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

Ziyu Meng; Jingya Wang; Yifang Yuan; Guangchao Cao; Shuobing Fan; Chao Gao; Li Wang; Zheng Li; Xiaoli Wu; Zhenzhou Wu; Liqing Zhao; Zhinan Yin

doi:10.1111/imm.12712

. 2017 Feb 20;151(1):43–55. doi: 10.1111/imm.12712

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

Ziyu Meng ¹, 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,^✉

PMCID: PMC5382349 PMID: 28092402

Summary

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, interleukin‐23, immune therapy, protective role, Vγ4 γδ T cells

Abbreviations

ALT: alanine aminotransferase
APC: allophycocyanin
Con A: concanavalin A
HBsAg: hepatitis B virus surface antigen
HBs‐Tg: hepatitis B virus transgenic mice
HBV: hepatitis B virus
IFN‐γ: interferon‐γ
IL: interleukin
MACS: magnetic cell sorting
MNC: mononuclear cell
NK: natural killer
PE: phycoerythrin
TCR: T‐cell receptor
TGF‐β: transforming growth factor‐β
Th1: T helper type 1
WT: wild‐type

Introduction

Hepatitis B virus (HBV) infection is a global health problem and has caused a tremendous burden with regard to public health. Approximately one million people die each year from HBV infection and HBV‐related diseases such as acute and chronic hepatitis, liver failure, liver cirrhosis and hepatocellular carcinoma.1, 2, 3 C57BL/6J‐TgN (AlblHBV) 44Bri transgenic (HBs‐Tg) mice over‐express HBV surface antigen (HBsAg) and mimic the HBV‐carrying condition but do not produce virus particles.4 Despite the presence of HBsAg in the serum and hepatocytes, these transgenic mice develop immune tolerance.5 HBs‐Tg mice do not exhibit pathological damage or liver dysfunction. Previous research has shown that HBs‐Tg mice were hypersensitive to liver injury induced by low doses of concanavalin A (Con A) due to natural killer (NK) cell activation via NKG2D ligands.6 The NK cells also played critical roles in polyinosinic : polycytidylic‐induced liver injury in HBs‐Tg mice in an interferon‐γ (IFN‐γ)‐dependent and interleukin‐12 (IL‐12) ‐independent manner.7 HBs‐Tg mice were also sensitive to low‐dose α‐galactosyl ceramide‐induced liver injury, and IFN‐β attenuated this liver injury by blocking the release of dendritic cell‐derived IL‐12 and suppressing IFN‐γ production by NKT cells.8

The liver is a unique organ that is not only responsible for removing toxins but also is rich in a large number of immune cells. Approximately 60–80% of the liver cell population is hepatocytes – the remainder including endothelial cells, Kupffer cells, biliary cells, stellate cells and lymphocytes. Liver lymphocytes comprise innate immune cells (NK/NKT, dendritic cells and γδ T cells) and adaptive immune cells (T and B cells).9, 10 Acute liver injury is a deadly syndrome and can directly reflect the sequelae of acute viral hepatitis, autoimmune hepatitis and alcohol consumption‐induced liver injury. Con A‐induced liver injury is mainly mediated by NKT cells,11, 12 CD4⁺ T cells13, 14 and Kupffer cells.15 The effector molecules include the following: IFN‐γ,16 IL‐4,11, 17 tumour necrosis factor‐α 18, 19 and Fas–Fas ligand.12 Some other cytokines, such as IL‐10,20 IL‐1521 and IL‐33,22 can negatively regulate Con A‐induced liver injury.

γδ T cells have special biological characteristics and functions; they have both innate immune cell characteristics and adaptive immune response properties, so are also called innate‐like lymphocytes. γδ T cells serve as a bridge between the innate and adaptive immune responses.23, 24 Depending on the usage of the T‐cell receptor (TCR) repertoire, peripheral γδ T cells mainly consist of two subsets, Vγ1 and Vγ4. Previous research results show that these two subsets have divergent cytokine signatures and play different roles in many immune responses. In coxsackievirus B3‐induced myocarditis, Vγ1⁺ cells tend to mediate a dominant T helper type 1 (Th1) response; however, Vγ4⁺ cells participate in a dominant Th2 response.25 Recent studies have shown that thymic selection can determine γδ T cells differentiation. Antigen‐stimulated γδ T cells mainly produce IFN‐γ, whereas antigen‐naive γδ T cells produce IL‐17.26 In addition, the CD70‐CD27 signal has been proven to affect γδ T‐cell differentiation in the thymus. CD27⁺ γδ T cells secrete IFN‐γ, but CD27⁻ γδ T cells produce IL‐17.27 However, these two subsets play different regulatory roles in diverse disease processes.

