Exploration Of NLRP3 Inflammasome In The Pathogenesis Of Rheumatoid Arthritis And Development Of A New Disease Activity Index For Rheumatoid Arthritis

BackgroundRheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation, autoantibody production, cartilage and bone destruction, and systemic inflammation and damage. There is an ongoing need for new therapeutic strategies, because a substantial number of patients remain to be suffered from active disease.Of all the cells implicated in the pathology of RA, neutrophils (also known as polymorphonuclear leukocytes or PMNs) possess the greatest cytotoxic potential, owing to their ability to release degradative enzymes and reactive oxygen species. Neutrophils in the inflammatory microenvironment of RA can extend their lifespan up to a few days. This extended lifespan enables neutrophils to perform their roles in inflammation before undergoing constitutive apoptosis or being cleared by macrophages.In recent years, a renewed interest in the role that neutrophils play in various systemic autoimmune diseases has emerged. However, because of the lack of availability of the human tissues from these RA patients, most of the investigations were carried out in animal models. Therefore authentic data from clinical RA patients are still lacking. In contrast to the difficulty in the availability of specimen from synovial joints, peripheral blood specimen is practically available for investigation. Peripheral blood is a highly dynamic environment, communicating with practically every tissue in the body, reflecting disease progression in the body. Furthermore, because the leukocytes in blood interact and communicate with practically every tissue, they bear rich informations regarding inflammation and immune responses. Neutrophils are the most abundant circulating leukocytes in human, accounting for around 60% of all leucocytes in the circulation. The roles that peripheral neutrophils play in RA progression remain to be clarified.Caspase-1 is a recognized important factor involved in the progression of autoimmune diseases. Though overactivation of caspase-1 was detected since the early phase of RA patients and was thought to be a potential therapeutic target for RA patients, its role in the RA disease progression remained to be clarified.Recent work established that caspase-1 activation required the formation of inflammasome. Inflammasome serves as a molecular platform to mediate caspase-1 activation, which further induces maturation and secretion of interleukin-1β (IL-1β) and interleukin-18 (IL-18). NLRP3 inflammasome is the most clarified one, and has been linked to many diseases. Caspase-1 activation induced by NLRP3 inflammasome has been reported to be involved in a lot of diseases; however, its role in the disease activity of RA has not been fully elucidated.ObjectiveTo investigate the expression of NLRP3 inflammasome in peripheral blood neutrophils of patients with RA and its correlation with disease activity.Methods1. RA patients (n=48) and healthy controls (n=41) were enrolled. Their blood samples and clinic data were collected for analysis.2. Protein expression of NLRP3 inflammasome components was detected by Western blot.3. Messenger RNA expression of NLRP3 inflammasome was detected by quantitative real-time reverse transcription-PCR.4. Sera levels of IL-1β and IL-18 were detected by enzyme-linked immunosorbent assay (ELISA).5. Correlations among NLRP3 inflammasome activation, sera cytokines and RA disease activities were analyzed. Results 1. Neutrophil count was correlated with disease activity of RA.Positive correlations were observed between neutrophil count and DAS28-ESR (r=0.3192, P=0.0186), neutrophil count and DAS28-CRP (r=0.3262, P=0.0171). PR3 levels in neutrophils were significantly increased in RA patients (P<0.0001) and higher in DAS28 high group (P<0.0129).2. Caspase-1 activation in PMN, but not in PBMC, was positively correlated with disease activity of RA patients.Activated caspase-1 in PMN was positively correlated with DAS28-ESR (r=0.4791, P=0.0178) and DAS28-CRP (r=0.4252, P=0.0383). But activated caspase-1 in PBMC did not have any significant correlation with disease activity as measured by DAS28-ESR (r=0.1843, P=0.3885) and DAS28-CRP (r=0.1557, P=0.4677).3. Caspase-1 activation in neutrophils was positively correlated with serum level of IL-18 but not IL-1β.A significant positive correlation