Sex bias in experimental immune-mediated, drug-induced liver injury in BALB/c mice: suggested roles for Tregs, estrogen, and IL-6 - PubMed (original) (raw)

Sex bias in experimental immune-mediated, drug-induced liver injury in BALB/c mice: suggested roles for Tregs, estrogen, and IL-6

Joonhee Cho et al. PLoS One. 2013.

Abstract

Background and aims: Immune-mediated, drug-induced liver injury (DILI) triggered by drug haptens is more prevalent in women than in men. However, mechanisms responsible for this sex bias are not clear. Immune regulation by CD4+CD25+FoxP3+ regulatory T-cells (Tregs) and 17β-estradiol is crucial in the pathogenesis of sex bias in cancer and autoimmunity. Therefore, we investigated their role in a mouse model of immune-mediated DILI.

Methods: To model DILI, we immunized BALB/c, BALB/cBy, IL-6-deficient, and castrated BALB/c mice with trifluoroacetyl chloride-haptenated liver proteins. We then measured degree of hepatitis, cytokines, antibodies, and Treg and splenocyte function.

Results: BALB/c females developed more severe hepatitis (p<0.01) and produced more pro-inflammatory hepatic cytokines and antibodies (p<0.05) than did males. Castrated males developed more severe hepatitis than did intact males (p<0.001) and females (p<0.05). Splenocytes cultured from female mice exhibited fewer Tregs (p<0.01) and higher IL-1β (p<0.01) and IL-6 (p<0.05) than did those from males. However, Treg function did not differ by sex, as evidenced by absence of sex bias in programmed death receptor-1 and responses to IL-6, anti-IL-10, anti-CD3, and anti-CD28. Diminished hepatitis in IL-6-deficient, anti-IL-6 receptor α-treated, ovariectomized, or male mice; undetectable IL-6 levels in splenocyte supernatants from ovariectomized and male mice; elevated splenic IL-6 and serum estrogen levels in castrated male mice, and IL-6 induction by 17β-estradiol in splenocytes from naïve female mice (p<0.05) suggested that 17β-estradiol may enhance sex bias through IL-6 induction, which subsequently discourages Treg survival. Treg transfer from naïve female mice to those with DILI reduced hepatitis severity and hepatic IL-6.

Conclusions: 17β-estradiol and IL-6 may act synergistically to promote sex bias in experimental DILI by reducing Tregs. Modulating Treg numbers may provide a therapeutic approach to DILI.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Female BALB/c mice develop more severe experimental anesthetic DILI than do males.

(A) Female mice (n = 8) had significantly more severe hepatitis 3 weeks after TFA-S100/CFA immunizations than did males (n = 7/group). (B) Representative liver sections from female and male mice (H&E, magnification 64×). (C) Numbers of hepatic CD4+, CD8+, NK+, and NKT+ cells were significantly higher in females than in males. mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.

Figure 2

Figure 2. Experimental anesthetic DILI is associated with higher serum antibodies and proinflammatory cytokines in female BALB/c mice.

(A) TFA IgG, IgG1, and IgG2a and S100 IgG1 and IgG2a antibodies were significantly higher in females than in males (values expressed as OD). (B) IL-2, IL-9, IL-12, and IL-17 (pg/g) were significantly higher in livers from females than in those from males. IL-5, IL-6, and IL-13 (pg/g) were significantly higher in spleens from females, but IL-10 was higher in spleens from males. mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.

Figure 3

Figure 3. TFA-S100 immunizations impair splenic Treg expansion in female BALB/c mice.

(A) Representative dot plot of gated CD4+ T-cells and scatter plot showing no sex differences in hepatic CD4+CD25+FoxP3+ cell (Treg) levels 3 weeks after TFA-S100 immunizations (n = 5/group). (B) Representative dot plot of gated CD4+ T-cells and scatter plot showing significantly elevated splenic Tregs in males 2 weeks after immunizations (Mann-Whitney U, n = 15/group *p<0.05, **p<0.01, ***p<0.001).

Figure 4

Figure 4. Anti-IL-10 antibody diminishes Tregs in TFA-S100 – immunized male and female splenocyte cultures in vitro.

IL-10 blocking antibody (rat anti-mouse, 10 µg/mL), similarly diminished Tregs in female (A) and male (B) BALB/c mice isolated from TFA-S100- immunized mouse splenocytes challenged with CYP2E1 or the TFA (TFA-OVA) (Mann-Whitney U, *p<0.05, **p<0.01, ***p<0.001, experiments run in duplicate, n = 3 mice/group).

Figure 5

Figure 5. No sex bias in baseline Treg function in BALB/c mice.

