Interleukin-17 signaling in inflammatory, Kupffer cells, and hepatic stellate cells exacerbates liver fibrosis in mice - PubMed (original) (raw)

. 2012 Sep;143(3):765-776.e3.

doi: 10.1053/j.gastro.2012.05.049. Epub 2012 Jun 8.

Kai Wang 2, Tomonori Aoyama 3, Sergei I Grivennikov 4, YongHan Paik 5, David Scholten 6, Min Cong 3, Keiko Iwaisako 7, Xiao Liu 3, Mingjun Zhang 3, Christoph H Österreicher 8, Felix Stickel 9, Klaus Ley 10, David A Brenner 3, Tatiana Kisseleva 11

Affiliations

Interleukin-17 signaling in inflammatory, Kupffer cells, and hepatic stellate cells exacerbates liver fibrosis in mice

Fanli Meng et al. Gastroenterology. 2012 Sep.

Abstract

Background & aims: Interleukin (IL)-17 signaling has been implicated in lung and skin fibrosis. We examined the role of IL-17 signaling in the pathogenesis of liver fibrosis in mice.

Methods: Using cholestatic and hepatotoxic models of liver injury, we compared the development of liver fibrosis in wild-type mice with that of IL-17RA(-/-) mice and of bone marrow chimeric mice devoid of IL-17 signaling in immune and Kupffer cells (IL-17RA(-/-) to wild-type and IL-17A(-/-) to wild-type mice) or liver resident cells (wild-type to IL-17RA(-/-) mice).

Results: In response to liver injury, levels of Il-17A and its receptor increased. IL-17A increased appeared to promote fibrosis by activating inflammatory and liver resident cells. IL-17 signaling facilitated production of IL-6, IL-1, and tumor necrosis factor-α by inflammatory cells and increased the expression of transforming growth factor-1, a fibrogenic cytokine. IL-17 directly induced production of collagen type I in hepatic stellate cells by activating the signal transducer and activator of transcription 3 (Stat3) signaling pathway. Mice devoid of Stat3 signaling in hepatic stellate cells (GFAPStat3(-/-) mice) were less susceptible to fibrosis. Furthermore, deletion of IL-23 from immune cells attenuated liver fibrosis, whereas deletion of IL-22 exacerbated fibrosis. Administration of IL-22 and IL-17E (IL-25, a negative regulator of IL-23) protected mice from bile duct ligation-induced liver fibrosis.

Conclusions: IL-17 induces liver fibrosis through multiple mechanisms in mice. Reagents that block these pathways might be developed as therapeutics for patients with cirrhosis.

Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

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Conflict of interest statement

Conflict of interests: none to declare

Figures

Figure 1

Figure 1. Development of liver fibrosis is strongly inhibited in IL-17RA−/− mice

(A)Expression of IL-17A and IL-17F, IL-17RA and IL-17C genes is upregulated during liver fibrosis. The data is fold induction of mRNA expression from whole liver of BDL (n=10) and CCl4-treated (n=10) mice compared to control mice, *p<0.01. Fibrogenic liver injury is associated with increased serum IL-17A detected in BDL (5 days, n=6; 10 days n=5) and CCl4-treated (8 weeks, n=5) mice by ELISA, **p<0.05. (B) Development of BDL- and CCl4-induced liver fibrosis is inhibited in IL-17RA−/− mice. Livers from wild type mice (untreated n=3), BDL (n=10) and CCl4-treated (n=10), and IL-17RA−/− mice (untreated n=3), BDL (n=9) and CCl4-treated (n=10), were analyzed by H&E, Sirius Red and α-SMA staining and quantified (*p<0.01). Bright field micrographs are shown using × 10 objectives. Liver function was assessed by ALT, *p<0.01. (C) Expression of ibrogenic genes is downregulated in whole livers from BDL and CCl4-treated IL-17RA−/− mice compared to wild type mice. The data are shown as fold mRNA induction compared with untreated mice, *p<0.02. (D)Reduced expression of α-SMA protein was detected in livers of BDL- and CCl4-treated IL-17RA−/− mice compared to the wild type mice.

Figure 2

Figure 2. IL-17A/IL-17RA signaling in fibrotic liver is mediated by immune cells, KC and HSCs

(A)Hepatic T cells are the major source of IL-17A in fibrotic livers. Lymphoid fraction from sham (n=3) and BDL (n=3) wild type mice were in vitro stimulated with PMA (100ng/ml)+Ionomycin (500ng/ml) or IL-23 (20 ng/ml) for 5 hours, and analyzed by flow cytometry for intracellular IL-17A and IL-17F expression. Expression of IL-17A was further analyzed in CD45+, CD3+, CD4+, CD8a+, TCRγδ+, TCRβ+, NK1.1+, CD11b+ cells. Representative dot plots are shown. (B) Primary wild type hepatocytes, quiescent and in vitro activated HSCs, and KC were stimulated with IL-17A (10ng/ml), IL-17F (10 ng/ml), or a combination of both. mRNA levels of IL-17 cytokine/receptors and inflammatory gene expression are shown as fold induction compared with untreated cells, *p<0.01, ** non significant.

