Suppression of Natural Killer Cell Activity by Regulatory NKT10 Cells Aggravates Alcoholic Hepatosteatosis - PubMed (original) (raw)
Suppression of Natural Killer Cell Activity by Regulatory NKT10 Cells Aggravates Alcoholic Hepatosteatosis
Kele Cui et al. Front Immunol. 2017.
Abstract
We and others have found that the functions of hepatic natural killer (NK) cells are inhibited but invariant NKT (iNKT) cells become activated after alcohol drinking, leaving a possibility that there exists interplay between NK cells and iNKT cells during alcoholic liver disease. Here, in a chronic plus single-binge ethanol consumption mouse model, we observed that NK cells and interferon-γ (IFN-γ) protected against ethanol-induced liver steatosis, as both wild-type (WT) mice treated with anti-asialo GM1 antibody and IFN-γ-deficient GKO mice developed more severe alcoholic fatty livers. As expected, IFN-γ could directly downregulate lipogenesis in primary hepatocytes in vitro. On the contrary, iNKT cell-deficient Jα18-/- or interleukin-10 (IL-10)-/- mice showed fewer alcoholic steatosis, along with the recovered number and IFN-γ release of hepatic NK cells, and exogenous IL-10 injection was sufficient to compensate for iNKT cell deficiency. Furthermore, NK cell depletion in Jα18-/- or IL-10-/- mice caused more severe hepatosteatosis, implying NK cells are the direct effector cells to inhibit liver steatosis. Importantly, adoptive transfer of iNKT cells purified from normal but not IL-10-/- mice resulted in suppression of the number and functions of NK cells and aggravated alcoholic liver injury in Jα18-/- mice, indicating that IL-10-producing iNKT (NKT10) cells are the regulators on NK cells. Conclusion: Ethanol exposure-triggered NKT10 cells antagonize the protective roles of NK cells in alcoholic hepatosteatosis.
Keywords: alcoholic fatty liver; interaction; interferon-γ; interleukin-10; invariant natural killer T cells; natural killer cells.
Figures
Figure 1
Deficiency of natural killer (NK) cells promoted liver steatosis but ameliorated inflammation in a chronic-binge model. Pair- or ethanol-fed wild-type C57BL/6 mice were treated with anti-asialo GM1 or control rabbit IgG once every 3 days as described in Section “Materials and Methods” to deplete NK cells. The mice were euthanized 9 h after gavage. (A) Liver mononuclear cells were isolated, and the depletion efficiency was verified by flow cytometry; liver injury and steatosis were evaluated based on (B) serum alanine aminotransferase and (C) liver triglyceride levels and (D) liver tissue hematoxylin and eosin staining (original magnification, 200×). The data are representative of two independent experiments and are shown as the mean ± SEM (n = 4–6). *P < 0.05; **P < 0.01; ***P < 0.005.
Figure 2
Deficiency of natural killer (NK) cells promoted liver steatosis but ameliorated inflammation at different time points. (A) Pair- or ethanol-fed wild-type C57BL/6 mice were treated with anti-asialo GM1 or control rabbit IgG once every 3 days as described in Section “Materials and Methods” to deplete NK cells. The mice were euthanized 3, 6, or 9 h after gavage. Liver injury and steatosis were evaluated based on (A) serum alanine aminotransferase and (B) liver triglyceride levels and (C) liver tissue hematoxylin and eosin staining (original magnification, 200×). The data are representative of two independent experiments and are shown as the mean ± SEM (n = 4–6). *P < 0.05; **P < 0.01.
Figure 3
Deficiency of interferon-γ promoted ethanol-induced liver injury. (A–C) wild-type or GKO mice were fed control or ethanol diets for 10 days and received a gavage. The mice were sacrificed 9 h after gavage, and liver injury and steatosis were evaluated based on (A) serum alanine aminotransferase and (B) liver triglyceride levels and (C) liver tissue hematoxylin and eosin staining (original magnification, 200×). The data are representative of more than three independent experiments and are shown as the mean ± SEM (n = 4–11). *P < 0.05; **P < 0.01; ****P < 0.0001.
