Significance of the balance between regulatory T (Treg) and T helper 17 (Th17) cells during hepatitis B virus related liver fibrosis

Abstract

Background: Hepatitis B virus-related liver fibrosis (HBV-LF) always progresses from inflammation to fibrosis. However, the relationship between these two pathological conditions is not fully understood. Here, it is postulated that the balance between regulatory T (Treg) cells and T helper 17 (Th17) cells as an indicator of inflammation may predict fibrosis progression of HBV-LF.

Methodology/principal findings: The frequencies and phenotypes of peripheral Treg and Th17 cells of seventy-seven HBeAg-positive chronic hepatitis B (CHB) patients who underwent liver biopsies and thirty healthy controls were determined by flow cytometry. In the periphery of CHB patients, both Treg and Th17 frequencies were significantly increased and correlated, and a lower Treg/Th17 ratio always indicated more liver injury and fibrosis progression. To investigate exact effects of Treg and Th17 cells during HBV-LF, a series of in vitro experiments were performed using purified CD4(+), CD4(+)CD25(+), or CD4(+)CD25(-) cells from the periphery, primary human hepatic stellate cells (HSCs) isolated from healthy liver specimens, human recombinant interleukin (IL)-17 cytokine, anti-IL-17 antibody and HBcAg. In response to HBcAg, CD4(+)CD25(+) cells significantly inhibited cell proliferation and cytokine production (especially IL-17 and IL-22) by CD4(+)CD25(-) cells in cell-contact and dose-dependent manners. In addition, CD4(+) cells from CHB patients, compared to those from HC subjects, dramatically promoted proliferation and activation of human HSCs. Moreover, in a dramatically dose-dependent manner, CD4(+)CD25(+) cells from CHB patients inhibited, whereas recombinant IL-17 response promoted the proliferation and activation of HSCs. Finally, in vivo evidence about effects of Treg/Th17 balance during liver fibrosis was obtained in concanavalin A-induced mouse fibrosis models via depletion of CD25(+) or IL-17(+) cells, and it's observed that CD25 depletion promoted, whereas IL-17 depletion, alleviated liver injury and fibrosis progression.

Conclusions/significance: The Treg/Th17 balance might influence fibrosis progression in HBV-LF via increase of liver injury and promotion of HSCs activation.

Figure 1. Phenotypes of Treg and Th17 cells in the periphery of CHB patients.

(A, B) Representative dot plots of peripheral Treg and Th17 cells with matched-isotype controls. (C) Representative co-expressions of CTLA-4, IL-10 and TGF-β in Treg cells of CHB patients. (D) Representative co-expressions of CCR6, IFN-γ and Foxp3 in Th17 cells of CHB patients. (E) The frequencies of CD4⁺CD25⁺Foxp3⁺CTLA-4⁺ cells are significantly higher in CHB patients (n = 28) than in HC subjects (n = 10). (F) The frequencies of CD4⁺Foxp3⁺IL-17⁺ cells are significantly higher in CHB patients (n = 29) than in HC subjects (n = 9). Data in subfigures D–E are presented as each value and the mean value. ***, p<0.001.

Figure 2. Treg and Th17 cells involved in fibrosis progression of HBV-LF.

(A) Representative dot plots of Treg (right panel) and Th17 (left panel) cells in age-and-sex matched HC and CHB subjects. (B) Subgroup analyses were performed in CHB patients with similar HBV loads (10⁵–10⁷ copies/mL) but with differing ALT levels (n = 57). With elevated ALT levels, Treg frequency decreased significantly whereas Th17 increased significantly. (C) Subgroup analyses were performed in CHB patients with normal ALT but differing virus loads (n = 13). With elevated virus loads, Treg frequency increased significantly whereas Th17 barely changed. (D) Subgroup analyses were performed in CHB patients with similar inflammation grades (G2–3) but with differing stages of fibrosis (n = 56). With elevated fibrosis stages, Treg frequency decreased significantly whereas Th17 increased significantly. Data in subfigures B–D are presented as each value and the mean value. **, p<0.01; ***, p<0.001; NS, not significant.

Figure 3. Peripheral Treg/Th17 ratios indicated liver injury and fibrosis progression of HBV-LF.

A) There is a significant correlation between peripheral Treg and Th17 frequencies in CHB patients. (n, case number; r, correlation coefficient; p value is shown). (B–D) Compared to HC subjects, peripheral Treg/Th17 ratio dramatically increased in CHB patients, however it dramatically decreased with enhanced inflammation grades (B) or elevated ALT levels (C) or advanced fibrosis stages (D). Data in subfigures B–D are presented as each value and the mean value. *, p<0.05; ***, p<0.001.

