Bile acid-induced IRF3 phosphorylation mediates cell death, inflammatory responses, and fibrosis in cholestasis-induced liver and kidney injury via regulation of ZBP1

Abstract

Background and aims: Cell death and inflammation play critical roles in chronic tissue damage caused by cholestatic liver injury leading to fibrosis and cirrhosis. Liver cirrhosis is often associated with kidney damage, which is a severe complication with poor prognosis. Interferon regulatory factor 3 (IRF3) is known to regulate apoptosis and inflammation, but its role in cholestasis remains obscure. In this study.

Approach and results: We discovered increased IRF3 phosphorylation in the liver of patients with primary biliary cholangitis and primary sclerosing cholangitis. In the bile duct ligation model of obstructive cholestasis in mice, we found that tissue damage was associated with increased phosphorylated IRF3 (p-IRF3) in the liver and kidney. IRF3 knockout ( Irf3-/- ) mice showed significantly attenuated liver and kidney damage and fibrosis compared to wide-type mice after bile duct ligation. Cell-death pathways, including apoptosis, necroptosis, and pyroptosis, inflammasome activation, and inflammatory responses were significantly attenuated in Irf3-/- mice. Mechanistically, we show that bile acids induced p-IRF3 in vitro in hepatocytes. In vivo , activated IRF3 positively correlated with increased expression of its target gene, Z-DNA-Binding Protein-1 (ZBP1), in the liver and kidney. Importantly, we also found increased ZBP1 in the liver of patients with primary biliary cholangitis and primary sclerosing cholangitis. We discovered that ZBP1 interacted with receptor interacting protein 1 (RIP1), RIP3, and NLRP3, thereby revealing its potential role in the regulation of cell-death and inflammation pathways. In conclusion.

Conclusions: Our data indicate that bile acid-induced p-IRF3 and the IRF3-ZBP1 axis play a central role in the pathogenesis of cholestatic liver and kidney injury.

Figure 1:. Increased IRF3 phosphorylation in the cholestatic liver and in bile acids treated hepatocytes and nonparenchymal cells.

(A) Liver lysates were probed for p-IRF and IRF3 from healthy controls and patients with PBC and PSC (n=4–5). The bottom panels indicate quantification. (B) H&E and Sirius red staining were performed on the liver sections of sham-or BDL-operated mice at the indicated time (n=4–6). Representative sections are shown (scale bar=100μm). (C-D) Liver lysates were probed for p-IRF and IRF3 from sham-or BDL-operated mice at day 14 (C) and day 28 (D) (n=4–8). The right panels indicate quantification. (E-F) Cell lysates were probed for p-IRF and IRF3 from primary human hepatocytes and human nonparenchymal cells (hNPC) (E), HepG2 and BMDM (F) treated with indicated bile acids for 1 hour (n=3–4). Right panels indicate quantification. (G) HepG2 and BMDM treated with GDCA for one hour as indicated concentration. *p<0.05, **p<0.01, ***p<0.001.

Figure 2:. Absence of IRF3 prevents BDL-induced liver injury and development of fibrosis.

(A-B) Bilirubin (A), ALT and AST (B) levels were measured in the serum of WT and Irf3^−/− mice at day 14 after Sham or BDL (n=3–8). (C-E) Liver section were stained with Sirius red (C) and Desmin (D) and α-SMA (E). Representative sections are shown (scale bar=100μm). The right panel indicate quantification. (F) Liver lysates were probed with α-SMA and Vimentin (n=3–6). Right panels indicate quantification. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3:. Inflammation and inflammasome activation are ameliorated in the liver of Irf3^−/− mice compared to WT mice after BDL.

(A-B) Serum levels of TGFβ (A) and MCP1, TNF, IL-6, IL-18 (B) were measured from WT and Irf3^−/− mice at day 14 after Sham or BDL (n=3–8). (C-D) Liver sections were stained with CD11b (C) and CD68 (D). Representative sections are shown (scale bar=100μm). The bottom panel indicates quantification. (E) Liver lysates were probed for NLPR3, Casp1 and IL-1β from healthy controls and patients with PBC and PSC (n=4–5). The bottom panels indicate quantification. (F) Liver lysates were probed for NLPR3, Casp1 and cleaved- IL-1β from WT and Irf3^−/− mice at day 14 after BDL (n=3–6). Bottom panels indicate quantification. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 4:. Irf3^−/− mice show attenuated apoptosis and necroptosis after BDL.

