Background and Aim: We have shown previously that endoplasmic reticulum (ER) stress may function in synergy with TLR4 in liver IRI to facilitate pro-inflammatory immune activation and hepatocellular death. To analyze molecular mechanisms of this synergy, we studied how intracellular signaling pathways of ER stress, in particular ATF6, regulated liver inflammatory immune responses against IR.

Methods: In a murine liver partial warm IR model, we first determined activations of intracellular signaling pathways of ER stress by Western blots. Their functions were studies by using gene-specific siRNAs to knock-down (KD) their expression in vivo (2mg/kg, i.v.) and in vitro. To increase cell targeting specificity, we utilized a mannose-conjugated polymer to deliver siRNA preferentially into phagocytes in vivo.

Results: Kinetic studies of ER stress signaling pathways showed that ischemia triggered a significant upregulation of cleaved-ATF6 and it was sustained throughout the reperfusion period; while increases of ATF4 and spliced XBP1 levels were only detected late during reperfusion. Functionally, in vivo KD of ATF6 in macrophages resulted in significant liver protections from IRI with much reduced sALT levels and better preserved liver architecture (H/E staining): sALT at 6h of reperfusion: ATF6siRNA 3684±353 vs. control siRNA 8165±341, or no siRNA 7250±599 (n=4/group, p<0.002). Additionally, liver pro-inflammatory immune responses against IR were significantly inhibited by ATF6 gene KD, as shown by lower intrahepatic TNF-a, IL-1b, IL-6 and CXCL10 gene expression levels in ATF6siRNA injected mice than those in controls, while the IL-10 gene expression was enhanced in the same animals. In vitro, ATF6 KD in macrophages resulted in a significant disruption of the synergy between ER stress response and TLR4 activation: pro-inflammatory cytokine productions in response to LPS and Thapsigargin, including TNF-a, IL-6, IL-12 and IL-23, were reduced, while anti-inflammatory cytokine IL-10 production was increased, in gene KD cells as compared with those in controls.

Conclusion: This study documented for the first time a key role of ATF6 activation in the pathogenesis of liver IRI, by its synergy with TLR-4 to facilitate pro-inflammatory immune response against IR.

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