Figure 4. Treg cells regulated Th17 response in HBV-LF

. (A) Compared to HC subjects (n = 30), Th17-associated cytokines such as IL-17, IL-21 and IL-22 significantly increased in serum of CHB patients (n = 77), however Treg-associated inhibitory cytokines IL-10 and TGF-β are similar in HC subjects and CHB patients. (B) After 1 µg/mL HBcAg stimulation for 5 days, CD4⁺ cells from CHB patients (n = 6) showed a significantly enhanced ability to secrete Th17-associated cytokines, including IL-17, IL-21, IL-22 and TNF-α, than do those from age-and-sex matched HC subjects (n = 6). (C) CD4⁺CD25⁺ cells from CHB patients (n = 6) were co-cultured with HLA-identical CD4⁺CD25⁻ cells in both contact and non-contact manners for 5 days. CD4⁺CD25⁺ cells significantly inhibited IL-17 and IL-22 production by CD4⁺CD25⁻ cells only in cell-contact manner. *, p<0.05 when compared to medium only group; ^#, p<0.05 when compared to uncontact co-culture group. (D) CD4⁺CD25⁺ cells from CHB patients (n = 6) significantly inhibited IL-17 and IL-22 production by CD4⁺CD25⁻ cells in a dose-dependent manner (p<0.05). No dramatic changes of IL-21 or TNF-α were observed after co-cultures with CD4⁺CD25⁺ cells. Each line represents a cytokine (p>0.05). (E) CD4⁺CD25⁺ cells from CHB patients (n = 6) inhibited proliferation of HLA-identical CD4⁺CD25⁻ cells in a dose-dependent manner. Each line represents a CHB patient. (F) On the left panel, HLA-identical CD4⁺CD25⁺ cells showed the greatest power to inhibit proliferation of CD4⁺CD25⁻ cells compared to allogenic CD4⁺CD25⁺ cells whether from other CHB patients or age-and-sex matched HC subjects (n = 6). On the right panel, the number of Foxp3⁺ cells among different groups of CD4⁺CD25⁺ cells used to co-culture with CD4⁺CD25⁻ cells was clarified. Data in subfigures A, B, C, and E are expressed as the mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 5. CD4⁺ cells during HBV-LF promoted activation and proliferation of primary HSCs.

(A) Co-cultures between HSCs and CD4⁺ cells were monitored by a Cell-IQ® system: HSCs were marked as blue dots and CD4⁺ cells as green ones. (B) Growth curves of HSCs cultured alone or with CD4⁺ cells from age-and-sex matched HC and CHB subjects for 128 consecutive hours (n = 6). (C) The concentrations of TGF-β, PDGF-BB, CTGF and EGF in the supernatant at day 6 after co-cultures between HSCs and CD4⁺ cells from age-and-sex matched HC and CHB subjects (n = 6). (D) The α-SMA proteins in HSCs that were contactly co-cultured with CD4⁺ cells from age-and-sex matched HC and CHB subjects (n = 6) were analyzed by western blot and the relative quantification was also shown. Data in subfigures C and D are expressed as the mean ± SEM. *, p<0.05 compared to medium only group; ^#, p<0.05 compared to the HC-CD4 group; ***, p<0.001.

Figure 6. Treg and Th17 responses during HBV-LF differently regulated primary HSCs in vitro.

(A) IL-17 blocking dramatically inhibited the proliferation of HSCs, and re-addition of recombinant IL-17 enhanced HSCs proliferation in a dose-dependent manner (n = 6). Each line represents a CHB patient. (B) Recombinant IL-17 promoted PDGF-BB production by HSCs (n = 6). Each line represents a growth factor. (C) The α-SMA proteins in HSCs after co-cultures with CD4⁺CD25⁻ cells from age-and-sex matched HC and CHB subjects at day six determined by western blot, with or without IL-17 blocking, are presented and the relative quantification is shown (n = 6). (D) The α-SMA proteins in HSCs after IL-17 incubation at different concentrations for six days determined by western blot are presented in the right panel and the relative quantification is in the left panel (n = 3). (E) CD4⁺CD25⁺ cells inhibit the proliferation of HSCs in cell contact manner (n = 3). Data are expressed as the mean ± SEM. (F) CD4⁺CD25⁺ cells from CHB patients inhibited cell proliferation of HSCs in a dose-dependent manner (n = 6). Each line represents a CHB patient. (G) CD4⁺CD25⁺ cells from CHB patients inhibited PDGF-BB and TGF-β production of HSCs in dose-dependent manners (n = 6). Each line represents a growth factor. (H) The α-SMA proteins in HSCs after co-cultures with CD4⁺CD25⁺ cells from age-and-sex matched HC and CHB subjects at day six are presented and the relative quantification is shown (n = 6). Data in subfigures C, D, E&H are expressed as the mean ± SEM *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 7. Treg/Th17 balance regulated liver fibrosis progression in mouse models

. (A) Experimental protocol: Mice were i.v. injected with ConA weekly for up to four weeks to establish liver fibrosis models. Depletion of CD25⁺ or IL-17⁺ cells during fibrosis progression was performed with i.p. injection of either anti-CD25 or anti-IL-17 mAb one day before and after ConA injection twice a week. (B) Representative dot plots of CD4⁺CD25⁺ or CD4⁺IL-17⁺ cells in mouse livers are shown at week 4 after the administration of anti-CD25 or anti-IL-17 or isotype control mAb. (C) The frequencies of CD4⁺CD25⁺ cells in mouse livers and spleens with administration of anti-CD25 or isotype control mAb were determined at week 0, 1, 2, 3 and 4 after ConA injection. ***, p<0.001 compared to isotype at week 0. (D) The frequencies of CD4⁺IL-17⁺ cells in mouse livers and spleens with administration of anti-IL-17 or isotype control mAb were determined at week 0, 1, 2, 3 and 4 after ConA injection. **, p<0.01; ***, p<0.001 compared to isotype at week 0. (E) Serum ALT levels at week 0, 1, 2, 3 and 4 after ConA injection. (F) Representative Masson staining of livers in ConA or PBS treated mice is shown at week 4 after the administration of anti-CD25 or anti-IL-17 or isotype Ab (× 100 and × 200 magnification). n = 6–8 per group. (G) The mRNA levels of α-SMA, PDGF-BB, TGF-β, collagen type I and III in mouse livers at week 4 after ConA injection are shown in four groups: isotype, isotype + ConA, anti-CD25+ ConA and anti-IL-17+ ConA groups. Data are expressed as the mean ± SEM; *, p<0.05; **, p<0.01; ***, p<0.001.