Interleukin-35 modulates the balance between viral specific CD4+CD25+CD127dim/- regulatory T cells and T helper 17 cells in chronic hepatitis B virus infection

Abstract

Background: Interleukin (IL)-35 regulates imbalance between regulatory T cells (Tregs) and T helper (Th) 17 cells, leading to an important modulator in autoimmune disorder, cancer, and infectious diseases. Our previous study revealed an immunosuppressive activity of IL-35 in chronic hepatitis B virus (HBV) infection. Thus, the aim of the current study was to investigate the role of regulatory function of IL-35 to viral specific Tregs/Th17 cells balance in chronic HBV infection.

Methods: A total of 44 HLA-A2 restricted chronic HBV infected patients, including 21 of chronic hepatitis B (CHB) and 23 of asymptomatic HBV carriers (ASC) were enrolled. Purified CD4⁺ T cells or CD4⁺CD25⁺CD127^dim/- Tregs were stimulated with recombinant IL-35. HBV core antigen specific Tregs and Th17 cells were determined by flow cytometry. FoxP3 and RORγt mRNA was measured by real-time PCR. Cytokines production (IL-10 and IL-17) was investigated by ELISA.

Results: Peripheral viral specific Tregs was comparable between CHB and ASC. However, increased percentage of viral specific Th17 cells was found in CHB, leading to the reduction of Tregs/Th17 ratio in CHB patients. IL-35 stimulation elevated viral specific Tregs, but not Th17 cells frequency, in both CHB and ASC, resulting in the elevation of Tregs/Th17 ratio in both groups. This process was accompanied by increased expression of FoxP3 mRNA and IL-10 production, and decreased IL-17 secretion and STAT3 phosphorylation in purified CD4⁺ T cells. Moreover, IL-35 stimulation inhibited viral specific Th17-like phenotype differentiation from Tregs in CHB patients. Effective anti-HBV therapy did not affect viral specific Tregs/Th17 cells frequency or IL-35 expression in CHB patients, however, reduced responsiveness of CD4⁺ T cells or Tregs to IL-35 stimulation in vitro.

Conclusion: Our findings indicated that IL-35 regulation to viral specific Tregs/Th17 balance may contribute to viral persistence in chronic HBV infection.

Keywords: Hepatitis B virus; Interleukin-35; Regulatory T cells; T helper 17 cells.

Fig. 1

HBV core-specific CD4⁺CD25⁺CD127^dim/− regulatory T cells (Tregs) and T helper 17 (Th17) cells in chronic hepatitis B (CHB) and asymptomatic HBV carriers (ASC). Peripheral blood mononuclear cells (PBMCs) were isolated from all enrolled HLA-A2 restricted chronic HBV infected patients (including 23 of CHB and 21 of ASC), and were stimulated with HBc 18–27 peptide for 12 h. Cells were then stained with anti-CD4, −CD25, −CD127, and -interleukin (IL)-17 for flow cytometry analysis. a Gating strategy and representative flow dots for CD4⁺CD25⁺CD127^dim/− Tregs and CD4⁺IL-17⁺ Th17 cells in both CHB and ASC were shown. b HBV core-specific CD4⁺CD25⁺CD127^dim/− Tregs percentage was comparable between CHB and ASC. c HBV core-specific CD4⁺IL-17⁺ Th17 cells percentage was significantly down-regulated in ASC when compared with CHB. d Ratio of Tregs/Th17 was notably increased in ASC when compared with CHB. Individual level for each subject was presented, and comparisons were made using Student t tests