Figure 3

Figure 3. Selective ablation of IL-17 signaling in inflammatory cells + KC and liver resident cells results in inhibition of BDL-induced liver fibrosis

(A) Development of liver fibrosis in BM chimeric mice was compared. Livers from BDL-injured Wt→wt (n=7), IL-17A−/− →wt (n=10), Wt→IL-17RA−/− (n=10), IL-17RA−/− →wt (n=10), and IL-17RA−/− → IL-17RA−/− (n=8) mice were analyzed by H&E, Sirius Red staining, and α-SMA immunohistochemistry. Representative bright field micrographs are shown using × 10 objectives. (B) Quantification of Sirius Red staining and α-SMA immunohistochemistry in BM chimeric mice. Results are shown as fold induction compared with the sham-control Wt→wt mice. (Each group of BMT mice is compared to the BDL-injured wt mice, p<0.01. For Wt→IL-17RA mice, p<0.05). (C) Fibrogenic mRNAs are downregulated in whole liver from BDL-operated IL-17A−/− →wt, Wt→IL-17RA−/−, or IL-17RA−/− →wt mice, compared to BDL-operated Wt→wt mice (p<0.01). The data are shown as fold induction compared with sham Wt→wt mice. (D) Expression of α-SMA protein, evaluated by Western blot, was progressively downregulated in Wt→IL-17RA−/− > IL-17A−/− →wt > IL-17RA−/− →wt > IL-17RA−/− →IL-17RA−/− mice. Representative images are shown.

Figure 4

Figure 4. IL-17A facilitates HSC activation into myofibroblasts via Stat3

(A)IL-17A induced nuclear translocation of p65 and Stat3 in primary murine HSCs. HSCs (5 × 105 cells) were stimulated with IL-17A (10 ng/ml), TNF-α (20 ng/ml) or IL-6 (30 ng/ml) for 0–30 min, translocation of p65 (NF-κB) or Stat3 was analyzed by fluorescent microscopy using × 40 objective. (B) IL-17A stimulates collagen-α1(I) expression by HSCs. Primary murine qHSCs were isolated from Col-GFP or Col-GFPIL-17RA−/− mice, and stimulated with IL-17A (10 ng/ml) or TGF-β1 (3 ng/ml ) in 1% FSCs for 48 hours. The percent of cells upregulated collagen-α1(I)-GFP was calculated, p<0.05. Representative images are shown using × 20 objective. (C) Fibrogenic properties of IL-17A, IL-6 and leptin. Fibrogenic mRNA expression was tested in HSCs treated with IL-17A (10 ng/ml), IL-6 (30 ng/ml), or leptin (100 ng/ml) for 4 h. IL-17A induced production of IL-6 in HSCs. The direct effect of IL-17A on collagen-α1(I) mRNA expression was assessed in IL-17A-stimulated HSC cultures depleted for IL-6 (using anti-IL-6 antibody; 0.5 μg/ml). *p<0.01, **p<0.05, ns – non significant. (D) IL-17A activates Stat3 in HSCs. Deletion of Stat3 in HSCs was achieved by crossing GFAP-Cre mice x floxed Stat3f/f mice = GFAPStat3−/− mice. qHSCs were isolated from GFAPStat3−/− or Stat3f/f wt mice, and stimulated with IL-17A (10 ng/ml) or TGF-β1 (3 ng/ml) for 15 min. Nuclear translocation of Stat3 (but not phosphor-Smad2/3) was detected in wild type HSCs but not in Stat3-deficient HSCs. Representative images are shown using × 20 objective. (E) IL-17-dependent collagen-α1(I) expression is impaired in Stat3-deficient HSCs. Primary wild type (Stat3f/f) and Stat3-deficient HSCs from GFAPStat3−/− mice were cultured in DMEM + 10% FCS for 18h and stimulated with IL-17A (10 ng/ml in 1% FSCs) for 8h, and mRNA levels of collagen-α1(I) expression were measured, *p<0.01.

Figure 5

Figure 5. Stat3-deficient HSCs have decreased activation

(A)Upregulation of Stat3, IL-17RA, IL-6RA, OSM-R, and LIF-1-R, but not Stat1 or IL-10Ra/b, was detected in aHSCs in comparison with qHSCs using the whole mouse genome microarray (Suppl. Methods). The mRNA level is the average of the multiple probes (p<0.01). (B) Development of liver fibrosis is attenuated in GFAPStat3−/− mice compared to wild type Stat3f/f mice. Livers from wild type mice (untreated (n=2), BDL (n=4) and CCl4-treated (n=4)), and GFAPStat3−/− mice (untreated (n=2), BDL (n=5) and CCl4-injured (n=6)), were analyzed by Sirius Red staining, and immunostaining for α-SMA and Desmin, and quantified (as percent of positive area, *p<0.01, **p<0.05). Representative bright field micrographs are shown using × 10 objectives. (C) Stat3-deficient and wild type primary HSCs were in vitro stimulated with IL-17A (10 ng/ml, 4 h). The ability to activate a-SMA and Col1a1 mRNA, and produce cytokines HGF, TGF-β1, PDGF, IL-6 mRNA was impaired in Stat3−/− HSCs compared to wt HSCs. *p<0.001, **p<0.005, ns - non significant.

