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. 2021 Feb 22:11:609060.
doi: 10.3389/fimmu.2020.609060. eCollection 2020.

TGR5 Regulates Macrophage Inflammation in Nonalcoholic Steatohepatitis by Modulating NLRP3 Inflammasome Activation

Yong Shi  1   2   3   4   5 , Wantong Su  1   2   3   4   5 , Lei Zhang  1   2   3   4   5 , Chengyu Shi  1   2   3   4   5 , Jinren Zhou  1   2   3   4   5 , Peng Wang  1   2   3   4   5 , Hao Wang  1   2   3   4   5 , Xiaoli Shi  1   2   3   4   5 , Song Wei  1   2   3   4   5 , Qi Wang  1   2   3   4   5 , Johan Auwerx  6 , Kristina Schoonjans  6 , Yue Yu  1   2   3   4   5 , Rui Pan  1 , Haoming Zhou  1   2   3   4   5 , Ling Lu  1   2   3   4   5
Affiliations

TGR5 Regulates Macrophage Inflammation in Nonalcoholic Steatohepatitis by Modulating NLRP3 Inflammasome Activation

Yong Shi et al. Front Immunol. .

Abstract

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease associated with dysregulation of liver metabolism and inflammation. G-protein coupled bile acid receptor 1 (TGR5) is a cell surface receptor that is involved in multiple metabolic pathways. However, the functions of TGR5 in regulating macrophage innate immune activation in NASH remain unclear. Here, we found that TGR5 expression was decreased in liver tissues from humans and mice with NASH. Compared to wild type (WT) mice, TGR5-knockout (TGR5-/-) mice exhibited exacerbated liver damage, increased levels of proinflammatory factors, and enhanced M1 macrophage polarization. Moreover, TGR5 deficiency facilitated M1 macrophage polarization by promoting NLRP3 inflammasome activation and caspase-1 cleavage. Taken together, our findings revealed that TGR5 signaling attenuated liver steatosis and inflammation and inhibited NLRP3-mediated M1 macrophage polarization in NASH.

