Age-dependent loss of hepatic SIRT1 enhances NLRP3 inflammasome signaling and impairs capacity for liver fibrosis resolution

Abstract

Our studies indicate that the longevity factor SIRT1 is implicated in metabolic disease; however, whether and how hepatocyte-specific SIRT1 signaling is involved in liver fibrosis remains undefined. We characterized a functional link of age-mediated defects in SIRT1 to the NLRP3 inflammasome during age-related liver fibrosis. In multiple experimental murine models of liver fibrosis, we compared the development of liver fibrosis in young and old mice, as well as in liver-specific SIRT1 knockout (SIRT1 LKO) mice and wild-type (WT) mice. Liver injury, fibrosis, and inflammation were assessed histologically and quantified by real-time PCR analysis. In a model of hepatotoxin-induced liver fibrosis, old mice displayed more severe and persistent liver fibrosis than young mice during liver injury and after injury cessation, as characterized by inhibition of SIRT1, induction of NLRP3, infiltration of macrophages and neutrophils, activation of hepatic stellate cells (HSCs), and excessive deposition and remodeling of the extracellular matrix. Mechanistically, deletion of SIRT1 in hepatocytes resulted in NLRP3 and IL-1β induction, pro-inflammatory response, and severe liver fibrosis in young mice, mimicking the ability of aging to impair the resolution of established fibrosis. In an aging mouse model, chronic-plus-binge alcohol feeding-induced liver fibrosis was attenuated by treatment with MCC950, a selective NLRP3 inhibitor. NLRP3 inhibition ameliorated alcoholic liver fibrosis in old mice by repressing inflammation and reducing hepatocyte-derived danger signaling-ASK1 and HMGB1. In conclusion, age-dependent SIRT1 defects lead to NLRP3 activation and inflammation, which in turn impairs the capacity to resolve fibrosis during aging.

Keywords: MCC950; NLRP3 inflammasome; aging; alcohol-associated liver disease; hepatic stellate cells (HSCs); hepatocyte-specific SIRT1 knockout; liver fibrosis.

FIGURE 1

Old mice are susceptible to liver fibrosis in an experimental model of CCl₄‐induced liver injury. (a) Schematic illustration for a mouse model of CCl₄‐induced liver fibrosis, which was used to evaluate the reparative response to liver injury in young and old mice. Mice at 4–6 months of age (young) and mice at 16–20 months of age (old) were subjected to injection with either olive oil or CCl₄ at a dose of 0.5 μL/g body weight twice weekly for 4 weeks. (b) Histopathologic characteristics of hepatocellular necrosis (green outline) and liver inflammation (green arrows) in young and old mice injected with either olive oil or CCl₄ were assessed by H&E staining. Notably, necrotic hepatocytes with cell swelling, plasma membrane rupture, or loss of the nucleus were seen in CCl₄‐injected young mice. Extensive necrosis involving larger numbers of necrotic hepatocytes was noted in CCl₄‐injected old mice. (c) Plasma ALT levels were measured. (d) Assessment of changes in body weight in mice. The change in body weight was expressed as a percentage change between body weight measurements of the mice from the initial and the final day of the experiment. (e) Representative images of Sirius Red staining (collagen is shown as red) revealed the fibrotic structural characteristics in the livers of young and old mice. Notably, collagen fibers were organized in bundles of various thicknesses. Thinner collagen fibrils were seen in young mice, whereas thicker collagen fibers were primarily located in immune cell‐abundant areas of old mice. (f) Positive areas of Sirius Red staining were calculated from five or six random fields of liver sections in each mouse using NIH Image J software. The bar graph represents the percentage of positively stained areas relative to the total tissue areas of liver sections. (g) Immunohistochemistry staining for collagen I revealed a similar pattern of collagen I distribution around the periportal regions in young and old mice, but the intensity of collagen I‐positive staining was increased in old mice in the CCl₄ model. (h) Hepatic mRNA levels of Col1a1 in mice were analyzed using quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR), normalized to those of GAPDH, and presented as relative levels to oil‐injected young mice. (i) qRT‐PCR analysis of mRNA levels of SIRT1. (j, k) Hepatic SIRT1 levels were negatively correlated with Sirius Red positive areas and Col1a1 levels in young and old mice injected with CCl₄. (l) Representative immunohistochemistry for α‐SMA, a marker for activated HSCs. (m) Real‐time PCR analysis of mRNA levels of α‐SMA. (n) Representative immunohistochemistry for TGF‐β1. Notably, positive staining for TGF‐β1 was visualized in hepatocytes (red arrows) and other cell types (green arrows) in fibrotic livers. (o) Real‐time PCR analysis showed that the induction of LOX was much higher in old mice than that in young mice after repeated liver injury. All images were acquired using 10×, 20×, or 40× objectives. The data were expressed as means ± SEM, n = 7–8. *p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001 between two groups.

