IL-17A Plays a Critical Role in the Pathogenesis of through Hepatic Stellate Cell Activation

This information is current as Zhongming Tan, Xiaofeng Qian, Runqiu Jiang, Qianghui of September 28, 2021. Liu, Youjing Wang, Chen Chen, Xuehao Wang, Bernhard Ryffel and Beicheng Sun J Immunol 2013; 191:1835-1844; Prepublished online 10 July 2013;

doi: 10.4049/jimmunol.1203013 Downloaded from http://www.jimmunol.org/content/191/4/1835

Supplementary http://www.jimmunol.org/content/suppl/2013/07/10/jimmunol.120301 Material 3.DC1 http://www.jimmunol.org/ References This article cites 47 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/191/4/1835.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-17A Plays a Critical Role in the Pathogenesis of Liver Fibrosis through Hepatic Stellate Cell Activation

Zhongming Tan,*,†,‡,x,1 Xiaofeng Qian,*,†,1 Runqiu Jiang,*,† Qianghui Liu,*,† Youjing Wang,*,† Chen Chen,*,† Xuehao Wang,*,† Bernhard Ryffel,‡,x and Beicheng Sun*,†

Liver fibrosis is a severe, life-threatening clinical condition resulting from nonresolving hepatitis of different origins. IL-17A is critical in inflammation, but its relation to liver fibrosis remains elusive. We find increased IL-17A expression in fibrotic from HBV-infected patients undergoing partial hepatectomy because of -related early-stage hepatocellular carcinoma in comparison with control nonfibrotic livers from uninfected patients with hepatic hemangioma. In fibrotic livers, IL-17A immu- noreactivity localizes to the inflammatory infiltrate. In experimental carbon tetrachloride–induced liver fibrosis of IL-17RA– deficient mice, we observe reduced neutrophil influx, proinflammatory cytokines, hepatocellular necrosis, inflammation, and

fibrosis as compared with control C57BL/6 mice. IL-17A is produced by neutrophils and T lymphocytes expressing the Th17 Downloaded from lineage–specific transcription factor Retinoic acid receptor–related orphan receptor gt. Furthermore, hepatic stellate cells (HSCs) isolated from naive C57BL/6 mice respond to IL-17A with increased IL-6, a-smooth muscle actin, , and TGF-b mRNA expression, suggesting an IL-17A–driven fibrotic process. Pharmacologic ERK1/2 or p38 inhibition significantly attenuated IL- 17A–induced HSC activation and collagen expression. In conclusion, IL-17A+ Retinoic acid receptor–related orphan receptor gt+ neutrophils and T cells are recruited into the injured liver driving a chronic, fibrotic hepatitis. IL-17A–dependent HSC activation may be critical for liver fibrosis. Thus, blockade of IL-17A could potentially benefit patients with chronic hepatitis and liver http://www.jimmunol.org/ fibrosis. The Journal of Immunology, 2013, 191: 1835–1844.