Keywords: G protein-coupled bile acid receptor 1; NLRP3 inflammasome; inflammation; macrophages; nonalcoholic steatohepatitis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
TGR5 deficiency exacerbates steatohepatitis. WT and TGR5-/- mice were fed a normal or MCD diet for 6 weeks (n = 5/group). (A) Hepatic mRNA levels were examined by real-time quantitative PCR. (B, C) Protein expressions were examined by western blotting and relative density ratio of protein levels of TGR5 was analyzed (TGR5/β-actin). (D) Representative images of H&E and Oil-Red O in liver sections from mice (scale bar, 100 μm). (E) NAFLD activity scores were calculated, and steatosis, inflammation, and ballooning were increased in TGR5-/-–MCD mice. N.D., not detected. (F–H) Levels of serum ALT, AST, and hepatic triglycerides were increased in TGR5-/-–MCD mice. Data represent mean ± SD; *P < 0.05.
Figure 2
Figure 2
TGR5 deficiency increases inflammation and promotes macrophage accumulation in mice with NASH. (A–D) Hepatic mRNA levels of TNF-α, IL-6, IL-4, and IL-10 were measured by real-time quantitative PCR. (E–H) Serum levels of TNF-α, IL-6, IL-4, and IL-10 were measured by ELISA in mice. (I, J) Macrophage infiltration was assessed by F4/80 immunostaining (scale bar, 100 μm). Positive cells per high magnification field (HMF) were counted using Image J in 8 fields per section (n = 5). (K) Representative immunofluorescence images of liver samples were stained with CD68 antibodies (scale bar, 50 μm). (L) Hepatic mRNA levels of CD68 were measured by real-time quantitative PCR (n = 5/group). Data represent mean ± SD; *P < 0.05.
Figure 3
Figure 3
Deficiency of TGR5 in BMDMs increases inflammation under PA stimulation. (A, B) mRNA levels and protein expressions of TGR5 detected by real-time quantitative PCR and western blotting, respectively (n = 3/group). (C) The relative quantities of TGR5 protein to β-actin. (D–G) mRNA levels of TNF-α, IL-6, IL-4, and IL-10 in BMDMs determined by real-time quantitative PCR. (H–K) Levels of TNF-α, IL-6, IL-4, and IL-10 were measured by ELISA in cell culture supernatants. Data represent mean ± SD; *P < 0.05.
Figure 4
Figure 4
TGR5 deficiency in NASH mediates macrophages polarization in response to PA treatment. (A, B) Representative immunofluorescence images of liver samples were stained with iNOS and CD206 antibodies (scale bar, 50 μm). (C–H) The mRNA levels of M1 markers (CD68, CD86 and iNOS) and M2 markers (CD163, Arg-1 and CD206) were analyzed by real-time qPCR (n = 3/group). (I) CD206, iNOS, p-STAT1, STAT1, p-STAT6, STAT6 and GAPDH protein levels were detected by western blotting. (J) The relative quantities of CD206, iNOS protein to GAPDH, p-STAT1 protein to STAT1, p-STAT6 protein to STAT6. Data represent mean ± SD; *P < 0.05.
Figure 5
Figure 5
Deficiency of TGR5 actives NLRP3 inflammasome both in mice and BMDMs with NASH. (A–D) Representative immunofluorescence images of liver samples were stained with NLRP3, caspase-1 and ASC antibodies (scale bar, 50 μm, 12.5 μm). Colocalization of NLRP3-Caspase-1 and NLRP3-ASC were analyzed by Image J. (E–G) mRNA levels of IL-1β, IL-18 and NLRP3 were measured by real-time qPCR in mice (n = 5/group). (H, I) Production of IL-1β, IL-18 was measured by ELISA in serum. (J) Protein levels of NLRP3, c-caspase-1, caspase-1, IL-1β and GAPDH were examined by western blotting in mice (n = 5/group). (K) Western blots analysis and relative density ratio of hepatic protein levels of NLRP3, c-caspase-1 and IL-1β. (L–N) mRNA levels of IL-1β, IL-18 and NLRP3 were measured by real-time qPCR in BMDMs (n = 3/group). (O, P) Production of IL-1β, IL-18 was measured by ELISA in cell culture supernatants. (Q) Protein levels of NLRP3, c-caspase-1, caspase-1, IL-1β and GAPDH were detected by western blotting in BMDMs. (R) Western blots analysis and relative density ratio of cellular protein levels of NLRP3, c-caspase-1 and IL-1β. Data represent mean ± SD; *P < 0.05.
Figure 6
Figure 6
Inhibition of the NLRP3 inflammasome reduces inflammation in BMDMs. (A–C) mRNA levels of IL-1β, IL-18 and NLRP3 were measured by real-time qPCR in BMDMs (n = 3/group). (D, E) Production of IL-1β and IL-18 was measured by ELISA in cell culture supernatants. (F) Protein levels of NLRP3, c-caspase-1, caspase-1, IL-1β and GAPDH were detected by western blotting in BMDMs. (G) Western blots analysis and relative density ratio of cellular protein levels of NLRP3, c-caspase-1 and IL-1β. (H, I) mRNA levels and production of TNF-α, IL-6, IL-4, and IL-10 were measured by real-time quantitative PCR in BMDMs and ELISA in cell culture supernatants, respectively. Data represent mean ± SD; *P < 0.05.
Figure 7
Figure 7
Inhibition of the NLRP3 inflammasome promotes macrophages polarization in TGR5-/- mice with NASH. (A–F) mRNA levels of M1 markers (CD68, CD86 and iNOS) and M2 markers (CD163, Arg-1 and CD206) were analyzed by real-time qPCR (n = 3/group). (G) CD206, iNOS, p-STAT1, STAT1, p-STAT6, STAT6, and GAPDH protein levels were detected by western blotting. (H) The relative quantities of CD206, iNOS protein to GAPDH, p-STAT1 protein to STAT1, p-STAT6 protein to STAT6. Data represent mean ± SD; *P < 0.05.
Figure 8
Figure 8
TGR5 inhibits macrophage M1 polarization and NLRP3 inflammasome activation in patients with NASH. (A) Human liver specimens were stained with H&E (scale bar, 100 μm and 50 μm, n = 6/group). (B) NAFLD activity scores were measured as mentioned in the text. N.D., not detected. (C) Expression of TGR5 protein was determined by western blotting. (D) Western blots analysis and relative density ratio of protein levels of TGR5. (E) Expression of TGR5 mRNA was measured by real-time quantitative PCR. (F–H) The mRNA levels of M1 markers (CD68, CD86 and iNOS), M2 markers (CD163, Arg-1 and CD206), IL-1β, IL-18 and NLRP3 were analyzed by real-time qPCR. (I) CD206, iNOS, p-STAT1, STAT1, p-STAT6, STAT6, and GAPDH protein levels were detected by western blotting. (J) The relative quantities of CD206, iNOS protein to GAPDH, p-STAT1 protein to STAT1, p-STAT6 protein to STAT6. (K) Protein levels of NLRP3, c-caspase-1, caspase-1, IL-1β and GAPDH were detected by western blotting in human liver. (L) Western blots analysis and relative density ratio of protein levels of NLRP3, c-caspase-1 and IL-1β. Data represent mean ± SD; *P < 0.05.
Figure 9
Figure 9
TGR5 regulates macrophage inflammation in nonalcoholic steatohepatitis by modulating NLRP3 inflammasome activation.
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