title: Nonreceptor tyrosine kinases ITK and BTK negatively regulate mast cell proinflammatory responses to lipopolysaccharide.
abstract: BACKGROUND
  Mast cells are indispensable for LPS-induced septic hypothermia, in which TNF-α 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-α 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-α 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-α 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.
authors.affiliation: Department of Microbiology and Immunology, Cornell University, Ithaca, NY.
authors.forename: Weishan
authors.initials: W
authors.lastname: Huang
authors.affiliation: Department of Veterinary and Biomedical Science, Center for Molecular Immunology and Infectious Disease, The Pennsylvania State University, University Park, Pa.
authors.forename: J Luis
authors.initials: JL
authors.lastname: Morales
authors.affiliation: Department of Microbiology and Immunology, Cornell University, Ithaca, NY.
authors.forename: Victor P
authors.initials: VP
authors.lastname: Gazivoda
authors.affiliation: Department of Microbiology and Immunology, Cornell University, Ithaca, NY. Electronic address: averyaugust@cornell.edu.
authors.forename: Avery
authors.initials: A
authors.lastname: August
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D016207:Cytokines / Q000378:metabolism; D007035:Hypothermia / Q000209:etiology / Q000276:immunology*; D008070:Lipopolysaccharides / Q000276:immunology*; D008407:Mast Cells / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000276:immunology* / Q000378:metabolism; D018805:Sepsis / Q000150:complications / Q000276:immunology*
chemical_list: D015415:Biomarkers; D016207:Cytokines; D008070:Lipopolysaccharides; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C080813:emt protein-tyrosine kinase
keywords: MAPKs; NF-κB; PI3K/Akt; TNF-α; Tec; lipopolysaccharide; mast cell; septic hypothermia