The PIANO project, Pathways Involved in the action of ANti-MDA5 antibodies: impact On innate immunity

Obiettivi del progetto

The project addresses the function of single anti-MDA5 antibody specificities targeted onto MDA5 epitopes and of ferritin in different in vitro models, focusing on the effect on type I interferons and NF-KB pathways. The working hypothesis is that anti-MDA5 antibodies and hyperferritinemia stimulate type I interferon and NF-KB pathways by activating MDA5, possibly triggering a ferritin-based inflammation loop. In this project, full-length recombinant human MDA5 will be used to isolate single antibody specificities by a microfluidic single B cell screening platform. The different anti-MDA5 antibodies formats obtained (full length, scFv and Fab) will be characterized and their epitopes mapped at least at the domain level by structural studies and molecular biology methods. Experiments on stabilized cell-lines and primary human cells (PBMCs, fibroblasts, macrophages, endothelial cells, bronchial epithelial cells, and cell-lines co-coltures) under different conditions, (e.g. stimulation with Poly (I:C), ferritin and/or its subunits) will be performed to compare the effect of:

• single or combined anti-MDA5 antibody specificities;
• anti-MDA5 antibody mixtures obtained from the serum of affected patients;
• B cells supernatants 

on type I interferons and NF-KB pathways, and on production of ferritin and its subunits. The possible occurrence of a ferritin-based inflammation loop will be specifically evaluated in macrophages. To establish if MDA5/anti-MDA5 antibody interactions only occur within or even outside cells, as observed in Anti-Synthetase Syndrome (ASSD), we will study anti-MDA5 antibody cell internalization and the presence of extracellular MDA5, its isoforms and subdomains. Finally, the latter, produced in recombinant form, will be used to develop a new, advanced ELISA kit to detect different anti-MDA5 antibodies specificities, that could be useful in the sub-phenotyping of affected patients. With this project, we will clarify the action of anti-MDA5 antibodies and ferritin on MDA5 activity, providing a general approach adaptable to pathogenic studies of similar conditions and identifying possible disease biomarkers/diagnostic kit and targets for therapy.

Data di inizio e fine

Responsabile del progetto

Istituzione coordinatrice del progetto

Altre Istituzioni coinvolte

• Università degli Studi dell’Aquila
• Università degli Studi di Pavia

Fonte/i di finanziamento