CARD8 SNP rs11672725 Identified as a Potential Genetic Variant for Adult-Onset Still's Disease.

Adult-onset Still's disease (AOSD), an autoinflammatory disorder, is related to the dysregulation of NLR3-containing a pyrin domain (NLRP3)-inflammasome signaling. We aimed to investigate the associations of genetic polymorphisms of NLRP3-inflammasome signaling with AOSD susceptibility and outcome and to examine their functional property. Fifty-three candidate single-nucleotide polymorphisms (SNPs) involved in NLRP3-inflammasome response were genotyped using Sequenom MassArray on the samples from 66 AOSD patients and 128 healthy controls. The significant SNPs were validated by direct sequencing using a TaqMan SNP analyzer. Serum levels of associated gene products were examined by ELISA. One SNP rs11672725 of CARD8 gene was identified to be significantly associated with AOSD susceptibility by using MassArray and subsequent replication validation (p = 3.57 × 10-7; odds ratio 3.02). Functional assays showed that serum CARD8 levels were significantly lower in AOSD patients (median, 10,524.6 pg/mL) compared to controls (13,964.1 pg/mL, p = 0.005), while levels of caspase-1, IL-1ß and IL-18 were significantly higher in patients (107.1 pg/mL, 2.1 pg/mL, and 1495.8 pg/mL, respectively) than those in controls (99.0 pg/mL, 1.0 pg/mL, and 141.4 pg/mL, respectively). Patients carrying rs11672725CC genotype had significantly higher serum caspase-1 and IL-18 levels (121.3 pg/mL and 1748.6 pg/mL) compared to those with CT/TT genotypes (72.6 pg/mL, p = 0.019 and 609.3 pg/mL, p = 0.046). A higher proportion of patients with rs11672725CC genotype had a systemic pattern of disease outcome, which was linked to low CARD8 levels. A novel variant, rs11672725, of the CARD8 gene was identified as a potential genetic risk for AOSD. Patients carrying the rs11672725CC genotype and C allele had low CARD8 levels, and were predisposed to a systemic pattern with an elevated expression of inflammasome signaling.

Genetic Association and Expression Correlation between Colony-Stimulating Factor 1 Gene Encoding M-CSF and Adult-Onset Still's Disease.

Adult-onset Still's disease (AOSD) is a rare and inflammatory disorder characterized by spiking fever, rash, arthritis, and multisystemic involvement. HLA has been shown to be associated with AOSD; however, it could not explain the innate immunity and autoinflammatory characteristics of AOSD. To assess the genetic susceptibility of AOSD, we conducted a genome-wide association study (GWAS) on a cohort of 70 AOSD cases and 688 controls following a replication study of 36 cases and 200 controls and meta-analysis. The plasma concentrations of associated gene product were determined. The GWAS, replication, and combined sample analysis confirmed that SNP rs11102024 on 5'-upstream of CSF1 encoding macrophage colony-stimulating factor (M-CSF) was associated with AOSD (P = 1.20 × 10-8, OR (95% CI): 3.28 (2.25~4.79)). Plasma levels of M-CSF increased in AOSD patients (n = 82, median: 9.31 pg/mL), particularly in the cases with activity score ≥ 6 (n = 42, 10.94 pg/mL), compared to the healthy donors (n = 68, 5.31 pg/mL) (P < 0.0001). Patients carrying rs11102024TT genotype had higher M-CSF levels (median: 20.28 pg/mL) than those with AA genotype (6.82 pg/mL) (P < 0.0001) or AT genotype (11.61 pg/mL) (P = 0.027). Patients with systemic pattern outcome were associated with elevated M-CSF and frequently observed in TT carriers. Our data suggest that genetic variants near CSF1 are associated with AOSD and the rs11102024 T allele links to higher M-CSF levels and systemic outcome. These results provide a promising initiative for the early intervention and therapeutic target of AOSD. Further investigation is needed to have better understandings and the clinical implementation of genetic variants nearby CSF1 in AOSD.

Impaired autophagic flux and its related inflammation in patients with adult-onset Still's disease.

The pathogenic role of autophagic immune regulation in adult-onset Still's disease (AOSD) is unclear. We investigated the relative levels of autophagy in AOSD patients and healthy controls, its association with disease activity or course, and the change in autophagy after 6 months of therapy. Autophagosome levels were determined from the mean fluorescence intensity of autophagosomotropic dye incorporated into circulating immune cells. The fluorescent signal from lymphocytes, monocytes, and granulocytes from AOSD patients was greater than from controls. Levels of p62 fluorescence measured using flow cytometry in lymphocytes and granulocytes from AOSD patients was greater than in the corresponding cells from healthy controls. Expression of Atg5 and LC3-II mRNA and protein levels of p62 and LC3-II were elevated in AOSD patients. Moreover, AOSD activity scores correlated positively with autophagosome levels in monocytes and granulocytes, p62 levels in circulating immune cells, and levels of Beclin-1, Atg5, and LC3-II mRNA. Autophagosome levels and Atg mRNA expression decreased with disease remission in AOSD patients. Elevated autophagosome formation and p62 levels suggest impaired autophagic flux in AOSD.

Human parvovirus B19 nonstructural protein NS1 activates NLRP3 inflammasome signaling in adult­onset Still's disease.

Dysregulation of inflammasomes serves a pathogenic role in autoinflammatory diseases (AIDs) and adult-onset Still's disease (AOSD) has been categorized as an AID. The present study investigated the expression of NLR family pyrin domain containing proteins (NLRPs) inflammasome in patients with AOSD, the effect of inflammasome inhibitors on NLRP3 signaling and whether human parvovirus B19­associated antigens can activate NLRP3 in patients with AOSD. mRNA expression levels of NLRPs in peripheral blood mononuclear cells (PBMCs) from 34 patients with AOSD and 14 healthy individuals were determined using reverse transcription­quantitative polymerase chain reaction. Protein expression of NLRP3 was evaluated by western blotting. Supernatant cytokine levels were measured by ELISA. Among the NLRPs investigated in the present study, NLRP3 transcripts were markedly elevated and expression of NLRP2, NLRP7 and NLRP12 was decreased in patients with AOSD compared with the controls. Treatment with NLRP3 inhibitors significantly reduced downstream NLRP3 signaling in PBMCs form patients with AOSD. B19­nonstructural protein (NS)1 stimulation of PBMCs from patients with AOSD induced significant upregulation of transcript levels of NLRP3, caspase­1 and interleukin (IL)­1ß compared with PBMCs from healthy controls. B19­NS1 stimulation of PBMCs from patients with AOSD induced significant increase in supernatant levels of IL­1ß and protein expression of NLRP3, caspase­1, IL­1ß, and IL­18 compared with healthy controls. Elevated expression of NLRP3 and its downstream inflammasome signaling components in patients with AOSD indicated a potential pathogenic role of B19­NS1. Thus, B19­NS1 may induce expression of IL­1ß and IL­18 through activation of caspase­1­associated NLRP3­inflammasome in AOSD.