iver fibrosis is a common outcome of chronic virus-in- liver failure, as well as the principal cause of morbidity and mor- duced hepatitis, autoimmune liver disease, drug abuse, tality (1). Accumulating evidence indicates that immune dysreg- L and , and it is a significant cause of ulations occur in liver fibrosis, leading to unbalanced extracellular matrix synthesis and degradation (2). Hepatic stellate cells (HSCs) are located in the spaces of Disse in *Liver Transplantation Center of the First Affiliated Hospital and State Key Labo- ratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu close contact with , sinusoidal endothelial cells, and Province 210029, People’s Republic of China; †The Key Laboratory of Living Donor autonomic nerve fibers (3). A critical role of activated HSCs in by guest on September 28, 2021 Liver Transplantation, Ministry of Health, Nanjing 210029, People’s Republic of ‡ collagen and extracellular matrix production has been previously China; Molecular and Experimental Immunology and Neurogenetics-Joint Research Unit 7355, Molecular Immunology, The National Center for Scientific Research- observed. HSC activation is characterized as a shift from the x University of Orleans, F-45071 Orleans Cedex 2, France; and Immunology, Institute quiescent “storing” phenotype to the highly proliferative “myofi- of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, Republic of South Africa broblast-like” phenotype (4) and leads to increased fibrillar extra- 1 cellular matrix production with reduced capabilities for degradation Z.T. and X.Q. contributed equally to this work. and remodeling (3). Several proinflammatory mediators are secreted Received for publication November 1, 2012. Accepted for publication June 8, 2013. from the activated HSCs, leading to further amplification of the This work was supported by the National Science Foundation for Distinguished Young Scholars of China (Grant 81225017 to B.S.), the National Natural Science inflammatory process (4, 5). Foundation (Grants 81072029 and 91029721 to B.S.), the National Basic Research Numerous mechanisms contribute to the activation of HSCs, Program of China (Grant 2012CB910800 to B.S.), the Program for Development of including matrix stiffness, altered E- to N-cadherin expression and Innovative Research Teams in the First Affiliated Hospital of Nanjing Medical Uni- versity, the Priority Academic Program of Jiangsu Higher Education Institutions, inflammatory mediators (4, 5). IL-17A is produced by specific Centre National de la Recherche Scientifique, the “Fondation pour la Recherche CD4+ Th cells (Th17) with proinflammatory properties that differ Me´dicale” (FRM Allergy DAL 2007 0822007), and the “Fond Europe´en de De´vel- from Th1 and Th2 cells in development and function. Differen- oppement Re´gional” (FEDER Asthme 1575-32168). tiation of Th17 cells requires the combined action of IL-1b, TGF- Z.T. conducted the study; X.Q., R.J., and X.W. contributed with clinical samples; Q.L. and Y.W. conducted in vitro study; C.C. performed advisory statistics analysis; b, IL-6, and IL-23 (6, 7). These cytokines, which are induced by and B.R. and B.S. designed and supervised the study. activated Kupffer and other cells, promote the expression of the Address correspondence and reprint requests to Prof. Beicheng Sun or Prof. Bernhard lineage-specific transcription factor orphan nuclear receptor reti- Ryffel, Liver Transplantation Center of the First Affiliated Hospital, Nanjing Medical noic acid receptor gt(RORgt; in mice) or RORc (in humans), University, Nanjing, Jiangsu Province 210029, People’s Republic of China (B.S.) or Unite´ Mixte de Recherche 7355, Immunologie et Neuroge´ne´tique Expe´rimentales et which is necessary and sufficient for the development of Th17 cells. Mole´culaires, Centre National de la Recherche Scientifique-University of Orleans, IL-17A plays a critical role in neutrophil recruitment, angiogenesis, 45071 Orle´ans Cedex 2, France (B.R.). E-mail addresses: [email protected] (B.S.) inflammation, and autoimmune disease that has been previously or [email protected] (B.R.) described extensively (6), including in pulmonary and cardiac fi- The online version of this article contains supplemental material. brosis via IL-17RA and MAPK signaling (8, 9). However, very few Abbreviations used in this article: ALT, alanine aminotransferase; CCl4, carbon tet- rachloride; EGFP, enhanced GFP; GBSS, Gey’s balanced salt solution; HSC, hepatic studies have investigated the role of IL-17A in liver fibrosis (10). stellate cell; MMP-9, matrix metalloproteinase 9; MPO, myeloperoxidase; rmIL- In this study, using IL-17RA KO mice, we report that upregu- 17A, recombinant murine IL-17A; RORgt, retinoic acid receptor gt; a-SMA, a-smooth lated expression of IL-17A by RORgt + neutrophils is crucial for muscle actin; TIMP-1, tissue inhibitor of metalloproteinase 1. carbon tetrachloride (CCl4)–induced liver injury leading to in- Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 flammation and fibrosis. Furthermore, IL-17A–dependent HSC www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203013 1836 THE ROLE OF IL-17A IN HSC ACTIVATION AND LIVER FIBROSIS activation and collagen production depend on phosphorylation OptiPrep interphase was gently aspirated, mixed with GBSS, and centri- of MAPK, especially ERK1/2 and p38. Therefore, we conclude fuged at 1400 3 g for 10 min at 4˚C. After another wash, the final cell that IL-17A–dependent HSC activation is critical in CCl -induced pellet was resuspended in DMEM containing penicillin, streptomycin, and 4 10% FBS (complete medium), and then plated on uncoated plastic at a liver injury and fibrosis. These findings provide a potential novel density of 5 3 106 per 10-cm diameter plate. After the first 24 h, nonad- treatment target that could benefit patients by reducing liver fi- herent cells and debris were removed by washing. Cell viability was brosis and leading to an improved prognosis. .90% as assessed by trypan blue exclusion. Purity was 90–95% as assessed by the typical light microscopic appearances. Then cells were digested and plated on 6-well plate at 2 3 105/well. Twenty-four hours Materials and Methods later, cells were considered to be quiescent by the light microscopic ap- Patients pearance of the lipid droplets. Recombinant murine IL-17A (rmIL-17A) was prepared in DMEM containing penicillin, streptomycin, and 10% FBS For this study, we enrolled patients who presented from December 2011 at 0, 1, 10, 30, or 100 ng/ml (PreproTech #210-17; PreproTech, Rocky Hill, to January 2012 at the First Affiliated Hospital of Nanjing Medical Uni- NJ). The restimulation was performed on the first passage of HSCs to versity (Nanjing, China). Informed consent for patient sample analysis was ensure the highest viability and plating efficiency. In some experiments, obtained from each subject before surgery; the study was approved by the ERK/MEK1 