Clinical Value Of Anti-MDA5 Antibody Subtypes And Study Of T Cell Abnormalities In The Pathogenesis Of Dermatomyositi

Part I.Evaluation and validation of the prognostic value of anti-MDA5 IgG subclasses in dermatomyositis-associated interstitial lung diseaseObjective:To investigate the association between the anti-melanoma differentiation associated gene 5(MDA5)IgG subclasses and prognosis of patients with dermatomyositis(DM)-associated interstitial lung disease(ILD).Methods:This retrospective study included 122 Anti-MDA5 positive DM-ILD patients admitted from October 2017 to October 2020 as training cohort,and additional 68 patients from August 2014 to September 2017 as validation cohort.The levels of anti-MDA5 total IgG and IgG subclasses were measured using in-house enzyme-linked immunosorbent assays,and analysed in association with the patient prognosis.Results:In the training cohort,the concentrations of anti-MDA5 IgG1 and IgG3 in nonsurvivors were significantly higher than survivors(P<0.05),whereas there were no significant differences in the IgG2 and IgG4 levels.Kaplan-Meier survival analysis revealed that the levels of anti-MDA5 total IgG,IgG1 and IgG3 were associated with mortality(P<0.05).Multivariate analysis revealed anti-MDA5 IgG1>13 U/mL and antiMDA5 IgG3>11 U/mL were independent risk factors for death of DM-ILD patients(P<0.05).Anti-MDA5 IgG1 was confirmed as an independent risk factor in the validation cohort,while anti-MDA5 IgG3 was not.Anti-MDA5 IgG1 showed greater discriminable power for patient prognosis(Youden index 0.494)than anti-MDA5 total IgG,IgG3 or the combination of IgGl and IgG3(Youden index as 0.356,0.32,0.447,respectively).Conclusion:Anti-MDA5 IgG1 and IgG3 are significantly associated with poor prognosis in DM-ILD patients,and anti-MDA5 IgG1 is more efficient as a prognostic biomarker in DM-ILD patients.Part Ⅱ.Expansion of exhausted CD8+T cells associates with increased pulmonary fungal infection risk in anti-MDA5+dermatomyositisObjective:To investigate the expression and clinical relevance of exhaustion markers on peripheral CD8+T cells from patients with idiopathic inflammatory myopathies(IIM).Methods:A total of 29 healthy controls and 88 IIM patients were enrolled,including 42 patients with anti-MDA5+ dermatomyositis(DM),18 with anti-MDA5-dermatomyositis(DM),17 with immune-mediated necrotizing myopathy(IMNM),11 with anti-synthetase syndrome(ASS).Flow cytometry analysis was used to detect the expression of PD-1,TIM3,LAG-3 on CD8+T cells in blood samples.Clinical associations of CD8+T cell exhaustion phenotype in anti-MDA5+DM patients were analyzed.Results:CD8+ T cells from anti-MDA5+DM patients showed significantly increased expression of LAG-3 and TIM-3 compared to patients with anti-MDA5-DM,IMNM,ASS or HCs(adjusted P all<0.05).The expression of PD-1 on CD8+T cells was increased in anti-MDA5+DM patients compared to anti-MDA5-DM or HCs(adjusted P all<0.05).CD8+ T cells consistent with low exhaustion(1+ marker for PD-1,TIM-3 and LAG-3),intermediate exhaustion(2+markers for PD-1,TIM-3 and LAG-3),and severe exhaustion phenotypes(3+markers for PD-1,TIM-3 and LAG-3)were all significantly increased in anti-MDA5+DM patients compared with HC group(P all<0.05).Patients with high levels of PD-1+TIM-3+LAG-3+CD8+T cells had a significant higher incidence of pulmonary fungal infections,and a higher level of LDH,serum ferritin and lower counts of CD4+T cells and CD8+cells.ROC analysis revealed that the frequency of PD-1+TIM-3+LAG-3+CD8+T cell significantly predicted pulmonary fungal infections of anti-MDA5+DM patients(AUC=0.828,P<0.05).Conclusion:CD8+ T cells from anti-MDA5+ DM patients show significant exhausted phenotype,and increased CD8+T cell exhaustion was associated with high risk of pulmonary fungal infection in anti-MDA5+DM patients.Part Ⅲ.Ferroptotic T cell death mediated by ACSL1 induces the increased risk of pulmonary fungal infection in anti-MDA5+dermatomyositisObjective:To investigate the association of T cell ferroptosis and anti-MDA5+dermatomyositis(DM),and analyzed the potential regulate mechanism of T cell ferroptosis in patients with anti-MDA5+dermatomyositis(DM).Methods:Western blotting and flow cytometry were used to detect the expression of GPX4 in T cells from 95 IIM patients(including 65 patients with anti-MDA5+DM)and 46 age-and sex-matched healthy controls.The characteristic of mitochondrial morphology in T cells from anti-MDA5+DM patients with T lymphonia were analyzed by transmission electron microscope(TEM).The lipid and iron metabolism of T cells were analyzed by live cell fluorescence staining.The level of MDA in plasma was measured by ELISA.The effect of GPX4 plasmid overexpression on ferroptosis of T cells was explored in vitro.Western blotting was used to analyze the protein expression levels of ACSL family(ACSL1,3,4)in T cells of anti-MDA5+ DM patients.The effect of ferroptotic cell death of anti-MDA5+DM patients triggered by α-ESA which mediated by ACSL1 was explored in vitro.Results:Western blotting and flow cytometry showed that the expression of GPX4 in the anti-MDA5+DM patients with T lymphopenia was significantly lower than those in the anti-MDA5+DM with T cell normal,other idiopathic inflammatory myopathy patients and healthy controls(P<0.05).Electron microscopic analysis showed that the peripheral blood T lymphocytes of the anti-MDA5+DM patients with T lymphopenia showed typical morphological characteristics of ferroptosis.Live cell fluorescence staining analysis showed that the intracellular Fe2+level and cellular lipid peroxide level of peripheral T cells in anti-MDA5+DM patients with T lymphopenia were significantly higher than those in healthy controls(P<0.05).ELISA showed that the level of MDA in plasma of antiMDA5+DM patients was significantly higher than that in healthy controls and other IIM(P<0.05).In vitro experiments,overexpression of GPX4 in T cell of anti-MDA5+DM patients with T lymphopenia could improve the resistance of peripheral T cells to ferroptosis.Western blot analysis showed that the expression of ACSL 1 was significantly up-regulated in anti-MDA5+DM patients.In vitro experiments,ferroptotic T cell death triggered byα-ESA witch mediated ACSL1 induced T lymphopenia in anti-MDA5+DM patients.Conclusion:Ferroptosis exists in peripheral T cells of anti-MDA5+ DM patients with T lymphopenia.Down-regulation of GPX4 expression,and abnormal lipid metabolism mediated by ACSL1 may be the cause of ferroptosis of peripheral T cells in anti-MDA5+DM.