CD4+CD25+ Regulatory T Cells Inhibit Natural Killer Cell Hepatocytotoxicity of Hepatitis B Virus Transgenic Mice via Membrane-Bound TGF-β and OX40 - PubMed (original) (raw)

CD4+CD25+ Regulatory T Cells Inhibit Natural Killer Cell Hepatocytotoxicity of Hepatitis B Virus Transgenic Mice via Membrane-Bound TGF-β and OX40

Yongyan Chen et al. J Innate Immun. 2016.

Abstract

CD4+CD25+ regulatory T cells (Tregs) are involved in the regulation of physiological and pathological hepatic immune responses, but the roles are not well explored in natural killer (NK) cell-mediated liver diseases. In this study, using the NK cell-mediated oversensitive liver injury model of hepatitis B virus transgenic (HBs-Tg) mice triggered by a low dose of concanavalin A, it was observed that an increased number of CD4+CD25+Foxp3+ Tregs were accumulated in the liver, along with the recovery of liver injury. Adoptive transfer of hepatic Tregs from HBs-Tg mice but not wild B6 mice could significantly attenuate the oversensitive liver injury via inhibiting liver accumulation and decreasing NK cell group 2D-mediated activation of NK cells in the recipient HBs-Tg mice. Furthermore, upregulated expression of membrane-bound TGF-β (mTGF-β) and OX40 on hepatic Tregs were demonstrated to account for inhibiting the NK cell-mediated hepatic injury in HBs-Tg mice through cell-cell contact, confirmed by antibody blockade and cell Transwell experiments in vivo and in vitro. Our findings for the first time indicated that CD4+CD25+ Tregs directly suppressed NK cell-mediated hepatocytotoxicity through mTGF-β and OX40/OX40L interaction in a cell-cell contact manner in HBV-associated liver disease.

© 2015 S. Karger AG, Basel.

PubMed Disclaimer

Figures

Fig. 1

Fig. 1

Higher content of CD4+CD25+ Tregs in the liver of HBs-Tg mice. MNCs prepared from thymus, spleen, liver and peripheral blood of HBs-Tg and B6 mice were analyzed by flow cytometry. a The percentages represent the net percentage of cells in the appropriate quadrant. Furthermore, CD4+CD25- and CD4+CD25+ cells were gated to analyze their Foxp3 expression. These are from a single experiment representative of 4 mice in each group. b Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01 compared with the control group.

Fig. 2

Fig. 2

Accumulation of CD4+CD25+ Tregs in NK cell-mediated liver injury of HBs-Tg mice. A Low dose of Con A (3 μg/g body weight; a) or high dose of Con A (15 μg/g body weight; b) was injected i.v. into HBs-Tg and B6 mice. At 24, 48, 72 and 96 h after injection, serum ALT activity was determined and MNCs were prepared from the liver and then analyzed by flow cytometry. The number of CD4+CD25+Foxp3+ Tregs was calculated according to the percentage of cells multiplied by total viable MNCs. Data are shown as the mean ± SEM in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control group.

Fig. 3

Fig. 3

CD4+CD25+ Tregs inhibited NK cell-mediated liver injury in HBs-Tg mice. a The procedures of adoptive CD4+CD25+ Treg transfer into the liver of HBs-Tg mice. Hepatic CD4+CD25+ Tregs were purified from HBs-Tg mice treated with Con A [3 μg/g body weight (wt)] for 48 h or from B6 mice treated with Con A (15 μg/g body weight) for 24 h. Tregs (2 × 105 or 3 × 105) were adoptively transferred into the liver of recipient HBs-Tg or B6 mice, followed by Con A injection (3 μg/g body weight for HBs-Tg and 15 μg/g body weight for B6). Sham group mice were transferred with 100 μl of medium followed by Con A injection. b At 18 h after Treg transfer, serum ALT levels were measured in the recipient mice. c At 18 h after Treg transfer, liver injury of the recipient mice was observed by liver pathology (HE stain. ×200). Data are shown as the mean ± SEM in each group. ** p < 0.01, *** p < 0.001 compared with the control group.

Fig. 4

Fig. 4

NK cell activation was inhibited by CD4+CD25+ Tregs from HBs-Tg mice. Hepatic CD4+CD25+ Tregs were isolated from HBs-Tg mice treated with a low dose of Con A (3 μg/g body weight) for 48 h, and then Tregs (2 × 105) were adoptively transferred into the liver of HBs-Tg mice, followed by a low dose of Con A (3 μg/g body weight) injection. At 18 h after Treg transfer, MNCs were prepared from the liver and analyzed by flow cytometry. a The percentages represent the net percentage of cells in the appropriate quadrant. b The percentages of hepatic NK cells (CD3-NK1.1+) and NKT cells (CD3+NK1.1+) were statistically analyzed in each group. c The number of liver MNCs, NK cells and NKT cells were calculated. d The expression of CD69 on hepatic NK cells and NKT cells are shown in the histogram. e The mean fluorescence (MFI) of CD69 expressed by hepatic NK cells and NKT cells were statistically analyzed in each group. Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control group.