(A) There was no sex bias in proliferation of T-effector cells (n = 10/group) co-cultured with naïve Tregs (Treg∶T-effector ratio 1∶1) in the presence of anti-CD3 (5 µg/mL) and anti-CD28 (2.5 µg/mL). (B) Using FACS analysis, of Tregs co-cultured as described in A, intracellular expression of inhibitory molecules PD-1(CD279, cloneJ43) was similar in female and male mice, and CTLA-4 (CD152, cloneUC10-489) was not detected in either sex (Experiments run in duplicate, n = 5–6 mice/group naive = filled histogram, stimulated = open histogram *p<0.05, **p<0.01, ***p<0.001).

Figure 6

Figure 6. IL-1β and IL-6 impair splenic Treg expansion in female BALB/c mice.

(A) Naïve mice exhibited no sex bias in IL-6 or IL-1β level or in number of Tregs; however, IL-6 and IL-1β were significantly elevated in splenocytes isolated from female mice 2 weeks after immunization and stimulated with CYP2E1 or TFA in vitro (10 µg/mL; Data are shown as mean ± SEM. Experiments run in duplicate n = 5/group). (B) The number of Tregs was significantly reduced in cultures from females when compared to males. (Experiments run in duplicate, n = 3 mice/group, Mann-Whitney U, * = p<0.05, ** = p<0.01, *** = p<0.001).

Figure 7

Figure 7. Increased T and B cells are associated with impaired splenic Treg expansion in female BALB/c mice following TFA-S100 immunizations.

Splenocytes were isolated from female mice 2 weeks after TFA-S100 immunizations, stimulated with CYP2E1 (A) or TFA (B) in vitro (10 µg/mL) and analyzed for Tregs as described in Figure 6. CD3+CD25 T cells and B220+ cells were significantly elevated in the same cultures from females when compared to males. (Experiments run in duplicate, n = 3 mice/group, Mann-Whitney U, * = p<0.05, ** = p<0.01, *** = p<0.001).

Figure 8

Figure 8. IL-6 supplementation diminishes Tregs in TFA-S100 – immunized male and female splenocyte cultures in vitro.

IL-6 supplementation (25 ng/mL) of in vitro splenocyte cultures from female (A) and male (B) TFA-S100-immunized BALB/c mice diminished Tregs in both groups. (Experiments run in duplicate, n = 3 mice/group, Mann-Whitney U, * = p<0.05, ** = p<0.01, *** = p<0.001).

Figure 9

Figure 9. Anesthetic DILI is diminished in IL-6 deficient (−/−) mice.

IL-6 −/− mice were immunized with TFA-S100 as described in the Methods section. (A) Twenty one days later, IL-6−/− mice developed significantly less hepatic injury (0.5±0.3, mean ± SEM) than did BALB/cBy mice (2.5±0.4). (B) IL-6−/− mice also produced lower TFA, S100, and CYP2E1 IgG1 and S100 and CYP2E1 IgG2a antibody levels than did BALB/cBy controls (n = 4/group, Mann-Whitney U, * = p<0.05).

Figure 10

Figure 10. IL-6Rα antibodies diminish anesthetic DILI in female BALB/c mice.

Female BALB/c mice were immunized with TFA-S100 as described in the Methods section. (A) IL-6Rα antibodies administered days 0 and 7 diminished hepatitis as demonstrated by decreased inflammation/injury scores (1.6±0.1, mean ± SE) when compared to TFA-S100 – immunized, female , BALB/c mice not treated with IL-6Rα but administered phosphate buffered saline at the same time points (2.3±0.2, mean ± SE). (B) Anti-TFA IgG and IgG2a subclass antibody levels measured in mouse sera were also decreased (p<0.05) when compared to mice immunized with TFA-S100 without blocking antibodies. (B) S100 and (C) CYP2E1 autoantibody levels were not significantly different when comparing sera of mice immunized with TFA-S100 ± anti-IL-6Rα. (Mann – Whitney U, n = 5/group, *p<0.05).

Figure 11

Figure 11. Ovariectomy reduces anesthetic DILI in female BALB/c mice.

Intact BALB/c females and males as well as females who had undergone ovariectomy were immunized with TFA-S100 as described in the Methods. By 3 weeks, the intact females had developed significantly more severe hepatitis (A, *p<0.05) and exhibited significantly higher levels of TFA IgG1 antibody levels (B) compared to ovariectomized (OVX, *p<0.05) or male mice (**p<0.01, n = 4 mice/group). (C) Splenic IL-6 levels were significantly higher in intact female mice than in OVX or male mice at 2 weeks after immunizations. (Mann-Whitney U, * = p<0.05, ** = p<0.01).

Figure 12

Figure 12. Elevated IL-6 levels are associated with more severe anesthetic DILI in castrated male BALB/c mice.

Intact BALB/c females and males, as well as males who that had undergone castration, were immunized with TFA-S100 as described in the Methods section (A) By 3 weeks, castrated males (CAS) had developed significantly more severe hepatitis than females (*p<0.05) and intact males (***p<0.001). (B) Intact males exhibited significantly lower levels of TFA IgG antibodies when compared to females or intact male mice (*p<0.05, n = 4 mice/group). (C) Splenic IL-6 levels were significantly higher in CAS than in intact male mice. (Mann-Whitney U, * = p<0.05, *** = p<0.001).

