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. 2018 Dec;155(6):1971-1984.e4.
doi: 10.1053/j.gastro.2018.09.010. Epub 2018 Sep 10.

Expression of STING Is Increased in Liver Tissues From Patients With NAFLD and Promotes Macrophage-Mediated Hepatic Inflammation and Fibrosis in Mice

Xianjun Luo  1 , Honggui Li  1 , Linqiang Ma  2 , Jing Zhou  1 , Xin Guo  1 , Shih-Lung Woo  1 , Ya Pei  1 , Linda R Knight  3 , Michael Deveau  3 , Yanming Chen  4 , Xiaoxian Qian  5 , Xiaoqiu Xiao  6 , Qifu Li  7 , Xiangbai Chen  8 , Yuqing Huo  9 , Kelly McDaniel  10 , Heather Francis  10 , Shannon Glaser  10 , Fanyin Meng  11 , Gianfranco Alpini  12 , Chaodong Wu  13
Affiliations

Expression of STING Is Increased in Liver Tissues From Patients With NAFLD and Promotes Macrophage-Mediated Hepatic Inflammation and Fibrosis in Mice

Xianjun Luo et al. Gastroenterology. 2018 Dec.

Abstract

Background & aims: Transmembrane protein 173 (TMEM173 or STING) signaling by macrophage activates the type I interferon-mediated innate immune response. The innate immune response contributes to hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). We investigated whether STING regulates diet-induced in hepatic steatosis, inflammation, and liver fibrosis in mice.

Methods: Mice with disruption of Tmem173 (STINGgt) on a C57BL/6J background, mice without disruption of this gene (controls), and mice with disruption of Tmem173 only in myeloid cells were fed a standard chow diet, a high-fat diet (HFD; 60% fat calories), or a methionine- and choline-deficient diet (MCD). Liver tissues were collected and analyzed by histology and immunohistochemistry. Bone marrow cells were isolated from mice, differentiated into macrophages, and incubated with 5,6-dimethylxanthenone-4-acetic acid (DMXAA; an activator of STING) or cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Macrophages or their media were applied to mouse hepatocytes or human hepatic stellate cells (LX2) cells, which were analyzed for cytokine expression, protein phosphorylation, and fat deposition (by oil red O staining after incubation with palmitate). We obtained liver tissues from patients with and without NAFLD and analyzed these by immunohistochemistry.

Results: Non-parenchymal cells of liver tissues from patients with NAFLD had higher levels of STING than cells of liver tissues from patients without NAFLD. STINGgt mice and mice with disruption only in myeloid cells developed less severe hepatic steatosis, inflammation, and/or fibrosis after the HFD or MCD than control mice. Levels of phosphorylated c-Jun N-terminal kinase and p65 and mRNAs encoding tumor necrosis factor and interleukins 1B and 6 (markers of inflammation) were significantly lower in liver tissues from STINGgt mice vs control mice after the HFD or MCD. Transplantation of bone marrow cells from control mice to STINGgt mice restored the severity of steatosis and inflammation after the HFD. Macrophages from control, but not STINGgt, mice increased markers of inflammation in response to lipopolysaccharide and cGAMP. Hepatocytes and stellate cells cocultured with STINGgt macrophages in the presence of DMXAA or incubated with the medium collected from these macrophages had decreased fat deposition and markers of inflammation compared with hepatocytes or stellate cells incubated with control macrophages.

Conclusions: Levels of STING were increased in liver tissues from patients with NAFLD and mice with HFD-induced steatosis. In mice, loss of STING from macrophages decreased the severity of liver fibrosis and the inflammatory response. STING might be a therapeutic target for NAFLD.

Keywords: Hepatic Stellate Cell; Interferon; Lipopolysaccharide; Nonalcoholic Steatohepatitis.

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Conflict of interest statement

CONFLICT OF INTEREST

This material, in part, is the result of work supported with resources and the use of facilities at the Central Texas Veterans Health Care System, Temple, Texas. The content is the responsibility of the author(s) alone and does not necessarily reflect the views or policies of the Department of Veterans Affairs or the United States Government.

The authors have nothing to declare.

