SIRT1 regulates hepatocyte programmed cell death via GSDME - IL18 axis in human and mouse liver transplantation

Abstract

Sirtuin 1 (SIRT1) is a histone/protein deacetylase in the cellular response to inflammatory, metabolic, and oxidative stressors. We previously reported that myeloid SIRT1 regulates the inflamed liver's canonical pyroptosis cell death pathway. However, whether/how hepatocyte SIRT1 is engaged in programmed cell death in the cold-stressed liver remains uncertain. Here, we undertook translational studies in human and mouse orthotopic liver transplantation (OLT) to interrogate the significance of hepatocyte-specific SIRT1 in cold-stored donor livers and liver grafts after reperfusion. In the clinical arm of sixty human OLT patients, hepatic SIRT1 levels in cold-preserved donor livers correlated with the anti-apoptotic Bcl-2 expression. After reperfusion, improved OLT function was accompanied by hepatic SIRT1 levels negatively associated with cleaved caspase-3 expression. In the experimental arm, we compared FLOX-control with hepatocyte-specific SIRT1-KO livers after orthotopic transplantation into WT mouse recipients, parallel with primary murine hepatocyte cultures subjected to cold activation with/without knockdown of SIRT1, GSDME, and IL18Rβ. Indeed, hepatocyte SIRT1 deficiency upregulated apoptosis and GSDME-mediated programmed cell death, deteriorating hepatocellular function and shortening OLT survival. Augmented GSDME processing, accompanied by increased secretion of IL18 by stressed hepatocytes, was prominent in SIRT1-deficient, cold-stored livers. Hepatocyte SIRT1 expression regulated anti-apoptotic Bcl-2/XIAP proteins, suppressed cold stress-triggered apoptosis, and mitigated GSDME licensing to release IL18. Notably, consistent with the ability of IL18 to depress hepatocyte SIRT1 and Bcl-2/XIAP in vitro, IL18 neutralization in vivo prevented hepatocellular damage and restored the anti-apoptotic phenotype in otherwise injury-prone SIRT1-deficient OLTs. In conclusion, this translational study identifies a novel hepatocyte SIRT1-IL18 molecular circuit as a therapeutic target in the mechanism underpinning hepatocyte death pathways in human and mouse liver transplantation.

Fig. 1. Hepatocyte SIRT1 regulates apoptosis and hepatocellular function in clinical OLT.

A Human liver biopsies (Bx; n = 60) were collected at the back-table before transplantation (after cold storage) and after liver transplantation at 2 h post-reperfusion (before abdominal closure). B WB-assisted detection of hepatic SIRT1, Bcl-2, and cCasp3 with β-actin normalization. Data are shown in bars indicative of mean ± SEM. Statistical analyses with 2-tailed Man–Whitney U test. *p < 0.05; **p < 0.01. C The correlation between SIRT1 and Bcl-2/cCasp3 levels in pre-transplant (left panel) and post-transplant (right panel) liver Bx samples was analyzed by nonparametric Spearman’s method. r, Spearman’s correlation coefficient. D Human OLT Bx samples collected at 2 h after reperfusion were divided into (E) low (n = 30) and high (n = 30) SIRT1 expression groups, based on the relative WB-assisted expression of SIRT1/β-actin. F Serum ALT levels at POD 1-7 in OLT recipients. ^#P < 0.05 (Mann–Whitney U test) (G) The cumulative probability of overall OLT survival based on hepatic SIRT1 levels (Kaplan–Meier method). Solid line: high-SIRT1; dotted line: low-SIRT1 group (log-rank test).

Fig. 2. Hepatocyte SIRT1 deficiency exacerbates apoptosis and GSDME processing in mouse OLT.

