Therapeutic And Single-cell Omics Studies In Rapidly Progressive Interstitial Lung Disease Associated With Anti-MDA5 Antibodies

[AIMS]To explore risk factors of anti-melanoma differentiation-associated gene 5(MDA5)antibody-associated rapidly progressive interstitial lung disease on the basis of the single-center retrospective cohort from Peking Union Medical College Hospital.To study the efficacy of To cilizumab on the cumulative survival rate of anti-MDA5 antibody-associated RP-ILD patients.Through single-cell RNA sequencing(scRNA-seq),our study aims at describing the landscape of transcriptomes from immune cells in patients' blood and lung,studying the differences among cell components from different patients,and exploring the differential gene expression of patients with different outcomes.We also compare the expression level of interferon stimulated genes in patients and health controls.Finally,through high through-put chips(ProcartaPlex(?)),the cytokine and chemokine levels of BALF and serum are tested.[METHODS]In this prognostic factor study,univariate and multivariate Cox proportional hazards analyses were carried out to determine the prognostic factors for anti-MDA5 antibody-associated RP-ILD.Propensity score matching was conducted to balance the baseline characteristics.Consequent survival analysis was completed using Kaplan-Meier analysis.As for scRNA-seq,principal component analysis was used to reduce the dimension of expression matrix.After removing batch effects and standardizing,cell type annotations were carried out according to pre-calculated specific marker genes.Consequent enrichment analyses were completed using GO and KEGG analysis.[RESULTS]A total of 152 patients diagnosed with anti-MDA5 antibody-associated RP-ILD were enrolled in our study,of which 40 patients were excluded because of infection before treatment.Univariate cox proportional hazards analysis demonstrated that elder age of disease onset,higher level of ?-glutamyl transferase,higher level of hypersensitive C-reaction protein,larger dosage of glucocorticoid and usage of pulse therapy were possible risk factors.Multivariate cox proportional hazards analysis revealed that elder age of disease onset and hypersensitive C-reaction protein were independent risk factors of poorer prognosis.Kaplan-Meier survival analysis after propensity score matching showed a tendency of curve separation but statistically insignificant.ScRNA-seq showed that cell type composition of PBMC among patients varied a lot while cell type composition of BALF demonstrated similar patterns.Subsequent gene functional enrichment revealed a prominent upregulation of genes associated with type ? interferon.We also found a prominent upregulation of chemokines in BALF,most of which were expressed by mono-macrophage systems and T helper cells.[Conclusions]This was the first retrospective study to evaluate the efficacy of Tocilizumab on prognosis among anti-MDA5 antibody associated RP-ILD.Our study demonstrated that Tocilizumab might serve as a rescue therapy for patients who were refractory to standard treatment and might worth subsequent study.Additionally,our team first explored single-cell transcriptome of myositis and anti-MDA5 antibody associated RP-ILD.Local inflammation,instead of systemic inflammation,might play a key role in pathogenesis of anti-MDA5 antibody associated RP-ILD.Mono-phagocytes and T helper cells expressed chemokines and might contribute to the local inflammation.Our study also proved from mRNA level that type I interferon associated signal pathway was up-regulated and might shed light on novel treatment of this virulent disease.