AB0134 IN-VITRO STUDY ON THE EFFECT OF SELECTIVE Jak-INHIBITORS ON PBMCs STAT3 PHOSPHORYLATION FROM SYSTEMIC SCLEROSIS PATIENTS

Abstract

BackgroundSystemic sclerosis (SSc) is a rare autoimmune connective tissue disease characterized by autoimmunity-driven damage and vasculopathy leading to fibrosis of the skin and internal organs (1). The Janus kinase (Jak) - signal transducer and activator of transcription (STAT) pathway has been evidenced markedly activated in SSc patients (2, 3), and its inhibition has been proved in preclinical and clinical trials (4), but no data on Jak selective inhibition are available.ObjectivesTo explore the effect of selective inhibition of Jak/STAT pathway in peripheral blood mononuclear cells (PBMC) from SSc patients.MethodsIn vitro Jak inhibition of the subunit 3 of phosphorylated (p) than activated STAT was measured by flow cytometry in peripheral blood mononuclear cells (PBMC) from SSc patients naïve to any immunosuppressive and/or corticosteroids (n.5). pSTAT3 activity was also assessed after stimulation with recombinant human 0.1 ng/ml IL-6 (Peprotech – NJ, USA). The PBMC were overnight incubated with IC50 concentrations of selective Jak1-, Jak2-, Jak3- and Tyk2-inhibitors (Biovision Inc. – CA, USA). Percentages of pSTAT3 positive cells were compared in presence of different compounds stimulation.ResultsAfter overnight incubation, percentage of pSTAT3 positive cells was significantly higher in CD14pos compared to CD4pos (16.3%; 95CI 10-22 vs 10.7%; 95CI 4--18, – p=0.02). pSTAT3posCD14pos cells were halved only by selective Jak3-inhibitor, while pSTAT3posCD4pos cells were reduced by 36% by selective Jak1-inhibitor. Selective Jak2- or Tyk2-inhibitors did not interfere with STAT3 phosphorylation in PBMC from SSc patients. After IL-6 stimulation, we observed a 2- and a 1.5-fold increase in percentage of pSTAT3posCD4pos and pSTAT3posCD14pos cells, respectively. pSTAT3posCD14pos cells were reduced in the PBMC co-culture with IL-6 and Jak-selective inhibitors, in contrast no effects were found in CD4pos cells. Specifically, selective Jak1- and Jak3-inhibitors reduced pSTAT3posCD14pos cells by an average of 37% and 25%, respectively. No effects were observed after co-culture with IL-6 and selective Jak2- or Tyk2-inhibitors.ConclusionJak/STAT3 pathway of PBMC from SSc patients with active disease may be differently modulated by specific inhibitors. Selectivity of Jak1- and Jak3-inhibitors seems more relevant, especially in CD14pos monocytes after IL-6 stimulation. These preliminary findings highlight some evidence for effectiveness of selective Jak-inhibitors in SSc treatment.