FIGURE 2

Deletion of SIRT1 in hepatocytes increases the susceptibility of young mice to CCl₄‐induced liver fibrosis. (a) Wild‐type (WT) and SIRT1 LKO mice at 3–5 months of age were subjected to injection with either olive oil or CCl₄ at a dose of 0.5 μL/g body weight twice weekly for 6 weeks. (b) Representative immunohistochemistry for collagen I. A similar pattern for collagen I positive staining was present primarily around the periportal regions in WT mice in the CCl₄ model, but higher collagen I deposition was noted in SIRT1 LKO mice. (c) Representative immunohistochemistry for α‐SMA. Intense staining for α‐SMA was detected in fibrotic areas of WT mice; the distribution and intensity of positive staining for α‐SMA were higher in SIRT1 LKO mice. (d) Representative immunohistochemistry for TGF‐β1. Immunostaining signals for TGF‐β1 were located primarily in hepatocytes (red arrows) and other cell types (green arrows). (e, f) Real‐time PCR analysis of mRNA levels of TGF‐β1 and MMP‐12. All images were acquired using 10×, 20×, or 40× objectives. The data were expressed as means ± SEM, n = 6–8. *p < 0.05, **p < 0.01, or ***p < 0.001 between two groups.

FIGURE 3

Persistent liver fibrosis in old mice after cessation of liver injury is associated with inflammation induction. (a) Schematic illustration of the time course of both liver fibrosis and resolution of established fibrosis in old mice in the CCl₄ model. (b) Assessment of plasma ALT levels. (c) Representative H&E staining of liver sections in oil‐injected old mice (Oil), in old mice 24 h post‐injection (CCl₄), and in old mice 5 days after the last CCl₄ injection (CCl₄ + Recovery). (d, e) Sirius Red staining and quantification showed that aging increased the susceptibility of mice to CCl₄‐induced liver fibrosis even after cessation of liver injury. (f) Real‐time PCR analysis showed that mRNA levels of Col1a1 and α‐SMA were increased by CCl₄ injections, which were not significantly altered upon cessation of CCl₄ administration. (g) Immunohistochemistry for collagen I showed a similar distribution and intensity for collagen deposition in old mice between 24 h post‐injection and 5 days after cessation of injury. (h) Immunohistochemistry for α‐SMA showed that elevated α‐SMA⁺ HSCs were primarily distributed around portal areas and fibrotic bands in old mice during liver injury and after cessation of injury. (i) Real‐time PCR analysis of hepatic expression of SIRT1. (j) Immunohistochemistry revealed positive staining for TGF‐β1 in hepatocytes (red arrows) and other cells (green arrows) in old mice. (k) Real‐time PCR analysis of ECM regulators including MMP‐12, TIMP1, and LOX. (l) Immunohistochemistry for F4/80⁺ macrophages and MPO⁺ neutrophils. (m) Real‐time PCR analysis showed that the expression of IL‐1β and MCP‐1 was increased in old mice that underwent CCl₄ injections and persisted upon cessation of CCl₄ administration. (n) Immunohistochemistry for collagen I and MPO. (o) Real‐time PCR analysis of fibrosis markers, including Col1a1, a‐SMA, MMP‐9, MMP‐12, TIMP‐1, and LOX. (p) Real‐time PCR analysis showed that upon cessation of CCl₄ administration, expression of inflammation regulators, including TNF‐α and MCP‐1, were upregulated in old mice. All images were acquired using 20× or 40× objectives. The data were expressed as means ± SEM, n = 6–8. *p < 0.05, **p < 0.01, or ***p < 0.001, between two groups.

FIGURE 4

SIRT1 deficiency in hepatocytes enhances NLRP3 signaling and mimics the effect of aging on NLRP3‐mediated inflammation. (a) Immunohistochemistry showed that cells positively stained for NLRP3 were mainly located in damaged hepatocytes with nuclear degradation (red arrows) and inflammatory cells (green arrows) in young WT mice after CCl₄ administration, and this elevation was further enhanced in young SIRT1 LKO mice. (b, c) Real‐time PCR analysis showed that mRNA levels of NLRP3 and IL‐1β were increased in WT mice, and this induction was further potentiated in SIRT1 LKO mice. (d) Real‐time PCR analysis showed that mRNA levels of NLRP3 were significantly increased in young and old mice after CCl₄ administration, and the induction of NLRP3 was higher in older mice than in younger mice. (e) Immunohistochemistry showed that cells positively stained for NLRP3 were visualized in damaged hepatocytes with nuclear degradation (red arrows) and inflammatory cells (green arrows). (f, g) Immunohistochemistry for F4/80, a macrophage marker, and MPO, a neutrophil marker. (h) Real‐time PCR analysis for mRNA levels of MCP‐1. (i) Immunohistochemistry revealed positive staining for ASK1 in damaged hepatocytes (red arrows). All images were acquired using 20×, or 40× objectives. The data were expressed as means ± SEM, n = 6–8. *p < 0.05 and **p < 0.01 or ***p < 0.001 between two groups.

