Its role as an early diagnostic marker of amyloidosis in FMF complicated with AA amyloidosis should be tested in larger patient groups.

The systemic autoinflammatory disorders for dermatologists. Part 1: overview.

The systemic autoinflammatory disorders (SAIDs) or periodic fever syndromes are disorders of innate immunity, which can be inherited or acquired. They are almost all very rare and easily overlooked; typically, patients will have seen multiple specialities prior to diagnosis, so a high level of clinical suspicion is key. It is important to note that these are 'high-value' diagnoses as the majority of these syndromes can be very effectively controlled, dramatically improving quality of life and providing protection against the development of irreversible complications such as AA amyloidosis. In this article, we take an overview of SAIDs and look at the common features; in Part 2, we take a more in-depth look at the better recognized or more dermatologically relevant conditions.

The Use of Interleukine-1 Inhibitors in Familial Mediterranean Fever Patients: A Narrative Review.

Purpose: Familial Mediterranean fever (FMF) is the most common monogenic auto-inflammatory disease characterized by recurrent attacks of fever and serositis. It is associated with mutation in pyrin inflammasome leading to interleukin-1 (IL-1) over secretion. Although colchicine is the first line treatment in FMF, 5-10% of patients are reported in literature as non-responders. Colchicine is not always well-tolerated due either to its direct toxicity or to co-morbidities that preclude the administration of its proper dosage. For these patients an alternative or additional treatment to colchicine is necessary. This literature review reports the published data regarding the use of IL-1 inhibitors in Familial Mediterranean Fever. Results: There is no uniform definition of colchicine resistance, but the different studies of treatment with IL-1 inhibitors provide evidence of IL-1 pathogenic role in colchicine-resistant FMF. IL-1 inhibition is an efficacious option for controlling and preventing flares -at least at the short term- in FMF patients who are insufficiently controlled with colchicine alone. Although canakinumab is the only approved drug in Europe for colchicine resistant FMF treatment, experience with anakinra is also substantial. In the absence of comparative studies both treatments seem to be an equal option for the management of these patients. Overall the safety profile of IL-1 inhibitors seems not different in FMF patients than in the other diseases and can be considered as globally safe. The main side effects are local injection site reactions and infections. Conclusion: IL-1 inhibitors have the potential to improve patient outcome even in FMF patients with co-morbidities or severe complications in whom inflammation control is difficult to achieve with colchicine alone. Nevertheless, current data are limited and further evaluation of long-term efficacy and safety of IL-1 inhibitors are necessary, in order to provide robust evidence in this domain.

Familial Mediterranean fever and atherosclerosis in childhood and adolescence.

Familial Mediterranean fever is a chronic inflammatory disease characterized by periodic and self-limited episodes of fever and aseptic polyserositis. Although colchicine treatment has altered the course of the disease, it is believed that subclinical inflammation is still present, leading to endothelial dysfunction and atherosclerosis in the course of time. In this review, following the published recommendations, we queried online databases such as MEDLINE Pubmed, Scopus, and Web of science for peer-reviewed studies and reviews written in English language, using the following keywords: familial Mediterranean fever, children, endothelial dysfunction, atherosclerosis, cardiovascular disease. The objective of this review is to highlight the correlation between familial Mediterranean fever and atherosclerosis, and moreover to describe new serum inflammatory markers and non-invasive methods of endothelial dysfunction, to detect the atherosclerosis process early starting from childhood.

Fecal calprotectin in children with familial Mediterranean fever in the attack-free period.

BACKGROUND: Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent episodes of fever and serosal inflammation. The aim of this study was to evaluate fecal calprotectin (FC) in children with FMF during the non-attack period. METHODS: A retrospective evaluation was made of the data of a total 66 patients diagnosed with FMF in an attack-free period and without amyloidosis or inflammatory bowel disease (IBD). FC level in the FMF patients was compared with that in the patients with IBD and healthy control subjects. RESULTS: The FMF patients consisted of 37 boys (56.1%) with a mean age of 10.1 ± 3.9 years. Mean FC was 192.5 µg/g (range, 19.5-800 µg/g) in the FMF group, 597.9 µg/g (range, 180-800 µg/g) in the IBD group, and 43.8 µg/g (range, 19.5-144 µg/g) in the control group. The FC level in the children with FMF was higher than in the control group (P < 0.001), and the FC level of the IBD patients was higher than both the FMF and the control groups (P = 0.020, P < 0.001, respectively). CONCLUSIONS: FC was higher in FMF patients compared with healthy children even in the absence of IBD/amyloidosis. Even though colonoscopy is the gold standard in identifying intestinal inflammation in FMF patients, FC, a non-invasive and inexpensive method, can be used for screening. The presence of subclinical intestinal inflammation was also quantitatively identified in children with FMF.