was observed between activated caspase-1 protein level and serum IL-18 level (r=0.6630, P=0.0005). But no significant correlation was observed between activated caspase-1 protein level and serum IL-1β level (r=0.0782, P=0.7162).4. Serum IL-18 level but not IL-1β level was positively correlated with disease activity of RA.Significant positive correlations were observed between serum IL-18 level and tender joint count (r=0.3637, P=0.0443), swollen joint count (r=0.3717, P=0.0395), DAS28-ESR (r=0.4473, P=0.0150) and DAS28-CRP (r=0.4739, P=0.0094). But no significant correlation was observed between serum IL-1β level and tender joint count (r=0.1322, P=0.4783), swollen joint count (r=-0.0345, P=0.8540), DAS28-ESR (r=0.1714, P=0.3739) and DAS28-CRP (r=0.1680, P=0.3837).5. Caspase-1 activation in neutrophils of RA patients was independent of NLRP3 inflammasomeThe protein expression levels of NLRP3, ASC and pro-caspase-1 were all significantly decreased in neutrophils of RA patients compared with that of controls (P=0.0466 for NLRP3; P=0.0017 for ASC; P<0.0001 for pro-caspase-1). But the activated caspase-1 level was significantly increased (P=0.0022). 6. Expression of NLRP3 inflammasome components was negatively correlated with disease activity of RA patients(1) The mRNA expression of NLRP3 and ASC decreased significantly in neutrophils of RA patients compared to healthy controls (P<0.0001 for NLRP3, P=0.0011 for ASC); whereas no significant difference was observed between mRNA expression of caspase-1 in neutrophils from RA patients and healthy controls (P=0.4467).(2) The mRNA levels of NLRP3, ASC and caspase-1 were positively correlated with the corresponding protein levels of NLRP3, ASC and pro-caspase-1.(3) Negative correlations between NLRP3 mRNA expression and clinical parameters including tender joint count (TJC), swollen joint count (SJC), ESR, CRP, DAS28-ESR, DAS28-CRP, RF and anti-CCP. Especially, two negative correlations achieved statistically significant differences (r=-0.3241, P=0.0246 for CRP; r=-0.3212, P=0.0260 for DAS28-CRP).(4) Neutrophil NLRP3 mRNA expression level was significantly decreased in DAS28-CRP high group (P=0.0258) and in anti-CCP high group (P=0.0197).ConclusionOur results indicated that over-activated caspase-1 in neutrophils of RA was likely to mediate IL-18 activation and thus promote the progression of RA in a NLRP3 inflammasome independent manner.BackgroundRheumatoid arthritis (RA) is a systemic autoimmune disease characterized by symmetric, progressive and erosive arthritis, which causes discomfort, disability, drug toxicity, dollar costs and death. The pathogenesis of RA is not yet clarified. It is the result of combined interaction of gene, environment and autoimmune. Among them, genetic factors have a substantial contribution to the pathogenesis of RA, accounting for approximately 60% of the variation in liability to disease.In recent years, studies about RA related susceptibility genes have showed a variety of genetic mutations are associated with the onset of RA. One of the most important genetic mutations is single nucleotide polymorphism (SNP) in related gene loci. SNP mainly refers to the DNA sequence polymorphism at the genome level caused by the variation of single nucleotide. It is one of the most common genetic mutations in human, accounting for more than 90% of all known polymorphism. Through the researches of the relationship between SNP and RA, it by far has been found that about 101 SNPs locus are associated with RA.NACHT, LRR and PYD domains-containing protein 3 (NALP3), also known by Cryopyrin, is the typical representative of NLR family, Pyrin domain containing (NLRPs) protein family. NALP3 together with apoptosis associated speck-like protein containing CARD (ASC) and cysteine-aspartic acid protease 1 precursor (pro-caspase-1) can assemble a complex compound-the NALP3 inflammasome. Then, activated caspase-1 can catalytic split precursor of interleukin 1β (IL-1β) and interleukin 18 (IL-18) proteins into their mature form and promote their releasing, causing a variety of immune inflammatory response. At present, the role of NALP3 inflammasome playing in autoimmune and auto-inflammatory pathological processes is attracting extensive attention.NLR family, pyrin domain containing 3 (NLRP3) gene, also known as PYPAF1 or CIAS1 gene, located on chromosome 1q44, encodes the NALP3 protein. This study