Interleukin‐17 is an important cytokine and has received considerable attention since the discovery of the Th17 cell subset. CD4⁺ αβ T cells, NKT cells and γδ T cells can produce IL‐17. γδ T cells are the main source of IL‐17 in many diseases, particularly in the early phases.28, 29 These IL‐17‐producing γδ T cells express IL‐23 receptors, and IL‐23 stimulates γδ T cells to produce IL‐17. The IL‐17⁺ γδ T cells can promote IL‐17 production from CD4⁺ T cells to amplify the loop of IL‐17 production and exacerbate experimental autoimmune encephalomyelitis.30 Interleukin‐23 is a member of the IL‐12 family of cytokines, and the IL‐23 p19/p40 heterodimer is stabilized by a disulphide bond. IL‐23 is highly expressed by activated dendritic cells, macrophages and epidermal Langerhans cells. It exerts its biological functions by interacting with IL‐23 receptors and activating downstream signalling pathways (nuclear factor‐κB, signal transducer and activator of transcription and Janus kinase 2).31, 32 Interleukin‐23 also promotes Th17 cell survival and IL‐17A, IL‐17F and IL‐22 secretion.33, 34 The IL‐23/IL‐17 axis plays an important role in experimental autoimmune encephalomyelitis, rheumatoid arthritis and inflammatory bowel disease models in mice.30, 35, 36

Our early research results have demonstrated that γδ T cells, especially Vγ4 γδ T cells, can suppress the production of IFN‐γ by NKT cells by secreting IL‐17A in Con A‐induced liver injury.37 We also found an inverse correlation between γδ T cells and liver damage in patients with HBV infection.38 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, we demonstrated that IL‐17‐producing γδ T cells played a protective role in Con A‐induced liver injury by inhibiting IFN‐γ release from CD4⁺ T cells in HBs‐Tg mice. The administration of exogenous IL‐23 suppressed hepatitis through promoting IL‐17 production by γδ T cells. γδ T cells are indispensable for IL‐23‐mediated protection against Con A‐induced hepatitis in HBs‐Tg mice. Hence, IL‐23 has therapeutic potential in the treatment of liver injury in carriers of HBV.

Materials and methods

Mice

Eight‐ to ten‐week‐old mice were used in all experiments. C57BL/6J‐TgN (AlblHBV) 44Bri transgenic (HBs‐Tg) mice were purchased from Peking University Health Science Centre (Beijing, China).5 TCR‐δ ^−/− and IFN‐γ ^−/− mice on the C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME). All the animals were maintained under specific pathogen‐free conditions according to the guidelines for experimental animals from the College of Life Sciences of Nankai University.

Isolation of liver mononuclear cells

The protocol for isolating murine liver mononuclear cells (MNCs) was described previously.21, 39 To obtain liver MNCs, murine livers were pressed through 200‐gauge stainless steel mesh and suspended in RPMI‐1640 medium (Hyclone) or PBS. Then, the cells were centrifuged at 50 g for 1 min. The cell suspension was collected and centrifuged at 974 g for 10 min. The MNCs were resuspended in 40% Percoll (GE Healthcare, Uppsala, Sweden), after which the cell suspension was overlaid on 70% Percoll and centrifuged at 1260 g for 30 min. The resulting cell pellets were collected from the interphase for two additional washings in PBS or RPMI‐1640 medium.

Serum transaminase activities

Con A (type IV) was purchased from Sigma Chemical Co (St Louis, MO) and dissolved in serum‐free 1× PBS. Con A at a concentration of 3 mg/kg body weight was injected intravenously into these mice.6 After Con A injection, serum samples were obtained at different time‐points. Serum alanine aminotransferase (ALT) was measured using the standard method (Rong Sheng, Shanghai, China) according to the manufacturer's instructions.

Cytokine detection with ELISA

Serum samples were collected at different time points after Con A injection. IFN‐γ, tumour necrosis factor‐α and IL‐17A (BioLegend, San Diego, CA, USA) levels in the serum were determined using ELISA kits according to the manufacturer's protocol.

Flow cytometry analysis

Liver MNCs were stimulated with PMA (50 ng/ml; Sigma), ionomycin (1 μg/ml; Sigma), and Golgi‐Plug (BD Bioscience, Franklin Lakes, NJ) for 4 hr.37, 40 The liver MNCs were first stained with antibodies against the following surface markers: FITC‐anti‐Vγ4 (Sungene Biotech, Tianjin, China), FITC‐anti‐TCR‐γδ (BD Biosciences), APC‐anti‐TCR‐γδ (Biolegend), PerCP‐anti‐CD3 (Sungene Biotech), phycoerythrin (PE) ‐conjugated anti‐IL‐23 receptor (BD Bioscience), FITC‐anti‐TCR‐β (Sungene Biotech), allophycocyanin (APC) ‐conjugated anti‐NK1.1 (Sungene Biotech), PECy5‐anti‐CD4 (Sungene Biotech) and APC‐anti‐CD4 (Sungene Biotech). For the intracellular cytokine staining, MNCs were fixed, permeabilized and stained with intracellular PE‐anti‐IL‐17A (Sungene Biotech) and PE‐anti‐IFN‐γ (Sungene Biotech) using a Cytofix/Cytoperm plus kit (BD Pharmingen, San Diego, CA, USA) according to the supplier's protocol.21

Cell depletion

To deplete CD4⁺ cells, doses of 200 μg anti‐CD4 monoclonal antibody (clone: GK1.5; Sungene Biotech) or 200 μg control antibodies (Rat IgG2b; Sungene Biotech) were intraperitoneally injected into mice 24 hr before Con A injections.13 With this protocol, maximum depletion of these subsets occurred after 3–5 days and resulted in a ≥ 90% decrease in the number of cells. The results were confirmed by flow cytometry.

