Suppressive and pro-inflammatory roles for IL-4 in the pathogenesis of experimental drug-induced liver injury

Abstract

The pathogenesis of immune-mediated drug-induced liver injury (DILI) following halogenated anesthetics, carbamazepine or alcohol has not been fully elucidated. Detecting cytochrome P450 2E1 (CYP2E1) IgG4 auto-antibodies in anesthetic DILI patients suggests a role for IL-4 in this hapten-mediated process. We investigated IL-4-mediated mechanisms using our model of experimental DILI induced by immunizing BALB/c (WT) and IL-4(-/-) (KO) mice with S100 liver proteins covalently modified by a trifluoroacetyl chloride (TFA) hapten formed following halogenated anesthetic metabolism by CYP2E1. WT mice developed more hepatitis, TFA and S100 antibodies (p<0.01), as well as T-cell proliferation to CYP2E1 and TFA (p<0.01) than KO mice. Additionally, WT CD4(+) T cells adoptively transferred hepatitis to naïve Rag(-/-) mice (p<0.01). Pro-inflammatory cytokines were expectedly decreased in TFA hapten-stimulated KO splenocyte supernatants (p<0.001); however, IL-2 and IFN-gamma (p<0.05), as well as IL-6 and IL-10 (p<0.001) levels were elevated in CYP2E1-stimulated KO splenocyte supernatants, suggesting dual IL-4-mediated pro-inflammatory and regulatory responses. Anti-IL-10 administered to KO mice increased hepatitis, TFA and CYP2E1 antibodies in KO mice confirming a critical role for IL-4. This is the first demonstration of dual roles for IL-4 in the pathogenesis of immune-mediated DILI by suppressing auto-antigen-induced regulatory responses while promoting hapten-induced pro-inflammatory responses.

Figure 1

Immune responses in WT splenocytes have a key role in experimental DILI. Stimulation indices were elevated in splenocyte cultures re-stimulated with the indicated concentrations of either (A) CYP2E1 or (B) KLH-TFA after isolation from TFA-S100 when compared to CFA-immunized WT mice (*p<0.05, Mann-Whitney U. Mean ± SEM of pooled data from individual mice. Experiments were performed in triplicate with n = 4 mice/group). (C) Hepatitis was evident in naïve female Rag −/− mice 10 days after receiving, by adoptive transfer, 1 × 10⁷ TFA-S100-immunized WT mixed splenocytes or CD4+ T cells re-stimulated with CYP2E1 (RAG 2E1 or RAG CD4+ T cells 2E1) or with TFA (RAG TFA or RAG CD4+ T cells TFA) when compared to RAG CFA or RAG Media (*p<0.05, **p<0.01, Mann-Whitney U. Experiments were performed in triplicate with n = 2 mice/group). (D) There were 30 and 20% increases in CD3+CD4+ T cell numbers, 8% increases and decreases in CD3+CD8+ T cell numbers, 20% decreases and 10% increases in B cell numbers, 24 % and 15% decreases in DX5+ NK cell numbers and 18 and 9 % decreases in DX5+CD3+ NKT cell numbers following stimulation with CYP2E1 and TFA, respectively. Shown are mean of pooled data from individual mice. Experiments were performed in duplicate with N = 4 mice/group. (E, F) Cytokine levels in splenocyte supernatants by ELISA prior to transfer, showed significantly elevated IL-1β, IL-2, IL-6 and IL-13 in supernatants from (E) CYP2E1-re-stimulated WT splenocytes (*p<0.05, **p<0.01), while supernatants from (F) TFA-stimulated WT splenocytes had significantly elevated IL-1β, IL-2, IL-6 and IFN-γ when compared to CFA-immunized mice (*p<0.05, **p<0.01, ***p<0.001, Mann-Whitney U. Mean ± SE of pooled data from individual mice. Experiments were performed in duplicate with N = 5 mice/group).

Figure 2

TFA-S100 significantly increases splenic and hepatic levels of IL-4 in WT mice. (A, B) WT mice demonstrated significantly more hepatitis three weeks following TFA-S100/CFA immunizations when compared to immunization with CFA alone. Shown are representative liver tissue sections stained with H&E (0.5 µm, Magnification 64x). (C) Analysis of supernatants from cultured mixed splenocytes obtained one week following TFA-S100 immunization showed significantly higher levels of IL-4 when re-stimulated with either CYP2E1 or TFA in comparison with non-stimulated splenocytes (media); *p<0.05, Mann-Whitney U. Mean ± SEM of pooled data from individual mice. Experiments were run in duplicate with N = 4 mice/group. (D) Cytokine expression in liver tissue supernatants from CFA and S100 controls as well as TFA-S100 – immunized BALB/c mice at 3 weeks confirmed mixed Th1 (IL-1β and IL-6) and Th2 (IL-4 and IL-13) cytokines, as well as increased expression of the neutrophil chemoattractant MIP-2 and diminished levels of IL-10 (**p<0.01, ***p <0.001, Mann-Whitney U. Mean ± SEM of pooled data from individual mice. Experiments were run in duplicate with N=5 mice/group).