Fig. 2

Interleukin (IL)-35 stimulation to HBV core-specific CD4⁺CD25⁺CD127^dim/− regulatory T cells (Tregs) and T helper 17 (Th17) cells in chronic hepatitis B (CHB) and asymptomatic HBV carriers (ASC). CD4⁺ T cells were purified from eighteen CHB and sixteen ASC, and were stimulated with or without IL-35 for 12 h in the presence of HBc 18–27 peptide. IL-12p35 and EBI3 mRNA expressions were investigated in unstimulated CD4⁺ T cells by real-time PCR. a IL-12p35 mRNA in CD4⁺ T cells was comparable between CHB and ASC. b EBI3 mRNA in CD4⁺ T cells was remarkably elevated in CD4⁺ T cells from CHB when compared with ASC. Individual level for each subject was presented, and comparisons were made using Student t tests. HBV core-specific CD4⁺CD25⁺CD127^dim/− Tregs and CD4⁺IL-17⁺ Th17 cells with or without IL-35 stimulation were investigated by flow cytometry. c HBV core-specific CD4⁺CD25⁺CD127^dim/− Tregs percentage was increased in response to IL-35 stimulation in both CHB and ASC. d HBV core-specific CD4⁺IL-17⁺ Th17 cells percentage was comparable between IL-35 stimulated and unstimulated CD4⁺ T cells in both CHB and ASC. e Ratio of Tregs/Th17 was increased in response to IL-35 stimulation in both CHB and ASC. Individual level for each subject was presented, and comparisons were made using paired t tests. mRNA relative expressions corresponding to Tregs transcriptional factor FoxP3 and Th17 transcriptional factor RORγt were measured by real-time PCR. f FoxP3 mRNA was notably increased in response to IL-35 stimulation in both CHB and ASC. g RORγt mRNA did not change significantly in response to IL-35 stimulation in either CHB or ASC. Individual level for each subject was presented, and comparisons were made using paired t tests. IL-10 and IL-17 production in cultured supernatants were measured by ELISA. h IL-10 expression in cultured supernatants was notably elevated in response to IL-35 stimulation in both CHB and ASC. i IL-17 expression in cultured supernatants was remarkably decreased in response to IL-35 stimulation only in CHB, but not in ASC. Individual level for each subject was presented, and comparisons were made using Wilcoxon matched pairs tests. j phosphorylated STAT3 (pSTAT3), STAT3, and GAPDH expression in CD4⁺ T cells with or without IL-35 stimulation was investigated by Western blot. pSTAT3 was significantly down-regulated in CD4⁺ T cells in response to IL-35 stimulation, however, total STAT3 expression was comparable in CD4⁺ T cells with or without IL-35 stimulation

Fig. 3

T helper 17 (Th17)-like phenotype differentiation from CD4⁺CD25⁺CD127^dim/− regulatory T cells (Tregs) in chronic hepatitis B (CHB) in response to interleukin (IL)-35 stimulation. CD4⁺CD25⁺CD127^dim/− Tregs were purified from thirteen CHB patients, and were stimulated with HBc 18–17 peptide in the presence or absence of IL-35 for 12 h. CCR4⁺ and CCR6⁺ cells in purified Tregs were investigated by flow cytometry. a CCR4⁺ Trges percentage was significantly down-regulated in response to IL-35 stimulation. b CCR6⁺ Trges percentage was significantly down-regulated in response to IL-35 stimulation. IL-17 and IL-22 production in cultured supernatants were measured by ELISA. c IL-17 expression in cultured supernatants was decreased in response to IL-35 stimulation. d IL-22 expression in cultured supernatants was decreased in response to IL-35 stimulation. Individual level for each subject was presented, and comparisons were made using paired t tests

Fig. 4

Influence of effective antiviral therapy to HBV core specific CD4⁺CD25⁺CD127^dim/− regulatory T cells (Tregs), T helper 17 (Th17) cells, and responsiveness to interleukin (IL)-35 stimulation. Ten of chronic hepatitis B (CHB) patients received tenofovir disoproxil fumarate (TDF, 300 mg once daily). Peripheral blood mononuclear cells (PBMCs), CD4⁺ T cells, and CD4⁺CD25⁺CD127^dim/− Tregs were isolated and purified three months post-therapy when reaching virological response. HBV core-specific CD4⁺CD25⁺CD127^dim/− Tregs and CD4⁺IL-17⁺ Th17 cells prior to and post TDF therapy were investigated by flow cytometry, and IL-35 expression in the serum was measured by ELISA. a CD4⁺CD25⁺CD127^dim/− Tregs percentage, (b) CD4⁺IL-17⁺ Th17 cells percentage, and (c) IL-35 expression did not change significantly in response to TDF-therapy. Purified CD4⁺ T cells from TDF-treated CHB patients were stimulated with HBc 18–17 peptide in the presence or absence of IL-35 for 12 h. HBV core-specific CD4⁺CD25⁺CD127^dim/− Tregs and CD4⁺IL-17⁺ Th17 cells were investigated by flow cytometry. d CD4⁺CD25⁺CD127^dim/− Tregs and (e) CD4⁺IL-17⁺ Th17 cells percentage in purified CD4⁺ T cells from TDF-treated patients did not change significantly in response to IL-35 stimulation. Purified CD4⁺CD25⁺CD127^dim/− Tregs from TDF-treated CHB patients were stimulated with HBc 18–17 peptide in the presence or absence of IL-35 for 12 h. f CCR4⁺ cells and (g) CCR6⁺ cells in purified Tregs from TDF-treated patients did not change significantly in response to IL-35 stimulation. Individual level for each subject was presented, and comparisons were made using paired t tests