Studies On Biomarkers Anti-Mi-2 Antibodies And Galectin-9 In Patients With Idiopathic Inflammatory Myopathy

Background:Myositis specific antibodies play important roles in the diagnosis,treatment and prognosis of patients with idiopathic inflammatory myopathy(IIM).The mortality rate was high and the prognosis was poor in IIM patients with rapidly progressive interstitial lung disease(RP-ILD).Multiple functions of galectin-9(Gal-9)were found in immune regulation.The first part was to explore the clinical phenotype and prognosis of anti-Mi-2-positive dermatomyositis(DM)patients;the second part was to explore the distribution and clinical significance of a new myositis-related serum biomarker Gal-9 in patients with IIM.Part Ⅰ.A study on the clinical features and prognosis of anti-Mi-2 antibodies in DM patientsObjective:To investigate the clinical features,pathological features,the correlation between serum levels of anti-Mi-2 antibodies and disease activity,and prognosis of DM patients carrying anti-Mi-2 antibodies.Methods:Three hundred and fifty-seven DM patients were included.Serum anti-Mi-2β antibodies were detected by enzyme-linked immunosorbent assays(ELISA).The clinical features associated with anti-Mi-2β antibodies were analyzed in cross-sectional and longitudinal studies.Results:Forty out of the 357(11.2%)DM patients were determined positive for anti-Mi-2β antibodies.Anti-Mi-2β-positive DM patients were found to have a higher incidence of V sign(P=0.001),shawl sign(P=0.003)and muscle weakness(P=0.013)compared with patients without anti-Mi-2β antibodies.In addition,they were found to have lower frequency of ILD(P=0.005)and malignancy(P=0.041)than patients witout anti-Mi-2β antibodies.The levels of anti-Mi-2β antibodies were positively correlated with physician’s global assessment(PGA)visual analog scale(VAS)scores(r=0.33,P=0.039,n=40),serum creatine kinase levels(r=0.52,P<0.001,n=40)and negatively correlated with manual muscle testing in 8 muscles scores(r=-0.34,P=0.029,n=40).The longitudinal study also revealed a positive correlation between anti-Mi-2β antibody levels and PGA VAS scores(β=0.039,P<0.001,n=16).The medium follow-up period was 44 months.Ninety-seven percent of patients got clinical remission.Among 26 patients whose disease course longer than 2 years,sixteen(61.5%)patients were monocyclic,five(15.1%)patients showed complete remission and they were drug-free for more than 3 months.The mortality rate of anti-Mi-2β-positive DM patients was lower than anti-Mi-2β-negative patients by using Kaplan-Meier survival curves(log-rank P=0.035).Interestingly,in the long-term longitudinal observation,anti-Mi-2β antibodies did not return negative in all patients.Conclusion:DM patients carrying anti-Mi-2β antibodies were frequently involved in cutaneous and muscle,and had low incidence of ILD and malignancy.In addition,the treatment response and outcome of anti-Mi-2β-positive DM patients were good.The correlation was observed between anti-Mi-2β levels and disease activities,but the anti-Mi-2β levels rarely became negative in the longitudinal study.Part Ⅱ.A study on the prevalence and related clinical significance of Gal-9 in IIM patientsObjective:To investigate the expression of Gal-9 in patients with DM and the association between Gal-9 and DM-ILD.Methods:One hundred and fifty-four patients with IIM and 30 healthy controls(HCs)were included in the current study.The association between serum levels of Gal-9 and clinical features were analyzed both in cross-sectional and longitudinal studies.The expression of Gal-9 in the sera and isolated peripheral blood mononuclear cells(PBMCs)were tested.The expression of Gal-9 and its ligand(T-cell immunoglobulin mucin(Tim)-3 and CD44)was tested in lung tissues from patients carrying anti-melanoma differentiation-associated gene 5(MDA5)antibodies by immunohistochemistry.The response of human lung fibroblasts(MRC-5)stimulated Gal-9 was tested in vitro.Results:Significantly differences of serum levels of Gal-9 were observed in IIM atients(median 19.8 ng/mL,interquartile range(IQR)10.0-33.6,n=154)than HCs(median 4.9 ng/mL,IQR 3.5-6.3,n=30)(P<0.001).Serum levels of Gal-9 were higher in DM patients(median 23.7 ng/mL,IQR 12.3-35.9,n=129)compared to those in immune-mediated necrotizing myopathy(IMNM)patients(median 7.4 ng/mL,IQR 5.2-10.8,n=25)and HCs(P<0.001).Serum levels of Gal-9 were significantly higher in anti-MDA5-positive DM patients(median 33.8 ng/mL,IQR 21.9-44.7 ng/mL,n=56)than in anti-MDA5-negative DM patients(median 16.2 ng/mL,IQR 10.0-26.9 ng/mL,n=73)(P<0.001).Among DM patients carrying anti-MDA5 antibodies,higher serum levels of Gal-9 were observed in patients with RP-ILD(median 42.4 ng/mL,IQR 34.9-68.5,n=20)than patients without RP-ILD(median 26.4 ng/mL,IQR 17.0-39.8,n=36)(P<0.001).In cross-sectional and longitudinal studies,the association was observed between serum levels of Gal-9 and disease activity in DM patients carrying anti-MDA5 antibodies.Higher levels of Gal-9 were produced from PBMCs isolated from DM patients than those from IMNM patients(P=0.022)and HCs(P=0.045).Positive correlation was observed between the Gal-9 mRNA levels and type I interferon-inducible genes MX1(r=0.659,P=0.020,n=12)and IFIH1(r=0.787,P=0.002,n=12)in PBMCs from DM patients carrying anti-MDA5 antibodies.The expression of Gal-9 and Tim-3 was increased in the lung tissues of DM patients with RP-ILD.The mRNA expression of monocyte chemoattractant protein-1 in MRC-5 fibroblasts stimulated with Gal-9 was up-regulated in vitro(P=0.024).Conclusion:Gal-9 might be a biomarker for monitoring disease activity and the severity of RP-ILD in DM patients carrying anti-MDA5 antibodies.Aberrant expression of the Gal-9 and its ligand Tim-3 might be involved in the DM-ILD immunopathogenesis.