Janus kinase inhibitors modify the fatty acid profile of extracellular vesicles and modulate the immune response

Background: Janus kinase inhibitors (jakinibs) are immunomodulators used for treating malignancies, autoimmune diseases, and immunodeficiencies. However, they induce adverse effects such as thrombosis, lymphocytosis, and neutropenia that could be mediated by extracellular vesicles (EVs). These particles are cell membrane-derived structures that transport cellular and environmental molecules and participate in intercellular communication. Jakinibs can modify the content of EVs and enable them to modulate the activity of different components of the immune response. Objective: to evaluate the interactions between immune system components of healthy individuals and EVs derived from monocytic and lymphoid lineage cells generated in the presence of baricitinib (BARI) and itacitinib (ITA) and their possible effects. Methods: EVs were isolated from monocytes (M) and lymphocytes (L) of healthy individuals, as well as from U937 (U) and Jurkat (J) cells exposed to non-cytotoxic concentrations of BARI, ITA, and dimethyl sulfoxide (DMSO; vehicle control). The binding to and engulfment of EVs by peripheral blood leukocytes of healthy individuals were analyzed by flow cytometry using CFSEstained EVs and anti-CD45-PeCy7 mAb-labeled whole blood. The effect of EVs on respiratory burst, T-cell activation and proliferation, cytokine synthesis, and platelet aggregation was evaluated. Respiratory burst was assessed in PMA-stimulated neutrophils by the dihydrorhodamine (DHR) test and flow cytometry. T-cell activation and proliferation and cytokine production were assessed in CFSE-stained PBMC cultures stimulated with PHA; expression of the T-cell activation markers CD25 and CD69 and T-cell proliferation were analyzed by flow cytometry, and the cytokine levels were quantified in culture supernatants by Luminex assays. Platelet aggregation was analyzed in platelet-rich plasma (PRP) samples by light transmission aggregometry. The EVs' fatty acid (FA) profile was analyzed using methyl ester derivatization followed by gas chromatography. Results: ITA exposure during the generation of EVs modified the size of the EVs released; however, treatment with DMSO and BARI did not alter the size of EVs generated from U937 and Jurkat cells. Circulating neutrophils, lymphocytes, and monocytes showed a 2-fold greater tendency to internalize ITA-U-EVs than their respective DMSO control. The neutrophil respiratory burst was attenuated in greater extent by M-EVs than by L-EVs. Autologous ITA-M-EVs reduced T-cell proliferation by decreasing IL-2 levels and CD25 expression independently of CD69. A higher accumulation of pro-inflammatory cytokines was observed in PHA-stimulated PBMC cultures exposed to M-EVs than to L-EVs; this difference may be related to the higher myristate content of M-EVs. Platelet aggregation increased in the presence of ITA-L/M-EVs by a mechanism presumably dependent on the high arachidonic acid content of the vesicles. Conclusions: Cellular origin and jakinib exposure modify the FA profile of EVs, enabling them, in turn, to modulate neutrophil respiratory burst, T-cell proliferation, and platelet aggregation. The increased T-cell proliferation induced by BARI-L/M-EVs could explain the lymphocytosis observed in patients treated with BARI. The higher proportion of arachidonic acid in the FA content of ITA-L/M-EVs could be related to the thrombosis described in patients treated with ITA. EVs also induced a decrease in the respiratory burst of neutrophils.