Role of fecal calprotectin in the assessment of intestinal inflammation in children with familial Mediterranean fever.

AIM: Familial Mediterranean fever (FMF) is the most common auto-inflammatory disease with recurrent fever and serositis episodes. In recent years, some cases with FMF were reported with gastrointestinal involvement without amyloidosis, vasculitis and inflammatory bowel disease (IBD). It is not yet known whether gastrointestinal involvement is a part of the disease or not. The aim of this study is to investigate the frequency of intestinal inflammation by using a noninvasive method, fecal calprotectin measurement, in pediatric FMF patients. METHOD: Sixty-five FMF patients, 30 healthy controls and 11 patients with acute ulcerative colitis were included in the study. A standard survey inquiring gastrointestinal and other clinical symptoms was completed. The medications, MEFV mutations, whole blood count and C-reactive protein levels were recorded. Fecal calprotectin was studied with the enzyme-linked immunosorbent assay method from the feces samples of the all subjects. RESULTS: None of the FMF patients had clinical signs of IBD. Fecal calprotectin levels of the FMF patients were found to be significantly higher than the healthy controls (174.8 ± 150.8 vs 52.9 ± 36.5, p < 0.001). Fecal calprotectin levels of the ulcerative colitis patients were significantly higher than the FMF patients (523.5 ± 183 vs 174.8 ± 150.8, p = 0.001). There was a correlation between fecal calprotectin levels and neutrophil/lymphocyte ratio (r = 0.324, p = 0.009). CONCLUSION: Our results supported subclinical intestinal inflammation in pediatric FMF patients. Further studies are needed to clarify the reason for intestinal inflammation in FMF patients.

The patient journey to diagnosis and treatment of autoinflammatory diseases.

BACKGROUND: Limited data are available on the experiences of patients with autoinflammatory diseases (AIDs) and their families along the path to diagnosis and treatment. We sought to describe these experiences in patients with AIDs including tumor necrosis factor receptor-associated periodic syndrome (TRAPS), mevalonate kinase deficiency/hyperimmunoglobulin D syndrome (MKD/HIDS), and familial Mediterranean fever (FMF). METHODS: Ninety-minute, semi-structured qualitative interviews and 5-day written/video diaries were used to gather information on the experiences of patients with AIDs and their families. RESULTS: Twelve families of patients from the US (TRAPS [n = 4], MKD/HIDS [n = 5], FMF [n = 5]) participated in this study from August to November 2015. The study included two families with multiple afflicted siblings. Patients' ages ranged from 1 to 28 years. Most parents reported realizing that something was seriously wrong with their child after medical emergencies and/or hospitalizations, which initiated the difficult path to diagnosis. For most, the process included multiple specialist visits, extensive and repeated testing, and many misdiagnoses. Over time, 92% of parents reported losing confidence in the healthcare system's ability to find an answer to their child's symptoms, while they also struggled with unsupportive school personnel and dismissive friends and relatives. Patients and their parents reported holding on to memories of "what life was like" before the onset of symptoms and mourning their subsequent loss of "normalcy." Even after diagnosis, patients and parents continued to feel uncertain about what to expect in the future. CONCLUSIONS: All families emphasized the need for efficient early diagnosis of AIDs. Initiatives that improve the speed and accuracy of diagnosis, provide more comprehensive patient education, and support patients and families through the illness have the potential to significantly improve the quality of life of patients with AIDs and their families. Healthcare providers should be aware of the impact of the long diagnostic journey on families and work to create an environment of trust and collaboration in the face of a difficult and prolonged diagnostic process.

GSDMD is critical for autoinflammatory pathology in a mouse model of Familial Mediterranean Fever.

