Rilzabrutinib, a potent and selective Bruton’s tyrosine kinase inhibitor, suppresses reactive oxygen species production and CD11b activation in human eosinophils

<bold>Introduction:</bold> Eosinophils are key innate immune cells contributing to the pathogenesis of asthma. Bruton’s tyrosine kinase (BTK) is expressed in eosinophils, and although murine data suggest that BTK does not play a functional role in eosinophils, the role of BTK in human eosinophils has not been investigated. <bold>Aim:</bold> To explore the functional impact of BTK inhibition in human eosinophils using a potent and highly selective inhibitor, rilzabrutinib, which is currently being investigated in clinical trials of asthma. <bold>Methods:</bold> Human eosinophils were isolated from healthy donors by magnetic isolation (MACSxpress® Whole Blood Eosinophil Isolation Kit, human) prior to activation by interleukin (IL)-5 and the toll-like receptor 4 agonist lipopolysaccharide (LPS). This was followed by flow cytometry analysis to determine reactive oxygen species (ROS) production and CD11b activation. To examine the role of BTK, isolated eosinophils were treated for 1 hour with rilzabrutinib at doses of 0.01, 0.1, and 1 µM before activation. <bold>Results:</bold> Activation by the combination of IL-5 and LPS increased ROS production (n=7 donors) and activated CD11b (n=6 donors) in human eosinophils. Pretreatment with rilzabrutinib significantly inhibited the response across all doses tested (p<0.001 ROS production; p<0.05 CD11b activation). <bold>Conclusions:</bold> This study is the first to demonstrate that BTK plays a novel role in human eosinophil activation and ROS generation elicited by IL-5 and LPS. These findings provide preclinical support for the therapeutic potential of rilzabrutinib in treating inflammatory diseases in which eosinophils play an integral role.