γδ T cells are indispensable for interleukin-23-mediated protection against Concanavalin A-induced hepatitis in hepatitis B virus transgenic mice - PubMed (original) (raw)

doi: 10.1111/imm.12712. Epub 2017 Feb 20.

Jingya Wang 1, Yifang Yuan 1, Guangchao Cao 2, Shuobing Fan 1, Chao Gao 1, Li Wang 1, Zheng Li 1, Xiaoli Wu 3, Zhenzhou Wu 1, Liqing Zhao 1, Zhinan Yin 1 2

Affiliations

γδ T cells are indispensable for interleukin-23-mediated protection against Concanavalin A-induced hepatitis in hepatitis B virus transgenic mice

Ziyu Meng et al. Immunology. 2017 May.

Abstract

Hepatitis B virus surface antigen (HBsAg) carriers are highly susceptible to liver injury triggered by environmental biochemical stimulation. Previously, we have reported an inverse correlation between γδ T cells and liver damage in patients with hepatitis B virus (HBV). However, whether γδ T cells play a role in regulating the hypersensitivity of HBsAg carriers to biochemical stimulation-induced hepatitis is unknown. In this study, using HBV transgenic (HBs-Tg) and HBs-Tg T-cell receptor-δ-deficient (TCR-δ-/- ) mice, we found that mice genetically deficient in γδ T cells exhibited more severe liver damage upon Concanavalin A (Con A) treatment, as indicated by substantially higher serum alanine aminotransferase levels, further elevated interferon-γ (IFN-γ) levels and more extensive necrosis. γδ T-cell deficiency resulted in elevated IFN-γ in CD4+ T cells but not in natural killer or natural killer T cells. The depletion of CD4+ T cells and neutralization of IFN-γ reduced liver damage in HBs-Tg and HBs-Tg-TCR-δ-/- mice to a similar extent. Further investigation revealed that HBs-Tg mice showed an enhanced interleukin-17 (IL-17) signature. The administration of exogenous IL-23 enhanced IL-17A production from Vγ4 γδ T cells and ameliorated liver damage in HBs-Tg mice, but not in HBs-Tg-TCR-δ-/- mice. In summary, our results demonstrated that γδ T cells played a protective role in restraining Con A-induced hepatitis by inhibiting IFN-γ production from CD4+ T cells and are indispensable for IL-23-mediated protection against Con A-induced hepatitis in HBs-Tg mice. These results provided a potential therapeutic approach for treating the hypersensitivity of HBV carriers to biochemical stimulation-induced liver damage.

Keywords: HBs-Tg mice; Vγ4 γδ T cells; immune therapy; interleukin-23; protective role.

© 2017 John Wiley & Sons Ltd.

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Figures

Figure 1

Figure 1

γδ T cells deficiency exacerbated concanavalin A (Con A) ‐induced hepatitis in HBs‐Tg mice. (a) A total of 3 mg/kg Con A was intravenously injected into T‐cell receptor‐δ_‐deficient (TCR‐_δ −/−), hepatitis B virus transgenic (HBs‐Tg), HBs‐Tg‐TCR‐δ −/− and their littermate wild‐type (WT) mice (n = 7 or n = 8/group). At 0, 6, 12, 18, 24, 36, 48 and 72 hr after Con A injection, serum alanine aminotransferase (ALT) levels were measured. (b) Liver tissues were fixed for haematoxylin & eosin staining; ‘N’ indicated the necrotic area, scale bars 200 μm. (c) Cytokine levels were measured by ELISA (n = 7 to n = 9/group). Data are representative of two independent experiments. Statistical comparisons between two groups were made using Student's _t_‐tests. One‐way analysis of variance with multiple comparisons was used for the bar graphs containing three or more groups and two‐way analysis of variance with multiple comparisons was used for ALT experiments. Data are shown as the mean ± SEM. *P < 0·05, **P < 0·01, ***P < 0·001.

Figure 2

Figure 2

Deficiency of γδ T cells accelerated interferon‐γ (IFN‐γ) release from CD4+ T cells in hepatitis B virus transgenic (HBs‐Tg) mice after concanavalin A (Con A) treatment. (a) A total of 3 mg/kg Con A was injected into wild‐type (WT), IFN‐γ −/−, HBs‐Tg and HBs‐Tg‐IFN‐γ −/− mice. Alanine aminotransferase (ALT) levels were measured 18 hr after Con A injection (n = 7 or n = 8/group). (b) Anti‐IFN‐γ (250 μg/mice) or control antibodies (250 μg/mice) were injected intraperitoneally into HBs‐Tg and HBs‐Tg‐TCR‐δ −/− mice 2 and 12 hr before Con A injection. ALT levels were measured 18 hr after Con A injection (n = 6 to n = 8/group). (c) WT, TCR‐δ −/−, HBs‐Tg and HBs‐Tg‐TCR‐δ −/− mice were killed and liver mononuclear cells (MNCs) were isolated 6 hr after Con A injection. Cells were cultured for 4 hr in the presence of Golgi‐Plug, and IFN‐γ production by CD4+ T cells was analysed. (d) Natural killer (NK) cells and NKT cells were investigated with flow cytometry (n = 8 to n = 10/group). (e) Two hours after Con A injection, WT, TCR‐δ −/−, HBs‐Tg and HBs‐Tg‐TCR‐δ −/− mice were killed, and liver CD4+ T cells were purified using magnetic beads. IFN‐γ mRNA levels were determined by quantitative real‐time PCR. Six mice were pooled together for each group and triplicate samples were used for statistical analysis. (f) Anti‐CD4 (200 μg/mice) or control antibodies (200 μg/mice) were injected into HBs‐Tg and HBs‐Tg‐TCR‐δ −/− mice 24 hr before Con A injection. Depletion efficiency was confirmed by flow cytometry. (g) ALT levels were measured 18 hr after Con A treatment (n = 8/group). (h) Serum IFN‐γ levels were measured 6 hr after Con A injection. (i) Liver tissues were fixed for haematoxylin & eosin staining; ‘N’ indicated the necrotic area, scale bars 200 μm. (j) Liver CD4+ T cells were isolated from HBs‐Tg mice. Cells (400 000 cells/200 μl) were seeded into 96‐well plates and were stimulated with a combination of Con A (2·5 μg/ml) and IL‐17A (30 or 100 ng/ml) for 24 hr and analysed by flow cytometry. Data are representative of two independent experiments. Statistical comparisons between two groups were made using Student's _t_‐tests. One‐way analysis of variance with multiple comparisons was used for the bar graphs containing three or more groups and two‐way analysis of variance with multiple comparisons was used for ALT experiments. Data are shown as the mean ± SEM. *P < 0·05, **P < 0·01, ***P < 0·001.