Figures

Figure 1.
Figure 1.. STING expression is increased in livers of human patients with NAFLD
(A) Liver sections of NAFLD patients (bottom two rows) and human subjects without NAFLD (Control, top row) were stained with H&E (the very left column) and Trichrome (the second left column), and examined for STING expression (the right three columns). (B) Degrees of hepatic steatosis. (C) STING abundance in liver sections. AU, arbitrary unit. For bar graphs, data are means ± SD. n = 6 – 8. ***, P < 0.001 NAFLD vs. Control.
Figure 2.
Figure 2.. STING disruption protects against HFD-induced NAFLD
Male STING-disrupted (STINGgt) mice and WT mice, at 5 – 6 weeks of age, were fed an LFD or HFD for 12 weeks. (A) Plasma levels of alanine aminotransferase (ALT). (B) Liver weight. (C) Liver levels of triglycerides. (D) Liver sections were stained with H&E (left column) or for F4/80 expression (right two columns). Bar graph, percentages of macrophages. (E) Liver lysates were examined for inflammatory signaling using Western blot analysis. Bar graphs, quantification of blots. (F) Liver mRNA levels were examined using real-time RT-PCR. For all bar graphs, data are means ± SD. n = 10 – 12. ‡, P < 0.05 and ‡‡, P < 0.01 HFD-WT vs. LFD-WT in A - C, and E; *, P < 0.05 and **, P < 0.01 HFD-STINGgt vs. HFD-WT (in A - E) for the same gene (in F).
Figure 3.
Figure 3.. Myeloid cell-specific STING disruption decreases the severity of HFD-induced NAFLD
Male WT C57BL/6J mice, at 5 – 6 weeks of age, were lethally irradiated and transplanted with bone marrow cells from STINGgt and/or WT mice. After recovery for 4 weeks, the chimeric mice were fed an HFD for 12 weeks. (A) Plasma levels of ALT. (B) Liver weight. (C) Liver levels of triglycerides. (D) Liver sections were stained with H&E (top row) or for F4/80 expression (bottom two rows). Bar graph, percentages of macrophages. (E) Liver lysates were examined for proinflammatory signaling using Western blot analysis. Bar graphs, quantification of blots. (F) Liver mRNA levels were examined using real-time RT-PCR. For A - F, WT/BMT-STINGgt, WT mice received STINGgt bone marrow cells; WT/BMT-WT, WT mice received WT bone marrow cells. For all bar graphs, data are means ± SD. n = 8 – 10. *, P < 0.05 and **, P < 0.01 WT/BMT-STINGgt vs. WT/BMT-WT (in A, and C- E) for the same gene (in F).
Figure 4.
Figure 4.. STING presence only in myeloid cells exacerbates HFD-induced NAFLD
Male STINGgt mice, at 5 – 6 weeks of age, were lethally irradiated and transplanted with bone marrow cells from WT and/or STINGgt mice. After recovery for 4 weeks, the chimeric mice were fed an HFD for 12 weeks. (A) Plasma levels of ALT. (B) Liver weight. (C) Liver levels of triglycerides. (D) Liver sections were stained with H&E (top row) or for F4/80 expression (bottom two rows). Bar graph, percentages of macrophages. (E) Liver lysates were examined for proinflammatory signaling using Western blot analysis. Bar graphs, quantification of blots. (F) Liver mRNA levels were examined using real-time RT-PCR. For A - F, STINGgt/BMT-WT, STINGgt mice received WT bone marrow cells; STINGgt/BMT-STINGgt, STINGgt mice received STINGgt bone marrow cells. For all bar graphs, data are means ± SD. n = 10 – 12. *, P < 0.05 and **, P < 0.01 STINGgt/BMT-WT vs. STINGgt/BMT-STINGgt (in C - E) for the same gene (in F).
Figure 5.
Figure 5.. STING enables macrophages to generate factors that promote hepatocyte fat deposition and proinflammatory responses
Macrophages and primary hepatocyte were prepared from male STINGgt mice and WT C57BL/6J mice as described in Methods. (A) Macrophage interferon beta (IFNβ) production. Bone marrow-derived macrophages (BMDM) were treated with DMXAA (75 µg/mL) or control (Ctrl, 7.5% NaHCO3) for 6 hr. BMDM-conditioned media were examined for IFNβ levels. (B) Macrophage proinflammatory signaling. BMDM were treated with or without DMXAA (75 µg/mL) for 24 hr in the absence or presence of LPS (100 ng/mL) for the last 30 min. (C) Hepatocyte fat deposition of co-cultures. Primary hepatocytes were incubated in the absence of macrophages or co-cultured with BMDM from WT or STINGgt mice for 48 hr, and treated with