Tim-3/galectin-9 regulate the homeostasis of hepatic NKT cells in a murine model of nonalcoholic fatty liver disease - PubMed (original) (raw)
Tim-3/galectin-9 regulate the homeostasis of hepatic NKT cells in a murine model of nonalcoholic fatty liver disease
Zhao-Hui Tang et al. J Immunol. 2013.
Abstract
T cell Ig and mucin domain (Tim)-3 is well known to interact with its natural ligand, Galectin-9 (Gal-9), to regulate T cell function. However, little is known about the function of Tim-3/Gal-9 signaling in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) mediated by hepatic NKT cells that also express Tim-3. In the current study, we define the role and the mechanism of Tim-3/Gal-9 signaling in hepatic NKT cell regulation in a mouse model of diet-induced NAFLD. Adult male wild-type or CD1d knockout C57BL/6 mice were fed a high-fat diet to induce steatosis. Some of the mice also received one or a combination of Gal-9, anti-IL-15R/IL-15 mAb, rIL-15, α-galactosylceramide, and multilamellar liposomes containing Cl(2)MDP. The expression of Tim-3 and various markers reflecting cell proliferation, activation, cytokine production, and apoptosis was analyzed. Liver histology, steatosis grade, and hepatic triglyceride content were also evaluated. In the liver, Tim-3(+) NKT cells are in an activated state, and Gal-9 directly induces Tim-3(+) NKT cell apoptosis and contributes to the depletion of NKT cells in diet-induced steatosis. However, Gal-9 also interacts with Tim-3-expressing Kupffer cells to induce secretion of IL-15, thus promoting NKT cell proliferation. Exogenous administration of Gal-9 significantly ameliorates diet-induced steatosis by modulating hepatic NKT cell function. In summary, the Tim-3/Gal-9-signaling pathway plays a critical role in the homeostasis of hepatic NKT cells through activation-induced apoptosis and secondary proliferation and, thus, contributes to the pathogenesis of NAFLD.
Figures
FIGURE 1.
Tim-3 expression on murine NKT cells. Total mononuclear cells (TMNCs) were obtained from the liver (L), spleen (S), thymus (T), peripheral blood (PB), and bone marrow (BM) of wild-type C57BL/6 mice fed ND and labeled with anti-mouse Abs to NK1.1, CD3, CD4, CD8, and Tim-3. (A) Representative dot plot of CD3 and NK1.1 staining (gated on hepatic TMNCs). (B) Representative dot plot of CD3 and CD1d tetramer staining (gated on hepatic TMNCs). (C) Representative graphs of Tim-3 labeling on hepatic NKT cells (gated on NK1.1+CD3+ cells). Anti–Tim-3: open graph; isotype control: filled graph). (D) Percentage of Tim-3+ cells (gated on NK1.1+CD3+ cells) from various tissues. (E) Percentage of Tim-3+ cells among CD4+ NKT cells (NK1.1+CD3+CD4+) and DN NKT cells (NK1.1+CD3+CD4−CD8−) in the liver. Data are mean ± SD of five to seven independent experiments.
FIGURE 2.
Characteristics of hepatic Tim-3+ NKT cells. HMNCs were isolated and labeled with various surface markers and intracellular cytokine staining. (A) Expression of CCR7, CD62L, CD25, and CD69 on Tim-3+ or Tim-3− NKT cells. Intracellular staining of IFN-γ (B) and IL-4 (C) among Tim-3+ or Tim-3− NKT, CD4+ NKT, and DN NKT cells. (D) Hepatic NKT cells were isolated and cocultured with splenic DCs pulsed or not with α-GalCer. Tim-3 expression was evaluated on NKT, CD4+ NKT, and DN NKT cells. (E) Wild-type C57BL/6 mice were injected i.p. with 2 μg of α-GalCer. HMNCs were isolated 3 d later, and Tim-3 expression on NKT, CD4+ NKT, and DN NKT cells was evaluated. (F) Hepatic NKT cells were isolated and stimulated with PHA (10 μg/ml) for 5 h. BrdU (1 mM) was then added for another hour. The proliferation of Tim-3+ or Tim-3− NKT, CD4+ NKT, and DN NKT cells was evaluated. (G) Wild-type C57BL/6 mice were injected i.p. with 2 μg of α-GalCer for 2 d, followed by i.p. BrdU during the last 24 h. HMNCs were isolated 3 d later, and the proliferation of Tim-3+ or Tim-3− NKT, CD4+ NKT, and DN NKT cells was evaluated. All results are mean ± SD of five independent experiments.
FIGURE 3.
Gal-9 induced Tim-3+ NKT cell apoptosis. Hepatic NKT cells were isolated and treated with different concentrations of Gal-9. NKT cell apoptosis assays were performed with Annexin V staining and concurrent incubation with 7-AAD. Apoptotic cells are Annexin V+/7-AAD−. (A) Representative dot plot of apoptosis assay of NKT cells. (B–F) Percentage of apoptotic cells among NKT cells. (B) Time and dose curve of Gal-9–induced NKT cell apoptosis. (C) Hepatic NKT cells were treated with Gal-9 or PBS in the presence or absence of anti-lactose, a Gal-9 antagonist. NKT cell apoptosis was evaluated. (D) Hepatic NKT cells were treated with Gal-9 or PBS in the presence of anti–Tim-3 Ab or isotype control. NKT cell apoptosis was evaluated. (E) Hepatic NKT cells were treated with Gal-9 or PBS. Apoptosis among Tim-3+ or Tim-3− NKT cells was evaluated. (F) Wild-type C57BL/6 mice were treated with Gal-9 (1.2 μg/g body weight, i.p.) or PBS once every 2 d for a total of 6 d. Hepatic NKT cells were isolated, and the percentage of apoptosis was evaluated. Data are mean ± SD of five independent experiments.