A Livers from FLOX control and hSIRT1KO mice were stored in UW solution (18 h/4 °C) and transplanted into WT mice, followed by OLT sampling at 6 h post-reperfusion. A separate group of OLT recipients was monitored for survival. B Representative OLT staining (H&E; original magnification ×100; scale bar: 200 μm). C sAST, sALT levels, and Suzuki’s histological score of liver IRI. D The cumulative OLT survival (Kaplan-Meier method). Dotted line: FLOX > WT; solid line: hSIRT1KO>WT (n = 9/group). E WB-assisted detection of SIRT1, Bcl-2, XIAP, Pro-Casp3, cCasp3, GSDME-FL, GSDME-N, Pro-IL18, and β-Actin in OLT. F The relative intensity ratios of Bcl-2, XIAP, cCasp3/Pro-Casp-3, GSDME-N/GSDME-FL, and Pro-IL18 normalized with β-Actin in OLT. G ELISA-assisted detection of serum IL18 levels. H qRT-PCR-assisted detection of mRNA coding for IL10, IL4, IL13, TNFα, and IL1β in OLT. Data were normalized to HPRT (n = 5-6/group). Data shown are mean ± SEM. *p < 0.05; **p < 0.01 by Student’s t-test.

Fig. 3. Cold preservation of mouse livers triggers GSDME processing in stressed hepatocytes.

WT livers stored in UW solution (18 h/4 °C) were transplanted into syngeneic hosts. Hepatic samples were collected after cold storage (pre-transplant; 0 h) and post-transplantation (1 h, 3 h). A WB-assisted detection of hepatic SIRT1, XIAP, Bcl-2, cCasp8, cCasp3, GSDME-FL, GSDME-N, Pro-Casp-1, cCasp1-p20, Pro-IL1β, Pro-IL18, and VCL. B Kinetics of relative intensity for hepatic XIAP, Bcl-2, cCasp8, cCasp3, GSDME-N, and cCasp1-p20 normalized with VCL. Data shown are mean ± SEM. *p < 0.05; **p < 0.01 by Student’s t-test. C The relative intensity in Pro-IL18 (solid line), and Pro-IL1β (dotted line) in mouse livers.

Fig. 4. Hepatocyte SIRT1 suppresses apoptosis and GSDME-mediated PCD in cold-stored mouse livers.

A Groups of FLOX and hSIRT1KO livers cold-stored in UW solution were perfused with physiological saline (0.5 ml) via a portal vein-cuff to collect liver flush from supra-hepatic inferior vena cava (n = 3/group). B WB-assisted detection of SIRT1, Bcl-2, XIAP, Pro-Casp3, cCasp3, GSDME-FL, GSDME-N, Pro-IL18, IL18, and β-actin in cold-stored livers (left panel). Some targets were evaluated in the liver flush by WB (right panel). C qRT-PCR assisted detection of mRNA coding for Bcl-2, Mcl1, XIAP, TNFα, IL10, NLRP3, IL1β, and IL18. D The relative intensity of Bcl-2, XIAP, GSDME-F, and GSDME-N normalized with β-actin in cold-stressed liver (left panel). The relative intensity of IL18, cCasp3, and GSDME-N in the liver flush (right panel). Data shown are mean ± SEM. *p < 0.05 by Student’s t-test.

Fig. 5. Hepatocyte SIRT1 deficiency depresses the anti-apoptotic gene program and promotes GSDME processing under cold stress in vitro.

A Primary mouse hepatocytes transfected with SIRT1 siRNA vs. control siRNA were subjected to cold stimulation (0–6 h). B qRT-PCR assisted detection of mRNA coding for SIRT1, Bcl-2, and XIAP. Green dots: siControl; purple dots; siSIRT1. C WB-assisted detection of SIRT1, Bcl-2, Pro-Casp3, cCasp3, GSDME-FL, GSDME-N, Pro-IL18, IL18, and β-actin in hepatocyte lysates (upper panels). The relative intensity ratios of Bcl-2, cCasp3/Pro-Casp3, and GSDME-N/GSDME-FL (lower panels). Green line: siControl; purple line: siSIRT1. D WB-assisted detection of Pro-Casp3, cCasp3. GSDME-FL, GSDME-N, Pro-IL18, and IL18 in the culture medium (upper panels). Ponceau S staining is shown as a loading control. The relative intensity ratios of cCasp3 and GSDME-N (lower panels). Green line: siControl; purple line: siSIRT1. E ELISA-assisted IL18 levels in cold-stressed hepatocytes. Data shown are mean ± SEM. *p < 0.05; **p < 0.01 by Student’s t-test. F Composite images of phase contrast and immunofluorescence propidium iodide staining of primary mouse hepatocytes transfected with control (upper panels) or SIRT1 (lower panels) siRNAs under cold stress (3 h, 6 h), followed by TNFα stimulation (4 h or 6 h; 25 ng/mL). Original magnification ×200; scale bar; 100 μM.