inhibitor PD98059 (20 mM; #9900; Cell Signaling), p38 in- appropriate Institutional Ethics Committee. Ten fibrotic livers were col- hibitor SB203580 (10 mM; #5633; Cell Signaling), and JNK inhibitor lected after partial hepatectomy due to early-stage hepatocellular carcinoma SP600125 (25 mM; #8177; Cell Signaling) were prepared in DMSO as which followed hepatitis B virus–related cirrhosis; five control livers are mother liquor before, and 2 ml mother liquor within 2 ml DMEM con- normal tissues obtained from hepatectomy for hepatic hemangioma. taining penicillin, streptomycin, and 10% FBS were used for culture for Clinical characteristics are described in Table I. 1 h and restimulated with 30 ng/ml rmIL-17A for another 24 h, respec- Animals tively, according to previous reports (14, 15). Downloaded from All mouse strains, bred or purchased, consisted of C57BL/6. IL-17RA– Collagen detection assay deficient mice (11) and RORgt-enhanced GFP (EGFP) reporter mice (12) Supernatants of liver homogenate samples were collected from mice including control C57BL/6 were bred at the Immunologie et Neuroge´ne´tique injected with 0.5 g/kg CCl or vehicle; cell media were obtained from 0, 1, Expe´rimentales et Mole´culaires, Orleans, France. The 6- to 10-wk-old 4 10, 30, or 100 ng/ml rmIL-17A restimulation for 24 h or from 30 ng/ml male mice were maintained in sterile, isolated, ventilated cages with rmIL-17A restimulation for 0, 1, 3, 12, and 24 h (1 3 105 cells/ml at 6-well controlled temperature (21–23˚C) and light (12-h light/12-h dark cycle) plate). The collagen concentration was measured using the Sircol assay conditions and ad libitum access to food and water. All experimental according to the manufacturer’s recommended procedure (Biocolor S1000; http://www.jimmunol.org/ procedures were performed according to the local Animal Care Committee Biocolor, Carrickfergus, U.K.). Note that collagen levels induced by dif- following guidelines that comply with those of the French and Chinese ferent concentrations of rmIL-17A were normalized by cell numbers and governments, and the experimental protocol was approved by the local are represented by percentage changes with respect to those of the HSCs ethics committee. without rmIL-17A restimulation. Animal model of acute liver injury and liver fibrosis Cytotoxicity and viability assays Mice received an i.p. injection of CCl4 (2 g/kg; catalog no. 488488; Sigma- Aldrich, St. Louis, MO) in a volume of corn oil equivalent to 200 ml/mouse Cell viability was assessed using the MTT reduction assay. In brief, the to create an acute liver injury model. Mice were sacrificed 24 h after the formation of blue formazan was measured spectrophotometrically from the metabolism of MTT by mitochondrial dehydrogenases that are active only challenge. For the chronic hepatitis and fibrosis model, mice received 3 4 in live cells. Isolated HSCs were seeded in 96-well plates (1 10 cells/ by guest on September 28, 2021 adoseofCCl4 (0.5 g/kg) in a volume of corn oil equivalent to 200 ml/mouse with i.p. injections performed every 3 d for 21 d. Twenty-four hours after the well) and then incubated in DMEM containing penicillin, streptomycin, last challenge, all mice were sacrificed. All control mice were injected with and 10% FBS for 24 h. The cells were then pretreated with various con- corn oil only. All procedures were performed under sterile conditions. centrations of rmIL-17A or with MAPK inhibitor for 24 h; then MTT reagent (5 mg/ml) was added to each of the wells, and the plate was in- Immunohistochemistry cubated for an additional 1 h at 37˚C. The media was then removed, and the intracellular formazan product was dissolved in 250 mlDMSO.The IL-17A immunohistochemistry was performed using 5-mm paraffin sec- absorbance of each well was measured at 540 nm using a microplate tions. The sections were deparaffinized in xylene and rehydrated in alcohol reader. OD values from untreated control cells were designated as 100% and distilled water. Ag retrieval was performed by heating sections in viability. Triplicate specimen was repeated three times independently. Com- solution (10 mM citrate buffer, pH 6.0) in a pressure cooker. Three percent parisons between groups were performed by one-way ANOVA followed by H2O2 was used to eliminate endogenous peroxidase. Slides were washed the Newman–Keuls posttest. with TBS-Triton three times and one time with TBS for 5 min. Nonspecific binding was blocked using 5% goat serum for 30 min. The blocking buffer Liver homogenization and myeloperoxidase activity was then removed and sections were incubated with a polyclonal anti–IL- 17A Ab (ab79056; Abcam, Cambridge, U.K.). Control experiments were Myeloperoxidase (MPO) activity assay was used as indirect index of the incubated with rabbit serum replacing the first Ab. After incubation over- neutrophil presence in lung, liver, and intestine (16). The 200-mg fresh night at 4˚C and washing, the sections were incubated with biotinylated goat samples in 1 ml PBS were homogenized and resuspended in PBS (1 ml) anti-rabbit IgG Ab. After washing, peroxidase-coupled Ab was applied for containing 0.5% hexadecyltrimethyl ammonium bromide and 5 mM 30 min at room temperature. Bound Ab was detected with 3,39-di- EDTA. After centrifugation, aliquots of 50 ml of supernatants were placed aminobenzidine tetrachloride (Sigma-Aldrich). All sections were then in test tubes containing 2 ml HBSS, 100 ml o-dianisidine dihydrochloride counterstained with hematoxylin. Brown-yellow staining was recog- (1.25 mg/ml), and 100 mlH2O2 0.05%. After 15-min incubation at 37˚C nized as positive in the cells. with shaking, the reaction was stopped adding 100 ml NaN3 1%. Absor- bance was determined at 460 nm using a microplate reader. HSC isolation and culture Microscopic investigation and TUNEL assay Primary mouse HSCs were isolated from livers of C57BL/6 mice accord- ing to a modified method described previously (13). In brief, livers were Fragments of liver tissues were fixed in 4% buffered formaldehyde. Sections perfused in situ with 45 ml Life Technologies Liver Perfusion Media (3 mm) of liver were cut and stained with H&E. Detection of apoptosis by (Invitrogen, Carlsbad, CA) followed by 45 ml Life Technologies Liver TUNEL assay was according to the protocol (Millipore S7100; Billerica, MA) (17). Liver H&E sections were graded for CCl4 injury under Leica Digestion Media (Invitrogen). The liver digests were filtered through a cell 3 3 strainer and washed with Gey’s balanced salt solution (GBSS; Sigma- DM4000 B Upright Research Microscope (200 and 400 ) using the system devised by Suzuki et al. (18) and as described previously. TUNEL+ Aldrich) containing DNase I (2 mg/ml; Roche Diagnostics). The homog- 3 enate was centrifuged at 25 3 g for 5 min at room temperature to remove cells were counted at 200 fields. All of the microscopic figures were the hepatocytes. The supernatant was transferred to a new tube