Bruton’s Tyrosine Kinase Is Required for TLR-Dependent Heme Oxygenase-1 Gene Activation via Nrf2 in Macrophages

Heme oxygenase (HO)-1 is the inducible isoform of the rate-limiting enzyme of heme degradation and provides cytoprotection against oxidative stress by its products carbon monoxide and biliverdin. More recently, HO-1 has also been shown to exert immunomodulatory functions via cell type-specific anti-inflammatory effects in myeloid/macrophage cells. In the current study, it is demonstrated that Bruton's tyrosine kinase (Btk), the gene of which is mutated in the human immunodeficiency X-linked agammaglobulinemia, is involved in the upregulation of HO-1 gene expression via TLR signaling in macrophages. The specific Btk inhibitor LFM-A13 blocked HO-1 induction by the classical TLR4 ligand LPS in cell cultures of RAW264.7 monocytic cells and primary mouse alveolar macrophages. Moreover, upregulation of HO-1 gene expression was abrogated in LPS-stimulated alveolar macrophages from Btk(-/-) mice. Transfection studies with luciferase reporter gene constructs demonstrated that LPS-dependent induction of HO-1 promoter activity was attenuated by pharmacological Btk inhibition and by an overexpressed dominant-negative mutant of Btk. This induction was mediated by the transcription factor Nrf2, which is a master regulator of the antioxidant cellular defense. Accordingly, nuclear translocation of Nrf2 in LPS-treated macrophages was reduced by Btk inhibition. The generation of reactive oxygen species, but not that of NO, was involved in this regulatory pathway. Btk-dependent induction of HO-1 gene expression was also observed upon macrophage stimulation with ligands of TLR2, TLR6, TLR7, and TLR9, suggesting that Btk is required for HO-1 gene activation by major TLR pathways.