Anti-Melanoma Differentiation-Associated protein 5 Auto-Antibodies Promote a Profibrotic Phenotype in a Human Lung Fibroblast Cell Line

Calandra, S.; Maggi, M.; Previtali, A.; Iamele, L.; Castellini, C.; Navarini, L.; Giacomelli, R.; Ruscitti, P.; Codullo, V.; Zanframundo, G.; Scotti, C.; Cavagna, L.

doi:10.64898/2026.05.31.727600

bioRxiv · 10.64898/2026.05.31.727600

Anti-Melanoma Differentiation-Associated protein 5 Auto-Antibodies Promote a Profibrotic Phenotype in a Human Lung Fibroblast Cell Line

Calandra, S.·Maggi, M.·Previtali, A.·Iamele, L.·Castellini, C.·Navarini, L.·Giacomelli, R.·Ruscitti, P.·Codullo, V.·Zanframundo, G.·Scotti, C.·Cavagna, L.

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Abstract

Anti-melanoma differentiation-associated protein 5 (anti-MDA5) autoantibodies identify a distinct dermatomyositis subset frequently associated with rapidly progressive interstitial lung disease (RP-ILD). While these antibodies are established disease markers, their direct contribution to pulmonary fibrosis is poorly defined. This study investigated the pathogenic effects of patient-derived polyclonal anti-MDA5 antibodies on IMR-90 human lung fibroblasts. Recombinant human MDA5 protein was produced in HEK293F cells and utilized to selectively isolate autoantibodies from a patients plasma via affinity chromatography. Fibroblasts were stimulated with MDA5, anti-MDA5 antibodies, or both. Real-Time Cell Analysis (RTCA) showed a statistically significant increase in cell impedance following treatment with an MDA5-anti-MDA5 mixture compared with controls, accompanied by a reduction in cell doubling time. MTT assays showed that neither MDA5 nor anti-MDA5, nor their immunocomplex, exerted acute cytotoxic effects in cell culture. Direct cell counting revealed a significant increase in fibroblast proliferation in response to the MDA5-anti-MDA5 combination. Molecular characterization by RT-qPCR revealed a significant alteration of TLR2, TLR7, and endothelin-1 (ET-1) mRNA levels. ELISA assays detected an increased secretion of pro-collagen and type I interferons in culture supernatants. All these results were mainly, but not only, observed in the MDA5/anti-MDA5-exposed cells. Our results suggest that anti-MDA5 autoantibodies and MDA5 antigen complex are not merely disease biomarkers, but active pathogenic drivers that stimulate proliferation and pro-fibrotic responses in lung fibroblasts. This mechanism may contribute to the rapid tissue remodeling characteristic of RP-ILD, supporting the development of targeted therapeutic strategies to mitigate fibrosis in this high-mortality patient subset.

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Calandra, S., Maggi, M., Previtali, A., Iamele, L., Castellini, C., Navarini, L., Giacomelli, R., Ruscitti, P., Codullo, V., Zanframundo, G., Scotti, C., Cavagna, L.. 2026-06-01. Anti-Melanoma Differentiation-Associated protein 5 Auto-Antibodies Promote a Profibrotic Phenotype in a Human Lung Fibroblast Cell Line. https://doi.org/10.64898/2026.05.31.727600

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