Pyroptosis is an inflammasome-induced lytic cell death mode, the physiological role of which in chronic inflammatory diseases is unknown. Familial Mediterranean Fever (FMF) is the most common monogenic autoinflammatory disease worldwide, affecting an estimated 150,000 patients. The disease is caused by missense mutations in Mefv that activate the Pyrin inflammasome, but the pathophysiologic mechanisms driving autoinflammation in FMF are incompletely understood. Here, we show that Clostridium difficile infection of FMF knock-in macrophages that express a chimeric FMF-associated MefvV726A Pyrin elicited pyroptosis and gasdermin D (GSDMD)-mediated interleukin (IL)-1ß secretion. Importantly, in vivo GSDMD deletion abolished spontaneous autoinflammatory disease. GSDMD-deficient FMF knock-in mice were fully protected from the runted growth, anemia, systemic inflammatory cytokine production, neutrophilia, and tissue damage that characterize this autoinflammatory disease model. Overall, this work identifies pyroptosis as a critical mechanism of IL-1ß-dependent autoinflammation in FMF and highlights GSDMD inhibition as a potential antiinflammatory strategy in inflammasome-driven diseases.

MicroRNA-204-3p inhibits lipopolysaccharide-induced cytokines in familial Mediterranean fever via the phosphoinositide 3-kinase γ pathway.

Objective: We sought to identify the microRNA (miRNA) profile and potential biomarkers in FMF and to clarify their gene targets to elucidate the pathogenesis of FMF. Methods: We performed an miRNA microarray using serum from FMF patients in attack and in remission. We then examined the expression of miRNAs in macrophages derived from THP-1 cells stimulated with toll-like receptor (TLR) ligands. Macrophages derived from THP-1 cells transfected with pre-miRNA were stimulated with lipopolysaccharides (LPSs) for the quantification of inflammatory cytokine production. To identify the target genes, we overexpressed their miRNA and performed a complementary DNA microarray. Transfection with reporter construct and the precursor miRNA was performed to confirm the suppression of target mRNA. Results: We found that miR-204-3p was greatly decreased in the serum from FMF patients in attack. The expression of miR-204-3p was suppressed by LPS stimulation in the macrophages derived from THP-1 cells and the inhibition of miR-204-3p significantly induced the production of TLR4-related cytokines. The bioinformatic analysis showed that miR-204-3p is predicted to target genes implicated in the TLR pathway through the regulation of PI3Kγ signalling. The reporter assay revealed that miR-204-3p directly suppressed the luciferase activity of 3'-UTR of PIK3CG reporter construct. The inhibition of PI3Kγ resulted in decreased amounts of IL-6 and IL-12p40 in monocytes from FMF patients. Conclusion: These data suggest that serum miR-204-3p has potential as a useful biomarker in FMF patients and that miR-204-3p serves as a suppressor of inflammatory cytokine production in FMF by targeting the PI3Kγ pathway.

RAC1 expression and role in IL-1ß production and oxidative stress generation in familial Mediterranean fever (FMF) patients.

Familial Mediterranean fever (FMF) is a recessively inherited autoinflammatory disorder. The caspase-1-dependent cytokine, IL-1ß, plays an important role in FMF pathogenesis, and RAC1 protein has been recently involved in IL-1ß secretion. This study aims to investigate RAC1 expression and role in IL-1ß and caspase-1 production and oxidative stress generation in FMF. The study included 25 FMF patients (nine during attack and remission, and 16 during remission only), and 25 controls. RAC1 expression levels were analyzed by real-time PCR. Ex vivo production of caspase-1, IL-1ß, IL-6 and markers of oxidative stress (malondialdehyde, catalase, and glutathione system) were evaluated respectively in supernatants of patients' and controls' PBMC and PMN cultures, in the presence and absence of RAC1 inhibitor. RAC1 gene expression and IL-1ß levels were increased in patients in crises compared to those in remission or controls. RAC1 expression levels were correlated with MEFV genotypes, patients carrying the M694V/M694V genotype having a two-fold increase in the expression levels compared to those carrying other genotypes. Caspase-1 levels were higher in LPS-induced PBMC of patients in remission than controls. Spontaneous and LPS-induced IL-1ß production were comparable in patients in remission and controls, whereas LPS-induced IL-6 production was enhanced in patients, compared to controls. RAC1 inhibition resulted in a decrease in caspase-1 and IL-1ß, but not IL-6, levels. Malondialdehyde levels produced by LPS-stimulated PMNs were increased in patients in remission compared to those in controls, but decreased following RAC1 inhibition. Catalase and GSH activities were reduced in unstimulated PMN culture supernatants of patients in remission compared to controls and were increased in the presence of RAC1 inhibitor. These results show the involvement of RAC1 in the inflammatory process of FMF by enhancing IL-1ß production, through caspase-1 activation, and generating oxidative stress, even during asymptomatic periods.