Figure 6

Figure 6. IL-23 and IL-22 kave opposing roles in liver fibrosis

(A) Serum IL-17A, IL-17E (IL-25), IL-17F, IL-6 and IL-22 were measured by ELISA in BDL (10 days) IL-17RA−/− mice versus wild type mice (*p<0.01, **p<0.05). (B) Hepatic levels of IL-17A, IL-17F, IL-23 and IL-22 mRNA were detected in BDL (10 days) IL-17RA−/− mice versus wild type mice (p<0.05) (C) Livers from BM chimeric CCl4-treated Wt→wt (n=5), IL-17A−/− →wt (n=6), IL-22−/− →wt (n=6), and IL-23−/− →wt (n=6) mice were analyzed by Sirius Red, α-SMA staining. Bright field micrographs are shown using × 10 objectives. Quantification of Sirius Red and α-SMA staining, mRNA expression is shown (p<0.05, compared to BDL wt mice). mRNA expression is fold induction compared to oil-control Wt→wt mice. (D) The percent of inhibition of liver fibrosis was calculated for BM chimeric mice in response to liver injury (in comparison Wt→wt mice, 100%, p<0.01) based on Sirius Red and α-SMA expression. The strongest inhibition of liver fibrosis was observed in IL-17RA−/− →IL-17RA−/− mice, the least inhibition of liver fibrosis was observed in Wt→IL-17RA−/− mice. Increased fibrosis was detected in IL-22−/− →wt mice. (E) Administration of IL-22 and IL-17E attenuates liver fibrosis. BDL-operated mice were daily treated with IL-22 (0.5 μg/mouse, n=4), IL-17E (0.5 μg/mouse n=5), or vehicle (PBS) for 7 days, tissues were analyzed by Sirius Red, and α-SMA staining; mRNA expression is shown (p<0.05, compared to BDL wt mice + PBS).

Figure 6

Figure 6. IL-23 and IL-22 kave opposing roles in liver fibrosis

(A) Serum IL-17A, IL-17E (IL-25), IL-17F, IL-6 and IL-22 were measured by ELISA in BDL (10 days) IL-17RA−/− mice versus wild type mice (*p<0.01, **p<0.05). (B) Hepatic levels of IL-17A, IL-17F, IL-23 and IL-22 mRNA were detected in BDL (10 days) IL-17RA−/− mice versus wild type mice (p<0.05) (C) Livers from BM chimeric CCl4-treated Wt→wt (n=5), IL-17A−/− →wt (n=6), IL-22−/− →wt (n=6), and IL-23−/− →wt (n=6) mice were analyzed by Sirius Red, α-SMA staining. Bright field micrographs are shown using × 10 objectives. Quantification of Sirius Red and α-SMA staining, mRNA expression is shown (p<0.05, compared to BDL wt mice). mRNA expression is fold induction compared to oil-control Wt→wt mice. (D) The percent of inhibition of liver fibrosis was calculated for BM chimeric mice in response to liver injury (in comparison Wt→wt mice, 100%, p<0.01) based on Sirius Red and α-SMA expression. The strongest inhibition of liver fibrosis was observed in IL-17RA−/− →IL-17RA−/− mice, the least inhibition of liver fibrosis was observed in Wt→IL-17RA−/− mice. Increased fibrosis was detected in IL-22−/− →wt mice. (E) Administration of IL-22 and IL-17E attenuates liver fibrosis. BDL-operated mice were daily treated with IL-22 (0.5 μg/mouse, n=4), IL-17E (0.5 μg/mouse n=5), or vehicle (PBS) for 7 days, tissues were analyzed by Sirius Red, and α-SMA staining; mRNA expression is shown (p<0.05, compared to BDL wt mice + PBS).

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References

    1. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest. 2005;115:209–18. - PMC - PubMed
    1. Seki E, De Minicis S, Osterreicher CH, et al. TLR4 enhances TGF-beta signaling and hepatic fibrosis. Nat Med. 2007;13:1324–32. - PubMed
    1. Friedman SL, Roll FJ, Boyles J, et al. Hepatic lipocytes: the principal collagen-producing cells of normal rat liver. Proc Natl Acad Sci U S A. 1985;82:8681–5. - PMC - PubMed
    1. Steinman L. A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue damage. Nat Med. 2007;13:139–45. - PubMed
    1. Kolls JK, Linden A. Interleukin-17 family members and inflammation. Immunity. 2004;21:467–76. - PubMed

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