Increased Tim-3 expression alleviates liver injury by regulating macrophage activation in MCD-induced NASH mice - PubMed (original) (raw)

. 2019 Nov;16(11):878-886.

doi: 10.1038/s41423-018-0032-0. Epub 2018 May 7.

Zhuanchang Wu 1, Yong Xu 1, Yuan Liu 2, Wen Liu 1, Tixiao Wang 1, Chunyang Li 3, Cuijuan Zhang 4, Fan Yi 1, Lifen Gao 1, Xiaohong Liang 1, Chunhong Ma 5 6

Affiliations

Increased Tim-3 expression alleviates liver injury by regulating macrophage activation in MCD-induced NASH mice

Xianhong Du et al. Cell Mol Immunol. 2019 Nov.

Abstract

As an immune checkpoint, Tim-3 plays roles in the regulation of both adaptive and innate immune cells including macrophages and is greatly involved in chronic liver diseases. However, the precise roles of Tim-3 in nonalcoholic steatohepatitis (NASH) remain unstated. In the current study, we analyzed Tim-3 expression on different subpopulations of liver macrophages and further investigated the potential roles of Tim-3 on hepatic macrophages in methionine and choline-deficient diet (MCD)-induced NASH mice. The results of flow cytometry demonstrated the significantly increased expression of Tim-3 on all detected liver macrophage subsets in MCD mice, including F4/80+CD11b+, F4/80+CD68+, and F4/80+CD169+ macrophages. Remarkably, Tim-3 knockout (KO) significantly accelerated MCD-induced liver steatosis, displaying higher serum ALT, larger hepatic vacuolation, more liver lipid deposition, and more severe liver fibrosis. Moreover, compared with wild-type C57BL/6 mice, Tim-3 KO MCD mice demonstrated an enhanced expression of NOX2, NLRP3, and caspase-1 p20 together with increased generation of IL-1β and IL-18 in livers. In vitro studies demonstrated that Tim-3 negatively regulated the production of reactive oxygen species (ROS) and related downstream pro-inflammatory cytokine secretion of IL-1β and IL-18 in macrophages. Exogenous administration of N-Acetyl-L-cysteine (NAC), a small molecular inhibitor of ROS, remarkably suppressed caspase-1 p20 expression and IL-1β and IL-18 production in livers of Tim-3 KO mice, thus significantly reducing the severity of steatohepatitis induced by MCD. In conclusion, Tim-3 is a promising protector in MCD-induced steatohepatitis by controlling ROS and the associated pro-inflammatory cytokine production in macrophages.

Keywords: NASH; ROS; Tim-3; macrophage.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1

Fig. 1

Augmented Tim-3 expression protected mice from MCD-induced liver injury. (a) Tim-3 expression in liver tissues of MCD mice was detected by IHC. b–e Compared with the WT group (n = 4), more severe liver damage was observed in Tim-3 KO (n = 4) mice after 2 weeks of MCD. b Colorimetric method analysis of ALT in serum from WT and Tim-3 KO mice. c HE-stained liver tissues from Tim-3 KO mice and WT mice. d Levels of TG in liver homogenate from WT and Tim-3 KO MCD mice were detected by enzymatic analysis. e ORO staining was applied to detect the lipid composition in liver tissues of WT and Tim-3 KO mice. f Masson staining and g qPCR results showed more collagen fibers in Tim-3 KO mice after 3 weeks of MCD treatment. The bars represent the mean values and the S.E.M.s. *p < 0.05, **p < 0.01. NASH nonalcoholic steatohepatitis, ND normal diet, MCD methionine-choline-deficient diet, WT wild type, Tim-3 KO Tim-3 TALEN, ORO oil red O, TG triglyceride

Fig. 2

Fig. 2

Oleic acid triggered Tim-3 upregulation in macrophages. a Peritoneal macrophages were stimulated with liver homogenate derived from ND or MCD mice; qPCR was used to evaluate Tim-3 expression. b–d BMDMs and peritoneal macrophages were treated with oleic acid at the indicated concentration. b Oil red O staining results showed increased lipid accumulation in peritoneal macrophages after oleic acid treatment. c qPCR was used to detect Tim-3 expression in peritoneal macrophages, and d western blot analysis was used to evaluate Tim-3 expression in BMDMs stimulated by oleic acid. BMDM bone marrow-derived macrophage

Fig. 3

Fig. 3

Tim-3 was increased on different subpopulations of hepatic macrophages in MCD mice. FCM analysis of Tim-3+ cells gated in (a) F4/80+CD11b+, (b) F4/80+CD68+, and (c) F4/80+CD169+ liver macrophages isolated from ND and MCD mice (n ≥ 3). Representative data are shown in the left panels, and statistical data are displayed in the right panels. The results were analyzed by a two-tailed test. Significance is shown as **p < 0.01, ***p < 0.001

Fig. 4

Fig. 4

Tim-3 inhibited ROS generation in macrophages. a NOX2 expression was analyzed by western blot in liver tissues of WT and Tim-3 KO mice after MCD treatment. b Western blotting was performed to analyze NOX2 expression in BMDMs pretreated with anti-Tim-3-neutralizing antibody. BMDMs were pretreated with anti-Tim-3-neutralizing antibody following LPS or PA stimulation (c). Peritoneal macrophages prepared from Tim-3 KO or WT mice were treated with LPS or PA (d, e). ROS (c, d) and mitochondrial ROS (e) were assayed with DCFH-DA or mitosox red as described in the Materials and methods. **p < 0.01, ***p < 0.001

Fig. 5

Fig. 5

Tim-3 regulated ROS-mediated pro-inflammatory cytokine liberation in macrophages. a, b Livers were isolated from MCD-treated Tim-3 KO and WT mice (n = 4). a Western blots were applied for the detection of NLRP3 and caspase-1 p20 in the liver tissues. b ELISA was used to detect the levels of IL-1β, IL-18, and TNFα in the liver homogenates. c, d BMDMs were pretreated with anti-Tim-3-neutralizing antibody (IgG as a control) and then stimulated by LPS. Western blot (c) and ELISA (d) were employed to detect the expression of the indicated proteins. e FCM analysis was used to analyze IL-1β generation in NAC-pretreated, HFD liver homogenate-stimulated peritoneal macrophages derived from WT or Tim-3 KO mice. The results were assessed by Student’s _t-_test. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

Fig. 6

Tim-3 alleviated liver injury by inhibiting ROS generation in NASH mice. WT and Tim-3 KO mice fed with MCD were administered with NAC as described in the Materials and methods (n = 4). Serum ALT (a) and liver TG (b) were tested. c HE was applied to determine the hepatic vacuolation. d Caspase-1 p20 expression was analyzed by western blot. e ELISAs were applied to detect the levels of IL-1β, IL-18, and TNFα in liver homogenates

Fig. 7

Fig. 7

Tim-3 protects against liver injury by regulating macrophage activation in NASH

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