A novel cluster of patients with Familial Mediterranean Fever (FMF) in southern Italy.

BACKGROUND: Familial Mediterranean Fever (FMF) is an autosomal recessive autoinflammatory disorder characterised by recurrent attacks of fever and serositis (peritonitis, pleuritic or synovitis) affecting mainly populations of Mediterranean origin. AIM: To describe a relatively new cluster of FMF subjects from Apulia and Basilicata regions (southern Italy). PATIENTS AND METHODS: Subjects were screened for FMF using the Tel-Hashomer criteria and genetic analysis. Demographic data were taken from patients' files and direct interviews. Patients were investigated about attack duration, intensity and site, body temperature, skin manifestations and overall quality of life before and after treatment with colchicine. Inflammatory parameters were also measured between these periods. RESULTS: Forty-nine subjects had FMF (M : F = 26 : 23, age 38 years ± 2 SE) and followed-up up to 8 years. The age at disease onset was 22·1 years ± 1·2SE and the diagnostic delay was 15·5 years ± 1·9SE. The majority of patients (82%) suffered from abdominal pain, and 35% had undergone prior abdominal surgery or laparotomy. Severity score (ISSF) was mild in 43% of patients and intermediate in 57% of patients. Serum amyloid A (SAA) was increased in 20% of patients (16·9 ± 3·7, normal range < 6·4 mg/dL). In over 95% of patients, inflammation markers, duration and intensity of febrile painful attacks, quality of life and ISSF score improved dramatically following colchicine treatment. CONCLUSION: The Apulia region represents a new endemic area for FMF. Clinical presentation of FMF can be misleading and requires a complete and early workup to recognise the disease and avoid unjustified surgery. Colchicine remains the gold standard therapy to prevent FMF attacks and fatal long-term complications.

A survey of resistance to colchicine treatment for French patients with familial Mediterranean fever.

BACKGROUND: Colchicine is the standard treatment for familial Mediterranean fever (FMF), preventing attacks and inflammatory complications. True resistance is rare and yet not clearly defined. We evaluated physicians' definition of colchicine resistance and report how they manage it. PATIENTS AND METHODS: We recruited patients with a clinical diagnosis of FMF, one exon-10 Mediterranean fever (MEFV) gene mutation and considered resistant to colchicine, via networks of expert physicians. Clinical, biological characteristics and information about colchicine treatment (dose adjustment, compliance) were collected. The severity of FMF was assessed by the Tel Hashomer criteria. RESULTS: We included 51 patients, most females (55%), mean age 34 ± 23.1 years years (range 4.7-86.3). Overall, 58% (27/47) patients had homozygous M694 MEFV gene mutations. Seventeen of 42 patients (40%) declared full adherence to colchicine treatment, greater for children (48%) than adults (22%). Physicians considered colchicine resistance with > 6 attacks/year (n = 21/51, 42%), > 4 attacks in the last 6 months (n = 13/51, 26%), persistent inflammation (n = 23/51, 45%), renal amyloidosis in (n = 6/28, 22%) of adult patients and intolerance to an increase in colchicine dose (n = 10/51, 19%), and other reasons (n = 13/51, 23%), including chronic arthralgia (n = 6/51, 12%). Interleukin 1-targeting drugs represented the only alternative treatments in addition to daily colchicine. CONCLUSION: Resistance to colchicine is rare (<10% of patients) and mostly observed in severe MEFV genotypes. The main reasons for physicians assessing resistance were severe clinical symptoms, persistent subclinical inflammation, and secondary amyloidosis. Low adherence to colchicine treatment is a key component of resistance.

Cytokine signatures in hereditary fever syndromes (HFS).

