*Purpose: Hepatic ischemia-reperfusion injury (IRI) represents a major risk factor for early allograft dysfunction and rejection episodes in liver transplantation (LT). The impact of Ikaros, an established tumor suppressor of lymphoid cell lineage, in IR-stressed LT remains to be elucidated. This translational from bench-to-bedside study focused on the relevance of Ikaros signaling in IRI-LT.

*Methods: In the clinical arm, human liver Bx collected at 2h after reperfusion from LT patients (n=55) were analyzed by RT-PCR and WB. In the experimental arm, bone marrow-derived macrophages (BMM) from myeloid-specific Sirtuin-1 (SIRT1) knockout (mSIRT1-KO), wild type (WT) mice and CD11b-DTR mice with/without Ikaros silencing (siRNA) were cultured with LPS. In parallel, mSIRT1 KO, WT and CD11b-DTR mice reconstituted with BMM with/without Ikaros silencing were subjected to hepatic warm ischemia (90min) followed by reperfusion (6h).

*Results: In clinical LT Bx, Ikaros levels (RT-PCR) correlated with sALT (r=0.3018, p=0.0266) at POD1. Then, we employed macrophage cultures to assess Ikaros function in innate-immune activation. Ikaros silencing (siRNA) in BMM decreased CXCL-10/IL-1β/p-IκBα and enhanced SIRT-1 under LPS stimulation. In contrast, Ikaros overexpression in BMM cultures increased CXCL-10/IL-1β and decreased SIRT-1 levels. Moreover, SIRT-1 deficiency in BMM increased CXCL-10/p-IκBα after LPS stimulation. In warm ischemia model, CD11b-depleted mice were lacking hepatic Ikaros after IR-stress. However, BMM reconstitution restored IR-induced Ikaros levels, implying recruited macrophages instrumental for hepatic Ikaros expression. Interestingly, reconstitution of CD11b-deficient mice with Ikaros-silenced BMM ameliorated hepatic IRI (n=5), evidenced by: 1/ decreased sALT/AST levels (p<0.05); 2/ reduced Suzuki's histological grading/frequency of TUNEL+ cells; 3/ decreased Ikaros but increased SIRT-1; 4/ suppressed IL-1β/CXCL-10/CXCL-2; and 5/ decreased CD11b+/Ly6G+ cells in peripheral blood. mSIRT-1 KO mice exacerbated hepatic IRI (n=7, p<0.05). In clinical LT Bx, Ikaros expression positively correlated with CXCL-10 (r=0.4420, p<0.001), TLR4 (r=0.3976, p<0.001), CD68 (r=0.2949, p=0.0289) and Cathepsin G (r=0.8193, p<0.0001), while negatively correlating with SIRT-1 (r=-0.3570, p=0.0075)

*Conclusions: This translational study documents Ikaros-mediated sterile inflammatory response in LT patients, and documents immune-regulatory function of Ikaros in macrophage activation. Thus, by negatively regulating SIRT1, Ikaros is essential in innate immune-driven hepatic IRI; and may represent a novel therapeutic target in LT recipients.

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