IL‐23 treatment and IFN‐γ neutralization

Doses of 4 μg/mouse recombinant IL‐23 or PBS were injected intraperitoneally into mice 2, 24 and 48 hr before Con A challenge. Anti‐IFN‐γ neutralizing monoclonal antibodies (clone: XMG1.2; Sungene Biotech) or control antibodies (Rat IgG1; Sungene Biotech) 250 μg/mouse were injected intraperitoneally into mice 2 and 12 hr before Con A injection.

mRNA expression analysis

Two hours after Con A injection, total liver RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Complemetary DNA synthesis was performed using a PrimeScript RT Reagent Kit (Takara, Shiga, Japan). The mRNA expression was quantified by real‐time PCR. Real‐Time PCR was completed with SYBR Premix Ex Taq II (Takara). Primer sequences are shown in the Supplementary material (Table S1).

Purification of CD4⁺ cells

Mice were killed 2 hr after Con A injection. Liver MNCs were isolated as previously described.21, 39 CD4⁺ T cells were enriched by positive magnetic cell sorting (MACS) according to the manufacturer's protocol. Biotin‐anti‐CD4 (Clone: GK1.5) was purchased from Sungene Biotech. Streptavidin Particles Plus‐DM was purchased from BD Biosciences. Approximately 90% of the magnetically sorted cells were CD4⁺ T cells.

Histopathological analysis

Mice were killed 18 hr after Con A injection. Liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 5‐μm sections, and stained with haematoxylin & eosin; pictures were acquired on a Leica DM3000 microscope (Leica, Germany).

Statistics and data presentation

Statistical comparisons between two groups were made using Student's t‐test. 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. A P‐value of < 0·05 was considered statistically significant.

Results

γδ T‐cell deficiency exacerbated Con A‐induced hepatitis in HBs‐Tg mice

To investigate the possible roles of γδ T cells in Con A‐induced hepatitis in HBs‐Tg mice, sex‐ and age‐matched wild‐type (WT), TCR‐δ ^−/−, HBs‐Tg and HBs‐Tg‐TCR‐δ ^−/− mice were challenged with different low doses of Con A. When mice were injected with 3 mg/kg body weight of Con A, the HBs‐Tg‐TCR‐δ ^−/− mice showed significantly higher ALT levels than the HBs‐Tg mice (Fig. 1a). However, WT and TCR‐δ ^−/− mice did not exhibit significant changes in ALT levels. Low doses of Con A could not induce obvious liver injury in WT and TCR‐δ ^−/− mice. To further dissect the roles of γδ T cells in this model, we administered varying low doses of Con A (5 or 7 mg/kg body weight) to these four types of mice and received similar results (data not shown). Therefore, we used a lower dose of Con A (3 mg/kg body weight) in this study.6 In histopathological examinations, necrosis of liver parenchymal cells was detected in HBs‐Tg mice; however, obvious necrotic areas were not detected in WT and TCR‐δ ^−/− mice. In contrast, larger necrotic areas were observed in HBs‐Tg‐TCR‐δ ^−/− mice, which was consistent with the elevated serum ALT levels in these mice (Fig. 1b).

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.

Subsequently, the serum levels of inflammatory cytokines such as IFN‐γ and tumour necrosis factor‐α were measured at different time‐points after Con A injection. A remarkable cytokine storm was observed after challenge with a low dose of Con A. The serum levels of these cytokines started to increase at approximately 2 hr. The serum IFN‐γ levels in HBs‐Tg‐TCR‐δ ^−/− mice were significantly increased relative to the levels observed in HBs‐Tg and WT mice, particularly at 6 hr, which correlated with ALT activity (Fig. 1c). These results suggested that in HBs‐Tg mice, γδ T cells played an important role in preventing Con A‐induced liver injury.

Deficiency of γδ T cells accelerated IFN‐γ release from CD4⁺ T cells in HBs‐Tg mice after Con A treatment

To determine the pathologic role of IFN‐γ in this model, HBs‐Tg mice were crossed with IFN‐γ ^−/− mice to obtain HBs‐Tg‐IFN‐γ ^−/− mice. When these mice were injected with 3 mg/kg Con A, the HBs‐Tg‐IFN‐γ ^−/− mice were highly resistant to Con A‐induced liver injury and ALT levels were much lower than those in HBs‐Tg mice. However, WT and IFN‐γ ^−/− mice did not exhibit significant changes in ALT levels (Fig. 2a). To confirm these results, HBs‐Tg mice and HBs‐Tg‐TCR‐δ ^−/− mice were treated with anti‐IFN‐γ monoclonal antibodies (250 μg/mice) before Con A injection. Similarly, neutralizing IFN‐γ suppressed Con A‐induced liver damage in HBs‐Tg and HBs‐Tg‐TCR‐δ ^−/− mice. The ALT values were much lower in the anti‐IFN‐γ‐treated HBs‐Tg and HBs‐Tg‐TCR‐δ ^−/− mice than in the control IgG‐treated mice, and the ALT levels did not differ significantly between anti‐IFN‐γ‐treated HBs‐Tg mice and anti‐IFN‐γ‐treated HBs‐Tg‐TCR‐δ ^−/− mice (Fig. 2b). These results collectively defined a critical pathogenic role of IFN‐γ in mediating Con A‐induced liver injury in HBs‐Tg mice. Additionally, γδ T cells might function upstream of IFN‐γ.