Hereditary fever syndromes (HFS) include a group of disorders characterized by recurrent self-limited episodes of fever accompanied by inflammatory manifestations occurring in the absence of infection or autoimmune reaction. Advances in the genetics of HFS have led to the identification of new gene families and pathways involved in the regulation of inflammation and innate immunity. The key role of several cytokine networks in the pathogenesis of HFS has been underlined by several groups, and supported by the rapid response of patients to targeted cytokine blocking therapies. This can be due to the direct effect of cytokine overproduction or to an absence of receptor antagonist resulting in dysbalance of downstream pro- and anti-inflammatory cytokine networks. The aim of this study was to present an overview and to discuss the major concepts regarding the cellular and molecular immunology of HFS, with a particular focus on their specific cytokine signatures and physiopathological implications. Based on their molecular and cellular mechanisms, HFS have been classified into intrinsic and extrinsic IL-1ß activation disorders or inflammasomopathies, and protein misfolding disorders. This review integrates all recent data in an updated classification of HFS.

DNA demethylation of inflammasome-associated genes is enhanced in patients with cryopyrin-associated periodic syndromes.

BACKGROUND: Inflammasomes are cytosolic multiprotein complexes in macrophages. They assemble after infection- or stress-associated stimuli, activating both caspase-1-mediated inflammatory cytokine secretion and pyroptosis. Increased inflammasome activity resulting from gene mutations is related to monogenic autoinflammatory syndromes. However, variable penetrance among patients with the same gene mutations suggests involvement of additional mechanisms associated with inflammasome gene regulation. OBJECTIVE: We sought to investigate the role of DNA demethylation in activating inflammasome genes during macrophage differentiation and monocyte activation in healthy control subjects and patients with autoinflammatory syndrome. METHODS: Inflammasome-related genes were tested for DNA methylation and mRNA levels by using bisulfite pyrosequencing and quantitative RT-PCR in monocytes in vitro differentiated to macrophages and exposed to inflammatory conditions. The contribution of Tet methylcytosine dioxygenase 2 (TET2) and nuclear factor κB to DNA demethylation was tested by using chromatin immunoprecipitation, small interfering RNA-mediated downregulation, and pharmacologic inhibition. RESULTS: We observed that inflammasome-related genes are rapidly demethylated in both monocyte-to-macrophage differentiation and on monocyte activation. Demethylation associates with increased gene expression, and both mechanisms are impaired when TET2 and nuclear factor κB are downregulated. We analyzed DNA methylation levels of inflammasome-related genes in patients with cryopyrin-associated periodic syndromes (CAPS) and familial Mediterranean fever, 2 archetypical monogenic autoinflammatory syndromes. Under the above conditions, monocytes from untreated patients with CAPS undergo more efficient DNA demethylation than those of healthy subjects. Interestingly, patients with CAPS treated with anti-IL-1 drugs display methylation levels similar to those of healthy control subjects. CONCLUSION: Our study is the first to demonstrate the involvement of DNA methylation-associated alterations in patients with monogenic autoinflammatory disease and opens up possibilities for novel clinical markers.

Canakinumab reverses overexpression of inflammatory response genes in tumour necrosis factor receptor-associated periodic syndrome.

OBJECTIVE: To explore whether gene expression profiling can identify a molecular mechanism for the clinical benefit of canakinumab treatment in patents with tumour necrosis factor receptor-associated periodic syndrome (TRAPS). METHODS: Blood samples were collected from 20 patients with active TRAPS who received canakinumab 150 mg every 4 weeks for 4 months in an open-label proof-of-concept phase II study, and from 20 aged-matched healthy volunteers. Gene expression levels were evaluated in whole blood samples by microarray analysis for arrays passing quality control checks. RESULTS: Patients with TRAPS exhibited a gene expression signature in blood that differed from that in healthy volunteers. Upon treatment with canakinumab, many genes relevant to disease pathogenesis moved towards levels seen in the healthy volunteers. Canakinumab downregulated the TRAPS-causing gene (TNF super family receptor 1A (TNFRSF1A)), the drug-target gene (interleukin (IL)-1B) and other inflammation-related genes (eg, MAPK14). In addition, several inflammation-related pathways were evident among the differentially expressed genes. Canakinumab treatment reduced neutrophil counts, but the observed expression differences remained after correction for this. CONCLUSIONS: These gene expression data support a model in which canakinumab produces clinical benefit in TRAPS by increasing neutrophil apoptosis and reducing pro-inflammatory signals resulting from the inhibition of IL-1ß. Notably, treatment normalised the overexpression of TNFRSF1A, suggesting that canakinumab has a direct impact on the main pathogenic mechanism in TRAPS. TRIAL REGISTRATION NUMBER: NCT01242813.