Figure 3

IL-4 has a critical role in the development of experimental anesthetic DILI. Representative H & E histological section from (A) WT and (B) IL-4−/− (KO) mice demonstrating significantly more hepatitis in WT mice (A and B, 0.5µm, representative data, 64x magnification). (C) Composite injury scores in WT and KO mice showed increased hepatitis in WT when compared to KO mice (**p<0.01, Mann-Whitney U. Experiments were performed in duplicate with N=5 mice/group). (D) Sera analysis for TFA and S100 antibodies showed significantly elevated TFA IgG1 antibodies as well as anti-S100 IgG1 and IgG2a antibodies in WT compared to KO mice (***p<0.001, ANOVA with Tukey’s post test. Mean ± SEM of pooled data from individual mice. Experiments were performed in duplicate with N=5 mice/group). (E, F) Analysis of cytokines in liver tissue supernatants showed that WT mice had significantly elevated levels of IL-1β, IL-4, IL-5, IL-6, IL-12p70, IL-13 and TNF-α when compared to KO mice (*p<0.05, **p<0.01, ***p<0.001, Mann-Whitney U. Mean ± SEM of pooled data from individual mice. Experiments were performed in duplicate with N=5 mice/group ).

Figure 4

IL-4 promotes T cell priming in experimental anesthetic DILI. After 72h, proliferation to (A) CYP2E1 and (B) TFA was significantly higher in WT splenocytes when compared to KO mice at all CYP2E1 or TFA doses (**p<0.01, ***p<0.001, Mann-Whitney U. Mean ± SEM of pooled data from individual mice. Experiments were run in triplicate with N = 5 mice/group). Supernatants from (C) CYP2E1-stimulated KO splenocyte cultures had significantly elevated concentrations of IL-2, IL-6, IL-10 and IFN-γ (*p<0.05, ***p<0.001 while (D) TFA-stimulated KO splenocyte cultures had significantly lower concentrations of IL-1β, IL-2, IL-6, IL-13 and IFN-γ, when compared to WT cultures (*p<0.05, **p<0.01, Mann-Whitney U. Mean ± SEM of pooled data from individual mice. Experiments were run in duplicate with N = 5 mice/group).

Figure 5

IL-4 suppression of responses to CYP2E1 does not involve regulatory T cells. (A) The percentages of Foxp3+CD4+CD25+ T cells were not significantly different in KO (9.4 ± 4.0%) when compared to WT (12.3 ± 3.2%) mice (Mean ± SEM of pooled data from individual mice. Experiments were run in duplicate with N = 4 mice/group). (B) TGF-β levels were not significantly different in splenocytes isolated from KO (1716 ± 156.3pg/ml) or WT (1511 ± 241.6pg/ml) mice and re-stimulated for 72h with 10µg/ml CYP2E1 Mean ± SEM of pooled data from individual mice. Experiments were run in duplicate with N = 5 mice/group. (C) Representative flow cytometric analysis of intracellular IL-10 and IFN-γ levels in splenocytes isolated from TFA-S100 – immunized KO mice and activated with PMA and ionomycin. No IFN-γ and IL-10 double positive cells were detected, although IFN-γ single positive and a few IL-10 single positive cells were seen.

Figure 6

Anti-IL-10 increases hepatitis, as well as CYP2E1 and TFA antibodies in KO mice. TFA-S100 immunization in combination with injection of (A) 200µl PBS or (B) 250µg anti-IL-10 blocking antibodies on days 14, 16, 18 and 20 increased the severity of hepatitis in comparison with the control. Representative data are shown (64x magnification). (C) Histology scores (2.2 ± 0.3 vs. 1.0 ± 0.5, mean ± SE, *p<0.05, (D) sera anti-CYP2E1 IgG autoantibodies (0.736 ± 0.111 vs. 0.374 ± 0.028, mean ± SE, *p<0.05) and (E) anti-TFA IgG1 antibodies (2.134 ± 0.319 vs. 1.089 ± 0.317, mean ± SE, *p<0.05) were observed for anti-IL-10– vs PBS -treated female KO mice respectively (Mann-Whitney U. Pooled data from individual mice. Experiments were performed in duplicate with N = 4 mice/group).

Figure 7

Proposed mechanism of immune-mediated DILI. In our model of experimental (murine), immune-mediated, anesthetic DILI, BALB/c mice develop an IL-4-promoted hypersensitive response to the TFA hapten and S100 liver proteins resulting in splenocyte TNF-α, IL-1β, IL-6 production. IL-4-dependent splenocyte proliferation is also demonstrated in response to human CYP2E1 and TFA through suppression of IL-10 and possibly IFN-γ regulatory responses. Immune responses to CYP2E1, S100 and TFA also activate mast cells that recruit neutrophils to the liver. Directly or indirectly through IL-10, a predominantly IL-4-driven Th2-type response leads to S100 and CYP2E1 autoantibody production as well TFA hapten antibodies.