*Purpose: Hepatic ischemia-reperfusion injury (IRI) represents a risk factor for early graft dysfunction and rejection in liver transplantation (LT). Despite the efficacy of Sirtuin 1 (SIRT1) to alleviate hepatic IRI, the underling mechanisms of innate immune activation in LT remain to be elucidated. This study focused on the clinical relevance of SIRT1-mediated hepatoprotection and crosstalk with Ikaros, a tumor suppressor of lymphoid lineage, in IR-stressed LT recipients.

*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, HMGB1-enriched liver flush (LF) was collected from cold-stored WT mouse livers. Bone marrow-derived neutrophils/macrophages (BMM) from myeloid-specific SIRT1 knockout (mSIRT1-KO) and WT mice were cultured with LPS/LF/IL4. In parallel, mSIRT1-KO and WT mice were subjected to hepatic warm ischemia (90min) followed by reperfusion (6h).

*Results: In clinical LT Bx, SIRT1 levels (WB) negatively correlated with sALT (r=-0.469, p<0.001) at POD1. Compared with low-SIRT1 expression Bx (n=27), high-SIRT1 LT recipients (n=28) had decreased sALT (225±26 vs 460±64 U/L, p=0.004) at POD1, improved LT survival (3-yr: 90.4 vs 76.1%) and superior rejection-free survival (3-y: 85.3 vs 73.3%). Then, we employed macrophage/neutrophil cultures to assess SIRT1 function in innate-immune activation. Addition of LPS (TLR4-ligand) markedly increased Ikaros in BMM, while Ikaros silencing (siRNA) suppressed IRF4/Arg1/Ym1 (M2) and enhanced p-IκBα/p-Stat1/MCP1 (M1) under LPS/IL4/LF stimulation, implying Ikaros represents inherent modulator of TLR4-dependant immune activation. SIRT1 deficiency in BMM cultures decreased Ikaros/IRF4/Arg1/Ym1 and enhanced p-IκBα/p-Stat1/MCP1 whereas Ikaros mRNA transfection increased Ikaros/IRF4/Arg1/Ym1. SIRT1 deficiency in LPS/LF-stimulated neutrophils triggered increased mRNA levels coding for TNFa/CXCL1/CXCR2 in vitro. Indeed, mSIRT1 in vivo deficiency exacerbated hepatic IRI (n=7-9) evidenced by: 1/ sALT/AST levels; 2/ Suzuki's histological grading and frequency of TUNEL+ cells; 3/ decreased Ikaros but increased p-IκBα; 4/ enhanced MCP1/CXCL10/TNFα but suppressed IRF4/Arg1/Ym1; and 5/ increased CD11b+/Ly6G+ cell infiltration (p<0.05). In clinical LT Bx, Ikaros expression positively correlated with IRF4 (r=0.691, p<0.001) while TLR4 positively correlated with Ikaros (r=0.495, p<0.001).

*Conclusions: This translational study documents SIRT1-mediated hepatoprotection in LT patients, and documents immunoregulatory function of SIRT1 in macrophage/neutrophil activation. Our data highlight a novel SIRT1/Ikaros/IRF4 signaling axis to regulate macrophage M2 shift and manage IR-stress, with implications for novel therapeutic strategies in LT recipients.

« Back to 2019 American Transplant Congress