and acquired by Leica Microsystems software (Leica, Germany). centrifuged at 400 3 g for 10 min at 4˚C. The cell pellet was then Cytokine analysis resuspended in 5 ml of 15% OptiPrep (Sigma-Aldrich) and loaded care- fully with 5 ml of 11.5% OptiPrep and 2 mL GBSS. After centrifuged at The homogenized tissue was centrifuged at 10,000 rpm for 15 min, and the 1400 3 g for 17 min at 4˚C, the cell fraction in the GBSS and 11.5% supernatant was recovered and stored at 280˚C until analyses. The levels The Journal of Immunology 1837 of cytokines were measured by ELISA using commercial kits (R&D, Results Abingdon, U.K.) according to manufacturer’s instructions. IL-17A expression is increased in patients with chronic Western blot hepatitis and cirrhosis Proteins were extracted from cultured cells and quantified using the Fresh fibrotic liver collected after partial hepatectomy due to early- Bradford assay (Bio-Rad Laboratories, Hercules, CA). Protein samples stage hepatocellular carcinoma and control liver tissues were ob- pretreated with protease and phosphatase inhibitors were fractionated by tained from The First Affiliated Hospital of Nanjing Medical SDS-PAGE, and upon electrophoresis, the proteins were transferred onto nitrocellulose membranes and blotted overnight as described previously University (Table I). All the samples were weighed and homoge- (19). The anti–a-smooth muscle actin (a-SMA; ab5694; Abcam), anti- nized following the methods previously described. Collagen levels ERK1 (sc-93), anti–p-ERK1/2 (sc-16982R, Thr202/Tyr204), anti-p38 (sc- were significantly increased in the fibrotic liver (Fig. 1A), and 182 728), anti–p-p38 (sc-7975R, Tyr ), JNK (sc-571), anti–p-JNK1 (sc-6254), a-SMA gene transcripts were upregulated (Fig. 1B). Masson tri- and anti–IL-17RA (ab134086) were used (Santa Cruz Biotechnology, chrome stain of liver sections confirmed a huge collagen deposi- Dallas, TX; Abcam; or Cell Signal Technology, Beverly, MA). Moreover, for computer-assisted semiquantitative analysis of MAPK phosphorylation tion as a result of disrupted normal metabolism and hepatocellular and a-SMA expression, ImageJ software (http:/rsbweb.nih.gov/ij/) was used necrosis with destruction of the normal hepatic lobular structure according to the manufacturer’s instructions. and formation of reparative pseudolobules (1) (Fig. 1C). Because Measurement of plasma alanine aminotransferase activity IL-17A exhibits the potential to recruit neutrophils and induces the expression of proinflammatory cytokines and chemokines (7), Blood was obtained by cardiac puncture at the time of sacrifice for analysis we investigated whether the development of clinical liver fibrosis of serum alanine aminotransferase (ALT) as an index of hepatocellular was associated with increased expression of IL-17A. Immuno- injury. Measurements of serum ALT were made using a diagnostic kit Downloaded from (Sigma-Aldrich). histochemistry analysis using IL-17A Ab revealed increased IL- 17A staining in the inflammatory foci in the margins of fibrotic Liver mononuclear cell isolation and flow cytometry analysis area between the regenerating pseudolobular structures. In par- Liver mononuclear cells were isolated from mice 24 h after the CCl4 ticular, IL-17A was found in Kupffer cells, activated HSCs, lym- challenge as described previously (20). In brief, liver mononuclear cells phoid cells, and neutrophils (Fig. 1D). Further, IL-1b and IL-17A 3 5 were collected and plated at 1 10 /ml and restimulated at 6 h in vitro gene expression were significantly upregulated in fibrotic liver with phorbol 12-myristate 13-acetate (50 ng/ml) and ionomycin (750 ng/ http://www.jimmunol.org/ ml) in complete medium (IMDM supplemented with 5% [v/v] FCS, L- (Fig. 1E, 1F), which suggested that IL-17A contribute to chronic glutamine [2 mM], penicillin [100 U/ml], streptomycin [100 mg/ml], and hepatitis-induced liver fibrosis. 2-ME [50 nM], all from Invitrogen). PBMCs were isolated from the clinic samples by Ficoll gradient and stimulated at 37˚C for 6 h. The Abs IL-17RA deficiency protects from CCl4-induced acute liver used for FACS analysis, FITC–anti-CD3e (clone 145-2C11), PerCP- injury in mice Cy5.5–anti–IL-17A (clone TC11-18H10), Pacific blue–anti-CD4 (cloneRMA- 5), allophycocyanin-Cy7–anti-CD8a (clone 53-6.7), PE-Cy7–anti–Gr-1 Because IL-17A is required for neutrophil recruitment (11) and (clone RB6-8C5), and isotype-matched controls were purchased from promotes proinflammatory cytokine and chemokine expression BD Pharmingen (San Diego, CA). Rabbit anti-mouse GFP IgG fraction (21), we investigated whether the IL-17A pathway plays a role in and Alexa Fluor 488–labeled goat anti-rabbit IgG were purchased from CCl4-induced acute liver injury. C57BL/6 and IL-17RA–deficient by guest on September 28, 2021 Invitrogen (Eugene, OR). The cells were counted and stained for ex- mice were both randomly divided into two groups and received tracellular and intracellular markers, and analyzed by FlowJo 7.6.4. (Tree Star, Ashland, OR). a single i.p. injection of CCl4 (2 g/kg) or the vehicle, corn oil, respectively. We found increased hepatic IL-17A expression after RT-PCR analysis for IL-1b, IL-6, IL-17A, TGF-b, and a-SMA CCl4 administration at 24 h in C57BL/6 and IL-17RA–deficient mRNA mice (Fig. 2A). Microscopically, pericentrolobular necrosis, micro- Total RNA of the frozen liver samples was extracted with TRIzol reagent vacuolar degeneration, and inflammation quantified by the Suzuki (Invitrogen) and quantified. RT-PCR was performed and analyzed in triplicate score were significantly lower in IL-17RA–deficient mice than in assays by SYBR Green master mix and either the ABI Prism 7700 Sequence C57BL/6 mice (Fig. 2B, 2D). Apoptosis of hepatocytes and Kupffer Detection System or the Step one plus (Applied Biosystems, Darmstadt, Germany). Different kinds of mRNA in the liver were quantified by using cells as assessed by TUNEL staining was dramatically attenuated in specific primer pairs (Supplemental Table I; Invitrogen Shanghai). PCR IL-17RA–deficient livers (Fig. 2C, 2E). Further, a significant re- products were analyzed by electrophoresis on a 1.5% agarose gel. duction of serum ALT levels as a marker of liver injury (Fig. 2F) and Statistical analysis of MPO activity reflecting neutrophil recruitment (Fig. 2G) were found in IL-17RA–deficient mice. Thus, our findings indicate that Data are expressed as mean 6 SEM. Comparisons between groups were IL-17A plays an important role in the development of CCl -induced performed by using a two-tailed unpaired Student t test or one-way ANOVA 4 with Newman–Keuls post hoc for individual subgroup comparison. The 4.0 acute liver damage and inflammation, as the absence of IL-17RA version (2005) of the GraphPad Prism software was used for this purpose. signaling largely protected mice from CCl4-induced liver injury and A p value ,0.05 indicates a significant result. inflammation-induced damage.

