Critical roles of conventional dendritic cells in promoting T cell-dependent hepatitis through regulating natural killer T cells

Abstract

Dendritic cells (DCs) play critical roles in initiating and regulating innate immunity as well as adaptive immune responses. However, the role of conventional dendritic cells (cDCs) in concanavalin A (ConA)-induced fulminant hepatitis is unknown. In this study, we demonstrated that depletion of cDCs using either CD11c-diphtheria toxin receptor transgenic mice (DTR Tg) mice or anti-CD11c antibody reduced the severity of liver injury significantly, indicating a detrimental role of cDCs in ConA-induced hepatitis. We elucidated further the pathological role of cDCs as being the critical source of interleukin (IL)-12, which induced the secretion of interferon (IFN)-γ by natural killer (NK) T cells. Reconstitution of cDCs-depleted mice with IL-12 restored ConA-induced hepatitis significantly. Furthermore, we determined that NK T cells were the target of DC-derived IL-12, and NK T cells contributed to liver inflammation and injury through production of IFN-γ. In summary, our study demonstrated a novel function of cDCs in mediating ConA-induced hepatitis through regulating IFN-γ secretion of NK T cells in an IL-12-dependent fashion. Targeting cDCs might provide potentially therapeutic applications in treating autoimmune related liver diseases.

Keywords: IFN-γ; IL-12; cDCs; hepatitis.

Figure 1

Liver conventional dendritic cell (cDC) depletion alleviates concanavalin A (ConA)‐induced fulminant hepatitis. (a) Administration of diphtheria toxin (DT) depletes major histocompatibility complex (MHC) class II⁺CD11c^high conventional dendritic cell populations from CD11c‐diphtheria toxin receptor transgenic mice (DTR Tg) mice. Sex and age‐matched C57BL/6 wild‐type and CD11c–DTR Tg mice were treated with intraperitoneal (i.p.) injections of DT (4 ng/g body weight); 24 h before ConA injections, liver cells were isolated and used for flow cytometry analysis. Results from one of the representative experiments are shown. (b) Complete resistance against lethal dose of ConA in the absence of cDCs. Sex‐ and age‐matched C57BL/6 wild‐type (wt) and CD11c–DTR Tg mice were treated with DT as described above, followed by a lethal dose of ConA (25 mg/kg). Survival rate was monitored and recorded for 72 h. Data shown are the combined results from three experiments (n = 10 per each group). (c) Depletion of cDCs reduced the severity of ConA‐induced hepatitis significantly. Sex and age‐matched B6 wt and DTR mice (n = 5 or 7 for each group) were treated with DT as above, then injected intravenously with subdoses of ConA (10 mg/kg). Serum samples were obtained at different time‐points post‐ConA injection to be used for measuring the level of alanine aminotransferase (ALT). Results from one typical experiment of many repeating ones are shown. (d) Similar results were obtained when mice were treated with anti‐CD11c antibodies. Sex‐ and age‐matched B6 wt and CD11c–DTR Tg mice (n = 5 or 7 for each group) were treated with DT as above; some wt mice were also treated with anti‐CD11c antibodies to deplete CD11c⁺ cells. Three groups of mice were injected intravenously with ConA (10 mg/kg). Serum samples were obtained at different time‐points post‐ConA injection to be used for measuring the level of ALT. Results from one representative experiment are shown. (e) Histology analysis showed similar results. Liver tissues at 12 h post‐ConA treatment were fixed and embedded in paraffin for haematoxylin and eosin staining and one representative tissue staining is shown. N = necrosis area; scale bars = 200 μm. (f) The percentage of necrosis is calculated and shown; n = 3 mice/group. *P < 0·05; **P < 0·01; ***P < 0·001. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Characterization of conventional dendritic cells (cDCs) in the liver during concanavalin A (ConA)‐induced hepatitis. C57BL/6 wild‐type (wt) mice were treated with ConA as described above. Mice were killed 0, 6, 12 and 18 h after ConA injections and hepatic mononuclear cells (MNCs) were collected. (a) Hepatic cDCs percentage decreased after ConA and began to recover at 6 h. Liver cDCs [CD11c^highmajor histocompatibility complex II (MHC class II⁺)] percentages of different time‐points were assessed by flow cytometry and are shown. (b) There was a correlation of alanine aminotransferase (ALT) level and hepatic cDCs percentage or numbers after ConA. Serum was collected at different time‐points to measure the level of ALT. The correlation of ALT (black) and hepatic cDCs percentages or numbers (red) are shown. (c) The maturation level of hepatic cDCs increased after ConA. The relative fold of mean fluorescence intensity (MFI) of CD80, CD86 and CD40 expressed by cDCs after ConA was detected by flow cytometry. Data show the combined results from two experiments with similar results. *P < 0·05; **P < 0·01; ***P< 0·001. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Conventional dendritic cells (cDCs) deficiency reduces the inflammatory cytokine storms upon concanavalin A (ConA) treatment. Serum interferon (IFN)‐γ and interleukin (IL)‐12 levels decreased in DTR mice after ConA injections. Sex and age‐matched C57BL/6 wild‐type (wt) and CD11c‐diphtheria toxin receptor transgenic (DTR Tg) mice (n = 6–10) were treated with diphtheria toxin (DT) as above, then injected intravenously with 10 mg/kg ConA. Blood serum was collected 0, 2, 4, 6, 12 and 24 h after ConA injections. The serum levels of IFN‐γ, tumour necrosis factor (TNF)‐α, IL‐4, IL‐12, IL‐10 and IL‐17 in C57BL/6 wt and CD11c–DTR Tg mice were detected by enzyme‐linked immunosorbent assay (ELISA). Data show the combined results from two experiments with similar results. *P < 0·05; **P < 0·01; ***P < 0·001.

