The Role Of NLRP3 Inflammasome And Downstream Factors In The Pathogenesis Of Idiopathic Inflammatory Myopathy

Backgrounds:Idiopathic inflammatory myopathy (IIM) is common acquired autoimmune myopathy with the incidence of 1-10/million. The clinical manifestation of IIM is acute or subacute proximal muscle weakness, myalgia, and muscle atrophy in the late stage, sometimes with lung and heart involved. The muscle biopsy reveals degeneration and necrosis of muscle fibers, usually with inflammatory cell infiltration in the perivascular and perifascicular area. The factors that trigger inflammatory muscle diseases remain unknown. Genetic risk factors regulating immune responses against undefined environmental agents have been proposed. Autoimmune is now implicated as the main pathogenesis of IIM, which is supported by the fact that the infiltration of inflammatory cells, autoantibody and over expression of major histocompatibility complex Ⅰ/Ⅱ. And now the current treatment strategy for patients with immune-mediated inflammatory myopathies involves first-line treatment with corticosteroids, alone or in combination with animmunosuppressant such as methotrexate, cyclophosphamide and azathioprine. The treatment above has great side effects. Some patients show relapse of IIM during the dose-reducing while some patients are resistant to the unspecific immunosuppression. Now the research of targeted therapy for IIM is extremely urgent.NLRP3 inflammasome is a complex that is comprised of NLRP3, ASC (apoptosis-associated speck-like protein containing CARD) and the aspartate protease Caspase-1. NLRP3 is one member of the NLRs families, which is a kind of cytosol PRRs (pattern-recognition receptors). NLRP3 can recognize the tissue damage and cell lysis (dangerous associated molecular pattern, DAMPs), participating the innate immunity. Once activation, NLRP3 forms inflammasome with ASC, which facilitates the autocatalytic activation of Pro-caspase-1 and the formation of an active Caspase-1 p10/20 tetramer. The activated Caspase-1 can process pro-IL-1β and pro-IL-18 into mature Iβand IL-18, which will activate the downstream inflammation.NLRPs inflammasome is now extensively studied, and the abnormal activation of NLRP3 inflammasome is involved in the pathogenesis of kinds of autoimmune diseases, such as gouts, alzheimer disease, multiple sclerosis and atherosclerosis. And there is no research on the role of NLRP3 inflammasome in the pathogenesis of IIM.Objectives:(1) To observe the expression of NLRP3 and downstream factors in PM and DM patients;(2) To explore the role of NLRPs in the pathogenesis of IIM by suppressing the expression of NLRP3 and downstream factors in EAM mice.Methods:(1) 10 cases of PM patients,12 cases of DM patients and 24 controls were enrolled in our study. And the blood and muscle samples of these three groups were collected. We observe the serum concentration of IL-1βand IL-18 with ELISA, mRNA expression of IL-1β、 IL-18 and NLRP3 in muscle with qRT-PCR, and protein expression of NLRP3 and Caspase-1 p20 in muscle with western blotting. All the clinical data was studied, including demographics data, medical history, clinical manifestation, creatine kinase (CK) and electromyography (EAM) results;(2) EAM was inducted by guinea pig skeletal homogenates.10 BALB/c mice were divided randomly into two groups:control group (n=5) were immunized by PBS and CFA for six times; EAM group (n=5) were immunized by guinea pig skeletal homogenates and CFA with the same protocol. Clinical manifestations and muscle pathology were observed and scored. Serum muscle enzymes spectrum analysis was detected by automatic biochemical analyzer, Muscles force were studied by muscle force detector;(3) The animals were randomly divided into control group (n=5) and EAM group (n=5) with the same protocols as above step. Collect the blood and muscle samples of the mice. We observe the serum concentration of IL-1β and IL-18 with ELISA, mRNA expression of IL-1β, IL-18, Caspase-1 and NLRP3 in muscle with qRT-PCR, and protein expression of NLRP3, Pro-caspase-1 and Caspase-1 p20 in muscle with western blotting;(4) 15 BALB/c mice were randomly divided into three groups:control group (n=5), EAM group (n=5) and EAM group with treatment. 10mg/kg/d of glucocorticoid were given with gavage administration for consecutive 7 days. Clinical manifestations and muscle pathology were observed and scored. Serum muscle enzymes spectrum analysis was detected by automatic biochemical analyzer, Muscles force were studied by muscle force detector. The blood and muscle sample were collected two weeks after the treatment. We observe the serum concentration of IL-1βand IL-18 with ELISA, mRNA expression of IL-1β, IL-18, Pro-caspase-land NLRP3 in muscle with qRT-PCR, and protein expression of NLRP3, Pro-caspase-1 and Caspase-1 p20 in muscle with western blotting;(5) The data was expressed as ±SD. SPSS 17.0 and GraphPad Prism 6.0 were used for statistically analysis and figure making. ANOVA and t test were used when the data obey normality and homogeneity of variance. Kruskal Wallis H and Wilcoxon test were used when the data didn’t obey normality and homogeneity of variance. Differences were considered statistically significant at P< 0.05.Results:(1) The serum concentrations of IL-1β for PM, DM and controls were 26.49±7.79 ng/ml, 25.02±8.29 ng/ml and 16.49±3.30 ng/ml (P<0.01); The serum concentrations of IL-18 for PM, DM and controls were 184.8±161.7ng/ml,214.74±198.11ng/ml and 50.90±37.62ng/ml (P<0.01); The mRNA expression of IL-1β, IL-18 and NLRP3 was increased in muscle samples of DM and PM when compared to controls (P<0.01), and the western blotting of muscle samples showed elevated protein expression of NLRP3 and Caspase-1 p20 (P<0.01), when the gray scale of test protein was normalized to that of GAPDH;(2) The EAM group showed high clinical score(EAM 2.3±0.2 vs. Control 0), having more inflammatory cell infiltration in muscle tissue. The concentration of CK was significantly increased in EAM group compared with control group (P<0.05). The four limbs and fore limbs force of EAM group were 7.1±0.9g/g and 3.8±0.4g/g, which were decreased compared to control group (P<0.05);(3) The serum concentrations of IL-1β and IL-18 for EAM group was 36.77±3.54 ng/ml, 74.28±3.36 ng/ml, and serum concentrations of IL-1β and IL-18 for control group were 6.26±0.77 ng/ml,22.02±1.07 ng/ml (P<0.001); The mRNA expression of IL-1β, IL-18, Caspase-1 and NLRP3 was increased in muscle samples(P<0.001), and the western blotting of muscle samples showed elevated protein expression of NLRP3, Pro-caspase-1 and Caspase-1 p20(P<0.001);(4) There were no differences on the clinical scores and muscle forces between EAM group and EAM treatment group, while the EAM treatment group showed decreased CK value(EAM treatment 1148±237.61U/L vs. EAM 4146±566.1 IU/L,P=0.0012) and inflammatory infiltration in muscle. The serum concentrations of IL-1β and IL-18 for EAM treatment group was 24.24±3.33ng/ml and 44.34±7.52ng/ml, which were significantly decreased compared to EAM group (P<0.05); The mRNA expression for EAM treatment group of IL-1β, IL-18, Caspase-1 and NLRP3 was decreased in muscle samples, and the western blotting of muscle samples showed elevated protein expression of NLRP3, Pro-caspase-1 and Caspase-1 p20 in EAM treatment group(P<0.01).Conclusions:(1) NLRP3 and downstream factors are elevated in PM and DM patients, which may be involved in the pathogenesis of IIM;(2) The immunosuppression therapy caused decreased expression of NLRP3 and downstream factors, which is a promising therapy target for IIM.