Fig. 6. SIRT1 regulates cold stress-induced apoptosis and GSDME processing to release IL18 in vitro.

A, B Primary mouse hepatocytes transfected with control or SIRT1 siRNA were subjected to cold stress (6 h). Some SIRT1-silenced cells were pretreated with a pan-caspase inhibitor zVAD-FMK (20 nM, 18 h) or transfected with GSDME siRNA. A WB-assisted detection of SIRT1, Bcl-2, XIAP, Pro-Casp3, and β-actin in lysates (left panels). qRT-PCR-assisted detection of mRNA coding for SIRT1 and GSDME (right upper panels). The relative intensity of SIRT1, Bcl-2, and XIAP in lysates (right lower panels). B cCasp3, GSDME-N, and IL18 in supernatants (left panels). The relative intensity of cCasp3/Pro-Casp3 ratio, GSDME-N, and IL18 in supernatants (right panels). C Primary mouse hepatocytes transfected with SIRT1 siRNA or/and Bcl-2 siRNA or/and XIAP siRNA were subjected to cold stress (6 h). WB-assisted detection of SIRT1, Bcl-2, XIAP, Pro-Casp3, and β-Actin in hepatocyte lysates (left upper panels) and cCasp3 in supernatants (left lower panels). The relative intensity of SIRT1, Bcl-2, XIAP, and cCasp3/Pro-Casp3 ratio (right panels). Data shown are mean ± SEM. *p < 0.05; **p < 0.01 by one-way ANOVA.

Fig. 7. IL18 suppresses hepatocyte SIRT1 and anti-apoptotic axis.

Primary hepatocytes transfected with control or IL18Rβ siRNA were subjected to cold stress (0–6 h). A WB-assisted detection of IL18Rβ, SIRT1, Bcl-2, XIAP, and β-actin (upper panels). The relative ratio of SIRT1, Bcl-2, and XIAP (lower panels). B qRT-PCR-assisted detection of mRNA coding for IL18Rβ, Bcl-2, and XIAP. C Primary hepatocytes transfected with control siRNA or SIRT1 siRNA and/or IL18Rβ siRNA were subjected to cold stress (6 h). WB-assisted detection of SIRT1, IL18Rβ, Bcl-2, XIAP, and β-Actin (upper panels). The relative intensity of SIRT1, IL18Rβ, Bcl-2, and XIAP (lower panels). Data shown are mean ± SEM. *p < 0.05; **p < 0.01 by one-way ANOVA.

Fig. 8. IL18 neutralization prevents liver IRI and promotes anti-apoptotic phenotype in SIRT1-deficient murine OLT.

Groups of FLOX control and hSIRT1KO livers, stored in UW solution (18 h/4 °C), were transplanted into WT mice that remained untreated or pretreated with anti-IL18 Ab (0.5 mg i.p. at -1 day), followed by sampling at 6 h post-OLT. A Representative H&E staining (original magnification ×100; scale bar 200 μm); B Suzuki’s histological score of liver IRI; frequency of TUNEL+ cells/HPF (original magnification ×200; scale bar 100 μm); and sALT levels. C WB-assisted detection of SIRT1, XIAP, Bcl-2, HMGB1, and β-actin in OLTs. D The relative intensity ratios of Bcl-2, XIAP, and HMGB1 normalized with β-actin. Data shown are for n = 5-7/group (sALT/Suzuki’s score) and n = 3/group (TUNEL staining/WB); mean ± SEM. *p < 0.05; **p < 0.01 by one-way ANOVA. E Schematic illustration of proposed cold stress-triggered hepatocyte cell death programs. SIRT1 regulates the activation of caspase3, followed by GSDME activation to release IL18 via the Bcl-2/XIAP anti-apoptotic axis. The secreted IL18 further down-regulates SIRT1, Bcl-2, and XIAP.