FIGURE 5

NLRP3‐mediated inflammation contributes to the pathogenesis of age‐associated persistent fibrosis even after cessation of liver injury. (a) Schematic illustration of two distinct phases of liver injury and cessation of injury in young and old mice in the CCl₄ mouse model. (b, c) Sirius Red staining and quantification showed that after the recovery period, fibrosis regression occurred in young mice, whereas severe fibrosis around the periportal regions was still seen in old mice. (d) Immunohistochemistry showed that the number of α‐SMA⁺ HSCs was significantly higher in older mice during liver injury. After injury recovery, clearance in α‐SMA⁺ HSCs was evident in young mice, whereas accumulation of α‐SMA⁺ HSCs was sustained in older mice. (e) Real‐time PCR analysis showed the induction of α‐SMA expression caused by CCl₄ administration was eliminated in young mice after the injury recovery, but the expression of α‐SMA was not significantly altered in old mice. (f) Immunohistochemistry for NLRP3 and MPO showed that after CCl₄ injection, NLRP3⁺ hepatocytes (red arrows) and NLRP3⁺other cell types (green arrows), as well as MPO⁺ neutrophils were higher in old mice than in young mice. After 5 days of recovery, these positively stained cells were reduced in young mice, but not in old mice. (g) Real‐time PCR analysis of NLRP3. (h) Immunohistochemistry showed that following CCl₄ injection, positively stained cells for ASK1 were detected in damaged hepatocytes with nuclear degradation (red arrows) in both young and old mice, but ASK1⁺ hepatocytes were higher in old mice. After the recovery period, ASK1⁺ hepatocytes were reduced in young mice, whereas a high number of ASK1⁺ hepatocytes were still present in old mice. All images were acquired using 10×, 20×, or 40× objectives. The data were expressed as means ± SEM, n = 6–8. *p < 0.05, or **p < 0.01 between two groups.

FIGURE 6

Inhibition of the NLRP3 inflammasome by MCC950 ameliorates age‐ and alcohol‐induced liver inflammation and fibrosis in mice. (a) Schematic illustration of an old mouse model of chronic‐plus‐binge alcohol feeding‐induced liver fibrosis and the design of MCC950 treatment (20 mg/kg/day), starting from the first day of the ethanol diet. (b) Real‐time PCR analysis showed that expression of NLRP3 and IL‐1β was markedly upregulated in old mice after chronic‐plus‐binge alcohol feeding but was significantly diminished by MCC950 treatment. (c) Immunohistochemistry for F4/80⁺ macrophages and MPO⁺ neutrophils. (d) Immunohistochemistry showed that positive staining for HMGB1 was primarily visualized in the nuclei of hepatocytes in control mice. Upregulation of HMGB1 and its translocation from the nucleus to the cytoplasm were observed in hepatocytes from old mice after chronic‐plus‐binge ethanol feeding. Positive staining for HMGB1 in both the cytoplasm and nucleus (red arrows) was detected in some hepatocytes, and nuclear export of HMGB1 (yellow arrows) was also found in other hepatocytes, indicating different stages of the cytoplasmic translocation of HMGB1. In particular, the cytoplasmic translocation of HMGB1 in hepatocytes was eliminated by MCC950 treatment. (e) Immunohistochemistry showed a high number of ASK1⁺ hepatocytes (red arrows) in old mice after chronic‐plus‐binge alcohol feeding, and this positive staining was reduced upon MCC950 treatment. (f) Assessment of plasma ALT levels. (g, h) Sirius Red staining and quantification. (i) Immunohistochemistry revealed excessive collagen I deposition in old mice after chronic‐plus‐binge alcohol feeding, and regression of liver fibrosis was observed in MCC950‐treated mice. (j) Real‐time PCR analysis for mRNA levels of pro‐fibrogenic genes, such as Col1a1 and α‐SMA. All images were acquired using 20× or 40× objectives. The data were expressed as means ± SEM, n = 6–8. *p < 0.05, **p < 0.01, or ***p < 0.001 between two groups.