Figure 4
Interferon-γ (IFN-γ) inhibits the expression of fat synthesis-associated genes in hepatocytes. (A) Primary hepatocytes (2 × 105) from the livers of wild-type (WT) mice were isolated and stimulated with 0, 1, 10, or 100 ng/mL IFN-γ, and the cells were collected to evaluate the mRNA levels of ACC, FAS, SCD-1, GPAT, SREBP-1, and FAT after 12 h culture. (B) Primary hepatocytes (2 × 105) from the livers of WT mice and GKO mice were isolated and stimulated with or without 100 ng/mL IFN-γ, and the cells were collected to evaluate the mRNA levels of ACC, FAS, SCD-1, GPAT, SREBP-1, and FAT after 12 h culture. The data are representative of two independent experiments and are shown as the mean ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.005.
Figure 5
Hepatic natural killer (NK) and NKT cells display negatively correlated dynamics in the chronic-binge model. C57BL/6 mice were fed control or ethanol diets for 10 days plus one intragastric dose of EtOH (5 g/kg body weight) or isocaloric dextran-maltose, followed by euthanasia 3, 6, or 9 h after gavage. Liver mononuclear cells were isolated, and the percentages and absolute numbers of (A) CD3−NK1.1+NK cells and (B) CD3+NK.1+T cells among all leukocytes in liver were detected by flow cytometry and were statistically analyzed. The data are representative of more than three independent experiments and are shown as the mean ± SEM (n = 3–6). *P < 0.05; **P < 0.01; ***P < 0.005.
Figure 6
The number and function of natural killer (NK) cells were markedly increased in Jα18−/− mice. (A–D) Wild-type or Jα18−/− mice were fed control or ethanol diets for 10 days and subsequently received gavage. The mice were sacrificed 9 h after gavage, and the liver mononuclear cells were then isolated. (A) The frequency of CD3−NK1.1+ NK cells among all leukocytes in the liver was analyzed by flow cytometry. (B) Statistical analysis of the percentage and absolute number of the NK cells shown in (A). (C) Representative CD107a expression (without PMA stimulation) and interferon-γ (IFN-γ) release (with PMA stimulation) in hepatic NK cells. (D) Statistical analysis of the percentages of CD107a+ and IFN-γ+ NK cells shown in (C). The data are representative of more than three independent experiments and are shown as the mean ± SEM (n = 3–6). *P < 0.05; **P < 0.01; ***P < 0.005.
Figure 7
The numbers and interferon-γ (IFN-γ) release of hepatic CD4+ and CD8+ T cells are not changed in Jα18 KO mice when exposed to ethanol. Wild-type or Jα18 KO mice were fed ethanol diets for 10 days, followed by gavage as described in Figure 1. The mice were sacrificed 9 h after gavage, and liver mononuclear cells were then isolated. The percentages, absolute numbers, and IFN-γ release levels of (A,B) CD4+ T cells and (C,D) CD8+ T cells among all leukocytes in the liver were detected by flow cytometry and were statistically analyzed. The data are representative of more than three independent experiments and are shown as the mean ± SEM (n = 3–6).
Figure 8
Natural killer (NK) cell deficiency promoted alcoholic hepatosteatosis in Jα18−/− mice. Pair- or ethanol-fed Jα18−/− mice were treated with anti-asialo GM1 or control rabbit IgG once every 3 days as described in Section “Materials and Methods” to deplete NK cells. The mice were euthanized 9 h after gavage. (A) Liver mononuclear cells were isolated, and the depletion efficiency was verified by flow cytometry; (B–D) liver injury and steatosis were evaluated by measuring (B) serum alanine aminotransferase and (C) hepatic triglyceride levels and (D) liver tissue by hematoxylin and eosin staining (original magnification, 200×). The data are representative of two independent experiments and are shown as the mean ± SEM (n = 3–6). *P < 0.05; **P < 0.01; ****P < 0.0001.
Figure 9
Hepatic invariant natural killer T (iNKT)-derived interleukin-10 (IL-10) was markedly increased after alcohol challenge. (A–C) C57BL/6 mice were fed control or ethanol diets for 10 days plus one intragastric dose of EtOH (5 g/kg body weight) or isocaloric dextran-maltose, followed by euthanasia 3, 6, or 9 h after gavage. The mRNA levels of IL-10 in (A) liver tissues and (B) purified hepatic iNKT cells were evaluated by quantitative PCR (q-PCR). (C) Liver mononuclear cells were isolated, and the frequency of CD3+CD1d tetramer+ iNKT cells among all leukocytes in the liver and the release of IL-10 by iNKT cells were analyzed by flow cytometry. (D) Wild-type or Jα18−/− mice were fed control or ethanol diets for 10 days, followed by gavage as described in Figure 3. The mice were euthanized 9 h after gavage, and the mRNA levels of IL-10 in liver tissues were detected by q-PCR. The data are representative of two independent experiments and are shown as the mean ± SEM (n = 4). *P < 0.05; ***P < 0.005.