Figure 13

Figure 13. Elevated 17β-estratiol levels are associated with more severe anesthetic DILI in castrated male BALB/c mice.

(A) Serum 17β-estradiol levels (pg/mL) were similar when comparing intact females, OVX and male mice (top panel); however serum 17β-estradiol levels were higher in CAS when compared to intact male mice (bottom panel, *p<0.05). (B) Serum testosterone levels (ng/mL) were higher in male mice when compared to intact female or OVX (top, ***p<0.001) and when compared to female or CAS (bottom, ***p<0.001), (Mann-Whitney U, * = p<0.05, *** = p<0.001).

Figure 14

Figure 14. 17β-estradiol (E2) induces pro-inflammatory cytokines in naïve splenocytes from BALB/c mice.

17β-estradiol (E2, 50 nM) increased IL-1β production in splenocytes cultured from naïve male BALB/c mice when compared to splenocytes cultured from naïve male BALB/c mice treated with β-cyclodextrin (BC, 5 mM) as a control (A, *p<0.05). E2 significantly increased IL-6 (B) and TNF-α (C) production in splenocytes cultured from naïve females when compared to splenocytes from naïve females treated with BC (*p<0.05). (Experiments run in duplicate, n = 5/group, Mann-Whitney U, * = p<0.05).

Figure 15

Figure 15. Tregs reduce experimental anesthetic DILI in female BALB/c mice.

Two weeks after the TFA-S100 immunizations, mice were given intravenous injections of 1×106 freshly isolated Tregs in PBS or PBS alone (control); the mice were killed 7 days later. The degree of hepatitis was significantly reduced in mice administered Tregs compared to those administered vehicle. Data are shown as mean ± SEM., n = 8–10 mice/group, Mann-Whitney U, *p<0.05, **p<0.01, ***p<0.001.

Figure 16

Figure 16. Tregs reduce hepatic pro-inflammatory cytokine levels following the induction of experimental anesthetic DILI in female BALB/c mice.

Liver supernatants were analyzed from BALB/c females immunized with TFA-S100 ± Tregs. IL-4, IL-5, IL-6, IL-7, IL-9, IL-12, IL-13, IL-15, IL-17, and TNF-α (pg/g) were significantly lower in livers of Treg-treated mice. Data are shown as mean ± SEM., n = 8–10 mice/group Mann-Whitney U, *p<0.05, **p<0.01, ***p<0.001.

Figure 17

Figure 17. Tregs reduce splenic IL-1β and IL-5 levels following the induction of experimental anesthetic DILI in female BALB/c mice.

Spleen supernatants were analyzed from BALB/c females immunized with TFA-S100 ± Tregs. IL-1β and IL-5 were significantly lower in spleens of Treg-treated mice when compared to levels in vehicle-treated mice. Data are shown as mean ± SEM., n = 8–10 mice/group, Mann-Whitney U, *p<0.05, **p<0.01, ***p<0.001.

Figure 18

Figure 18. Tregs reduce serum S100 and TFA antibody levels following the induction of experimental anesthetic DILI in female BALB/c mice.

Spleen supernatants were analyzed from BALB/c females immunized with TFA-S100 ± Tregs. S100 IgG and IgG1 (A), TFA IgG and IgG1, and TFA IgG2a. (B) Antibodies were lower in the sera from Treg-treated group than in controls. Data are shown as mean ± SEM., n = 8–10/group, Mann-Whitney U, *p<0.05, **p<0.01, ***p<0.001.

References

    1. Bourdi M, Chen W, Peter RM, Martin JL, Buters JT, et al. (1996) Human cytochrome P450 2E1 is a major autoantigen associated with halothane hepatitis. Chem Res Toxicol 9: 1159–1166. - PubMed
    1. Neuberger J, Vergani D, Mieli-Vergani G, Davis M, Williams R (1981) Hepatic damage after exposure to halothane in medical personnel. Br J Anaesth 53: 1173–1177. - PubMed
    1. Njoku D, Laster MJ, Gong DH, Eger EI, Reed GF, et al. (1997) Biotransformation of halothane, enflurane, isoflurane, and desflurane to trifluoroacetylated liver proteins: association between protein acylation and hepatic injury. Anesth Analg 84: 173–178. - PubMed
    1. Pohl LR (1990) Drug-induced allergic hepatitis. Semin Liver Dis 10: 305–315. - PubMed
    1. Njoku DB, Talor MV, Fairweather D, Frisancho-Kiss S, Odumade OA, et al. (2005) A novel model of drug hapten-induced hepatitis with increased mast cells in the BALB/c mouse. Exp Mol Pathol 78: 87–100. - PubMed

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