Table I. Clinical characteristics

Characteristics Fibrotic Liver Control Liver n 10 5 Sex 10 male 5 male Mean age (minimum–maximum), y 55.1 (43–61) 46.6 (36–51) HBV infected, n 10 0 Mean ALT level (minimum-maximum), IU/l 42.4 (15–120) 21.1 (13–68) Mean AST level (minimum-maximum), IU/l 38.8 (14–112) 18.6 (11–44) Mean total bilirubin level (minimum-maximum) 16.5 (4–25) 12.7 (5–19) Mean serum albumin (minimum-maximum), g/l 31 (26–36) 34 (31–36) Mean primary mass size (minimum-maximum), cm 5.1 (3.8–9.1) (tumor) 8.5 (6.7–12) (Hemangioma) AST, aspartate aminotransferase; HBV, hepatitis B virus. 1838 THE ROLE OF IL-17A IN HSC ACTIVATION AND LIVER FIBROSIS Downloaded from

FIGURE 1. IL-17A is increased in clinical liver fibrosis. Fibrotic and normal liver tissues were collected from patients undergoing partial hepatectomy for early-stage hepatocellular carcinoma or hepatic hemangioma, respectively. (A) Collagen concentration was measured in liver homogenate (0.1 g) in fibrotic or normal livers. (B) a-SMA RNA was assessed by real-time PCR. (C) Collagen deposition in liver using Masson’s trichrome stain (original magnification 3200). (D) Immunostaining of liver with anti–human IL-17A Ab (3400 original magnification is shown). One representative result is shown from 10 specimens. http://www.jimmunol.org/ Negative controls are shown in Supplemental Fig. 1. (E and F) The transcription of IL-1b and IL-17A genes was measured by real-time PCR in fibrotic or normal liver separately. Data are representative of 10 liver specimens and expressed as mean 6 SEM (n = 10; *p , 0.05, **p , 0.01, ***p , 0.001).

Inflammatory cytokine expression levels after acute liver injury the inflammatory cytokine expression profiles in C57BL/6 and IL- 2/2 are dependent on IL-17RA signaling 17RA mice injected CCl4 or corn oil. IL-1b is the principal Although IL-17RA deficiency largely protected the mice from proinflammatory cytokine that activates several cytokines and CCl4-induced acute liver injury, the exact mechanisms underlying chemokines leading to inflammatory cell recruitment and cell this protective capacity remain unclear. Therefore, we investigated damage (22). IL-1b expression in the liver from IL-17RA–deficient by guest on September 28, 2021

FIGURE 2. IL-17RA deficiency protects from CCl4-induced acute liver injury. C57BL/6 and IL-17RA–deficient mice were subjected to a single dose of 2 g/ml CCl4 or vehicle by i.p. administration, and the livers were analyzed 24 h after administration. (A) IL-17A in liver homogenates (ELISA) from C57BL/6 or IL-17RA2/2 mice. (B) Microscopic sections showing inflammation and necrosis (H&E, original magnification 3200 and 3400). (C) Rep- resentative TUNEL-stained sections showing apoptosis (original magnification 3200 and 3400), (D) Suzuki score estimating the liver lesion and (E) TUNEL-positive cells were counted from 20 random microscope fields and the mean values 6 SEM were presented. (F) Serum ALT levels. (G) Hepatic 2/2 MPO activity from CCl4-injected C57BL/6 and IL-17RA mice compared with mice after vehicle administration. Data represent the mean values 6 SEM (n = 6–8 mice per group) and are representative of three independent experiments (**p , 0.01, ***p , 0.001). The Journal of Immunology 1839 Downloaded from

FIGURE 3. IL-17RA–dependent cytokines produced after acute liver injury. Cytokines expressed in the liver in C57BL/6 and IL-17RA2/2 mice 24 h after CCl4 injection–induced liver injury. (A–D) Reduced IL-1b, IL-6, CXCL1/KC, and TNF-a protein in liver homogenate and (E, F) MMP-9 and TIMP-1 in IL-17RA2/2 mice as compared with C57BL/6 mice (by ELISA). Data are presented as means 6 SEM (n = 6–8 mice per group) and are representative of three independent experiments (*p , 0.05, **p , 0.01, ***p , 0.001). http://www.jimmunol.org/ mice was significantly lower after the CCl4 challenge as compared remodeling enzymes, were increased in C57BL/6 mice and were with C57BL/6 mice (Fig. 3A). Moreover, IL-6, an important cy- IL-17RA signaling dependent (9) (Fig. 3E, 3F). Therefore, IL-1b tokine for Th17 differentiation, was attenuated in IL-17RA2/2 and several key proinflammatory mediators were significantly mice (Fig. 3B), suggesting that IL-6 may contribute to the re- diminished in the absence of IL-17RA signaling. sponse in accordance with previous reports (7, 23). CXCL1/KC Neutrophils are the principal IL-17A–producing cells after and TNF-a upregulation in CCl4 injected in C57BL/6 mice was significantly attenuated in IL-17RA2/2 mice, indicating that IL- CCl4-induced liver injury 17RA deficiency largely limits inflammation in tissue (Fig. 3C, Hepatic-infiltrating mononuclear cells were obtained from mice at by guest on September 28, 2021 3D). Levels of matrix metalloproteinase 9 (MMP-9) and tissue 24 h after CCl4 administration (2 g/kg i.p.), and IL-17A–producing inhibitor of metalloproteinase 1 (TIMP-1), which reflect matrix cells were analyzed by flow cytometry. First, among CD3+ cells,

