Bruton’s tyrosine kinase is a possible therapeutic target in myeloperoxidase-antineutrophil cytoplasmic antibody-associated vasculitis

Abstract

Abstract Background Bruton’s tyrosine kinase (Btk) is an enzyme expressed in leukocytes other than T lymphocytes and plasma cells and involved in B-cell receptor- and Fcγ receptor (FcγR)-mediated signal transduction. Btk inhibitors potentially suppress autoantibody production due to the expected inhibitory ability of B lymphocyte differentiation into antibody-producing plasma cells and reduce FcγR-mediated neutrophil activation, including the release of neutrophil extracellular traps (NETs). Myeloperoxidase (MPO)-antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (MPO-AAV) is a systemic small-vessel vasculitis characterized by the pathogenic autoantibody, MPO-ANCA. MPO and MPO-ANCA immune complex (MPO-ANCA-IC)-induced FcγR-mediated NETs are critically involved in MPO-AAV pathogenesis. This study aimed to demonstrate the therapeutic efficacy of the Btk inhibitor tirabrutinib on MPO-AAV. Methods Various doses of tirabrutinib or vehicle were orally administered to Sprague-Dawley rats daily. Four weeks later, the number of peripheral B lymphocytes was counted, and Btk phosphorylation in B lymphocytes was evaluated by flow cytometry. Human peripheral blood neutrophils were stimulated by MPO-ANCA-ICs, and Btk and its downstream Vav phosphorylation was assessed by western blotting. The effects of tirabrutinib on MPO-ANCA-IC-induced NET formation were examined in vitro. Wistar Kyoto rats were immunized with human MPO to induce MPO-AAV and given drug-free or tirabrutinib-containing feed (0.0037% or 0.012%) from day 0 or 28. All rats were euthanized on day 42 for serological and histological evaluation. Results Tirabrutinib inhibited Btk phosphorylation without decreasing B lymphocytes in vivo. Neutrophil Btk and Vav were phosphorylated when stimulated with MPO-ANCA-ICs. Tirabrutinib suppressed MPO-ANCA-IC-induced NET formation in vitro and ameliorated MPO-AAV in a dose-dependent manner. Although MPO-ANCA production was not affected, NET-forming neutrophils in the blood were significantly reduced by tirabrutinib. Conclusions The Btk inhibitor tirabrutinib suppressed MPO-ANCA-IC-induced NET formation in vitro and ameliorated MPO-AAV by reducing NET-forming neutrophils but not decreasing MPO-ANCA titer in vivo. This study suggests that Btk is a possible therapeutic target in MPO-AAV.