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 involved in cellular senescence, inflammation, and stress resistance. We previously reported that myeloid SIRT1 signaling 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 signaling 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 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 signaling. Hepatocyte SIRT1 deficiency upregulated apoptosis and GSDME-mediated programmed cell death, which in turn deteriorated the hepatocellular function and shortened OLT survival. Augmented GSDME processing, accompanied by increased secretion of IL18 by stressed hepatocytes, was prominent in SIRT1-deficient, cold-stored livers. Hepatocyte SIRT1 signaling regulated anti-apoptotic Bcl-2/XIAP proteins, suppressed cold stress-triggered apoptosis, and mitigated GSDME licensing to release IL18. Notably, while crosslinking IL18R depressed SIRT1 and Bcl-2/XIAP signaling 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 signaling circuit as a therapeutic target in the mechanism underpinning hepatocyte death in human and mouse liver transplantation.

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

(A) Human liver biopsies (Bx; n=60) were collected at the back-table (after cold-storage) and 2h 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<0.001. (C) The correlation between SIRT1 and Bcl-2/cCasp3 in pre-transplant (left panel) and post-transplant (right panel) liver Bx was analyzed by nonparametric Spearmansmethod. r, Spearmans correlation coefficient. (D) Representative Western blots from SIRT1-low and SIRT1-high liver Bx with β-actin normalization. (E) Human OLT Bx samples collected 2h after reperfusion (n=60) were divided into (F) low (n=30) and high (n=30) SIRT1 expression groups, based on the relative SIRT1/β-actin levels. (G) Serum ALT levels in OLT recipients. ^#P<0.05 (Mann-Whitney U test) (H) The cumulative probability of overall graft survival (Kaplan-Meier method). Solid line: high-SIRT1 group; dotted line: low-SIRT1 group (log-rank test).

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

(A) Groups of FLOX control and hSIRT1KO livers, stored in UW solution (18h/4°C), were transplanted into WT mice, followed by OLT sampling at 6h. A separate OLT group was monitored for survival. (B) Representative OLT staining (H&E; original magnification ×100; scale bar: 200μm). (C) sAST, sALT levels and Suzukìs score of liver IRI. (D) The cumulative OLT survival (Kaplan-Meier method). Dotted line: FLOX>WT; solid line: hSIRT1KO>WT (n=9/group). (E) Western-assisted detection of SIRT1, Bcl-2, XIAP, Pro-Casp3, cCasp3, GSDME-FL, GSDME-N, Pro-IL18 and β-Actin in OLT at 6h. The red arrow indicates IL18. (F) The relative intensity ratios of Bcl-2, XIAP, cCasp3/Pro-Casp-3, GSDME-N/GSDME-FL and Pro-IL18 normalized with β-Actin. (G) Serum IL18 levels. (H) qRT-PCR-assisted detection of mRNA coding for IL10, IL4, IL13, TNFα and IL1β. Data were normalized to HPRT (n=5–6/group). Data shown are mean±SEM. *p<0.05; **p<0.01 ; ****p<0.0001 by Student`s t-test.

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

WT livers stored in UW solution (18h/4C) were transplanted into syngeneic hosts. Hepatic samples were collected after cold-storage (pre-transplant) and post-transplantation. (A)WB-assisted detection of SIRT1, XIAP, Bcl-2, cCasp8, cCasp3, GSDME-FL, GSDME-N, Pro-Casp-1, cCasp1-p20, Pro-IL1β and Pro-IL18 and VCL. (B) Kinetics of relative intensity for 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).

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

(A) Groups of FLOX and hSIRT1KO livers cold-stored in UW solution were perfused with physiological saline (0.5ml) 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, GSDME-N in the liver flush (right panel).

Figure 5.. Disruption of hepatocyte SIRT1 signaling depresses the anti-apoptotic gene program and promotes GSDME processing under cold stress in vitro:

(A) Primary mouse hepatocyte cultures conditioned with SIRT1 vs. control siRNA were subjected to cold stimulation. (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, 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; ***p<0.001 by Student`s t-test. (F) Composite images of phase contrast and immunofluorescence propidium iodide staining of primary mouse hepatocytes conditioned with control (upper panels) or SIRT1 (lower panels) siRNAs under cold stress (3, 6h), followed by TNFα stimulation (4, 6h; 25ng/mL). Original magnification ×200; scale bar; 100μM.

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

(A-B) Primary mouse hepatocytes pretreated with control or SIRT1 siRNA were subjected to cold stress. Some SIRT1-silenced cells were pretreated with a pan-caspase inhibitor zVAD-FMK (20nM, 18h) 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 pretreated with SIRT1 or/and Bcl2 or/and XIAP siRNA were subjected to cold stress. 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; ***p<0.001;****p<0.0001 by one-way ANOVA.

Figure 7.. IL18 signaling suppresses hepatocyte SIRT1 and anti-apoptotic axis:

Primary hepatocytes pretreated with control or IL18Rβ siRNA were subjected to cold stress. (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 conditioned with control or SIRT1 and/or IL18Rβ siRNA were subjected to cold stress. 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; ****p<0.0001 by one-way ANOVA.

Figure 8.. IL18 neutralization prevents liver IR damage and promotes anti-apoptotic phenotype in SIRT1-deficient murine OLT:

Groups of FLOX control and hSIRT1KO livers, stored in UW solution (18h/4°C), were transplanted into WT mice that remained untreated or pretreated with anti-IL18 Ab (0.5mg i.p. at −1 day), followed by sampling at 6h. (A) Representative H&E staining (original magnification ×100; scale bar 200μm); (B) Suzukìs score of liver IRI; frequency of TUNEL+ cells/HPF (original magnification ×200; scale bar 100μm); and sALT levels. (C) Western-assisted detection of SIRT1, XIAP, Bcl-2, HMGB1 and β-actin in OLTs. (D) The relative intensity ratios of XIAP, Bcl-2 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.