We have shown that activation of toll-like receptor 4 (TLR4) and its interferon regulatory factor 3 (IRF3)-dependent downstream signaling pathway are required for the development of liver ischemia/reperfusion injury (IRI). This study focused on the role of TLR4-IRF3 activation pathway products, in particular, chemokine (C-X-C motif) ligand 10 (CXCL10). The induction of CXCL10 by liver IR was rapid (1 hour postreperfusion), restricted (ischemic lobes), and specific (no CXCL9 and CXCL11 induction). I schemia/reperfusion injury (IRI) develops in the absence of exogenous Ag, and innate immunity has been thought to play a dominant pathogenic role. [1][2][3] Liver ischemia activates Kupffer cells, and to a lesser degree endothelial cells as well as hepatocytes, leading to the formation of reactive oxygen species and secretion of proinflammatory cytokines/chemokines. The oxidant stress directly damages endothelial cells/hepatocytes, whereas the soluble factors are largely responsible for leukocyte recruitment and activation, leading to the full development of intrahepatic inflammation causing further organ damage. Although excessive pro-inflammatory response has been recognized as the key element leading to IRI, the mechanisms that initiate and regulate liver inflammation cascade remain to be elucidated.We and others have reported that mammalian sentinel receptor toll-like receptor 4 (TLR4) is involved in the initiation of IRI. [4][5][6][7] We found that livers in TLR4 knockout (KO) mice were protected from IRI and associated inflammation. 4-7 Furthermore, MyD88-independent signaling mediated by IRF3, downstream of TLR4 activation, was critical, because mice deficient in interferon regulatory factor 3 (IRF3) but not MyD88 were protected from IRI. 5 MyD88-dependent signaling in TLR4 activation pathway leads to direct nuclear factor kappa B activation and induction of pro-inflammatory cytokines, whereas the IRF3-mediated signaling induces type 1 IFN

Dysregulation of the mitochondrial fission machinery has been linked to cell death following ischemia. Fission is largely dependent on recruitment of Dynamin-related protein 1 (Drp1) to the receptor Mitochondrial fission factor (Mff) located on the mitochondrial outer membrane (MOM). Drp1 is a target for SUMOylation and its deSUMOylation, mediated by the SUMO protease SENP3, enhances the Drp1-Mff interaction to promote cell death in an oxygen/glucose deprivation (OGD) model of ischemia. Another interacting partner for Drp1 is the Bcl-2 family member Bcl-xL, an important protein in cell death and survival pathways. Here we demonstrate that preventing Drp1 SUMOylation by mutating its SUMO target lysines enhances the Drp1-Bcl-xL interaction in vivo and in vitro. Moreover, SENP3-mediated deSUMOylation of Drp1 promotes the Drp1-Bcl-xL interaction. Our data suggest that Mff primes Drp1 binding to Bcl-xL at the mitochondria and that Mff and Bcl-xL can interact directly, independent of Drp1, through their transmembrane domains. Importantly, SENP3 loss in cells subjected to OGD correlates with reduced Drp1-Bcl-xL interaction, whilst recovery of SENP3 levels in cells subjected to reoxygenation following OGD correlates with increased Drp1-Bcl-xL interaction. Expressing a Bcl-xL mutant with defective Drp1 binding reduces OGD plus reoxygenation-evoked cell death. Taken together, our results indicate that SENP3-mediated deSUMOlyation promotes an Mff-primed Drp1-Bcl-xL interaction that contributes to cell death following ischemia.

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