FIGURE 4.
Upregulation of Tim-3 expression among hepatic NKT cells of mice fed HF diet. Mononuclear cells were isolated from the liver or the spleen of C57BL/6 mice fed ND or HF diet for up to 24 wk and stained with Abs to NK1.1, CD3, CD4, CD8, and Tim-3. (A) Representative graphs of Tim-3 labeling on hepatic NKT cells from mice fed ND (dashed line), mice fed HF diet for 24 wk (open graph), or isotype control (filled graph) (gated on NK1.1+CD3+ cells). Percentage of Tim-3+ cells among hepatic (B) or splenic (C) NKT cells (gated on CD3+NK1.1+ cells). (D) Percentage of apoptotic cells (Annexin V+/7-AAD−) among Tim-3+ NKT cells (gated on Tim-3+CD3+NK1.1+ cells) or Tim-3− NKT cells (gated on Tim-3−CD3+NK1.1+ cells). (E) Percentage of apoptotic NKT cells (gated on CD3+NK1.1+Annexin V+7-AAD− cells) among Tim-3+ T cells (gated on Tim-3+CD3+ cells) or Tim-3− T cells (gated on Tim-3−CD3+ cells). Percentage of Tim-3+ cells among hepatic CD4+ NKT cells (gated on CD3+CD4+NK1.1+ cells) (F) or DN NKT cells (gated on CD3+CD4−CD8−NK1.1+ cells) (G). Results are mean ± SD of five independent experiments.
FIGURE 5.
Gal-9 increased hepatic IL-15 expression and NKT cell proliferation in animals fed HF diet. C57BL/6 mice were fed ND or HF diet for 16 wk. Some of the mice fed HF diet also received Gal-9 (0.6 or 1.2 μg/g body weight, i.p.) every three days for the last 4 wk of feeding, with or without anti–IL-15 Ab (1 μg/g body weight, i.p.). Other mice fed HF diet received rIL-15 protein (25 ng/g body weight) once every 3 d for 9 d before sacrifice. HMNCs were isolated and stained with Abs to NK1.1, CD3, CD4, CD8, and Tim-3. Gal-9 treatment caused a significant increase in NKT cells (A), reversal of CD4+/DN NKT cell ratio (B), and expansion of Tim-3− NKT cells (C) in the liver of mice fed HF diet. Gal-9 treatment significantly increased both protein (D) and mRNA (E) expression of IL-15 in the liver of mice fed HF diet. Anti–IL-15 Ab treatment abolished Gal-9–induced NKT cell proliferation (F), reversal of CD4+/DN NKT cell ratio (G), and Tim-3− NKT cell expansion (H) in the liver of mice fed HF diet. rIL-15 protein treatment had similar effects on Gal-9 that caused NKT cell proliferation (I), reversal of CD4+/DN NKT cell ratio (J), and expansion of Tim-3− NKT cells (K) in the liver of mice fed HF diet. Results are mean ± SD of five independent experiments.
FIGURE 6.
Gal-9 induced Kupffer cell secretion of IL-15 in mice fed HF diet. C57BL/6 mice were fed ND or HF diet for 16 wk. Some mice also received Gal-9 (1.2 μg/g body weight, i.p.) every 3 d for the last 4 wk of feeding. In addition, some mice also received clodronate- or PBS-loaded liposomes (40 mg/g body weight, i.v.) every 3 d during the Gal-9 treatment. (A) Kupffer cells isolated from animal fed ND were cultured with Gal-9 (0.5 μM), α-lactose (40 mM), anti–Tim-3 mAb (20 μg/ml), or LPS (5 μg/ml), alone or in combination, for 24 h. IL-15 released to the media was measured by ELISA. Hepatic expression of IL-15 from animals fed HF diet treated with liposomes loaded with clodronate or PBS and stimulated or not with Gal-9 were determined by ELISA as pg/mg of liver protein (B) and quantitative PCR (C). (D) Percentage of hepatic NKT cells (gated on CD3+ T cells) from animals treated as above. (E) Kupffer cells were isolated from mice fed ND or HF diet. Percentage of Tim-3+ cells was determined by FACS. (F) Kupffer cells were isolated from mice fed ND or HF diet and cultured with LPS (5 μg/ml), with or without Gal-9 (0.5 μM), for 24 h. IL-15 released to the media was measured by ELISA. Results are mean ± SD of five independent experiments.
FIGURE 7.
Gal-9 treatment ameliorated HF diet–induced obesity and hepatic steatosis. C57BL/6 wild-type or CD1dko mice were fed ND or HF diet for 16 wk. Some mice also received Gal-9 (0.6 or 1.2 μg/g body weight, i.p.) every 3 d for the last 4 wk of feeding. Hepatic expression of IFN-γ (A) and IL-4 (B) was determined by quantitative PCR. (C and F) Representative H&E staining of liver histology [original magnification ×100 (C) and ×200 (F)]. (D) Hepatic triglyceride contents. (E) Animal weight. (G) NAFLD histological score. Results are mean ± SD of five independent experiments (n = 5/group).
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