FIGURE 4. Neutrophils are the principal IL-17A–producing cells in CCl4-induced liver injury. Infiltrating mononuclear cells were isolated from the liver 24 h after CCl4 or vehicle injection. Cells were permeabilized, stained by IL-17A and cell lineage–specific Abs, and analyzed by flow cytometry. (A) Proportion of IL-17A+ cells is presented in CD3+ populations. (B) CD4+ T cells and CD8+ T cells were analyzed based on CD3+ gate. (C) Using RORgt– EGFP mice, we investigated the IL-17A–related transcription factor expressed in CD4+ and CD8+ subpopulations. (D) IL-17A expression in Gr-1hi neutrophils. (E) FACS analysis indicating that IL-17A expression in Gr-1hi neutrophils is RORgt–EGFP dependent. (F) Total cell counts of CD4+, CD8+,or hi Gr-1 subpopulations expressing IL-17A of liver-infiltrating mononuclear cells from vehicle or CCl4 challenge. Data are presented as mean value 6 SEM (**p , 0.01, ***p , 0.001). Results from flow cytometry are from one representative experiment of six independent studies. 1840 THE ROLE OF IL-17A IN HSC ACTIVATION AND LIVER FIBROSIS

IL-17A expression did not increase (Fig. 4A). Using the CD3+ suggesting that the mechanism underlying the protection of the + + gate, we investigated IL-17A expression in CD4 and CD8 liver from CCl4-induced fibrosis in IL-17RA–deficient mice might T cells. We found a slight increase of IL-17A expression in CD4+ be partly due to downregulation of TGF-b. T cells that typically express IL-17A (24) (from 4.11 to 5.56%), Activation of HSCs and production of collagen are IL-17A but also in CD8 T cells (from 3.43 to 5.78%) after CCl admin- 4 dependent istration (Fig. 4B). Furthermore, we asked whether the Th17 lin- eage marker is expressed in CD4+ and CD8+ T cells. We induced We then asked whether HSCs contribute to IL-17A–dependent acute liver injury in RORgt–EGFP reporter mice and found that liver fibrosis. First, we confirmed that isolated HSCs express IL- the recruited CD4+ and CD8+ T cells expressed RORgt (Fig. 4C). 17RA and respond to rIL-17A stimulation (Supplemental Fig. Because we established abundant neutrophil recruitment in IL- 1B). We found that in vitro activation of HSCs is IL-17A de- 17A–dependent CCl4 injury-induced acute liver inflammation, we pendent. rIL-17A was used to activate isolated quiescent HSCs for asked whether neutrophils may also be a source of IL-17A in 24 h at 0, 1, 10, 30, or 100 ng/ml. We demonstrated that IL-6 and acute liver injury (25, 26). Therefore, we labeled IL-17A+ cells a-SMA gene transcripts were dose dependently increased by with the Gr-1 (Ly-6G) Ab and found a dramatic increase of Gr-1hi rmIL-17A stimulation, whereas TGF-b gene expression exhibited neutrophils (27) upon CCl4 injury (1.2–10.3%) (Fig. 4D). Fur- significant increase only upon 100 ng/ml rmIL-17A restimulation thermore, the Gr-1hi IL-17A+ neutrophils exhibited the Th17 lin- (Fig. 6A). Moreover, collagen production in the medium was eage transcription factor RORgt (Fig. 4E). Based on the different upregulated by the increasing concentration of rmIL-17A stimu- IL-17A+ proportions of cells and of total infiltrating cell counts lation (Fig. 6B, Supplemental Fig. 1C). Increased collagen pro-

(Table II), we calculated the numbers of IL-17A producing cells in duction was observed already 1 h after the culture and increased Downloaded from each population (Fig. 4F). These absolute numbers reveal a major during the 48-h culture period at 30 ng/ml rmIL-17A in quiescent contribution by neutrophils and, to a lesser extent, by CD4+ T and HSCs (Fig. 6C), which supports the notion that IL-17A activates CD8+ cells to IL-17A production. Therefore, in addition to a direct HSCs to produce collagen. We further investigated the mecha- effect of neutrophils on hepatocytes and nonparenchymal hepatic nisms underlying IL-17A promotion of liver fibrosis. We found cells, neutrophils at the injury site potentially augment the in- that the phosphorylation of MAPK members, such as ERK1/2,

flammation and liver injury through the IL-17A production. The JNK, and p38, are IL-17A signaling dependent in HSCs, and http://www.jimmunol.org/ fact that Gr-1hi neutrophils express the RORgt lineage marker that the phosphorylation was distinct at 30 min and plateaued for and represent the major source of IL-17A is biologically signifi- p-ERK1/2 between 6 and 24 h (Fig. 6D). We further used nontoxic cant, suggesting an amplification of the inflammatory response by concentrations of MAPK inhibitors to investigate whether IL- neutrophils. 17A–induced collagen production is MAPK signaling dependent and which pathways are potentially involved. Isolated HSCs were CCl4-induced chronic inflammation and liver fibrosis is IL- cultured in complete medium with JNK inhibitor SP600125 17RA dependent (25mM), ERK/MEK1 inhibitor PD98059 (20 mM), or p38 inhib-

Because IL-17RA deficiency largely protected mice from CCl4- itor SB203580 (10mM) for 1 h. The cells were stimulated with associated liver injury, and upregulated MMP-9 and TIMP-1 were rmIL-17A for 24 h and absence of a cytotoxic effect of the phar- by guest on September 28, 2021 also attenuated in IL-17RA KO mice (28), we used IL-17RA– macologic inhibitors was confirmed by MTT viability assay (Sup- deficient mice to investigate the contribution of IL-17A signaling plemental Fig. 1D). We found that SB203580 or PD98059 sig- to CCl4-induced fibrosis (9). In brief, CCl4 was injected into nificantly reduced collagen production in IL-17A–stimulated or C57BL/6 or IL-17RA KO mice at 0.5 g/kg every 3 d. After 21 d, quiescent HSCs, whereas the inhibitory effects of SP600125 were mice were sacrificed. The microscopic liver sections revealed at- observed only in quiescent HSCs (Fig. 6E). Moreover, a-SMA tenuated hepatocellular damage, inflammation, and fibrosis in IL- production appears to be ERK1/2 and p38 dependent (Fig. 6F). 17RA KO mice as compared with C57BL/6 mice (Fig. 5A, 5C). These results strongly suggest that IL-17A activate HSCs in vitro Using Masson staining, we found that fibrous septa form bridges JNK independently, whereas IL-17A–dependent collagen expres- between adjacent vascular structures, portal to portal and portal sion is largely dependent on ERK1/2 and p38 pathway. to central areas, and some parenchymal nodules were completely surrounded by fibrosis, indicating early cirrhosis, which was Discussion drastically reduced in IL-17RA KO mice (Fig. 5B). Liver injury, Liver fibrosis is a serious medical condition that occurs upon measured by serum ALT activity, exhibited a significant increase chronic hepatitis virus infection, drug abuse, alcoholic liver dis- in CCl4-challenged mice that was attenuated in IL-17RA–deficient ease, and autoimmune hepatitis, and potentially leads to portal mice (Fig. 5D). Collagen concentrations in liver homogenate were hypertension, hepatic failure, and liver cancer (1). It has been also diminished in IL-17RA KO mice, which is consistent with the widely reported that activated HSCs are the principal source of the reduced Masson staining results (Fig. 5E). TGF-b, the typical extracellular matrix and collagen production (29), and upon acti- fibrosis-associated cytokine, is IL-17RA dependent (Fig. 5F), vation they are instrumental in liver fibrosis and cirrhosis. We used

Table II. Different infiltrating mononuclear cells counting

Number (3105)

Population Names Vehicle CCl4 Total mononuclear cells, mean 6 SD 1.38 6 0.282 3.64 6 0.46a Neutrophils, mean 6 SD 0.0352 6 0.031 0.6443 6 0.081b CD4+ T cells, mean 6 SD 0.1808 6 0.037 0.5913 6 0.074a CD8+ T cells, mean 6 SD 0.0931 6 0.019 0.3662 6 0.046b Data are mean 6 SD (n =10). a b p , 0.01 or p , 0.001, significantly different from control or CCl4 group, respectively, using Student t test. The Journal of Immunology 1841 Downloaded from http://www.jimmunol.org/

FIGURE 5. CCl4-induced liver fibrosis is IL-17RA dependent. C57BL/6 and IL-17RA–deficient mice were administered i.p. 0.5 g/kg CCl4 (diluted in corn oil) or oil only twice a week for 21 d, and mice were sacrificed at 24 h after the last challenge. (A) Liver sections from C57BL/6 and IL-17RA– deficient mice injected with CCl4 or oil were stained with H&E (original magnification 3200, 3400). (B) Collagen deposition in liver sections (Masson trichrome, original magnification 3100). (C) Microscopic liver lesions were graded by Suzuki scores. (D) Serum ALT levels indicating liver injury. (E) Sircol assay was performed to measure the collagen in the liver homogenate supernatant. (F) KC in liver homogenates (ELISA). Data are mean 6 SEM (n = 6–8 mice/group) and are representative of three independent experiments (*p , 0.05, **p , 0.01, ***p , 0.001). by guest on September 28, 2021 gene-deficient mice and evaluated the role of IL-17A in CCl4- inflammatory response (39); neutrophils also represent an im- induced acute and chronic injury and HSC activation. portant source of IL-17A in inflamed synovium (40) and renal IL-17A/IL-17RA signaling plays a critical role in autoimmune ischemia/reperfusion (41). In liver, Kupffer cells were reported to diseases such as asthma, arthritis, ulcerative colitis, and Crohn’s produce IL-17A, which could potentially promote liver fibrosis in disease (30–33). IL-17A is involved in neutrophil recruitment and the ligation and CCl4 model (42). Our findings indicated angiogenesis (34), and IL-17A expression is partially dependent that CD4+ and CD8+ T cells both exhibited increased IL-17A on IL-1 and IL-23 signaling (8, 35). However, the relationship expression. However, Gr-1hi neutrophils represent the major source between IL-17A and liver fibrosis remains elusive and further of IL-17A among the cells infiltrating the liver 24 h after CCl4 investigation is warranted. In this study, we used clinical samples injection. The upregulation of transcription factor RORgtin and found that IL-17A was upregulated in fibrotic liver tissue. This neutrophils suggests that neutrophils express the Th17 transcrip- finding is consistent with the elevated collagen and a-SMA levels tion factor, and hence enabling those cells to produce IL-17A, that were found in the liver. Furthermore, we evaluated the con- which is consistent with our previous findings in hepatic ischemia/ tribution of IL-17A signaling in experimental CCl4-induced acute reperfusion injury (20). Therefore, IL-17A–expressing neutrophils liver injury via genetic analysis. Compared with the wild type potentially exhibit an autoamplification effect. T cells as the typical C57BL6 mice, IL-17RA–deficient mice exhibited a protective IL-17A–producing cells also present and contribute to IL-17A pro- effect. Hepatic necrosis and inflammation, serum ALT levels, duction, whereas NK and NKT cells did not exhibit upregulation hepatic MMP-9, TIMP-1 and collagen content, and MPO activity of IL-17A or RORgt–EGFP (data not shown). were attenuated in IL-17RA KO mice, indicating the CCl4-in- The IL-17RA–deficient mice exhibited dampened MMP-9 and duced liver injury and inflammation is likely IL-17A signaling TIMP-1 expression levels, which are essential to extracellular dependent. IL-17A levels were not significantly reduced in IL- remodeling, indicating that IL-17A potentially plays a profibrotic 17RA KO mice upon 2-g/kg CCl4 challenge compared with WT role. Thus, by injecting mice with CCl4 at indicated intervals, we mice. However, in the absence of IL-17RA, signaling of CCl4- successfully induced liver fibrosis in C57BL/6 and IL-17RA– induced liver injury and inflammation is attenuated, supporting the deficient mice. We demonstrated that IL-17RA deficiency largely notion of an IL-17–dependent effect. protects mice from repeated challenges that cause liver injury, Because IL-17A plays an important role in the regulation of profibrotic cytokine expression, and fibrosis with collagen depo- inflammation, we aimed to verify the sources of IL-17A in ex- sition. TGF-b, the hallmark of fibrotic processes, enhances col- perimental liver lesions. Previous reports suggested that CD4+ lagen and matrix deposition by activation of HSCs through the T cells, NKT cells, and gd T cells produce IL-17A (26, 36, 37). Smad2 and Smad3 pathways. This is further supported by the fact Specifically, CD8+ cells produce IL-17A in patients with chronic that neutralization of the biological activity of TGF-b ameliorates obstructive pulmonary disease (38). For innate immune cells, IL- experimental liver fibrosis (2). IL-17A was reported to exhibit the 17A expressed by Paneth cells has been implicated in the systemic ability to promote pulmonary fibrosis via TGF-b (43). In this 1842 THE ROLE OF IL-17A IN HSC ACTIVATION AND LIVER FIBROSIS Downloaded from http://www.jimmunol.org/

FIGURE 6. HSC activation is IL-17A dependent. Mouse HSCs were isolated from naive C57BL/6 mice and stimulated as previously described. (A) IL-6, TGF-b, and a-SMA RNA expression in HSCs was activated in vitro by rmIL-17A; qPCR expressed as fold RNA expression over GAPDH. (B) Collagen released into the supernatant upon 0, 1, 10, 30, and 100 ng/ml rmIL-17A exhibited dose dependency. Results were normalized to the cell viability and presented by percentage changes with respect to those of the cells without rmIL-17A restimulation. (C) Collagen released into the supernatant upon 30 ng/ by guest on September 28, 2021 ml rmIL-17A restimulation was time dependent. (D) Western blot analysis at 0 min, 5 min, 10 min, 30 min, 1 h, 3 h, 12 h, and 24 h after 30 ng/ml rm-IL- 17A stimulation to investigate whether the activation of HSCs is relying on ERK/MEK1, MAPK p38, or JNK phosphorylation. Quiescent HSCs (0 min) were taken as negative control. ImageJ was used for computer-assisted semiquantitative analysis of ERK1/2, JNK, or p38 phosphorylation. (E and F) HSCs were cultured with JNK inhibitor SP600125, ERK/MEK1 inhibitor PD98059, or p38 inhibitor SB203580 for 1 h, and stimulated with 30 ng/ml rmIL-17A or those inhibitors for another 24 h. The supernatant and cell proteins were collected, respectively. (E) Collagen concentrations were measured by Sircol assay. (F) a-SMA expression was examined by Western blot. ImageJ was used for computer-assisted semiquantitative analysis of a-SMA expression. Data represent mean 6 SEM. Experiments were repeated three times; comparisons between subgroups were performed by one-way ANOVA followed by the Newman–Keuls posttest: *p , 0.05, **p , 0.01, ***p , 0.001 (compared with medium cultured cells); #p , 0.05, ##p , 0.01 (compared with rmIL-17A– treated cells). study, we revealed IL-17RA–dependent TGF-b upregulation in found that Stat3 are critical in IL-17A–induced HSC activation CCl4-induced liver fibrosis. Because TGF-b is the key cytokine and collagen production, and rmIL-17A could promote the ex- that promotes IL-17A+ cell differentiation (7), further investiga- pression of a-SMA in isolated HSCs (42). Because Stat3 could be tion of the signaling that drives the positive feedback of TGF-b phosphorylated by activated ERK1/2, JNK, or p38 (at Ser727), we and IL-17A is warranted. further investigated which MAPK pathway may be involved. We In chronic liver disease, HSC differentiates into a myofibroblast- observed that ERK1/2, p38, and JNK exhibit time-dependent like phenotype characterized by proliferation and the deposition of phosphorylation in HSCs upon IL-17A restimulation, and their abnormal matrix (4). HSCs that express multiple receptors are the response to rmIL-17A restimulation is rapid (within 30 min), like primary targets of fibrogenic stimuli in the diseased liver. Among previously observed in human airway smooth muscle cells (45, these, IL-17RA is the newly discovered receptor expressed in 46). Furthermore, pharmacological inhibition of ERK1/2 or HSC-mediated alcoholic liver fibrosis (24). We also demonstrated p38 decreased collagen and a-SMA expression in rmIL-17A– that HSCs from C57BL/6 mice constitutively express IL-17RA challenged or quiescent HSCs. This is a novel finding that may (Supplemental Fig. 1B) and responded to rIL-17A restimulation by partially explain how IL-17A signaling induced profibrotic protein expressing IL-6, a-SMA, and TGF-b mRNA. expression. JNK phosphorylation appeared to be rmIL-17A de- MAPKs are activated within the protein kinase family, which pendent, but JNK inhibition by the selective inhibitor SP600125 respond to extracellular stimuli (mitogens, heat shock, or proin- could reduce collagen and a-SMA production only in inactivated flammatory cytokines) and modulate cellular activities, such as HSCs, but not on rmIL-17A–induced collagen deposition and gene expression, proliferation, differentiation, mitosis, cell sur- a-SMA production. The disparity between JNK and ERK1/2 or vival, and apoptosis (44). By using GFAPStat32/2 mice (glial p38 was consistent with the similar findings on rat HSCs (47). 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