Tofacitinib Decreases Autophagy of Fibroblast-Like Synoviocytes From Rheumatoid Arthritis Patients.

M Vomero,C Alessandri,T Colasanti,F Ceccarelli,F Ucci,C Barbati,F Conti,C Ciancarella,G Buoncuore,E Corsiero,E Putro,M Bombardieri,A I Celia,M Caliste,M Speziali,F R Spinelli

doi:10.3389/fphar.2022.852802

Abstract

The pathway of Janus tyrosine kinases (JAKs) has a central role in the pathogenesis of Rheumatoid Arthritis (RA) by regulating multiple immune functions and cytokine production. The JAK inhibitor tofacitinib is effective in RA patients not responding to methotrexate or TNF-inhibitors. Since hyperactive autophagy has been associated with impaired apoptosis of RA fibroblast-like synoviocytes (FLS), we aimed to investigate the role of tofacitinib in modulating autophagy and apoptosis in these cells. FLS isolated from RA biopsies were cultured with tofacitinib in presence of autophagy inducer rapamycin and in serum deprivation condition. Levels of autophagy, apoptosis, and citrullinated proteins were analyzed by western blot, flow cytometry, immunocytofluorescence, and Real-Time PCR. Rapamycin induced an increase in RA-FLS autophagy while the levels of autophagy marker LC3-II were reduced after in vitro treatment with tofacitinib. The analysis of autophagic flux by specific fluorescence dye confirmed the reduction of autophagy in RA FLS. The treatment with tofacitinib did not influence apoptosis of RA FLS. Modulation of the autophagic process by tofacitinib did not significantly change citrullination. The results of this study demonstrate that tofacitinib is able to modulate autophagy of FLS contributing to its effectiveness in RA patients.

Highlights

Rheumatoid arthritis (RA) is an inflammatory disease characterized by an autoimmune response against self-antigens promoted by adaptive and innate immune cells
We investigated whether the addition of the Janus tyrosine kinases (JAKs) inhibitor tofacitinib in cell cultures of fibroblast-like synoviocytes (FLS) obtained from Rheumatoid Arthritis (RA) patients could affect autophagy and apoptosis
Based on preliminary doseresponse experiments performed on FLS isolated from OA patients (Supplementary Figure S1), cells were cultured in the presence of tofacitinib at a concentration of 1uM for 20 h

Summary

Introduction

Rheumatoid arthritis (RA) is an inflammatory disease characterized by an autoimmune response against self-antigens promoted by adaptive and innate immune cells. RA fibroblast-like synoviocytes (RA-FLS) are subjected to complex molecular changes that lead to an aggressive and invasive phenotype characterized by the release of inflammatory cytokines, chemokines and matrixdegrading enzymes (McInnes and Schett, 2020). Autophagy is a metabolic process involved in the degradation of intracellular components via lysosomal machinery. Several autophagy types are described in mammalian cells, macro-autophagy, hereafter referred to as autophagy, is the most studied (Nakatogawa et al, 2009). Autophagy is stimulated by starvation and it is characterized by proteins and organelles engulfment in double-membrane vesicles called autophagosomes.

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