Progression of chronic liver inflammation and fibrosis driven by activation of c-JUN signaling in Sirt6 mutant mice

Abstract

The human body has a remarkable ability to regulate inflammation, a biophysical response triggered by virus infection and tissue damage. Sirt6 is critical for metabolism and lifespan; however, its role in inflammation is unknown. Here we show that Sirt6-null (Sirt6(-/-)) mice developed chronic liver inflammation starting at ∼2 months of age, and all animals were affected by 7-8 months of age. Deletion of Sirt6 in T cells or myeloid-derived cells was sufficient to induce liver inflammation and fibrosis, albeit to a lesser degree than that in the global Sirt6(-/-) mice, suggesting that Sirt6 deficiency in the immune cells is the cause. Consistently, macrophages derived from the bone marrow of Sirt6(-/-) mice showed increased MCP-1, IL-6, and TNFα expression levels and were hypersensitive to LPS stimulation. Mechanistically, SIRT6 interacts with c-JUN and deacetylates histone H3 lysine 9 (H3K9) at the promoter of proinflammatory genes whose expression involves the c-JUN signaling pathway. Sirt6-deficient macrophages displayed hyperacetylation of H3K9 and increased occupancy of c-JUN in the promoter of these genes, leading to their elevated expression. These data suggest that Sirt6 plays an anti-inflammatory role in mice by inhibiting c-JUN-dependent expression of proinflammatory genes.

FIGURE 1.

Liver inflammation in Sirt6 mutant mice. A–E, gross morphological changes of Sirt6 mutant (MT) (A-D) and WT (E) liver. Liver foci (A) in a 5-month-old animal and nodules (B–D) in 9–15-month-old animals. F–H, hematoxylin and eosin staining showing necrotic foci surrounded by inflammatory cells (F) and massive inflammation (G) in MT and WT (H) liver sections. I and M, CD3 staining in MT (I) and WT (M) liver section. The green signals in M are nonspecific staining of red blood cells. J–L and N–P, F4/80, MPO, and IL-1β immunohistochemistry staining in Sirt6 MT (J–L) and WT (N–P) liver. Q, hepatic expression of proinflammatory genes. The error bars indicate standard error of the mean. *, p < 0.05. Scale bar, 100 μm (F); 50 μm (G–P). For A–P, at least five pairs of mice were analyzed. For Q, three pairs of 8–9-month-old mice were analyzed.

FIGURE 2.

Sirt6 loss results in liver fibrosis, elevated liver proliferation, and global decline of body condition. A and B, Sirius Red staining images in WT (A) and mutant (MT) (B) mice liver. Eight pairs of mice were analyzed. C and D, PCNA and CD3 staining in MT (C) and WT liver (D). The white arrow indicates PCNA positive only cell (most likely hepatocyte according to the morphology). The arrowhead indicates PCNA and CD3 double positive cell (leukocyte). The red arrows indicate nonspecific staining of the red blood cells. At least five pairs of mice were analyzed for C and D. Scale bars, A and B, left panel, 250 μm; right panel, 50 μm. C and D, 20 μm. E, body weight of WT and MT mice. F, percentage of liver weight (LW) versus body weight (BW) in three time points of WT and MT mice. The error bars indicate the standard error of the mean. *, p < 0.05. At least four pairs of mice were analyzed for E and F.

FIGURE 3.

Sirt6 loss activates immune cells leading to the liver inflammation. A, CD4⁺, CD8⁺, IFN-γ, and IL-17 secretion in CD4⁺ liver lymphocytes. B, IL-6 and TNFα secretion from Kupffer cells isolated from mouse liver. C, thymidine uptake of splenocytes under the stimulation of anti-CD3 alone and anti-CD3+CD28. MT, mutant.

FIGURE 4.

Activation of Sirt6 mutant macrophages. A and B, cytokines secreted from the macrophage cell lines with and without LPS stimulation. C–K, proinflammatory gene expression in the macrophage cell lines. E and F, levels of LPS-induced up-regulation of Il-6 and Mcp-1 mRNAs with or without actinomycin D. The error bars indicate standard error of the mean. *, p < 0.05. MT, mutant.

FIGURE 5.

SIRT6 interacts with c-JUN and binds to and epigenetically modifies H3K9 in the promoter of proinflammatory genes. Immortalized WT or MT macrophages were utilized for ChIP analysis. A and B, in silico analysis to show AP-1 or AP-1-related binding sites on IL-6 and MCP-1 promoters from both mouse and human genes. The numbers indicate the positions of the fragments relative to translational start codon (ATG), which were amplified by PCR during ChIP assay. C, ChIP assay showing enhanced Ac-H3K9 occupancy on Il-6 and Mcp-1 promoters upon Sirt6 deletion. D and E, ChIP assay showing Me-H3K4 occupancy on Il-6 and Mcp-1 promoters. F, ChIP assay indicates SIRT6 binds to Il-6 and Mcp1 promoters in wild type cells. The background binding levels in mutant cells were set at 1. G, immunoprecipitation assay displays that c-JUN and SIRT6 interact with each other. H, ChIP assay using c-JUN antibody demonstrates that upon Sirt6 deletion, the binding of c-JUN to Il-6 and Mcp-1 promoters was extremely enhanced. I, expression of Il-6 and Mcp-1 upon c-JUN knockdown in both WT and MT immortalized macrophages. The inset indicates Western blot of c-JUN upon c-JUN knockdown.