Fig. 5

Fig. 5

NKG2D function of NK cells was regulated by CD4+CD25+ Tregs in HBs-Tg mice. a A low dose of Con A (3 μg/g body weight) and a high dose of Con A (15 μg/g body weight) was injected i.v. into HBs-Tg mice and B6 mice, respectively. At 48 or 24 h after Con A injection, MNCs were prepared from the livers. The expression of surface NKG2D on NK cells (CD3-NK1.1+) was analyzed by flow cytometry. b Hepatic CD4+CD25+ Tregs were isolated from HBs-Tg mice treated with a low dose of Con A (3 μg/g body weight) for 48 h, and then Tregs (2 × 105) were adoptively transferred into the liver of HBs-Tg mice, followed by a low-dose Con A (3 μg/g body weight) injection. At 18 h after Treg transfer, MNCs were prepared from the livers and the expression of surface NKG2D on NK cells (CD3-NK1.1+) was analyzed by flow cytometry. The mean fluorescence intensity (MFI) of surface NKG2D expressed on hepatic NK cells was statistically analyzed in each group. Data are presented as the mean ± SEM. * p < 0.05 compared with the control group.

Fig. 6

Fig. 6

CD4+CD25+ Tregs inhibited hepatocytotoxicity of NK cells through mTGF-β and OX40/OX40L interaction in HBs-Tg mice. a Hepatic CD4+CD25+ Tregs were analyzed for the expression of mTGF-β and OX40 in HBs-Tg mice treated with a low dose of Con A (3 μg/g body weight) for 48 h, or in B6 mice treated with a high dose of Con A (15 μg/g body weight) for 24 h. b Hepatic NK cells (CD3-NK1.1+) were analyzed for the expression of OX40L in HBs-Tg mice treated with a low dose of Con A (3 μg/g body weight) or in B6 mice treated with a high dose of Con A (15 μg/g body weight) for 24 and 48 h. Mean fluorescence intensity (MFI) data are shown as the mean ± SEM. c, d At 24 h after the low-dose Con A (3 μg/g body weight) injection, anti-TGF-β antibody or anti-OX40L antibody was injected i.p. into HBs-Tg mice, and then at each indicated time point (48, 72 and 96 h) serum ALT activity was determined. At 96 h after Con A injection, liver samples were collected for HE staining. e Four-hour AST release assay was performed to test the effect of Tregs on the cytotoxicity of hepatic NK cells against hepatocytes. Hepatic NK cells purified from 2-hour Con A-treated HBs-Tg mice were added to the freshly isolated hepatocytes (1 × 104) from 2-hour Con A-treated HBs-Tg mice at the E/T of 10/1. The Treg/NK cell ratio = 2:1. A dose of 40 μg/ml anti-TGF-β mAb or 20 μg/ml anti-OX40L mAb was added to the culture. Data are presented as the mean ± SEM from triplicates in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control group.

References

    1. Chen Y, Wei H, Sun R, Dong Z, Zhang J, Tian Z. Increased susceptibility to liver injury in hepatitis B virus transgenic mice involves NKG2D-ligand interaction and natural killer cells. Hepatology. 2007;46:706–715. - PubMed
    1. Wei HX, Chuang YH, Li B, Wei H, Sun R, Moritoki Y, Gershwin ME, et al. CD4+CD25+ Foxp3+ regulatory T cells protect against T cell-mediated fulminant hepatitis in a TGF-β-dependent manner in mice. J Immunol. 2008;181:7221–7229. - PubMed
    1. Erhardt A, Biburger M, Papadopoulos T, Tiegs G. IL-10, regulatory T cells, and Kupffer cells mediate tolerance in concanavalin A-induced liver injury in mice. Hepatology. 2007;45:475–485. - PubMed
    1. Miethke AG, Saxena V, Shivakumar P, Sabla GE, Simmons J, Chougnet CA. Post-natal paucity of regulatory T cells and control of NK cell activation in experimental biliary atresia. J Hepatol. 2010;52:718–726. - PMC - PubMed
    1. Xu D, Fu J, Jin L, Zhang H, Zhou C, Zou Z, Zhao JM, et al. Circulating and liver resident CD4+CD25+ regulatory T cells actively influence the antiviral immune response and disease progression in patients with hepatitis B. J Immunol. 2006;177:739–747. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources