International Immunopharmacology 25 (2015) 293–301
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Regulatory T cells ameliorate acetaminophen-induced immune-mediated liver injury Xuefu Wang a, Rui Sun a,b, Yongyan Chen a, Zhe-Xiong Lian a,b, Haiming Wei a,b, Zhigang Tian a,b,c,⁎ a Institute of Immunology and CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center, University of Science & Technology of China, Hefei, Anhui 230027, China b Hefei National Laboratory for Physical Sciences at Microscale, Hefei, Anhui 230027, China c Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China
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Article history: Received 16 September 2014 Received in revised form 1 January 2015 Accepted 4 February 2015 Available online 14 February 2015 Keywords: Acetaminophen Adaptive immune cells Regulatory T cells CXCL10 Liver injury
a b s t r a c t The contribution of innate immune cells to acetaminophen (APAP)-induced liver injury has been extensively investigated. However, the roles of T cell populations among adaptive immune cells in APAP-induced liver injury remain to be elucidated. Herein, we found that distinct CD4+ T cell subsets but not CD8+ T cells modulated APAPinduced liver injury in mice. After APAP challenge, more CD62LlowCD44hiCD4+ T cells appeared in the liver, accompanied by increased IFN-γ. The removal of CD4+ T cells by either antibody depletion or genetic deficiency markedly compromised pro-inflammatory cytokine levels and ameliorated liver injury. Meanwhile, we also found that the frequency and absolute number of Treg cells also increased. Treg cell depletion increased hepatic CD62LlowCD44hiCD4+ T cells, augmented pro-inflammatory cytokines, and exacerbated liver injury, while adoptive transfer of Treg cells ameliorated APAP-induced liver injury. Furthermore, the recruitment of Treg cells into the liver through specific expression of CXCL10 in the liver could ameliorate APAP-induced liver injury. Our investigation suggests that Th1 and Treg subsets are involved in regulating APAP-induced liver injury. Thus, modulating the Th1/Treg balance may be an effective strategy to prevent and/or treat APAP-induced liver injury. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that is generally safe to use at therapeutic doses. However, there are many cases in which careless use of APAP leads to overdose and subsequent acute liver injury. Mortality by APAP overdose has even been reported in the USA [1], and the Federal Drug Administration (FDA) in the USA has now limited the amount of APAP allowed in prescription combination products. Mechanistically, an overdose of APAP generates an excess of N-acetyl-p-benzoquinone imine (NAPQI), leading to hepatocyte death [2,3]. Studies in murine models of APAP overdose show that the inflammatory response induced by damageassociated molecular pattern molecules (DAMPs) released from dead hepatocytes exaggerates liver injury [4]. The pro-inflammatory cytokine IFN-γ plays a critical role in mediating APAP-induced liver injury [5]. In terms of cellular mediators, innate immune cells such as neutrophils and macrophages are known to mediate APAP-induced liver injury [6, 7]. However, whether and how adaptive immune cells—especially ⁎ Corresponding author at: School of Life Sciences, University of Science and Technology of China, #443 Huangshan Road, Hefei 230027, China. Tel.: +86 551 6360 0845; fax: +86 551 6360 6783. E-mail address: [email protected] (Z. Tian).
http://dx.doi.org/10.1016/j.intimp.2015.02.008 1567-5769/© 2015 Elsevier B.V. All rights reserved.
CD8+ and CD4+ T cells and their functional subsets—play a role in APAP-induced liver injury has not been well documented. The inhibition of acute inflammation shortly after it begins is generally accepted as an active process that is quickly initiated by inflammatory responses in order to minimize immune-mediated damage to host tissues [8]. The regulatory T cell (Treg) subset of CD4+ T cells is an important regulator in the resolution phase of inflammation. Various chemokine receptors are expressed on Treg cells for the organ-specific localization of Treg cells [9]. The CCL22 signaling-induced Treg Cell recruitment promotes venous metastases of HBV-positive hepatocellular carcinoma [10]. Therefore, modulating the recruitment of Treg cells via the chemokine–receptor axis has been advised to treat the organ-specific inflammation or tumor. CCL22-mediated Treg recruitment to the pancreatic islets prevents murine autoimmune diabetes [11]. The recruitment of CCR5+ Treg cells prevents the exacerbation of chronic inflammation in the intestine [12]. The CXCR3+ Treg recruitment protects against αGalCer- and Con A-induced liver injury [13,14]. Up to now, whether Treg cells are involved in APAP-induced liver injury remains unclear. Moreover, whether Treg recruitment into the inflamed liver can help to treat APAP-induced liver injury is worth investigating. In this study, we explored the roles of adaptive immune cells in the progression of APAP-induced liver injury. The data presented here demonstrated that Th1 cells but not CD8+ T cells exacerbated APAP-
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induced liver injury and that Treg cells attenuated APAP-induced liver injury. Moreover, the recruitment of Treg cells into the liver is helpful to suppress APAP-induced liver injury. Thus, modulating Treg cells may be a useful strategy to ameliorate drug-induced immuneexacerbated liver injury.
2. Materials and methods 2.1. Mice Male C57BL/6 mice (6–8 weeks old) were purchased from the Shanghai Laboratory Animal Center at the Chinese Academy of Sciences. Rag-1−/− mice were purchased from the Model Animal Research Center at Nanjing University. All mice, including CD4−/−, and CD8−/− mice, were housed in microisolator cages under humidityand temperature-controlled specific pathogen-free conditions in the animal facility at the School of Life Sciences of the University of Science and Technology of China. Mice were maintained on an irradiated sterile diet and given autoclaved water. All experiments were performed according to the animal care regulations of the University of Science and Technology of China. The study was approved by the Local Ethics Committee for Animal Care and Use at University of Science and Technology of China.
2.3. Immune cell depletion and IL-10 blocking 0.2 mg/mouse anti-CD4 mAb (clone TIB-207, ATCC) was i.v. injected into mice to deplete CD4+ T cells, 0.2 mg/mouse anti-CD25 mAb (clone TIB-222, ATCC) to deplete CD25+ Treg cells, 0.2 mg/mouse PK136 (clone HB-191, ATCC) to deplete NK cells and NKT cells, and 30 μl/mouse anti-ASGM1 antibody (WAKO Pure Chemical Industries, Ltd, Osaka, Japan) to deplete NK cells, 48 h before APAP treatment. 0.2 mg/mouse anti-IL-10 antibody (eBioscience, San Diego, CA, USA) was i.v. injected into mice to block IL-10 at 1 h before APAP treatment. The isotype antibody was used as control. 2.4. Isolation and adoptive transfer of Treg cells Spleens were excised from normal mice, made into single-cell suspensions, and treated with red blood cell (RBC) lysis buffer to lyse erythrocytes. The pellets were resuspended, and cell number was determined. CD4+CD25+ Treg cells were isolated using the mouse CD4+CD25+ Regulatory T Cell Isolation Kit (Order no. 130-091-041, Miltenyi Biotec Inc., Bergisch Gladbach, Germany) according to the manufacturer's instruction manual. The purity of isolated Treg cells was N90%. Purified cells (2 × 106 cells/mouse) were adoptively transferred (i.v.) into recipient mice, and an equal volume of PBS was used as a control.
2.2. APAP treatment
2.5. Overexpression of chemokines in the liver
Experimental mice were fasted for 16 h and subsequently injected with PBS control or freshly prepared APAP (dissolved in PBS) intraperitoneally (i.p.) (400 mg/kg body weight, Sigma-Aldrich, St. Louis, MO, USA). At the indicated time points, mice were anesthetized, bled, and sacrificed. Sera were isolated for testing alanine aminotransferase (ALT) levels and cytokines. Liver tissues were excised for analyzing cell populations, extracting RNA, and cutting tissue sections.
The expression vectors pLIVE-CXCL10 and pcDNA3.0-hIL-10 were constructed in our lab. The expression vector pCMV3-TGF-β was purchased from Sino Biological Incorporation (Beijing, China). These vectors were hydrodynamically injected into the tail vein in a 2-ml volume at indicated times before APAP treatment respectively, as described previously [15]. The null vector (pLIVE-negative, pcDNA3.0negative and pCMV3-negative) was used as the control.
Fig. 1. Adaptive immune cells aggravate APAP-induced liver injury. Mice were fasted for 16 h and injected i.p. with acetaminophen (APAP) at 400 mg/kg body weight or equal volumes of PBS as a control (n = 5). (A) ALT levels increased in APAP-challenged mice. Sera were collected 24 h after APAP challenge. (B) IFN-γ and TNF-α levels increased in the sera after APAP challenge. (C) ALT levels were similar between mice treated with PK136 (to deplete NK cells and NKT cells) and control mAb, or αASGM1 (to deplete NK cells) and control mAb. Mice were treated with mAb 48 h before APAP challenge. (D) ALT levels decreased in Rag−/− mice. The data are representative of three independent experiments and are shown as the mean ± SEM. **, P b 0.01; ***, P b 0.005.
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Fig. 2. CD8+ T cells do not contribute to APAP-induced liver injury. (A) ALT levels were similar between C57BL/6 and CD8−/− mice. Sera were collected 24 h after APAP challenge (n = 5). (B) Systemic IFN-γ and TNF-α levels were similar between C57BL/6 and CD8−/− mice. The data are representative of three independent experiments and are shown as the mean ± SEM.
2.6. Assessment of liver injury Liver injury was evaluated by ALT levels, immunohistochemical staining, and mouse survival. ALT levels were measured using a diagnostic kit (Rongsheng, Shanghai, China). Liver specimens were fixed with 4% paraformaldehyde, dehydrated with graded alcohol, embedded in paraffin, cut into tissue sections, and stained with hematoxylin and eosin (H&E). Mouse survival was observed at the indicated hours after APAP treatment. 2.7. RNA isolation and quantitative RT-PCR (qRT-PCR) analysis RNA was isolated from liver tissue using commercially available solutions for purifying total RNA (Invitrogen, Shanghai, China) and was reverse transcribed to cDNA at 37 °C for 50 min and 70 °C for 10 min using Reverse Transcription Kits (Sangon Biotech Company, Shanghai). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to assess TNF-α, IFN-γ, CXCL9, and CXCL10 mRNA expression using commercially available SYBR Premix Ex Taq (TaKaRa Biotechnology, Dalian, China) and specific primers. An optimal number of PCR cycles was used at 95 °C for 10 s and 60 °C for 30 s in a Corbett Rotor-Gene 3000 real-time PCR system (Corbett Research). Gene-of-interest expression levels were calculated relative to the housekeeping gene, β-actin. The specific primers are as follows: CXCL9 (sense 5′-GATCAAACCTGCCTAGATCC-3′; antisense 5′-GG CTGTGTAGAACACAGAGT-3′). CXCL10 (sense 5′-ACCATGAACCCAAGTGCTGCCGTC-3′; antisense 5′CTTCACTCCAGTTAAGGAGCCCT-3′). IL-10 (sense 5′-TGAATTCCCTGGGTGAGAAG-3′; antisense 5′-GCT CCACTGCCTTGCTCTTA-3′
β-actin (sense 5′-TGACGTTGACATCCGTAAAGACC-3′; antisense 5′CTCAGGAGGAGCAATGATCTTGA-3′). 2.8. Measurement of cytokines in sera The concentrations of mouse IFN-γ, TNF-α, TGF-β, and hIL-10 in sera were measured using enzyme-linked immunosorbent assay (ELISA) kits (Dakewe Biotech Company, Shenzhen, China). 2.9. Flow cytometric analysis Spleens and livers were excised from experimental mice at the indicated time points. Splenocytes were made into single-cell suspensions through a 200-gauge stainless steel mesh and suspended in PBS. Cell suspensions were centrifuged at 450 ×g for 5 min. The pellet was resuspended and erythrocytes were lysed with lysis buffer (0.155 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH7.4) on ice for 5 min. Cell suspensions were added with PBS to stop lysis and centrifuged at 450 ×g for 5 min. The pellet was resuspended with PBS for counting cell number and labeling antibodies. Liver mononuclear cells (MNCs) were prepared as described previously [16]. Livers were pressed through a 200-gauge stainless steel mesh and suspended in PBS. Cell suspensions were centrifuged at 50 ×g for 1 min. The supernatant was then transferred into a new tube and centrifuged at 800 ×g for 10 min. The pellet was resuspended in 40% Percoll (Gibco BRL) solution, gently overlaid onto 70% Percoll solution, and centrifuged at 1260 ×g for 30 min at room temperature. The interface cells between the Percoll solutions were aspirated and washed twice with PBS. Liver MNCs were resuspended with PBS for counting cell number and labeling antibodies. Cells (1 × 106) were stained with FITC–anti-CD62L, PE– anti-NK1.1, PerCp-Cy5.5–anti-CD44, and APC–anti-CD4 to assess
Fig. 3. CD62LlowCD44hiCD4+ T cells increase in the liver after APAP challenge. (A and B) The frequency of CD62LlowCD44hiCD4+ T cells increased in the livers of APAP-treated mice. Lymphocytes were isolated from mouse liver and spleen 24 h after treatment with APAP or PBS and analyzed by flow cytometry. Lymphocytes were gated on CD4+NK1.1− cells, and the frequency of CD62LlowCD44hi cells was measured (A) and statistically analyzed (B). The data are representative of three independent experiments and are shown as the mean ± SEM. **, P b 0.01.
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Fig. 4. Depletion of CD4+T cells alleviates APAP-induced liver injury. (A) ALT levels decreased in mice treated with anti-CD4 mAb compared to those treated with control mAb. Anti-CD4 or control mAb was administered 48 h before APAP challenge. Sera and liver specimens were harvested 24 h after APAP challenge. ALT values were measured and H&E staining (arrows indicated pathological areas) was performed and analyzed (n = 5). (B) IFN-γ and TNF-α levels decreased in mice treated with anti-CD4 mAb. The data are representative of three independent experiments and are shown as the mean ± SEM. *, P b 0.05; **, P b 0.01.
activated T cells. Cells (1 × 106) were stained with FITC–anti-CD4, PE– anti-CD25, and Percp-Cy5.5–anti-NK1.1, and then intracellularly stained with APC–anti-Foxp3 after fixation and permeabilization using the Fixation/Permeabilization Diluent (eBioscience, San Diego, CA, USA) to identify Treg cells. Cells (1 × 106) were stained with FITC– anti-CD4, PE–anti-CXCR3, and PerCp-Cy5.5–anti-NK1.1, and intracellularly stained with APC–anti-Foxp3 after fixation and permeabilization to detect CXCR3 expression on Treg cells. All mAbs and isotype controls used were purchased from BD Pharmingen (San Jose, CA, USA). The stained cells were analyzed using a FACSCalibur (BD Biosciences, San Jose, CA, USA) or BD LSR II (BD Biosciences). The acquired data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).
2.10. Statistical analysis Data presented as the mean ± standard error of the mean (SEM) were analyzed using Student's t-test. Differences were considered significant when P b 0.05 and were marked in the figures as *P b 0.05; **P b 0.01; and ***P b 0.005. All analyses were performed with Prism 4 software (GraphPad Software, San Diego, CA, USA).
3. Results 3.1. Adaptive immune cells aggravate APAP-induced liver injury To begin assessing whether adaptive immune cells were involved in APAP-induced liver injury, we first confirmed APAP-induced liver injury by assessing ALT levels and observing pathological damage in the livers of APAP-treated mice. ALT levels in the sera significantly increased in APAP-treated mice compared to those of PBS-treated mice (Fig. 1A). In line with the report by Ishida et al. [5], the levels of the proinflammatory cytokines IFN-γ and TNF-α were elevated in APAPtreated mice compared to PBS-treated mice (Fig. 1B), which suggested that inflammatory response was involved in APAP-induced liver injury. However, neither NK nor NKT cells were required for APAP-induced liver injury, as depletion of NK/NKT cells using the PK136 (anti-NK1.1) antibody or depletion of NK cells with αASGM1 antibody did not reduce APAP-induced liver injury (Fig. 1C), in line with the finding by Masson et al. [17]. To assess the role of the adaptive immune cells in the pathogenesis, we evaluated liver injury in Rag1−/− mice that lack adaptive immune cells after APAP challenge. ALT levels in Rag1−/− mice were markedly less than those in wild-type (WT) mice (Fig. 1D). Thus,
Fig. 5. CD4+ T cell deficiency alleviates APAP-induced liver injury. (A) ALT levels decreased in CD4−/− mice. Sera were collected 24 h after APAP challenge (n = 5). (B) IFN-γ and TNF-α levels were decreased in CD4−/− mice. The data are representative of three independent experiments and are shown as the mean ± SEM. **, P b 0.01; ***, P b 0.005.
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Fig. 6. Treg cells increase in the spleen and liver after APAP challenge. (A and B) The frequency and absolute number of Treg cells increased in the spleen and liver of APAP-treated mice compared to PBS-treated mice. MNCs were isolated from mouse spleen and liver 24 h after treatment and analyzed by flow cytometry. Cells were gated on CD4+NK1.1− cells, the frequency of Foxp3+ cells was analyzed (A), and the absolute number of Treg cells was calculated (B) (n = 5). The data are representative of three independent experiments and are shown as the mean ± SEM. *, P b 0.05; **, P b 0.01.
these findings reveal that adaptive immune cells aggravate APAPinduced liver injury. 3.2. CD4+ but not CD8+ T cells mediate APAP-induced liver injury Since both CD8+ and CD4+ T cells could produce pro-inflammatory cytokines to mediate inflammatory response, we next assessed their respective roles in APAP-induced liver injury using gene-deficient mice or depleting antibodies. After APAP challenge, ALT levels and
pro-inflammatory cytokine levels in CD8−/− mice were similar to those in WT control mice, suggesting that CD8+ T cells were not involved in the production of pro-inflammatory cytokines (Fig. 2). In contrast, we observed a significant increase in the frequency of NK1.1−CD62LlowCD44hiCD4+ T cells in the liver of APAP-treated WT mice, but not in the spleen (Fig. 3). Moreover, the depletion of CD4+ T cells with an anti-CD4 antibody significantly reduced ALT levels and hepatic necrotic areas, and decreased pro-inflammatory cytokines compared to that of isotype control-treated mice (Fig. 4). CD4−/−
Fig. 7. Treg cell deletion exacerbates APAP-induced liver injury. (A) ALT levels increased in mice treated with anti-CD25 mAb. Anti-CD25 or control mAb was administered 48 h before APAP challenge. Sera were collected 12 h after APAP challenge (n = 10). (B) IFN-γ and TNF-α levels increased in mice treated with anti-CD25 mAb. Anti-CD25 or control mAb was administered 48 h before APAP challenge. Sera were collected 24 h after APAP challenge. (C) The frequency of CD62LlowCD44hiCD4+ T cells increased in the livers of mice treated with anti-CD25 mAb. The frequency of CD62LlowCD44hi cells was analyzed 24 h after APAP challenge. The data are representative of three independent experiments and are shown as the mean ± SEM. *, P b 0.05; ***, P b 0.005.
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Fig. 8. Adoptive transfer of Treg cells ameliorates APAP-induced liver injury. (A) ALT levels decreased in mice receiving adoptively transferred Treg cells compared to those receiving PBS control. Purified Treg cells (2 × 106) were adoptively transferred into recipient mice 24 h before APAP challenge; PBS was used as the control. Sera were collected 24 h after APAP challenge (n = 5). (B) IFN-γ and TNF-α levels decreased in mice receiving adoptively transferred Treg cells compared to those receiving PBS control. The data are representative of three experiments and are shown as the mean ± SEM. *, P b 0.05; **, P b 0.01.
mice treated with APAP also exhibited significantly reduced ALT levels and decreased pro-inflammatory cytokines compared to WT control mice (Fig. 5). Taken together, these data reveal that among adaptive immune cells, CD4+ but not CD8+ T cells contribute to APAP-induced liver injury. 3.3. Regulatory T cells attenuate APAP-induced liver injury As an important subset of CD4+ T cells, Treg cells play a critical role in modulating the inflammatory responses triggered by pathogen infection or an autoimmune reaction. Therefore, the roles of Treg cells in the pathogenesis were also assessed. Firstly, we observed that the absolute number of Treg cells increased in both the liver and the spleen 24 h after APAP challenge (Fig. 6). To determine the role of Treg cells in APAP-induced hepatic inflammation, Treg cells were depleted using an anti-CD25 mAb. In the absence of Treg cells, mice exhibited more severe liver injury. ALT levels in the mice treated with anti-CD25 mAb were much higher than those in control mice even at 12 h after APAP
challenge (Fig. 7A). IFN-γ and TNF-α levels were also higher compared to control mice at 24 h after APAP challenge (Fig. 7B). Moreover, the frequency of the CD62LlowCD44hiCD4+ T cells increased in the liver after Treg cells depletion (Fig. 7C). To confirm whether Treg cells were sufficient to attenuate APAP-induced liver injury, purified Treg cells from the spleen were adoptively transferred into recipient mice before APAP treatment. Indeed, adoptive transfer of Treg cells ameliorated APAP-induced liver injury, with lower ALT levels, and decreased proinflammatory cytokine levels over that of PBS-treated control mice (Fig. 8). Additionally, we found the IL-10 levels in the liver significantly decreased after Treg cell depletion (Fig. 9A). To assess the role of IL-10 in the pathogenesis, the pcDNA3.0-hIL-10 vector was hydrodynamically injected. The treatment of the pcDNA3.0-hIL-10 vector increased the hIL-10 levels in the sera (Fig. 9B). Overexpression of hIL-10 protected against APAP-induced liver injury, demonstrated by decreased ALT levels and lower IFN-γ levels (Fig. 9C–D). Moreover, the treatment with anti-IL-10 neutralizing antibody significantly aggravated APAPinduced liver injury, characterized by higher ALT levels and IFN-γ levels
Fig. 9. IL-10 and TGF-β suppress APAP-induced liver injury. (A) IL-10 levels decreased in the liver of mice treated with anti-CD25 mAb (n = 10). (B) hIL-10 levels in sera from mice treated with pcDNA3.0-hIL-10 (20 μg/mouse) at indicated time points after hydrodynamic injection. (C) ALT levels decreased in mice treated with pcDNA3.0-hIL-10 compared to those with pcDNA3.0-negative. The vector (20 μg/mouse) was hydrodynamically injected into recipient mice 3 days before APAP challenge (n = 5). (D) IFN-γ levels decreased in mice treated with pcDNA3.0-hIL-10 compared to those with pcDNA3.0-null. (E) ALT levels increased in mice treated with anti-IL-10 antibody compared to those with isotype antibody. Anti-IL-10 antibody and isotype antibody were injected 1 h before APAP challenge (n = 5). (F) IFN-γ levels increased in mice treated with anti-IL-10 antibody compared to those with isotype antibody. (G) TGF-β levels increased in mice adoptively transferred with Treg cells compared to those with PBS after APAP challenge (n = 5). (H) ALT levels decreased in mice treated with pCMV3-TGF-β compared to those with pCMV3-negative. The data are representative of three independent experiments and are shown as the mean ± SEM. *, p b 0.05; **, P b 0.01.
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Fig. 10. Treg cells express CXCR3, and chemokines for CXCR3 increase after APAP challenge. (A) Treg cells expressed CXCR3. MNCs were isolated from the spleen and liver. Treg cells were gated as CD4+Foxp3+ cells and analyzed for CXCR3 expression by flow cytometry (n = 5). (B) CXCL9 and CXCL10 chemokine mRNA levels increased in the liver 24 h after APAP treatment (n = 5). The data are representative of three independent experiments.
in sera (Fig. 9E–F). To confirm whether Treg cells also function via TGFβ, we measured the TGF-β levels after adoptive transfer of Treg cells and the ALT levels after overexpression of TGF-β vector. Adoptive transfer of Treg cells enhanced the TGF-β levels in the sera after APAP challenge (Fig. 9G). Moreover, overexpression of TGF-β in vivo significantly alleviated APAP-induced liver injury (Fig. 9H). Therefore, these results demonstrate that Treg cells play a protective role in APAP-induced liver injury through IL-10 and TGF-β during the pathogenesis.
3.4. The recruitment of regulatory T cells to the liver ameliorates APAPinduced liver injury Given that Treg cells could suppress APAP-induced liver injury, we wondered whether the APAP-induced liver injury could be ameliorated by actively recruiting Treg cells to the inflamed hepatic microenvironment. We found that Treg cells in the spleen and liver expressed CXCR3 (Fig. 10A). Moreover, we found that CXCL9 and CXCL10 were
Fig. 11. Recruitment of Treg cells into the liver attenuates APAP-induced liver injury. The pLIVE-CXCL10 vector (20 μg/mouse) was hydrodynamically injected into recipient mice 3 days before APAP challenge; pLIVE-negative was used as the control. Mice were then injected i.p. with APAP at 400 mg/kg body weight (n = 5). (A and B) The frequency and absolute number of Treg cells increased in the livers of mice treated with pLIVE-CXCL10 compared to those treated with pLIVE-negative. The frequency of Foxp3+ cells was measured and statistically analyzed (A); and the absolute number of Treg cells was calculated (B) 24 h after APAP treatment. (C) ALT levels decreased in mice treated with pLIVE-CXCL10 compared to those with pLIVEnegative. (D) IFN-γ and TNF-α levels decreased in mice treated with pLIVE-CXCL10 compared to those with pLIVE-negative. The data are representative of three independent experiments and are shown as the mean ± SEM. *, P b 0.05; **, P b 0.01.
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increased in the liver after APAP challenge (Fig. 10B), which might partially explain the increase of Treg cells in the liver after APAP challenge. To test the possibility that the expression of chemokines for CXCR3 in the liver could recruit Treg cells and subsequently suppress APAP-induced liver injury, we overexpressed CXCL10 in the liver by hydrodynamically injecting pLIVE-CXCL10 vectors before APAP challenge. Expectedly, the frequency and absolute number of Treg cells increased in the liver of pLIVE-CXCL10-treated mice compared to control mice treated with pLIVE-negative vector (Fig. 11A–B). More importantly, pretreatment with pLIVE-CXCL10 ameliorated liver injury and reduced pro-inflammatory cytokine levels (Fig. 11C–D). Therefore, the recruitment of regulatory T cells to the liver by the CXCL10–CXCR3 axis protects against drug-induced fulminant liver injury. 4. Discussion The inflammatory response is responsible for the second damaging pathological hit to the liver following APAP-induced hepatocyte necrosis. This study revealed that Th1 cells were pathogenic in APAPinduced liver injury and Treg cells exerted a crucial protective role in APAP-induced liver injury. Therefore, modulating Treg cells may provide novel insights into controlling APAP-induced liver injury. IFN-γ has a pathogenic role in several murine models of liver disease [5,18,19]. As previously described by Ishida et al. [5], we found that IFNγ was locally and systemically increased in the liver and sera, respectively, after APAP challenge and that it was required for APAP-induced liver injury. As the findings by Masson et al. [17], we found that NK and NKT cells were not main producers of IFN-γ in the pathogenesis. We further established that CD4+, but not CD8+, T cells were the main producers of IFN-γ in APAP-induced liver injury. We also found an increase of CD44hiCD4+ T cells in the liver after APAP challenge. IL12 has previously been reported to induce activation, IFN-γ production, and proliferation of innate-like CD44hiCD4+ T cells by bystander activation [20,21]. However, APAP-induced liver injury was independent of IL-12, as IL-12p35−/− mice and their intact WT counterparts had similar liver injury to APAP challenge (data not shown). Given that IL-12 is a potent activator of NK cell effector function [22], this may help to explain why NK and CD8+ T cells are not activated after APAP challenge. Thus, the precise mechanisms underlying the activation of CD4+ T cells during the pathological process of APAP-induced liver injury remain to be understood. Interestingly, CD4+ T cells overexpressing suppressor of cytokine signaling 3 (SOCS3Tg-CD4+ T cells) cause exaggerated APAP-induced liver injury [23]. This is in contrast to their effect in lipopolysaccharide (LPS)-, and Con A-induced liver injury in which SOCS3 suppresses signal transducer and activator of transcription 4 (STAT4) signaling activation [24,25]. Thus, similar to what we observe in the present study for the distinct role of Treg cells, the exaggerated liver injury mediated by SOCS3Tg-CD4+ T cells might not be attributed to an enhanced ability of CD4+ effector T cells but to the impaired suppressive function of Treg cells, as SOCS3 overexpression in Treg cells impairs Treg suppressive function, decreases proliferation, and reduces CTLA-4 and Foxp3 expression [26]. Recent studies demonstrate that some innate immune cells play protective roles in APAP-induced liver injury [27–29]. Additionally, BALB/c mice, which harbor Th2 bias, are not susceptible to APAP-induced liver injury in an IL-6-dependent manner, indicating that Th2 cells may help to regulate the inflammatory response [30]. Here, we are the first to reveal that the Treg subset is necessary and sufficient to control APAP-induced liver injury mediated, indicating that CD4+ T cell subsets play a pivotal role in the extent of APAPinduced liver injury. Depletion of Treg cells caused more severe liver injury even at 12 h and lower survival ratio at 24 h after APAP challenge (Fig. 7A and data not shown). Additionally, the IL-10 level increased accompanied with the increase of hepatic and splenic Treg cells. The depletion of Treg cells reduced the hepatic IL-10 level. Moreover, over-expression of IL-10 in the liver alleviated APAP-induced liver
injury and neutralizing IL-10 with anti-IL-10 antibody aggravated APAP-induced liver injury (Fig. 9), in line with the conclusion using IL10-deficient mice [31]. Additionally, we found that Treg cells also played a protective role through TGF-β. Thus, Treg cells might suppress inflammatory response in IL-10- and TGF-β-dependent manner. IL-2 is essential for Treg cell homeostasis, proliferation, and function [32]. Treg cells do not produce IL-2 but they constitutively express CD25 to efficiently compete for IL-2 from activated Th1 cells [33]. In addition, IFN-γ has been found to promote CXCR3+ Treg cells to limit Th1 cellmediated pathology [34]. IL-2 and IFN-γ indeed increased during APAP-induced liver injury (Fig. 1B and data not shown). Moreover, Treg cells in the spleen and liver increased after APAP challenge (Fig. 6). Higher percentages of CXCR3+ Treg cells appeared in the liver compared to spleen (Fig. 10A). Therefore, we speculated that IL-2 and/or IFN-γ produced by Th1 cells induced Treg cell activation and promoted their accumulation at inflammatory sites via chemokines. In return, Treg cells inhibited Th1 cell-mediated inflammation via producing inhibitory cytokines including IL-10 and TGF-β. However, the precise mechanisms by which Treg cells activate and function in the pathogenesis require further investigation. Compartmentalization and trafficking of Treg cells are indispensible for their efficient function. IFN-γ acts as a pro-inflammatory signal in T cell-mediated hepatitis via induction of multiple chemokines, such as CXCL-10 [35,36]. However, it has been demonstrated that IFNγ-induced chemokines can also recruit CXCR3+ Treg cells to murine liver and protect against alpha galactosyl-C18-ceramide (αGal-C18Cer)- and Con A-induced liver injury [13,14]. Here, we found that hepatic and splenic Treg cells expressed CXCR3 and that the IFN-γ-induced chemokines and Treg cells increased after APAP-induced liver injury. Based on this discovery, we hypothesized that chemokine-induced recruitment of Treg cells to the liver might be a useful therapeutic regimen to resolve APAP-induced liver injury. As predicted, exogenous CXCL10 expression in the liver before APAP administration efficiently inhibited APAP-induced liver injury via recruiting Treg cells into the liver. Additionally, the findings by Bone-Larson et al. showing that recombinant CXCL10 has hepatoprotective effects through inducing CXCR2 on hepatocytes may be another mechanism by which CXCL10 functions in APAP-induced liver injury [37]. Chemokines besides CXCL10 have also been demonstrated to be important for compartmentalization and trafficking of Treg cells into inflammatory microenvironment [9], and it will be worthwhile to further identify other Treg-recruiting chemokines during APAP-induced liver injury in future studies. In conclusion, our study provides evidence to support that T cell populations among adaptive immune cells are involved in APAPinduced hepatic inflammation, where Th1 cells mediate APAP-induced liver injury and Treg cells alleviate APAP-induced liver injury. Thus, the results of the present study may provide new insights into the treatment of drug-induced liver injury. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This work was supported by the Natural Science Foundation of China (#31390433, #81302863) and China Postdoctoral Science Foundation. References [1] Larson AM, Polson J, Fontana RJ, Davern TJ, Lalani E, Hynan LS, et al. Acetaminopheninduced acute liver failure: results of a United States multicenter, prospective study. Hepatology 2005;42:1364–72. [2] Dahlin DC, Miwa GT, Lu AY, Nelson SD. N-acetyl-p-benzoquinone imine: a cytochrome P-450-mediated oxidation product of acetaminophen. Proc Natl Acad Sci U S A 1984;81:1327–31.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Regulatory T cells ameliorate acetaminophen-induced immune-mediated liver injury Xuefu Wang a, Rui Sun a,b, Yongyan Chen a, Zhe-Xiong Lian a,b, Haiming Wei a,b, Zhigang Tian a,b,c,⁎ a Institute of Immunology and CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center, University of Science & Technology of China, Hefei, Anhui 230027, China b Hefei National Laboratory for Physical Sciences at Microscale, Hefei, Anhui 230027, China c Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China
a r t i c l e
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Article history: Received 16 September 2014 Received in revised form 1 January 2015 Accepted 4 February 2015 Available online 14 February 2015 Keywords: Acetaminophen Adaptive immune cells Regulatory T cells CXCL10 Liver injury
a b s t r a c t The contribution of innate immune cells to acetaminophen (APAP)-induced liver injury has been extensively investigated. However, the roles of T cell populations among adaptive immune cells in APAP-induced liver injury remain to be elucidated. Herein, we found that distinct CD4+ T cell subsets but not CD8+ T cells modulated APAPinduced liver injury in mice. After APAP challenge, more CD62LlowCD44hiCD4+ T cells appeared in the liver, accompanied by increased IFN-γ. The removal of CD4+ T cells by either antibody depletion or genetic deficiency markedly compromised pro-inflammatory cytokine levels and ameliorated liver injury. Meanwhile, we also found that the frequency and absolute number of Treg cells also increased. Treg cell depletion increased hepatic CD62LlowCD44hiCD4+ T cells, augmented pro-inflammatory cytokines, and exacerbated liver injury, while adoptive transfer of Treg cells ameliorated APAP-induced liver injury. Furthermore, the recruitment of Treg cells into the liver through specific expression of CXCL10 in the liver could ameliorate APAP-induced liver injury. Our investigation suggests that Th1 and Treg subsets are involved in regulating APAP-induced liver injury. Thus, modulating the Th1/Treg balance may be an effective strategy to prevent and/or treat APAP-induced liver injury. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that is generally safe to use at therapeutic doses. However, there are many cases in which careless use of APAP leads to overdose and subsequent acute liver injury. Mortality by APAP overdose has even been reported in the USA [1], and the Federal Drug Administration (FDA) in the USA has now limited the amount of APAP allowed in prescription combination products. Mechanistically, an overdose of APAP generates an excess of N-acetyl-p-benzoquinone imine (NAPQI), leading to hepatocyte death [2,3]. Studies in murine models of APAP overdose show that the inflammatory response induced by damageassociated molecular pattern molecules (DAMPs) released from dead hepatocytes exaggerates liver injury [4]. The pro-inflammatory cytokine IFN-γ plays a critical role in mediating APAP-induced liver injury [5]. In terms of cellular mediators, innate immune cells such as neutrophils and macrophages are known to mediate APAP-induced liver injury [6, 7]. However, whether and how adaptive immune cells—especially ⁎ Corresponding author at: School of Life Sciences, University of Science and Technology of China, #443 Huangshan Road, Hefei 230027, China. Tel.: +86 551 6360 0845; fax: +86 551 6360 6783. E-mail address: [email protected] (Z. Tian).
http://dx.doi.org/10.1016/j.intimp.2015.02.008 1567-5769/© 2015 Elsevier B.V. All rights reserved.
CD8+ and CD4+ T cells and their functional subsets—play a role in APAP-induced liver injury has not been well documented. The inhibition of acute inflammation shortly after it begins is generally accepted as an active process that is quickly initiated by inflammatory responses in order to minimize immune-mediated damage to host tissues [8]. The regulatory T cell (Treg) subset of CD4+ T cells is an important regulator in the resolution phase of inflammation. Various chemokine receptors are expressed on Treg cells for the organ-specific localization of Treg cells [9]. The CCL22 signaling-induced Treg Cell recruitment promotes venous metastases of HBV-positive hepatocellular carcinoma [10]. Therefore, modulating the recruitment of Treg cells via the chemokine–receptor axis has been advised to treat the organ-specific inflammation or tumor. CCL22-mediated Treg recruitment to the pancreatic islets prevents murine autoimmune diabetes [11]. The recruitment of CCR5+ Treg cells prevents the exacerbation of chronic inflammation in the intestine [12]. The CXCR3+ Treg recruitment protects against αGalCer- and Con A-induced liver injury [13,14]. Up to now, whether Treg cells are involved in APAP-induced liver injury remains unclear. Moreover, whether Treg recruitment into the inflamed liver can help to treat APAP-induced liver injury is worth investigating. In this study, we explored the roles of adaptive immune cells in the progression of APAP-induced liver injury. The data presented here demonstrated that Th1 cells but not CD8+ T cells exacerbated APAP-
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induced liver injury and that Treg cells attenuated APAP-induced liver injury. Moreover, the recruitment of Treg cells into the liver is helpful to suppress APAP-induced liver injury. Thus, modulating Treg cells may be a useful strategy to ameliorate drug-induced immuneexacerbated liver injury.
2. Materials and methods 2.1. Mice Male C57BL/6 mice (6–8 weeks old) were purchased from the Shanghai Laboratory Animal Center at the Chinese Academy of Sciences. Rag-1−/− mice were purchased from the Model Animal Research Center at Nanjing University. All mice, including CD4−/−, and CD8−/− mice, were housed in microisolator cages under humidityand temperature-controlled specific pathogen-free conditions in the animal facility at the School of Life Sciences of the University of Science and Technology of China. Mice were maintained on an irradiated sterile diet and given autoclaved water. All experiments were performed according to the animal care regulations of the University of Science and Technology of China. The study was approved by the Local Ethics Committee for Animal Care and Use at University of Science and Technology of China.
2.3. Immune cell depletion and IL-10 blocking 0.2 mg/mouse anti-CD4 mAb (clone TIB-207, ATCC) was i.v. injected into mice to deplete CD4+ T cells, 0.2 mg/mouse anti-CD25 mAb (clone TIB-222, ATCC) to deplete CD25+ Treg cells, 0.2 mg/mouse PK136 (clone HB-191, ATCC) to deplete NK cells and NKT cells, and 30 μl/mouse anti-ASGM1 antibody (WAKO Pure Chemical Industries, Ltd, Osaka, Japan) to deplete NK cells, 48 h before APAP treatment. 0.2 mg/mouse anti-IL-10 antibody (eBioscience, San Diego, CA, USA) was i.v. injected into mice to block IL-10 at 1 h before APAP treatment. The isotype antibody was used as control. 2.4. Isolation and adoptive transfer of Treg cells Spleens were excised from normal mice, made into single-cell suspensions, and treated with red blood cell (RBC) lysis buffer to lyse erythrocytes. The pellets were resuspended, and cell number was determined. CD4+CD25+ Treg cells were isolated using the mouse CD4+CD25+ Regulatory T Cell Isolation Kit (Order no. 130-091-041, Miltenyi Biotec Inc., Bergisch Gladbach, Germany) according to the manufacturer's instruction manual. The purity of isolated Treg cells was N90%. Purified cells (2 × 106 cells/mouse) were adoptively transferred (i.v.) into recipient mice, and an equal volume of PBS was used as a control.
2.2. APAP treatment
2.5. Overexpression of chemokines in the liver
Experimental mice were fasted for 16 h and subsequently injected with PBS control or freshly prepared APAP (dissolved in PBS) intraperitoneally (i.p.) (400 mg/kg body weight, Sigma-Aldrich, St. Louis, MO, USA). At the indicated time points, mice were anesthetized, bled, and sacrificed. Sera were isolated for testing alanine aminotransferase (ALT) levels and cytokines. Liver tissues were excised for analyzing cell populations, extracting RNA, and cutting tissue sections.
The expression vectors pLIVE-CXCL10 and pcDNA3.0-hIL-10 were constructed in our lab. The expression vector pCMV3-TGF-β was purchased from Sino Biological Incorporation (Beijing, China). These vectors were hydrodynamically injected into the tail vein in a 2-ml volume at indicated times before APAP treatment respectively, as described previously [15]. The null vector (pLIVE-negative, pcDNA3.0negative and pCMV3-negative) was used as the control.
Fig. 1. Adaptive immune cells aggravate APAP-induced liver injury. Mice were fasted for 16 h and injected i.p. with acetaminophen (APAP) at 400 mg/kg body weight or equal volumes of PBS as a control (n = 5). (A) ALT levels increased in APAP-challenged mice. Sera were collected 24 h after APAP challenge. (B) IFN-γ and TNF-α levels increased in the sera after APAP challenge. (C) ALT levels were similar between mice treated with PK136 (to deplete NK cells and NKT cells) and control mAb, or αASGM1 (to deplete NK cells) and control mAb. Mice were treated with mAb 48 h before APAP challenge. (D) ALT levels decreased in Rag−/− mice. The data are representative of three independent experiments and are shown as the mean ± SEM. **, P b 0.01; ***, P b 0.005.
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Fig. 2. CD8+ T cells do not contribute to APAP-induced liver injury. (A) ALT levels were similar between C57BL/6 and CD8−/− mice. Sera were collected 24 h after APAP challenge (n = 5). (B) Systemic IFN-γ and TNF-α levels were similar between C57BL/6 and CD8−/− mice. The data are representative of three independent experiments and are shown as the mean ± SEM.
2.6. Assessment of liver injury Liver injury was evaluated by ALT levels, immunohistochemical staining, and mouse survival. ALT levels were measured using a diagnostic kit (Rongsheng, Shanghai, China). Liver specimens were fixed with 4% paraformaldehyde, dehydrated with graded alcohol, embedded in paraffin, cut into tissue sections, and stained with hematoxylin and eosin (H&E). Mouse survival was observed at the indicated hours after APAP treatment. 2.7. RNA isolation and quantitative RT-PCR (qRT-PCR) analysis RNA was isolated from liver tissue using commercially available solutions for purifying total RNA (Invitrogen, Shanghai, China) and was reverse transcribed to cDNA at 37 °C for 50 min and 70 °C for 10 min using Reverse Transcription Kits (Sangon Biotech Company, Shanghai). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to assess TNF-α, IFN-γ, CXCL9, and CXCL10 mRNA expression using commercially available SYBR Premix Ex Taq (TaKaRa Biotechnology, Dalian, China) and specific primers. An optimal number of PCR cycles was used at 95 °C for 10 s and 60 °C for 30 s in a Corbett Rotor-Gene 3000 real-time PCR system (Corbett Research). Gene-of-interest expression levels were calculated relative to the housekeeping gene, β-actin. The specific primers are as follows: CXCL9 (sense 5′-GATCAAACCTGCCTAGATCC-3′; antisense 5′-GG CTGTGTAGAACACAGAGT-3′). CXCL10 (sense 5′-ACCATGAACCCAAGTGCTGCCGTC-3′; antisense 5′CTTCACTCCAGTTAAGGAGCCCT-3′). IL-10 (sense 5′-TGAATTCCCTGGGTGAGAAG-3′; antisense 5′-GCT CCACTGCCTTGCTCTTA-3′
β-actin (sense 5′-TGACGTTGACATCCGTAAAGACC-3′; antisense 5′CTCAGGAGGAGCAATGATCTTGA-3′). 2.8. Measurement of cytokines in sera The concentrations of mouse IFN-γ, TNF-α, TGF-β, and hIL-10 in sera were measured using enzyme-linked immunosorbent assay (ELISA) kits (Dakewe Biotech Company, Shenzhen, China). 2.9. Flow cytometric analysis Spleens and livers were excised from experimental mice at the indicated time points. Splenocytes were made into single-cell suspensions through a 200-gauge stainless steel mesh and suspended in PBS. Cell suspensions were centrifuged at 450 ×g for 5 min. The pellet was resuspended and erythrocytes were lysed with lysis buffer (0.155 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH7.4) on ice for 5 min. Cell suspensions were added with PBS to stop lysis and centrifuged at 450 ×g for 5 min. The pellet was resuspended with PBS for counting cell number and labeling antibodies. Liver mononuclear cells (MNCs) were prepared as described previously [16]. Livers were pressed through a 200-gauge stainless steel mesh and suspended in PBS. Cell suspensions were centrifuged at 50 ×g for 1 min. The supernatant was then transferred into a new tube and centrifuged at 800 ×g for 10 min. The pellet was resuspended in 40% Percoll (Gibco BRL) solution, gently overlaid onto 70% Percoll solution, and centrifuged at 1260 ×g for 30 min at room temperature. The interface cells between the Percoll solutions were aspirated and washed twice with PBS. Liver MNCs were resuspended with PBS for counting cell number and labeling antibodies. Cells (1 × 106) were stained with FITC–anti-CD62L, PE– anti-NK1.1, PerCp-Cy5.5–anti-CD44, and APC–anti-CD4 to assess
Fig. 3. CD62LlowCD44hiCD4+ T cells increase in the liver after APAP challenge. (A and B) The frequency of CD62LlowCD44hiCD4+ T cells increased in the livers of APAP-treated mice. Lymphocytes were isolated from mouse liver and spleen 24 h after treatment with APAP or PBS and analyzed by flow cytometry. Lymphocytes were gated on CD4+NK1.1− cells, and the frequency of CD62LlowCD44hi cells was measured (A) and statistically analyzed (B). The data are representative of three independent experiments and are shown as the mean ± SEM. **, P b 0.01.
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Fig. 4. Depletion of CD4+T cells alleviates APAP-induced liver injury. (A) ALT levels decreased in mice treated with anti-CD4 mAb compared to those treated with control mAb. Anti-CD4 or control mAb was administered 48 h before APAP challenge. Sera and liver specimens were harvested 24 h after APAP challenge. ALT values were measured and H&E staining (arrows indicated pathological areas) was performed and analyzed (n = 5). (B) IFN-γ and TNF-α levels decreased in mice treated with anti-CD4 mAb. The data are representative of three independent experiments and are shown as the mean ± SEM. *, P b 0.05; **, P b 0.01.
activated T cells. Cells (1 × 106) were stained with FITC–anti-CD4, PE– anti-CD25, and Percp-Cy5.5–anti-NK1.1, and then intracellularly stained with APC–anti-Foxp3 after fixation and permeabilization using the Fixation/Permeabilization Diluent (eBioscience, San Diego, CA, USA) to identify Treg cells. Cells (1 × 106) were stained with FITC– anti-CD4, PE–anti-CXCR3, and PerCp-Cy5.5–anti-NK1.1, and intracellularly stained with APC–anti-Foxp3 after fixation and permeabilization to detect CXCR3 expression on Treg cells. All mAbs and isotype controls used were purchased from BD Pharmingen (San Jose, CA, USA). The stained cells were analyzed using a FACSCalibur (BD Biosciences, San Jose, CA, USA) or BD LSR II (BD Biosciences). The acquired data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).
2.10. Statistical analysis Data presented as the mean ± standard error of the mean (SEM) were analyzed using Student's t-test. Differences were considered significant when P b 0.05 and were marked in the figures as *P b 0.05; **P b 0.01; and ***P b 0.005. All analyses were performed with Prism 4 software (GraphPad Software, San Diego, CA, USA).
3. Results 3.1. Adaptive immune cells aggravate APAP-induced liver injury To begin assessing whether adaptive immune cells were involved in APAP-induced liver injury, we first confirmed APAP-induced liver injury by assessing ALT levels and observing pathological damage in the livers of APAP-treated mice. ALT levels in the sera significantly increased in APAP-treated mice compared to those of PBS-treated mice (Fig. 1A). In line with the report by Ishida et al. [5], the levels of the proinflammatory cytokines IFN-γ and TNF-α were elevated in APAPtreated mice compared to PBS-treated mice (Fig. 1B), which suggested that inflammatory response was involved in APAP-induced liver injury. However, neither NK nor NKT cells were required for APAP-induced liver injury, as depletion of NK/NKT cells using the PK136 (anti-NK1.1) antibody or depletion of NK cells with αASGM1 antibody did not reduce APAP-induced liver injury (Fig. 1C), in line with the finding by Masson et al. [17]. To assess the role of the adaptive immune cells in the pathogenesis, we evaluated liver injury in Rag1−/− mice that lack adaptive immune cells after APAP challenge. ALT levels in Rag1−/− mice were markedly less than those in wild-type (WT) mice (Fig. 1D). Thus,
Fig. 5. CD4+ T cell deficiency alleviates APAP-induced liver injury. (A) ALT levels decreased in CD4−/− mice. Sera were collected 24 h after APAP challenge (n = 5). (B) IFN-γ and TNF-α levels were decreased in CD4−/− mice. The data are representative of three independent experiments and are shown as the mean ± SEM. **, P b 0.01; ***, P b 0.005.
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Fig. 6. Treg cells increase in the spleen and liver after APAP challenge. (A and B) The frequency and absolute number of Treg cells increased in the spleen and liver of APAP-treated mice compared to PBS-treated mice. MNCs were isolated from mouse spleen and liver 24 h after treatment and analyzed by flow cytometry. Cells were gated on CD4+NK1.1− cells, the frequency of Foxp3+ cells was analyzed (A), and the absolute number of Treg cells was calculated (B) (n = 5). The data are representative of three independent experiments and are shown as the mean ± SEM. *, P b 0.05; **, P b 0.01.
these findings reveal that adaptive immune cells aggravate APAPinduced liver injury. 3.2. CD4+ but not CD8+ T cells mediate APAP-induced liver injury Since both CD8+ and CD4+ T cells could produce pro-inflammatory cytokines to mediate inflammatory response, we next assessed their respective roles in APAP-induced liver injury using gene-deficient mice or depleting antibodies. After APAP challenge, ALT levels and
pro-inflammatory cytokine levels in CD8−/− mice were similar to those in WT control mice, suggesting that CD8+ T cells were not involved in the production of pro-inflammatory cytokines (Fig. 2). In contrast, we observed a significant increase in the frequency of NK1.1−CD62LlowCD44hiCD4+ T cells in the liver of APAP-treated WT mice, but not in the spleen (Fig. 3). Moreover, the depletion of CD4+ T cells with an anti-CD4 antibody significantly reduced ALT levels and hepatic necrotic areas, and decreased pro-inflammatory cytokines compared to that of isotype control-treated mice (Fig. 4). CD4−/−
Fig. 7. Treg cell deletion exacerbates APAP-induced liver injury. (A) ALT levels increased in mice treated with anti-CD25 mAb. Anti-CD25 or control mAb was administered 48 h before APAP challenge. Sera were collected 12 h after APAP challenge (n = 10). (B) IFN-γ and TNF-α levels increased in mice treated with anti-CD25 mAb. Anti-CD25 or control mAb was administered 48 h before APAP challenge. Sera were collected 24 h after APAP challenge. (C) The frequency of CD62LlowCD44hiCD4+ T cells increased in the livers of mice treated with anti-CD25 mAb. The frequency of CD62LlowCD44hi cells was analyzed 24 h after APAP challenge. The data are representative of three independent experiments and are shown as the mean ± SEM. *, P b 0.05; ***, P b 0.005.
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Fig. 8. Adoptive transfer of Treg cells ameliorates APAP-induced liver injury. (A) ALT levels decreased in mice receiving adoptively transferred Treg cells compared to those receiving PBS control. Purified Treg cells (2 × 106) were adoptively transferred into recipient mice 24 h before APAP challenge; PBS was used as the control. Sera were collected 24 h after APAP challenge (n = 5). (B) IFN-γ and TNF-α levels decreased in mice receiving adoptively transferred Treg cells compared to those receiving PBS control. The data are representative of three experiments and are shown as the mean ± SEM. *, P b 0.05; **, P b 0.01.
mice treated with APAP also exhibited significantly reduced ALT levels and decreased pro-inflammatory cytokines compared to WT control mice (Fig. 5). Taken together, these data reveal that among adaptive immune cells, CD4+ but not CD8+ T cells contribute to APAP-induced liver injury. 3.3. Regulatory T cells attenuate APAP-induced liver injury As an important subset of CD4+ T cells, Treg cells play a critical role in modulating the inflammatory responses triggered by pathogen infection or an autoimmune reaction. Therefore, the roles of Treg cells in the pathogenesis were also assessed. Firstly, we observed that the absolute number of Treg cells increased in both the liver and the spleen 24 h after APAP challenge (Fig. 6). To determine the role of Treg cells in APAP-induced hepatic inflammation, Treg cells were depleted using an anti-CD25 mAb. In the absence of Treg cells, mice exhibited more severe liver injury. ALT levels in the mice treated with anti-CD25 mAb were much higher than those in control mice even at 12 h after APAP
challenge (Fig. 7A). IFN-γ and TNF-α levels were also higher compared to control mice at 24 h after APAP challenge (Fig. 7B). Moreover, the frequency of the CD62LlowCD44hiCD4+ T cells increased in the liver after Treg cells depletion (Fig. 7C). To confirm whether Treg cells were sufficient to attenuate APAP-induced liver injury, purified Treg cells from the spleen were adoptively transferred into recipient mice before APAP treatment. Indeed, adoptive transfer of Treg cells ameliorated APAP-induced liver injury, with lower ALT levels, and decreased proinflammatory cytokine levels over that of PBS-treated control mice (Fig. 8). Additionally, we found the IL-10 levels in the liver significantly decreased after Treg cell depletion (Fig. 9A). To assess the role of IL-10 in the pathogenesis, the pcDNA3.0-hIL-10 vector was hydrodynamically injected. The treatment of the pcDNA3.0-hIL-10 vector increased the hIL-10 levels in the sera (Fig. 9B). Overexpression of hIL-10 protected against APAP-induced liver injury, demonstrated by decreased ALT levels and lower IFN-γ levels (Fig. 9C–D). Moreover, the treatment with anti-IL-10 neutralizing antibody significantly aggravated APAPinduced liver injury, characterized by higher ALT levels and IFN-γ levels
Fig. 9. IL-10 and TGF-β suppress APAP-induced liver injury. (A) IL-10 levels decreased in the liver of mice treated with anti-CD25 mAb (n = 10). (B) hIL-10 levels in sera from mice treated with pcDNA3.0-hIL-10 (20 μg/mouse) at indicated time points after hydrodynamic injection. (C) ALT levels decreased in mice treated with pcDNA3.0-hIL-10 compared to those with pcDNA3.0-negative. The vector (20 μg/mouse) was hydrodynamically injected into recipient mice 3 days before APAP challenge (n = 5). (D) IFN-γ levels decreased in mice treated with pcDNA3.0-hIL-10 compared to those with pcDNA3.0-null. (E) ALT levels increased in mice treated with anti-IL-10 antibody compared to those with isotype antibody. Anti-IL-10 antibody and isotype antibody were injected 1 h before APAP challenge (n = 5). (F) IFN-γ levels increased in mice treated with anti-IL-10 antibody compared to those with isotype antibody. (G) TGF-β levels increased in mice adoptively transferred with Treg cells compared to those with PBS after APAP challenge (n = 5). (H) ALT levels decreased in mice treated with pCMV3-TGF-β compared to those with pCMV3-negative. The data are representative of three independent experiments and are shown as the mean ± SEM. *, p b 0.05; **, P b 0.01.
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Fig. 10. Treg cells express CXCR3, and chemokines for CXCR3 increase after APAP challenge. (A) Treg cells expressed CXCR3. MNCs were isolated from the spleen and liver. Treg cells were gated as CD4+Foxp3+ cells and analyzed for CXCR3 expression by flow cytometry (n = 5). (B) CXCL9 and CXCL10 chemokine mRNA levels increased in the liver 24 h after APAP treatment (n = 5). The data are representative of three independent experiments.
in sera (Fig. 9E–F). To confirm whether Treg cells also function via TGFβ, we measured the TGF-β levels after adoptive transfer of Treg cells and the ALT levels after overexpression of TGF-β vector. Adoptive transfer of Treg cells enhanced the TGF-β levels in the sera after APAP challenge (Fig. 9G). Moreover, overexpression of TGF-β in vivo significantly alleviated APAP-induced liver injury (Fig. 9H). Therefore, these results demonstrate that Treg cells play a protective role in APAP-induced liver injury through IL-10 and TGF-β during the pathogenesis.