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.

To elucidate the cellular source of IFN‐γ and the downstream target of γδ T cells, we treated WT, TCR‐δ ^−/−, HBs‐Tg and HBs‐Tg‐TCR‐δ ^−/− mice with Con A as described earlier. Six hours after Con A injection, liver MNCs were isolated from these mice and cultured in the presence of Golgi‐Plug for 4 hr, after which intracellular IFN‐γ staining was performed. Compared with HBs‐Tg mice, a significantly higher percentage of IFN‐γ‐producing CD4⁺ T cells was observed in HBs‐Tg‐TCR‐δ ^−/− mice (Fig. 2c). However, there were no significant differences in the percentages of IFN‐γ‐producing NKT cells or NK cells among these mice (Fig. 2d). To further confirm this conclusion, CD4⁺ T cells were isolated from the livers of WT, TCR‐δ ^−/−, HBs‐Tg and HBs‐Tg‐TCR‐δ ^−/− mice 2 hr after Con A (3 mg/kg) injection, and purified using magnetic beads. Interestingly, the relative IFN‐γ mRNA expression levels in HBs‐Tg‐TCR‐δ ^−/− mice were significantly higher than those in HBs‐Tg mice (Fig. 2e). To verify this conclusion, HBs‐Tg and HBs‐Tg‐TCR‐δ ^−/− mice were injected intraperitoneally with anti‐CD4 (200 μg/mice) or control antibodies (200 μg/mice) before Con A injection. The cell population targeted by these antibodies was depleted effectively (Fig. 2f). Surprisingly, the depletion of CD4⁺ T cells partially but significantly reduced ALT levels in HBs‐Tg and HBs‐Tg‐TCR‐δ ^−/− mice (Fig. 2g). Moreover, after CD4⁺ T cells depletion, there were no significant differences in ALT levels between HBs‐Tg and HBs‐Tg‐TCR‐δ ^−/− mice. The serum IFN‐γ levels and liver histology were also consistent with ALT activity (Fig. 2h,i). These findings suggested that CD4⁺ T cells were a source of increased IFN‐γ, and that these cells mediated the pathogenesis of Con A‐induced hepatitis in HBs‐Tg mice in the downstream of γδ T cells.

γδ T cells‐derived IL‐17A played a protective role in Con A‐induced hepatitis and negatively regulated IFN‐γ production by NKT cells.37 To verify the role of IL‐17A in the production of IFN‐γ by CD4⁺ T cells, liver CD4⁺ T cells from HBs‐Tg mice were isolated and enriched by positive magnetic cell sorting (MACS) and stimulated with Con A (2·5 μg/ml) and IL‐17A (30 or 100 ng/ml) or PBS. After 24 hr, the cells were assayed for IFN‐γ production with flow cytometry. The combination of Con A and IL‐17A (30 or 100 ng/ml) significantly suppressed IFN‐γ release by CD4⁺ T cells relative to control cells; in contrast, there were no significant differences in the percentages of IFN‐γ ⁺/CD4⁺ found in the 30 and 100 ng/ml IL‐17A treatments. These results suggested that IL‐17A could partially suppress IFN‐γ production by CD4⁺ T cells (Fig. 2j). Taken together, CD4⁺ T cells played a partially pathological role in Con A‐induced hepatitis in HBs‐Tg mice, and γδ T cells negatively regulated IFN‐γ production by CD4⁺ T cells.

HBs‐Tg mice showed an enhanced IL‐17 signature in the livers and γδ T cells highly expressed IL‐23 receptor

The two subsets of γδ T cells (Vγ1and Vγ4) played diverse roles in different disease models.23 Previous studies have demonstrated that γδ T cells mainly produce IFN‐γ and IL‐17A and that these cells are the primary sources of IL‐17A in its early phases.26, 27, 28, 29 To further analyse the cytokines produced by γδ T cells, liver MNCs were isolated from WT and HBs‐Tg mice 2 hr after Con A injection and stained for intracellular cytokines as previously described. γδ T cells from HBs‐Tg mice produced significantly more IL‐17A than those from WT mice. The main subset of γδ T cells that produces IL‐17A is Vγ4 γδ T cells (Fig. 3a). There was no significant change in the IL‐17A production by Vγ1 γδ T cells between WT and HBs‐Tg mice (see Supplementary material, Fig. S1). Approximately 30% of the hepatic γδ T cells in HBs‐Tg mice were IL‐17A⁺ but only 15% of the hepatic γδ T cells in the WT mice expressed IL‐17A. Few CD4⁺ T cells were IL‐17A⁺ in the WT and HBs‐Tg mice (Fig. 3b). These findings suggested that γδ T cells, but not CD4⁺ T cells, were the predominant source of IL‐17A in Con A‐induced hepatitis. The larger proportion of γδ T cells (mainly Vγ4 γδ T cells) in HBs‐Tg mice produced a greater amount of IL‐17A than the γδ T cells from WT mice.

