Nonreceptor tyrosine kinases ITK and BTK negatively regulate mast cell proinflammatory responses to lipopolysaccharide

Background: Mast cells are indispensable for LPS-induced septic hypothermia, in which TNF-alpha plays an essential role to initiate septic responses. ITK and BTK regulate mast cell responses to allergens, but their roles in mast cell responses in LPS-induced sepsis are unclear. Objective: We sought to investigate the roles of ITK and BTK in mast cell responses during LPS-induced septic inflammation. Methods: Mice (genetically modified or bone marrow-derived mast cell-reconstituted Sash) were given LPS to induce septic hypothermia in the presence or absence of indicated inhibitors. Flow cytometry was used to determine LPS-induced cell influx and TNF-alpha production in peritoneal cells. Microarray was used for genomewide gene expression analysis on bone marrow-derived mast cells. Quantitative PCR and multiplex were used to determine transcribed and secreted proinflammatory cytokines. Microscopy and Western blotting were used to determine activation of signal transduction pathways. Results: The absence of ITK and BTK leads to exacerbation of LPS-induced septic hypothermia and neutrophil influx. Itk(-/-)Btk(-/-) mast cells exhibit hyperactive preformed and LPS-induced TNF-alpha production, and lead to more severe LPS-induced septic hypothermia when reconstituted into mast cell-deficient Sash mice. LPS-induced nuclear factor kappa B, Akt, and p38 activation is enhanced in Itk(-/-)Btk(-/-) mast cells, and blockage of phosphatidylinositol-4,5-bisphosphate 3-kinase, Akt, or p38 downstream mitogen-activated protein kinase interacting serine/threonine kinase 1 activation significantly suppresses TNF-alpha hyperproduction and attenuates septic hypothermia. Conclusions: ITK and BTK regulate thermal homeostasis during septic response through mast cell function in mice. They share regulatory function downstream of Toll-like receptor 4/LPS in mast cells, through regulating the activation of canonical nuclear factor kappa B, phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt, and p38 signaling pathways.