DMXAA (75 µg/mL) or control in the presence of palm itate (250 µM) for the last 24 hr. Prior to harvest, hepatocytes or co-cultures were stained with oil red O for 1 hr. Bar graph, quantification of fat content. (D, E, F) Macrophage factors regulation of hepatocyte proinflammatory responses and gene expression. Conditioned media (CM) were collected from DMXAA (DMX)-treated WT BMDM, DMX-treated STINGgt BMDM, and Ctrl-treated WT BMDM (described in B) and designated as DMX/WT BMDM-CM, DMX/STINGgt BMDM-CM, and Ctrl/WT BMDM-CM, respectively. CM were mixed with fresh media at a 1:1 ratio and supplemented to hepatocytes for 48 hr. Prior to harvest, CM-incubated hepatocytes were treated with or without LPS (100 ng/mL) for 30 min (D) or LPS (20 ng/mL) for 6 hr (E). For B and D, cell lysates were examined for proinflammatory signaling using Western blot analysis. Bar graphs, quantification of blots. For E and F, the mRNA levels were examined using real-time RT-PCR. For bar graphs in A - F, data are means ± SD. n = 6 – 8 (A, E, and F) or 4 – 6 (B, C, and D). *, P < 0.05, **, P < 0.01, and ***, P < 0.001 STINGgt vs. WT with the same treatment (Ctrl or DMXAA in A and C; Ctrl/LPS or DMX/LPS in B) or DMX/STINGgt BMDM-CM vs. DMX/WT BMDM-CM under the same condition (PBS or LPS in D) or for the same gene (in E and F); †, P < 0.05, ††, P < 0.01, and †††, P < 0.001 DMXAA vs. Ctrl (in A and C) or DMX/LPS vs. Ctrl/LPS (in B) within the same genotype, or DMX/WT BMDM-CM vs. Ctrl/WT BMDM-CM under LPS-stimulated condition (in D) or for the same gene (in E and F); ‡‡, P < 0.01 hepatocytes co-cultured with WT BMDM vs. hepatocytes alone in the presence of DMXAA (in C).
Figure 6.
Figure 6.. STING disruption decreases the severity of MCD-induced NASH
Male STINGgt mice and WT C57BL/6J mice, at 11 – 12 weeks of age, were fed an MCD for 5 weeks. (A) Plasma levels of ALT. (B) Liver weight. (C) Liver levels of triglycerides. (D) Liver sections were stained with H&E (top row), for F4/80 expression (middle two rows), or with Trichrome (bottom row). Bar graph, percentages of macrophages. (E) Liver lysates were examined for inflammatory signaling and αSMA amount using Western blot analysis. Bar graphs, quantification of blots. (F) Hepatic expression of genes related to fibrosis was examined using real-time RT-PCR. MMP2, matrix metalloproteinase 2. For bar graphs in A - F, data are means ± SD. n = 8 – 10 (A - D) or 6 – 8 (E and F)., P < 0.05 and, P < 0.01 MCD-STINGgt vs MCD-WT (in A - E) for the same gene (in F).
Figure 7.
Figure 7.. STING increases HSC activation status and enables macrophage factors to enhance HSC activation (A, B, C)
Activating STING enhances HSC activation. For A - C, LX2 cells were treated with or without DMXAA (75 µg/mL) in the absence or presence of TGFβ1 (8 ng/mL) for 24 hr (A and C (top panel)) or TGFβ1 (2.5 ng/mL) for 48 hr (B and C (bottom panel)). (D, E) STING-driven macrophage factors enhance HSC activation. For D, LX2 cells were co-cultured with STINGgt BMDM and/or WT BMDM that were pre-treated with DMXAA (75 µg/mL) or Ctrl for 24 hr. The co-cultures were incubated for 48 hr in the absence or presence of TGFβ1 (2.5 ng/mL). For E, LX2 cells were treated with conditioned media (CM) of DMXAA (DMX)- or Ctrl-treated WT BMDM and/or STINGgt BMDM for 48 hr. For A, B, and D, cell lysates were subjected to Western blot analysis. Bar graphs, quantification of blots. For C and E, the expression of fibrogenic genes was examined using real-time RT-PCR. For bar graphs in A - E, data are means SD. n = 4 – 6 (A, B, and D) or 6 – 8 (C and E). *, P < 0.05 and **, P < 0.01 DMXAA vs. Control (Ctrl) under the same condition (in A and B) or for the same gene (in C), co-cultures with DMX/WT BMDM vs. co-cultures with Ctrl/WT BMDM under the same condition (in D), or DMX/WT BMDM-CM-treated HSCs vs. Ctrl/WT BMDM-CM-treated HSCs for the same gene (in E); †, P < 0.05 and ††, P < 0.01 TGFβ1 vs. Ctrl with the same treatment (in A, B, and D); ‡, P < 0.05 DMX/STINGgt BMDM-CM-treated HSCs vs. DMX/WT BMDM-CM-treated HSCs under TGFβ1-stimulated condition (in D) or for the same gene (in E).
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