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.

To further study the mechanism of enhanced IL‐17A production in HBs‐Tg mice, we selected WT, HBs‐Tg and HBs‐Tg‐TCR‐δ ^−/− mice injected with Con A (3 mg/kg). Two hours after Con A injection, liver tissues were collected for RNA extractions, and cDNA samples were prepared for real‐time PCR analysis. In comparison with WT mice, IL‐17A and IL‐17F were expressed at significantly higher levels in HBs‐Tg mice. However, IL‐17A, IL‐17F and IL‐22 were expressed at significantly lower levels in the liver tissues of HBs‐Tg‐TCR‐δ ^−/− mice than the liver tissues of HBs‐Tg mice. We then discovered that IL‐23p19, IL‐23 receptor and IL‐1β levels were significantly higher in HBs‐Tg mice than WT mice. Furthermore, the IL‐23 receptor levels in HBs‐Tg‐TCR‐δ ^−/− mice were significantly lower than those in HBs‐Tg mice. Nonetheless, there were no significant differences between these two groups of mice with regard to IL‐23p19 and IL‐1β levels. Moreover, no significant differences in IL‐23p40, IL‐6 and transforming growth factor‐β (TGF‐β) levels were observed among these three groups of mice (Fig. 3c).

Interleukin‐23 and IL‐1β were required for the production of IL‐17A by γδ T cells, and γδ T cells also expressed the IL‐23 receptor.30, 41 To further investigate IL‐23 receptor expression levels on γδ T cells, liver MNCs were isolated and analysed by flow cytometry 2 hr after Con A treatment. The IL‐23 receptor levels were significantly higher in the HBs‐Tg mice than in the WT mice, which was consistent with the previous real‐time PCR results (Fig. 3d). Taken together, these results demonstrated that HBs‐Tg mice showed an enhanced IL‐17 signature in their livers in the Con A‐induced hepatitis model and γδ T cells highly expressed IL‐23 receptor. In addition, IL‐23 played a critical role in producing IL‐17A by γδ T cells in HBs‐Tg mice.

Administration of exogenous IL‐23 suppressed hepatitis in HBs‐Tg mice

Previous studies have reported that γδ T cells‐derived IL‐17A plays a protective role in Con A‐induced hepatitis by suppressing IFN‐γ production by NKT cells.37 Interleukin‐23 is essential for IL‐17A secretion by γδ T cells.30, 34, 41 Based on previous described results that Con A‐induced liver injury was more serious in HBs‐Tg mice than in WT mice, HBs‐Tg mice showed an enhanced IL‐17 signature in their livers. We hypothesized that the IL‐17A produced by HBs‐Tg mice was not sufficient to resist liver injury. To test our hypothesis, we treated sex‐ and age‐matched WT and HBs‐Tg mice with Con A (3 mg/kg) plus PBS or Con A (3 mg/kg) plus IL‐23 (4 μg/mouse). Serum samples were collected at different time‐points for measuring ALT levels. Interestingly, pretreatment with IL‐23 partially but significantly inhibited the Con A‐induced elevation of serum ALT levels in HBs‐Tg mice. However, there were no significant differences in ALT levels between IL‐23‐ and PBS‐treated WT mice (Fig. 4a). We next explored whether IL‐23 influenced the production of the inflammatory cytokine IFN‐γ. Pretreatment with IL‐23 significantly reduced serum levels of IFN‐γ 6 hr after Con A injection (Fig. 4b). The relative expression levels of IL‐17A, IL‐17F, IL‐22 and IL‐23 receptor were significantly increased in IL‐23‐treated HBs‐Tg mice relative to PBS‐treated HBs‐Tg mice (Fig. 4c). These results suggested that IL‐23 administration exerted protective effects on Con A‐induced hepatitis in the HBs‐Tg mice.

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.

IL‐23 protected against Con A‐induced liver injury through an IL‐17A‐producing γδ T cell‐dependent manner

