Background/Aim: Sirtuin1 (SIRT1), a NAD⁺-dependent type III histone/protein deacetylase, is essential for resistance against liver ischemia-reperfusion injury (IRI). We asked whether SIRT1 may affect cytoprotective functions of myeloid cell-specific heme oxygenase-1 (HO-1). Methods/Results: Tissue-specific HO-1 KO mice in myeloid cells were developed by breeding HO-1 floxed KO mice with myeloid-specific Cre Tg mice under the control of the LysM promoter. First, we used a model of hepatic IRI in which myeloid-specific HO-1 (Mø-HO-1) KO mice and WT mice (BL6; n=6/gr.) underwent warm ischemia (90min). Sera and liver samples were harvested at 6h of reperfusion. Ablation of myeloid HO-1 exacerbated IRI, as compared with controls and evidenced by liver enzyme levels (sALT: 25521±4468 vs. 10363±4299 IU/L, p<0.05; sAST: 16378±3239 vs. 10764±3238 IU/L, p<0.05); and Suzuki's score of histological liver IRI (7.1±1.9 vs. 4.0±0.8, p<0.05). RT-PCR analysis showed the higher (p<0.05) mRNA levels coding for proinflammatory IRI signature (IL-6, IL-1β, IFNγ, TNFα, MCP-1, IP-10, MIP-2, CXCL-1) in Mø-HO-1 KO livers; while the mRNA levels of cytoprotective IL-10/TGF-β were diminished (p<0.05). Western analysis of IR-livers revealed diminished expression of SIRT1 and autophagy-related LC3B and Beclin1 markers selectively in Mø-HO-1 KO group. Second, we contrasted LPS-stimulated bone marrow-derived macrophages (BMM) from Mø-HO-1 KO vs. WT mice. Indeed, consistent with our in vivo data, HO-1 deficiency repressed the expression of macrophage SIRT1 and autophagy-related proteins (LC3B, Beclin1, SQSTM1). Interestingly, addition of a chemical SIRT1 activator rescued autophagy pathway, evidenced by enhanced LC3B, Beclin1, SQSTM1 levels in Mø-HO-1 KO BMM cultures. Third, to validate our Mø-HO-1 KO in vivo/in vitro findings, we used gene silencing approach (siRNA) to disrupt HO-1 signaling in LPS-activated Raw 264.7 macrophage-like cell cultures. Strikingly, addition of HO-1 (but not scrambled) siRNA diminished the expression of SIRT1, LC3B, Beclin1 (Western blot) and decreased mRNA levels (qRT-PCR) of anti-inflammatory M2 phenotype markers, such as Mrc-1, Arg-1 and IL-10. Conclusion: Macrophage SIRT1 signaling is critical for HO-1 driven anti-inflammatory program, sufficient autophagy function, and M2 polarization in IR-stressed livers.

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