(A) Liver lysates were probed for GSDMD and N terminal-GSDMD from WT and Irf3^−/− mice at day 14 after BDL (n=3–6). (B) Serum levels of ASC were measured from WT and Irf3^−/− mice at day 14 after Sham or BDL (n=3–8). (C) Liver tissue homogenates from healthy controls and patients with PBC and PSC were assessed for expression of Casp8 and Casp3 (n=4–5). (D) Liver tissue homogenates from WT and Irf3^−/− mice at day 14 after BDL were assessed for expression of Casp8 and Casp3 (n=3–6). The right panels indicate quantification. (E) Liver tissue sections were stained with TUNEL. Representative sections are shown (scale bar=100μm). The right panel indicates quantification. (F) RIP3, p-MLKL and MLKL expression were assessed in the liver tissue from healthy controls and patients with PBC and PSC (n=4–5). (G) RIP3 and MLKL expression were assessed in the liver tissue from WT and Irf3^−/− mice (n=3–4). The right panels indicate quantification. (H) Liver section were stained with p-MLKL. Representative sections are shown (scale bar=50μm). The right panels indicate quantification. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 5:. Irf3^−/− mice display reduced severity of kidney injury and fibrosis.

(A) p-IRF3 and IRF3 expression was measured in the kidney tissue after 14 days of sham and BDL (n=4–6). The bottom panel indicates quantification (n=3). (B) Cell lysates were probed for p-IRF and IRF3 from HEK293T cells treated with indicated bile acids for 1hr. The bottom panel indicates quantification (n=3). (C) Serum levels of Creatinine, BUN and KIM-1 were measured from WT and Irf3^−/− mice at day 14 after Sham or BDL (n=3–8). (D) Kidney injury score at day 14 after Sham or BDL. (E) α-SMA and Vimentin expression were assessed in the kidney tissue (n=3–6). The bottom panels indicate quantification. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6:. Lack of IRF3 attenuates inflammation, inflammasome activation, apoptosis and necroptosis in the kidney after BDL.

(A) MCP1, IL-6 and IL-18 gene expression were quantified in the kidney tissue from WT and Irf3^−/− mice at day 14 after Sham or BDL (n=3–8). (B) Kidney sections were stained with CD11b (B) and CD68 (C) (n=3–4). Representative sections are shown (scale bar=100μm). The bottom panel indicates quantification. (D) Casp1, cleaved-IL-1b and NLRP3 expression were measured in the kidney tissue (n=3–6). The bottom panels indicate quantification. (E) Representative images of TUNEL staining (scale bar=100μm). Bottom panel indicates quantification. (F-G) Kidney lysates were probed with Casp8 and Casp3 (F), and RIP3 and MLKL (G) (n=3–6). The bottom panel indicates quantification. (H) Representative images of p-MLKL staining (scale bar=50μm). The bottom panel indicates quantification. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 7:. Increased ER stress induced by BDL is independent of IRF3.

(A) Serum levels of endotoxin were measured from WT and Irf3^−/− mice at day 14 after Sham or BDL (n=3–8). (B) STING expression were measured in the liver and kidney tissue (n=3–6). The right panels indicate quantification. (C) STING expression were measured in the livers of healthy controls and human patients with PBC and PSC (n=4–5). The bottom panel indicates quantification. (D) PERK expression were measured in the liver and kidney tissue (n=3–6). The bottom panels indicate quantification. (E) CHOP gene expression was quantified in the kidney tissue from WT and Irf3^−/− mice at day 14 after Sham or BDL (n=3–8). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 8:. IRF3 upregulates ZBP1 expression to promote cholestatic liver injury.

(A) IFNb, Ifit1, Ifit3 and ISG15 gene expression were quantified in the liver and kidney from WT and Irf3^−/− mice at day 14 after Sham or BDL (n=3–8). (B) IRF3 was immunoprecipitated using IRF3 antibody from liver of Sham- or BDL-operated mice, followed by immunoblotting of Ubiquitin (Ub), IgG as control. (C) ZBP1 was identified from publicly available RNA-seq and IRF3 CUT&RUN data (17) analyzed by DAVID. (D) ZBP1 expression was assessed in the liver and kidney from WT and Irf3^−/− mice at day 14 after Sham or BDL (n=3–8), and in the livers of healthy controls and human patients with PBC and PSC (n=4–5). The bottom panels indicate quantification. (E) ZBP1 was immunoprecipitated followed by immunoblotting of ZBP1, RIP3, NLRP3 and RIP1, IgG as control. (F) Primary hepatocytes were isolated from WT and Irf3^−/− mice, ZBP1 and IRF3 expression was analyzed (n=4). (G) Primary hepatocytes were treated with DCA, CDCA and LPS for 6hrs, and ZBP1 and IRF3 expression was analyzed (n=3). The bottom panel indicates quantification. (H) ZBP1 and IRF3 expression was analyzed in BMDM from WT and Irf3^−/− mice without or with LPS treatment for 6hrs (n=3). (I) LDH was measured in the supernatant of primary hepatocytes after DCA treatment for 6hrs. (J) Cell lysates of primary hepatocytes were assessed for expression of ZBP1, IRF3, and Casp3.