Interleukin‐23 is important for IL‐17A secretion by γδ T cells.30, 34 We then used flow cytometry to analyse intracellular IL‐17A production in hepatic Vγ4 γδ T cells and CD4⁺ T cells. Liver MNCs were isolated and analysed 2 hr after Con A injection. Compared with PBS‐treated HBs‐Tg mice, a significantly higher percentage of IL‐17A‐producing Vγ4 γδ T cells was observed in the IL‐23‐treated HBs‐Tg mice; in contrast there was no difference between these two groups of mice with regard to percentage of IL‐17A‐producing CD4⁺ T cells (Fig. 5a). Next, to investigate whether IL‐23 protects against Con A‐induced liver injury in a γδ T‐cell‐dependent manner, HBs‐Tg and HBs‐Tg‐TCR‐δ ^−/− mice were treated with Con A (3 mg/kg) plus PBS or Con A (3 mg/kg) plus IL‐23 (4 μg/mouse). Eighteen hours after Con A injection, the serum ALT levels of IL‐23‐treated HBs‐Tg mice were much lower than those in PBS‐treated HBs‐Tg mice. In contrast to PBS‐treated HBs‐Tg‐TCR‐δ ^−/−mice, serum ALT activity was not reduced in IL‐23‐treated HBs‐Tg‐TCR‐δ ^−/−mice (Fig. 5b). Two hours after Con A injection, the serum IL‐17A levels in IL‐23‐treated HBs‐Tg mice were significantly higher than those in PBS‐treated HBs‐Tg mice. However, the serum IL‐17A levels of IL‐23‐treated HBs‐Tg‐TCR‐δ ^−/− mice were slightly higher than those in PBS‐treated HBs‐Tg‐TCR‐δ ^−/− mice, and there were no significant differences between these two groups (Fig. 5c). Moreover, liver histology was only minimally ameliorated and was consistent with the ALT and ELISA results (Fig. 5d). The above results indicated that IL‐23 protected against Con A‐induced hepatitis through an IL‐17A‐producing γδ T cell‐dependent mechanism.

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.

Discussion

As a bridge that links the innate immune system and adaptive immune system, γδ T cells have various functional roles. During collagen‐induced arthritis, Vγ4 γδ T cells promoted the progress of collagen‐induced arthritis by producing IL‐17A.42 The IL‐17A‐producing γδ T cells also played an important role in protective immunity against Listeria monocytogenes infection in the liver.43 The specificity of tissue distribution, local microenvironment and antigen‐receptor structure can influence γδ T cells to exert various functions.44 In this study, we focused on the role of γδ T cells in Con A‐induced acute hepatitis in HBs‐Tg mice, which mimicked the HBV‐carrying condition. As shown in Figs 1 and 2, γδ T cells from HBs‐Tg mice played a protective role in the immune responses to low‐dose Con A‐induced liver injury by suppressing IFN‐γ production by CD4⁺ T cells. Additionally, previous clinical studies have also demonstrated that the frequency and number of Vδ2T cells decrease in immune‐activated patients and that the changes in Vδ2T cell frequency were negatively correlated with serum ALT and aspartate aminotransferase levels.38 Therefore, our study successfully provided a disease model for studying the protective mechanism of γδ T cells in a state of immune tolerance and acute HBV infection.

What are the differences of hepatic γδ T cells between WT and HBs‐Tg mice? Compared with WT mice, HBs‐Tg mice showed an enhanced IL‐17 signature in the liver. γδ T cells were the predominant source of IL‐17A, and the main subset of γδ T cells producing IL‐17A was Vγ4 γδ T cell subset (Fig. 3). We did check the IL‐17A production of Vγ1 γδ T cells in HBs‐Tg mice and did not find significant changes when compared with WT controls (see Supplementary material, Fig. S1). There were no differences in the percentages of IFN‐γ ⁺ γδ T cells or Vγ1 γδ T cells between WT and HBs‐Tg mice (data not shown). However, our previous studies demonstrated that Vγ1 γδ T cells played a negative regulatory role in Vγ4 γδ T‐cell‐mediated anti‐tumour immune responses.45 Whether Vγ1 γδ T cells and IL‐17A‐producing Vγ4 γδ T cells interact in Con A‐induced hepatitis needs further investigation. Next, we analysed some key factors driving IL‐17 production. The combination of IL‐6 and TGF‐β could induce Th17 cell differentiation from naive CD4⁺ T cells, and TGF‐β ₁ could control IL‐17‐producing γδ T‐cell expression in the thymus.46 But IL‐17 production by γδ T cells is independent of IL‐6 in the lung and skin.47 Interleukin‐1‐signalling regulated the early differentiation of Th17 cells,48 and IL‐23 promoted Th17 cell proliferation and maintained the stability of these cells. Moreover, IL‐1 and IL‐23 were also potent drivers of IL‐17 production by γδ T cells. As shown in Fig. 3, the relative expression levels of IL‐23p19, IL‐1β and the IL‐23 receptor were significantly higher in HBs‐Tg mice than in WT mice. However, the relative expression level of the IL‐23 receptor in HBs‐Tg‐TCR‐δ ^−/− was significantly lower than that in HBs‐Tg mice. Nevertheless, there were no differences in the IL‐23p19 and IL‐1β relative expression levels between HBs‐Tg and HBs‐Tg‐TCR‐δ ^−/− mice. In addition, we also found that IL‐23 could enhance IL‐17 production by Vγ4 γδ T cells of HBs‐Tg mice in vitro (see Supplementary material, Fig. S2). These results suggested that γδ T cells were not the source of IL‐23, but that IL‐23 might be the main targets of γδ T cells in the liver and important for both induction and maintenance of IL‐17 by γδ T cells in HBs‐Tg mice. Indeed, γδ T cells expressed the IL‐23 receptor. Interleukin‐23 bound to the IL‐23 receptor on the membrane surface of activated γδ T cells to form a complex and stimulate the intracellular signal transduction system.31, 49

