IRG1 induced by heme oxygenase-1/carbon monoxide inhibits LPS-mediated sepsis and pro-inflammatory cytokine production.

The immunoresponsive gene 1 (IRG1) protein has crucial functions in embryonic implantation and neurodegeneration. IRG1 promotes endotoxin tolerance by increasing A20 expression in macrophages through reactive oxygen species (ROS). The cytoprotective protein heme oxygenase-1 (HO-1), which generates endogenous carbon monoxide (CO), is expressed in the lung during Lipopolysaccharide (LPS) tolerance and cross tolerance. However, the detailed molecular mechanisms and functional links between IRG1 and HO-1 in the innate immune system remain unknown. In the present study, we found that the CO releasing molecule-2 (CORM-2) and chemical inducers of HO-1 increased IRG1 expression in a time- and dose-dependent fashion in RAW264.7 cells. Furthermore, inhibition of HO-1 activity by zinc protoporphyrin IX (ZnPP) and HO-1 siRNA significantly reduced expression of IRG1 under these conditions. In addition, treatment with CO and HO-1 induction significantly increased A20 expression, which was reversed by ZnPP and HO-1 siRNA. LPS-stimulated TNF-α was significantly decreased, whereas IRG1 and A20 were increased by CORM-2 application and HO-1 induction, which in turn were abrogated by ZnPP. Interestingly, siRNA against IRG1 and A20 reversed the effects of CO and HO-1 on LPS-stimulated TNF-α production. Additionally, CO and HO-1 inducers significantly increased IRG1 and A20 expression and downregulated TNF-α production in a LPS-stimulated sepsis mice model. Furthermore, the effects of CO and HO-1 on TNF-α production were significantly reversed when ZnPP was administered. In conclusion, CO and HO-1 induction regulates IRG1 and A20 expression, leading to inhibition of inflammation in vitro and in an in vivo mice model.

A functional link between heme oxygenase-1 and tristetraprolin in the anti-inflammatory effects of nicotine.

Nicotine stimulates the cholinergic anti-inflammatory pathway and prevents excessive inflammation by inhibiting the release of inflammatory cytokines from macrophages. We have previously reported that heme oxygenase-1 (HO-1) and tristetraprolin (TTP) are induced by nicotine and mediate the anti-inflammatory function of nicotine in macrophages. However, it was not clear whether the two molecules are functionally linked. In this study, we sought to determine whether HO-1 associates with TTP to mediate the anti-inflammatory effects of nicotine. Inhibition of HO-1 activity or HO-1 expression attenuated the effects of nicotine on STAT3 activation, TTP induction, and TNF-α production in LPS-treated macrophages. Induction of HO-1 expression increased the level of TTP in the absence of nicotine. In an LPS-induced endotoxemia model, HO-1 deficiency blocked the effects of nicotine on the STAT3 phosphorylation, TTP induction, and LPS-induced TNF-α production in the liver. Downregulation of STAT3 by siRNA attenuated the effect of nicotine on TTP expression and TNF-α production but did not affect the nicotine-mediated induction of HO-1. In TTP knockout mice, nicotine treatment enhanced HO-1 expression and STAT3 activation but failed to inhibit LPS-induced TNF-α production. Our results suggest that HO-1 and TTP are functionally linked in mediating the anti-inflammatory effects of nicotine; HO-1 is necessary for the induction of TTP by nicotine. This novel nicotine-HO-1-TTP signaling pathway provides new possibilities for the treatment of inflammatory diseases.