Figure 4

Conventional dendritic cells (cDCs) deficiency leads to decreased interleukin (IL)‐12 levels in diphtheria toxin receptor (DTR) mice. (a) Liver lymphocytes of CD11c–DTR transgenic (Tg) mice expressed significantly lower levels of IL‐12 and IL‐12 receptor (IL‐12r). Sex‐ and age‐matched C57BL/6 wild‐type (wt) and CD11c–DTR Tg mice (n = 6–8) were treated with diphtheria toxin (DT) and ConA as above, and liver mononuclear cells (MNCs) were isolated; gene expression was analysed by real‐time polymerase chain reaction (PCR). (b) CD11c⁺ cells in DTR mice, other than other cell subsets, expressed lower levels of IL‐12 than wt after ConA. Sex‐ and age‐matched B6 wt and DTR mice were treated with DT as mentioned above, then treated with ConA and killed 2 h after injection. CD11c⁺, CD11c^–CD11b⁺ and CD11c^–CD11b^– cells from the two groups of mice were sorted for RNA isolation (10 mice pooled together per group) and gene expression of IL‐12 p40 was analysed by real‐time PCR. (c) The expression of IL‐12 in hepatic cDCs increased after ConA. C57BL/6 wt mice were treated with ConA or phosphate‐buffered saline (PBS) and killed 2 h after injections; cDCs were sorted (20 mice pooled together per group) for real‐time PCR analysis. Data are representative of two independent experiments. *P < 0·05; **P < 0·01; ***P < 0·001.

Figure 5

Essential role of interleukin (IL)‐12 in mediating interferon (IFN)‐γ production of natural killer T cells (NKT cells) in hepatitis. (a) The ability of NKT cells to produce IFN‐γ after concanavalin A (ConA) treatment was abolished in CD11c‐diphtheria toxin receptor transgenic (DTR Tg) mice, which can be restored by IL‐12 reconstitution. Sex and age‐matched C57BL/6 wild‐type (wt) and CD11c–DTR Tg mice were treated with diphtheria toxin (DT) as mentioned above, then these mice (n = 5–8) were treated with intraperitoneal (i.p.) injections of either 400 ng recombinant mouse (rm)IL‐12 or phosphate‐buffered saline (PBS) followed by ConA injection. Liver lymphocytes were prepared 2 h after ConA for intracellular cytokine staining. NKT cells [gated on T cell receptor (TCR) β⁺ NK1.1⁺] and CD4⁺ T cells (CD4⁺ NK1.1^–) were analysed for IFN‐γ production by flow cytometry. Statistical analysis of IFN‐γ‐secreting NK T and CD4⁺ T cells of wt, DTR and IL‐12‐treated DTR mice are shown. (b) One representative staining result is shown. (c) NK1.1 antibody depleted NK and NK T cells effectively. The liver mononuclear cells (MNCs) of NK1.1 antibody‐ or PBS‐treated mice were analysed by flow cytometry, NKT cells (gated on CD3⁺ NK1.1⁺) and NK cells (gated on CD3^–NK1.1⁺) were depleted in NK1.1 antibody‐treated mice. (d,e) IL‐12 played a proinflammation role in hepatitis, which was dependent upon NK1.1⁺ cells. Sex‐ and age‐matched B6 wt were treated with or without anti‐IL‐12, and DTR mice were treated with diphtheria toxin (DT) as above, followed with IL‐12 only or IL‐12 plus anti‐NK1.1 antibodies. Alanine aminotransferase (ALT) values of five groups of mice were measured at different time‐points after ConA injections. (f) Similar results were obtained by histology analysis. Five groups of mice were killed 12 h after ConA, liver tissues were collected, fixed and subjected to haematoxylin and eosin (H&E) staining. The percentage of necrosis is calculated and shown and one representative tissue staining is shown. N = necrosis area; scale bars = 200 μm; n = 3 mice/group. (g) NKT cells were the target of IL‐12 in hepatitis. Sex and age‐matched DTR mice were treated with DT, IL‐12 and anti‐NK1.1 antibodies as mentioned above. Liver MNCs from wt mice or CD1d^–/– mice were transferred to these mice as described in the Materials and methods section. PBS was given as control. These mice were injected intravenously with ConA (10 mg/kg). ALT values of three groups of mice were measured at different time‐points after ConA injections. (h,i) Similar results were obtained by histology analysis. Three groups of mice were killed 12 h after ConA, liver tissues were collected, fixed and subjected to haematoxylin and eosin (H&E) staining. The percentage of necrosis is calculated and shown and one representative tissue staining is shown. N = necrosis area; scale bars, 200 μm; n = 3 mice/group. *P < 0·05; **P < 0·01; ***P< 0·001; n.s. = not significant. [Colour figure can be viewed at wileyonlinelibrary.com]