focuses on the NLRP3 p.Q705K (rs35829419) mutation, which is a missense mutation. The Q705K mutation of NLRP3 gene is also a gain of function mutation, which can change its conformation and function, cause the NALP3 protein autoactivation, and thus promote the maturity and release of IL-1β and IL-18 precursor protein.Caspase recruitment domain family member 8 (CARD8), also known as TUCAN or CARDINAL, is a protein involved in pathways leading to activation of caspases or nuclear factor kappa-B (NF-κB), which plays a role in apoptosis and inflammatory process. This protein is also a component of the inflammasome involved in activation of caspase-1 as well as maturity and release of IL-1β and IL-18 precursor protein. CARD8 gene located on chromosome 19q13.33 encodes the CARD8 protein. The SNP rs2043211 comprises a T to A transversion, and changes a cysteine residue (TGT) to a stop codon (TGA) at amino acid position 10 (p.ClOX) which, presumably leads to a severely truncated protein.and this rather profound change could have consequences for the function of the protein in both inflammasome-mediated processes and NF-κB suppression.Combined analysis of single nucleotide polymorphisms of different genetic locus can help to find out the genetic susceptibility which can not be explained use single nucleotide polymorphism of one single gene locus. Carriers with both NLRP3 p.Q705K and CARD8 p.C10X mutations showed higher activity of inflammasome, and are more prone to immune response. By pooling the studies about NLRP3 p.Q705K and CARD8 p.C10X single nucleotide polymorphisms, our meta analysis investigated their associations with the susceptibility of RA.Objective1. To investigate the association of NLRP3 p.Q705K SNP with the susceptibility of RA.2. To investigate the association of CARD8 p.C10X SNP with the susceptibility of RA.3. To investigate if the compound polymorphism of NLRP3 p.Q705K and CARD8 p.C10X to be associated with increased RA susceptibility.4. To investigate if the individuals carrying at least one variant allele compared with those carrying only wild-type alleles at both the NLRP3 p.Q705K and CARD8 p.C10X locus are more susceptible to RA.Methods1. Search strategy:To identify relevant published studies, a comprehensive literature search was conducted without language restriction using the following computerized bibliographic databases:China National Knowledge Internet, WanFang database, the Weipu Journal, Chinese biological and medical database, PubMed, Cochrane library, EMBase, Elsevier science direct (SD), Web of science, SpringerLink (last updated search in December 2015).2. Literature screening and quality evaluation:The retrieved studies were assessed for their suitability to this meta-analysis using the previously prescribed inclusion and exclusion criteria. The Newcastle-Ottawa Scale (NOS) criteria were employed to assess the methodological quality of the included trials.3. Data extraction:Collected information included the first author, publication year or submission year, country, journal, ethnicity, language, diseases type, source of controls, confirmation of diagnosis, and demographic variables of subjects, genotype frequencies, HWE test, and detection method.4. Statistical analysis:STATA 12.0 statistical software was applied for statistical analyses. The effect sizes representing the unadjusted ORs for the presence of the target gene SNP in patients and controls were calculated. A Z test was conducted to assess the significance of the overall effect size. Pooled ORs were generated for comparisons using the allelic (M allele vs W allele), genotype (MM vs WW、MM vs WM、WM vs WW), dominant model (MM+MW vs WW), recessive model (MM vs MW+WW).5. Heterogeneity analysis:The heterogeneity between all the studies was evaluated with Cochran’s Q-statistic and the I2 test. A random effects model was used in instances of significant heterogeneity; otherwise, a fixed effect model was applied.6. Sensitivity analysis:A sensitivity analysis was performed to evaluate whether removal of any single study could influence the overall outcomes.7. Publication bias analysis:Egger’s linear regression test and a funnel plot were applied to assess publication bias.Results1. Included studies:A total of 44 potentially eligible articles were identified fromthe literature. Finally,5 case-control studies were included into this meta-analysis, that contained a total