In HBs‐Tg mice, IL‐17A played a protective role against Con A‐induced hepatitis. However, in spite of elevated IL‐17A levels, HBs‐Tg mice still developed much more serious liver injury than WT mice. We sought to identify the reason for this phenomenon. We speculated that the hepatocytes of HBs‐Tg mice were more sensitive to CD4/NK/NKT‐mediated damage upon Con A administration, and that the elevation of protective cytokines such as IL‐17A, IL‐22 and IL‐23 was a compensatory reaction following aggravated stress. Nevertheless, these elevated protective cytokines were still not enough to resist liver injury in HBs‐Tg mice. Indeed, supplementation with exogenous IL‐23 in HBs‐Tg mice restrained the elevation in serum ALT levels and liver necrosis after Con A treatment. Interleukin‐23 protected against Con A‐induced hepatitis through an IL‐17A‐producing γδ T cell‐dependent mechanism. It promoted the production of IL‐17A by γδ T cells, but not CD4⁺ T cells (Fig. 5). γδ T cells are indispensable for IL‐23‐induced protection against Con A‐induced hepatitis in HBs‐Tg mice. Nonetheless, in another previous study of Con A‐induced hepatitis, endogenous IL‐23 played a protective role in an IL‐22‐dependent manner, but exogenous IL‐23 played a pathological role in IL‐17‐dependent and IL‐17‐independent manners.50 The specific reason for such a discrepancy is unclear. We considered that the possible reasons for such a discrepancy could include the following: differences in the doses of IL‐23 administered, different strains of mice, and diverse intestinal microbes. Differences in these factors would probably lead to different phenotypes. Crossing HBs‐Tg mice with IL‐23p19^−/− or IL‐23p40^−/− mice would help to elucidate the true role of IL‐23 in regulating Con A‐induced liver injury in HBV carriers. Furthermore, previous studies have suggested that antigen‐presenting cells were the main source of IL‐23, such as dendritic cells51 and macrophages.52 Investigation of the source of endogenous IL‐23 will greatly extend our understanding of the liver immune system and provide potential targets for combating acute liver injury in the further study. In addition to IL‐23, several other Th17‐related cytokines were also reported to have protective roles in Con A‐induced hepatitis. Interleukin‐6 alleviated Con A‐induced hepatitis by inhibiting NKT cell activation.53 The IL‐22 protected mice from Con A‐induced liver injury and also functioned as a survival factor for hepatocytes through signal transducer and activator of transcription 3 activation.54

Natural killer T cells, CD4⁺ T cells and Kupffer cells play important roles in the pathogenesis of Con A‐induced liver injury. Previous studies suggested that the hypersensitivity of HBs‐Tg mice to Con A‐induced liver injury was due to NK cell activation by NKG2D ligands and support from NKT cells.7 The imbalance between Th1 and Th2 cell immune responses was one of the important mechanisms of chronic HBV infection,55 and CD4⁺ T cells were also critical regulators in the adaptive immune response to HBV infection.56 In our study, we checked the IFN‐γ production of NK/NKT cells and CD4⁺ T cells (Fig. 2), only IFN‐γ ⁺ CD4 cells were significantly changed between HBs‐Tg and HBs‐Tg‐TCR‐δ ^−/− mice. Besides, the total number of IFN‐γ ⁺ CD4⁺ T cells in the liver of HBs‐Tg mice was significantly higher than WT mice (see Supplementary material, Fig. S3), which was consistent with the pathological data. When CD4⁺ T cells were depleted, the levels of ALT and IFN‐γ in HBs‐Tg and HBs‐Tg‐TCR‐δ ^−/− mice were significantly lower than those in control mice. The liver damage of both HBs‐Tg and HBs‐Tg‐TCR‐δ ^−/− mice was reduced to a similar level. These results indicated that at least in HBs‐Tg mice, CD4⁺ T cells were downstream of γδ T cells and that the protective function of γδ T cells occurs mainly through targeting CD4⁺ T cells. What could be the conjunction between γδ T cells and CD4⁺ T cells? Previously, we found that γδ T cells expressed IL‐17A‐suppressed IFN‐γ production from NKT cells after Con A treatment. We hypothesized that γδ T cells might also suppress IFN‐γ production from CD4⁺ T cells through IL‐17A. Some previous studies have demonstrated that Th1 cells and Th17 cells can regulate each other's activity in some autoimmune disease models. Interleukin‐17A played a protective role in T‐cell‐mediated intestinal inflammatory diseases by suppressing Th1 differentiation,57 and T‐bet deficiency also resulted in enhanced IL‐17 production in experimental autoimmune myocarditis.58 In this study, our hypothesis was supported by the results that recombinant IL‐17A significantly suppressed IFN‐γ production by Con A‐treated liver CD4⁺ T cells from HBs‐Tg mice (Fig. 2i). These results were also consistent with previous studies showing that IL‐17A inhibited the development of α‐galactosyl ceramide‐induced liver injury.59

In summary, this study has demonstrated that γδ T cells from HBs‐Tg mice played a protective role in Con A‐induced hepatitis by inhibiting IFN‐γ production by CD4⁺ T cells. Pretreatment with IL‐23 ameliorated Con A‐induced liver injury in HBs‐Tg mice. The protective roles of IL‐23 were mediated through IL‐17A‐producing γδ T cells and resulted in inhibiting IFN‐γ production. γδ T cells were indispensable for IL‐23‐mediated protection against Con A‐induced hepatitis in HBs‐Tg mice. This study provided important evidence that γδ T cells and IL‐23 signalling may be potential targets for the development of new drugs and treatment for combating acute liver injury in HBV carriers.