Figure 3

Figure 3

Hepatitis B virus transgenic (HBs‐Tg) mice showed an enhanced interleukin‐17 (IL‐17) signature in the livers and γδ T cells highly expressed IL‐23 receptor. (a, b) Wild‐type (WT) and HBs‐Tg mice were injected intravenously with concanavalin A (Con A; 3 mg/kg). Mice were killed 2 hr after Con A injection, and liver MNCs were isolated and analysed by flow cytometry (n = 11 to n = 13/group). (c) WT, HBs‐Tg and HBs‐Tg‐T‐cell receptor‐δ_‐deficient (TCR‐_δ −/−) mice were treated with Con A (3 mg/kg) as described earlier (n = 8 to n = 10/group), and liver tissues were isolated 2 hr after Con A injection. The cDNA was prepared for analysis of the expression levels of the indicated genes with real‐time PCR. Values from WT mice were set at 1. (d) WT and HBs‐Tg mice were killed and liver MNCs were isolated and analysed by flow cytometry 2 hr after Con A (3 mg/kg) treatment (n = 6 or n = 7/group). Data are representative of two independent experiments. Statistical comparisons between two groups were made using Student's _t_‐tests. One‐way analysis of variance with multiple comparisons was used for the bar graphs containing three or more groups and two‐way analysis of variance with multiple comparisons was used for ALT experiments. Data are shown as the mean ± SEM. *P < 0·05, **P < 0·01.

Figure 4

Figure 4

Administration of exogenous interleukin‐23 (IL‐23) suppressed hepatitis in hepatitis B virus transgenic (HBs‐Tg) mice. (a) Wild‐type (WT) and HBs‐Tg mice were intraperitoneally injected with IL‐23 (4 μg/mouse) or PBS at 2, 24 and 48 hr before concanavalin A (Con A; 3 mg/kg) injection. Alanine aminotransferase activity was determined at different time‐points (n = 8 to n = 10/group). (b) Six hours after Con A treatment, interferon‐γ (IFN‐γ) levels were measured by ELISA (n = 8 to n = 10/group). (c) Liver tissues from WT, PBS‐treated HBs‐Tg and IL‐23 (4 μg/mice) ‐treated HBs‐Tg mice were isolated 2 hr after Con A injection. The cDNA was prepared for analysis of the expression levels of the indicated genes with real‐time PCR. Values from WT mice were set at 1. Data are representative of two independent experiments. Statistical comparisons between two groups were made using Student's _t_‐tests. One‐way analysis of variance with multiple comparisons was used for the bar graphs containing three or more groups and two‐way analysis of variance with multiple comparisons was used for ALT experiments. Data are shown as the mean ± SEM.*P < 0·05, **P < 0·01, ***P < 0·001.

Figure 5

Figure 5

Interleukin‐23 protected against concanavalin A (Con A) ‐induced liver injury via an interleukin‐17A (IL‐17A)‐producing γδ T‐cell‐dependent manner. (a) Wild‐type (WT) and HBs‐Tg mice were intraperitoneally injected with IL‐23 (4 μg/mouse) or PBS at 2, 24 and 48 hr before Con A (3 mg/kg) injection. Two hours after Con A challenge, liver mononuclear cells (MNCs) were isolated and analysed by flow cytometry (n = 7 or n = 8/group). (b) HBs‐Tg and HBs‐Tg‐T‐cell receptor‐δ_‐deficient (TCR‐_δ −/−) mice were treated as described earlier. Eighteen hours after Con A injection, serum alanine aminotransferase (ALT) levels were measured (n = 7 or n = 8/group). (c) Two hours after Con A injection, IL‐17A levels were detected with ELISA (n = 7 or n = 8/group). (d) Liver tissues were fixed for haematoxylin & eosin staining; ‘N’ indicated necrotic area, scale bars 200 μm. Data are representative of two independent experiments. Statistical comparisons between two groups were made using Student's _t_‐tests. One‐way analysis of variance with multiple comparisons was used for the bar graphs containing three or more groups and two‐way analysis of variance with multiple comparisons was used for ALT experiments. Data are shown as the mean ± SEM. *P < 0·05, **P < 0·01, ***P < 0·001.

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