of 2705 RA patients and 2711 healthy controls.2. Literature quality:Among the five studies, one scored 6 stars, three scored 7 stars, and another one scored 8 stars, according to the NOS criteria.3. NLRP3 p.Q705K SNP meta analysis results:No obvious heterogeneity was found between studies. The fixed effects model was used. The pooled result suggest NLRP3 p.Q705K gene polymorphism have no statistical significant correlation with the occurrence of RA (P>0.05). After kicking out the data which do not meet the Hardy-Weinberg equilibrium, the pooled result still suggest NLRP3 p.Q705K gene polymorphism have no statistical significant correlation with the occurrence of RA (P>0.05).4. CARD8 p.C10X SNP meta analysis results:No obvious heterogeneity was found between studies. The fixed effect model was used. The pooled result suggest CARD8 p.C10X gene polymorphism have no statistical significant correlation with the occurrence of RA (P>0.05).5. NLRP3 p.Q705K and CARD8 p.C10compound polymorphism meta analysis results:(1) When compared with combined genotype CARD8/NLRP3 AA/CC, none of the pooled ORs of other combined genotypes has statistical significant (P>0.05).(2) When compared with combined genotype carrying only wild-type alleles at both the NLRP3 p.Q705K and CARD8 p.C10X locus, the pooled OR of combined genotype carrying at least one variant allele at both locus has no more susceptible to RA (OR=1.056,95%CI=0.545-2.046, P=0.872). Moderate heterogeneity existed (Cochran’s Q=7.58, P=0.056, I2%=60.4%). The random effect model was used and subgroup analysis was performed. In subgroup analysis stratified by population, there was no difference between subgroups. But in subgroup analysis stratified by method, the subgroup of SNuPe method showed susceptibility to RA (OR=2.178> 95%CI=1.085-4.373, P=0.029), which was different from the pooled OR. Sensitivity analysis showed kicking out any part of the research data can not yield a substantial impact on the overall conclusion.Conclusion1. NLRP3 p.Q705K SNP does not associated with the susceptibility of RA.2. CARD8 p.C10X SNP does not associated with the susceptibility of RA.3. The compound polymorphism of NLRP3 p.Q705K and CARD8 p.C10X does not associated with the susceptibility of RA.4. The individuals carrying at least one variant allele compared with those carrying only wild-type alleles at both the NLRP3 p.Q705K and CARD8 p.C10X locus show no susceptible to RA.ObjectiveRheumatoid arthritis (RA) is characterized by systemic inflammation. Thrombocytosis and anemia are common extra-articular manifestations of RA, which reflect the abnormality of platelet and hemoglobin. In this study, a novel disease activity marker, platelet to hemoglobin ratio (PHR) was investigated for the first time to evaluate its predictive value for RA patients.Methods250 patients with active RA and 100 healthy controls were recruited to investigate the value of PHR. The Disease Activity Score in 28 joints (DAS28) was calculated and its correlation with PHR was statistically analyzed in these RA patients. Correlations of PHR with systemic inflammatory markers and cardiovascular disease related biomarkers were evaluated. Response of PHR to treatment was further investigated in a group of patients.ResultsPHR was increased in RA patients as compared with healthy controls (P<0.001). PHR showed a positive correlation with RA disease activity as measured by DAS28 based on erythrocyte sedimentation rate (DAS28-ESR) (r=0.428, P<0.001)and DAS28 based on C-reactive protein (DAS28-CRP) (r=0.376, P<0.001). PHR was significantly correlated with systemic inflammatory markers, including serum interlrukin-6, erythrocyte sedimentation rate, C-reactive protein, albumin to globulin ratio, immunoglobulin G and complement component 3 (all P<0.001). PHR was significantly positively correlated with serum sialic acid, a known cardiovascular risk factor (P<0.001). PHR was significantly negatively correlated with cardiovascular protective factors, including superoxide dismutase, total bilirubin, and apolipoprotein A-Ⅰ (all P<0.001). After effective treatment, increased PHR value was significantly rectified (P<0.01) in these patients responding well to treatment.ConclusionPHR is a novel systemic inflammatory marker associated with disease activities in RA patients, which indicates its great potential in cardiovascular disease prediction and therapeutic evaluation.