Disclosures

Authors have no financial conflicts of interest.

Supporting information

Figure S1. There were no significant changes in the interleukin‐17A production of Vγ1 γδ T cells between wild‐type (WT) and hepatitis B virus transgenic (HBs‐Tg) mice.

Click here for additional data file.^{(67.5KB, pdf)}

Figure S2. Interleukin‐23 (IL‐23) could enhance IL‐17 production by Vγ4 γδ T cells of hepatitis B virus transgenic (HBs‐Tg) mice in vitro.

Click here for additional data file.^{(65.2KB, pdf)}

Figure S3. The total number of interferon‐γ⁺ CD4⁺ T cells in the liver of hepatitis B virus transgenic (HBs‐Tg) mice was increased.

Click here for additional data file.^{(42KB, pdf)}

Table S1. RT‐PCR primer sequences used in this study

Click here for additional data file.^{(12.9KB, docx)}

Click here for additional data file.^{(26.5KB, doc)}

Acknowledgements

This work was supported by a Major programme of the National Natural Science Foundation of China (Grant 31230025) and Tianjin Science and Technology Foundation (Grant 08QTPTJC28400) to Dr Zhinan Yin, the National Natural Science Foundation of China (Grant 31170858; 31370883) to Dr Liqing Zhao, and the National Natural Science Foundation of China (Grant 31500723) to Dr Xiaoli Wu. We thank Charron Cote for revision of the manuscript. ZM conceived the project, designed and performed all experiments, analysed data and wrote the manuscript; JW, YY, GC, SF, CG, LW and ZL performed the experiments and mouse model; GC and XW helped to design the experiments and write the manuscript. ZW supervised participants. LZ and ZY designed the overall research, helped conceive the project, wrote the manuscript, mentored and supervised participants. All authors have read, commented on, and approved the final manuscript.

Contributor Information

Liqing Zhao, Email: lqzhao@nankai.edu.cn.

Zhinan Yin, Email: zhinan.yin@yale.edu.

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59. Wondimu Z, Santodomingo‐Garzon T, Le T, Swain MG. Protective role of interleukin‐17 in murine NKT cell‐driven acute experimental hepatitis. Am J Pathol 2010; 177:2334–46. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. There were no significant changes in the interleukin‐17A production of Vγ1 γδ T cells between wild‐type (WT) and hepatitis B virus transgenic (HBs‐Tg) mice.

Click here for additional data file.^{(67.5KB, pdf)}

Figure S2. Interleukin‐23 (IL‐23) could enhance IL‐17 production by Vγ4 γδ T cells of hepatitis B virus transgenic (HBs‐Tg) mice in vitro.

Click here for additional data file.^{(65.2KB, pdf)}

Figure S3. The total number of interferon‐γ⁺ CD4⁺ T cells in the liver of hepatitis B virus transgenic (HBs‐Tg) mice was increased.

Click here for additional data file.^{(42KB, pdf)}

Table S1. RT‐PCR primer sequences used in this study

Click here for additional data file.^{(12.9KB, docx)}

Click here for additional data file.^{(26.5KB, doc)}

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PERMALINK

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

Ziyu Meng

Jingya Wang

Yifang Yuan

Guangchao Cao

Shuobing Fan

Chao Gao

Li Wang

Zheng Li

Xiaoli Wu

Zhenzhou Wu

Liqing Zhao

Zhinan Yin

Summary

Abbreviations

Introduction

Materials and methods

Mice

Isolation of liver mononuclear cells

Serum transaminase activities

Cytokine detection with ELISA

Flow cytometry analysis

Cell depletion

IL‐23 treatment and IFN‐γ neutralization

mRNA expression analysis

Purification of CD4+ cells

Histopathological analysis

Statistics and data presentation

Results

γδ T‐cell deficiency exacerbated Con A‐induced hepatitis in HBs‐Tg mice

Figure 1.

Deficiency of γδ T cells accelerated IFN‐γ release from CD4+ T cells in HBs‐Tg mice after Con A treatment

Figure 2.

HBs‐Tg mice showed an enhanced IL‐17 signature in the livers and γδ T cells highly expressed IL‐23 receptor

Figure 3.

Administration of exogenous IL‐23 suppressed hepatitis in HBs‐Tg mice

Figure 4.

IL‐23 protected against Con A‐induced liver injury through an IL‐17A‐producing γδ T cell‐dependent manner

Figure 5.

Discussion

Disclosures

Supporting information

Acknowledgements

Contributor Information

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Purification of CD4⁺ cells

Deficiency of γδ T cells accelerated IFN‐γ release from CD4⁺ T cells in HBs‐Tg mice after Con A treatment