3.4. The recruitment of regulatory T cells to the liver ameliorates APAPinduced liver injury Given that Treg cells could suppress APAP-induced liver injury, we wondered whether the APAP-induced liver injury could be ameliorated by actively recruiting Treg cells to the inflamed hepatic microenvironment. We found that Treg cells in the spleen and liver expressed CXCR3 (Fig. 10A). Moreover, we found that CXCL9 and CXCL10 were
Fig. 11. Recruitment of Treg cells into the liver attenuates APAP-induced liver injury. The pLIVE-CXCL10 vector (20 μg/mouse) was hydrodynamically injected into recipient mice 3 days before APAP challenge; pLIVE-negative was used as the control. Mice were then injected i.p. with APAP at 400 mg/kg body weight (n = 5). (A and B) The frequency and absolute number of Treg cells increased in the livers of mice treated with pLIVE-CXCL10 compared to those treated with pLIVE-negative. The frequency of Foxp3+ cells was measured and statistically analyzed (A); and the absolute number of Treg cells was calculated (B) 24 h after APAP treatment. (C) ALT levels decreased in mice treated with pLIVE-CXCL10 compared to those with pLIVEnegative. (D) IFN-γ and TNF-α levels decreased in mice treated with pLIVE-CXCL10 compared to those with pLIVE-negative. The data are representative of three independent experiments and are shown as the mean ± SEM. *, P b 0.05; **, P b 0.01.
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increased in the liver after APAP challenge (Fig. 10B), which might partially explain the increase of Treg cells in the liver after APAP challenge. To test the possibility that the expression of chemokines for CXCR3 in the liver could recruit Treg cells and subsequently suppress APAP-induced liver injury, we overexpressed CXCL10 in the liver by hydrodynamically injecting pLIVE-CXCL10 vectors before APAP challenge. Expectedly, the frequency and absolute number of Treg cells increased in the liver of pLIVE-CXCL10-treated mice compared to control mice treated with pLIVE-negative vector (Fig. 11A–B). More importantly, pretreatment with pLIVE-CXCL10 ameliorated liver injury and reduced pro-inflammatory cytokine levels (Fig. 11C–D). Therefore, the recruitment of regulatory T cells to the liver by the CXCL10–CXCR3 axis protects against drug-induced fulminant liver injury. 4. Discussion The inflammatory response is responsible for the second damaging pathological hit to the liver following APAP-induced hepatocyte necrosis. This study revealed that Th1 cells were pathogenic in APAPinduced liver injury and Treg cells exerted a crucial protective role in APAP-induced liver injury. Therefore, modulating Treg cells may provide novel insights into controlling APAP-induced liver injury. IFN-γ has a pathogenic role in several murine models of liver disease [5,18,19]. As previously described by Ishida et al. [5], we found that IFNγ was locally and systemically increased in the liver and sera, respectively, after APAP challenge and that it was required for APAP-induced liver injury. As the findings by Masson et al. [17], we found that NK and NKT cells were not main producers of IFN-γ in the pathogenesis. We further established that CD4+, but not CD8+, T cells were the main producers of IFN-γ in APAP-induced liver injury. We also found an increase of CD44hiCD4+ T cells in the liver after APAP challenge. IL12 has previously been reported to induce activation, IFN-γ production, and proliferation of innate-like CD44hiCD4+ T cells by bystander activation [20,21]. However, APAP-induced liver injury was independent of IL-12, as IL-12p35−/− mice and their intact WT counterparts had similar liver injury to APAP challenge (data not shown). Given that IL-12 is a potent activator of NK cell effector function [22], this may help to explain why NK and CD8+ T cells are not activated after APAP challenge. Thus, the precise mechanisms underlying the activation of CD4+ T cells during the pathological process of APAP-induced liver injury remain to be understood. Interestingly, CD4+ T cells overexpressing suppressor of cytokine signaling 3 (SOCS3Tg-CD4+ T cells) cause exaggerated APAP-induced liver injury [23]. This is in contrast to their effect in lipopolysaccharide (LPS)-, and Con A-induced liver injury in which SOCS3 suppresses signal transducer and activator of transcription 4 (STAT4) signaling activation [24,25]. Thus, similar to what we observe in the present study for the distinct role of Treg cells, the exaggerated liver injury mediated by SOCS3Tg-CD4+ T cells might not be attributed to an enhanced ability of CD4+ effector T cells but to the impaired suppressive function of Treg cells, as SOCS3 overexpression in Treg cells impairs Treg suppressive function, decreases proliferation, and reduces CTLA-4 and Foxp3 expression [26]. Recent studies demonstrate that some innate immune cells play protective roles in APAP-induced liver injury [27–29]. Additionally, BALB/c mice, which harbor Th2 bias, are not susceptible to APAP-induced liver injury in an IL-6-dependent manner, indicating that Th2 cells may help to regulate the inflammatory response [30]. Here, we are the first to reveal that the Treg subset is necessary and sufficient to control APAP-induced liver injury mediated, indicating that CD4+ T cell subsets play a pivotal role in the extent of APAPinduced liver injury. Depletion of Treg cells caused more severe liver injury even at 12 h and lower survival ratio at 24 h after APAP challenge (Fig. 7A and data not shown). Additionally, the IL-10 level increased accompanied with the increase of hepatic and splenic Treg cells. The depletion of Treg cells reduced the hepatic IL-10 level. Moreover, over-expression of IL-10 in the liver alleviated APAP-induced liver
injury and neutralizing IL-10 with anti-IL-10 antibody aggravated APAP-induced liver injury (Fig. 9), in line with the conclusion using IL10-deficient mice [31]. Additionally, we found that Treg cells also played a protective role through TGF-β. Thus, Treg cells might suppress inflammatory response in IL-10- and TGF-β-dependent manner. IL-2 is essential for Treg cell homeostasis, proliferation, and function [32]. Treg cells do not produce IL-2 but they constitutively express CD25 to efficiently compete for IL-2 from activated Th1 cells [33]. In addition, IFN-γ has been found to promote CXCR3+ Treg cells to limit Th1 cellmediated pathology [34]. IL-2 and IFN-γ indeed increased during APAP-induced liver injury (Fig. 1B and data not shown). Moreover, Treg cells in the spleen and liver increased after APAP challenge (Fig. 6). Higher percentages of CXCR3+ Treg cells appeared in the liver compared to spleen (Fig. 10A). Therefore, we speculated that IL-2 and/or IFN-γ produced by Th1 cells induced Treg cell activation and promoted their accumulation at inflammatory sites via chemokines. In return, Treg cells inhibited Th1 cell-mediated inflammation via producing inhibitory cytokines including IL-10 and TGF-β. However, the precise mechanisms by which Treg cells activate and function in the pathogenesis require further investigation. Compartmentalization and trafficking of Treg cells are indispensible for their efficient function. IFN-γ acts as a pro-inflammatory signal in T cell-mediated hepatitis via induction of multiple chemokines, such as CXCL-10 [35,36]. However, it has been demonstrated that IFNγ-induced chemokines can also recruit CXCR3+ Treg cells to murine liver and protect against alpha galactosyl-C18-ceramide (αGal-C18Cer)- and Con A-induced liver injury [13,14]. Here, we found that hepatic and splenic Treg cells expressed CXCR3 and that the IFN-γ-induced chemokines and Treg cells increased after APAP-induced liver injury. Based on this discovery, we hypothesized that chemokine-induced recruitment of Treg cells to the liver might be a useful therapeutic regimen to resolve APAP-induced liver injury. As predicted, exogenous CXCL10 expression in the liver before APAP administration efficiently inhibited APAP-induced liver injury via recruiting Treg cells into the liver. Additionally, the findings by Bone-Larson et al. showing that recombinant CXCL10 has hepatoprotective effects through inducing CXCR2 on hepatocytes may be another mechanism by which CXCL10 functions in APAP-induced liver injury [37]. Chemokines besides CXCL10 have also been demonstrated to be important for compartmentalization and trafficking of Treg cells into inflammatory microenvironment [9], and it will be worthwhile to further identify other Treg-recruiting chemokines during APAP-induced liver injury in future studies. In conclusion, our study provides evidence to support that T cell populations among adaptive immune cells are involved in APAPinduced hepatic inflammation, where Th1 cells mediate APAP-induced liver injury and Treg cells alleviate APAP-induced liver injury. Thus, the results of the present study may provide new insights into the treatment of drug-induced liver injury. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This work was supported by the Natural Science Foundation of China (#31390433, #81302863) and China Postdoctoral Science Foundation. References [1] Larson AM, Polson J, Fontana RJ, Davern TJ, Lalani E, Hynan LS, et al. Acetaminopheninduced acute liver failure: results of a United States multicenter, prospective study. Hepatology 2005;42:1364–72. [2] Dahlin DC, Miwa GT, Lu AY, Nelson SD. N-acetyl-p-benzoquinone imine: a cytochrome P-450-mediated oxidation product of acetaminophen. Proc Natl Acad Sci U S A 1984;81:1327–31.
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