Figure 10
Interleukin-10 (IL-10) deficiency promoted natural killer (NK) cell accumulation and activation and alleviated alcoholic hepatosteatosis. Wild-type or IL-10−/− mice were fed control or ethanol diets for 10 days, followed by gavage, and the mice were euthanized 9 h after gavage. (A–C) Liver mononuclear cells were isolated. (A) The frequency and numbers of CD3−NK1.1+ NK cells among all leukocytes in the liver were analyzed by flow cytometry. (B,C) Statistical analysis of the percentages of (B) CD107a+ (with PMA stimulation) and (C) interferon-γ+ NK cells (with PMA stimulation). (D–F) Liver injury and steatosis were evaluated based on (D) serum alanine aminotransferase and (E) hepatic triglyceride levels and (F) liver tissue hematoxylin and eosin staining (original magnification, 200×). The data are representative of two independent experiments and are shown as the mean ± SEM (n = 4–10). *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001.
Figure 11
Natural killer (NK) cell deficiency promoted alcoholic hepatosteatosis in interleukin-10 (IL-10) KO mice. Ethanol-fed wild-type or IL-10 KO mice were treated with anti-asialo GM1 or control rabbit IgG once every 3 days as described in Section “Materials and Methods” to deplete NK cells. The mice were euthanized 9 h after gavage. (A) Liver mononuclear cells were isolated, and the depletion efficiency was verified by flow cytometry; (B–D) liver injury and steatosis were evaluated based on (B) serum alanine aminotransferase and (C) hepatic triglyceride levels and (D) liver tissue hematoxylin and eosin staining (original magnification, 200×). The data are representative of two independent experiments and are shown as the mean ± SEM (n = 4–5). *P < 0.05; **P < 0.01; ****P < 0.0001.
Figure 12
Exogenous interleukin-10 (IL-10) inhibited natural killer (NK) cell activities and promoted liver steatosis in Jα18−/− mice. Jα18−/− mice were treated with daily intraperitoneal injection of 100 ng of recombinant murine IL-10 or an equal volume of PBS along with 10-day ethanol or control diets. The mice were euthanized 9 h after gavage. (A–C) Liver mononuclear cells were isolated. (A) The frequency and numbers of CD3−NK1.1+ NK cells among all leukocytes in the liver were analyzed by flow cytometry. (B,C) Statistical analysis of the percentages of (B) CD107a+ (with PMA stimulation) and (C) interferon-γ+ NK cells (with PMA stimulation). (D–F) Liver injury and steatosis were evaluated based on (D) serum alanine aminotransferase and (E) hepatic triglyceride levels and (F) liver tissue hematoxylin and eosin staining (original magnification, 200×). The data are representative of three independent experiments and are shown as the mean ± SEM (n = 4–5). *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001.
Figure 13
Adoptive transfer of interleukin-10 (IL-10)−/− cells, but not wild-type (WT) invariant natural killer T (iNKT) cells, restored NK cell accumulation, and function in Jα18−/− mice. Jα18−/− mice were fed ethanol diets for 10 days, followed by gavage with a single dose of ethanol and the intrasplenic adoptive transfer of iNKT cells (5 × 105 cells) purified from the livers of WT or IL-10−/− mice. The mice were sacrificed 9 h after gavage. (A–C) Liver mononuclear cells were isolated. (A) The percentage and absolute number of NK cells among all leukocytes in the liver. (B) CD107a expression (with PMA stimulation) and (C) interferon-γ release (with PMA stimulation) from hepatic NK cells were detected by flow cytometry and were statistically analyzed. (D–F) Liver injury and steatosis were evaluated based on (D) serum alanine aminotransferase and (E) liver triglyceride levels and (F) liver tissue hematoxylin and eosin staining (original magnification, 200×). The data are representative of two independent experiments and are shown as the mean ± SEM (n = 3–7). *P < 0.05; **P < 0.01.
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