Abstract
IL-17 is closely associated with inflammation in intrahepatic cholestasis (IHC). Targeting IL-17 ameliorates IHC in mice. Invariant natural killer T (iNKT) cells are predominantly enriched in the liver and they mediate drug-induced liver injury through their secreted cytokines. However, whether iNKT17 cells are involved in ethinylestradiol (EE)-induced IHC remains unclear. In the present study, the administration of EE (10 mg/kg in vivo and 6.25 μM in vitro) promoted the activation and expansion of iNKT17 cells, which contributed to a novel hepatic iNKT17/Treg imbalance. iNKT cell-deficient Jα18−/− mice and the RORγt inhibitor digoxin (20 μg) alleviated EE-induced cholestatic hepatotoxicity and downregulated the IL-17 signalling pathway. In contrast, the co-administration of EE with recombinant IL-17 (1 μg) to Jα18−/− mice induced cholestatic hepatotoxicity and increased the infiltration of hepatic neutrophils and monocytes. Importantly, the administration of IL-17−/− iNKT cells (3.5 × 105) to Jα18−/− mice resulted in the attenuation of hepatotoxicity and the recruitment of fewer hepatic neutrophils and monocytes than the adoptive transfer of wild-type iNKT cells. These results indicated that iNKT17 cells could exert pathogenic effects. The recruitment and activation of iNKT17 cells could be attributed to the high level of CXCR3 expression on their surface. CXCL10 deficiency ameliorated EE-induced cholestatic liver damage, reduced hepatic CXCR3+ iNKT cells and inhibited RORγt expression. These findings suggest that iNKT17 cells play a key role in EE-induced cholestatic liver injury via CXCR3-mediated recruitment and activation. Our study provides new insights and therapeutic targets for cholestatic diseases.
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Abbreviations
- ALP:
-
Alkaline phosphatase
- ALT:
-
Alanine transaminase
- AST:
-
Aspartate transaminase
- BDL:
-
Bile duct ligation
- CXCL10:
-
C-X-C motif chemokine ligand 10
- DAB:
-
Diaminobenzidine
- DGX:
-
Digoxin
- EE:
-
Ethinylestradiol
- Egr1:
-
Early growth response factor 1
- ELISA:
-
Enzyme linked immunosorbent assay
- FXR:
-
Farnesoid X receptor
- H&E:
-
Hematoxylin and eosin
- HRP:
-
Horseradish peroxidase
- ICAM-1:
-
Intercellular adhesion molecule-1
- ICP:
-
Intrahepatic cholestasis of pregnancies
- IFN-γ:
-
Interferon gamma
- IHC:
-
Intrahepatic cholestasis
- IL-4:
-
Interleukin 4
- iNKT cell:
-
Invariant natural killer T cell
- NKT cell:
-
Natural killer T cell
- NPC cell:
-
Non-parenchymal cell
- Oatp:
-
Organic anion transporting polypeptide
- OC:
-
Oral contraceptives
- PBC:
-
Primary biliary cirrhosis
- RORγt:
-
Retinoid-related orphan receptor-γt
- TBA:
-
Total bile acid
- TCR:
-
T cell receptor
- UDCA:
-
Ursodeoxycholic acid
References
Allen K, Jaeschke H, Copple BL (2011) Bile acids induce inflammatory genes in hepatocytes: a novel mechanism of inflammation during obstructive cholestasis. Am J Pathol 178(1):175–186. https://doi.org/10.1016/j.ajpath.2010.11.026
Azuma T, Takahashi T, Kunisato A, Kitamura T, Hirai H (2003) Human CD4+ CD25+ regulatory T cells suppress NKT cell functions. Cancer Res 63(15):4516–4520
Blackstock R, Murphy JW (2004) Age-related resistance of C57BL/6 mice to Cryptococcus neoformans is dependent on maturation of NKT cells. Infect Immun 72(9):5175–5180. https://doi.org/10.1128/IAI.72.9.5175-5180.2004
Bonacchi A, Romagnani P, Romanelli RG et al (2001) Signal transduction by the chemokine receptor CXCR3: activation of Ras/ERK, Src, and phosphatidylinositol 3-kinase/Akt controls cell migration and proliferation in human vascular pericytes. J Biol Chem 276(13):9945–9954. https://doi.org/10.1074/jbc.M010303200
Chappell LC, Bell JL, Smith A et al (2019) Ursodeoxycholic acid versus placebo in women with intrahepatic cholestasis of pregnancy (PITCHES): a randomised controlled trial. Lancet 394(10201):849–860. https://doi.org/10.1016/S0140-6736(19)31270-X
Choi J, Selmi C, Leung PS, Kenny TP, Roskams T, Gershwin ME (2016) Chemokine and chemokine receptors in autoimmunity: the case of primary biliary cholangitis. Expert Rev Clin Immunol 12(6):661–672. https://doi.org/10.1586/1744666X.2016.1147956
Chuang YH, Lian ZX, Cheng CM et al (2005) Increased levels of chemokine receptor CXCR3 and chemokines IP-10 and MIG in patients with primary biliary cirrhosis and their first degree relatives. J Autoimmun 25(2):126–132. https://doi.org/10.1016/j.jaut.2005.08.009
Chuang YH, Lian ZX, Yang GX et al (2008) Natural killer T cells exacerbate liver injury in a transforming growth factor beta receptor II dominant-negative mouse model of primary biliary cirrhosis. Hepatology 47(2):571–580. https://doi.org/10.1002/hep.22052
Crosby CM, Kronenberg M (2018) Tissue-specific functions of invariant natural killer T cells. Nat Rev Immunol 18(9):559–574. https://doi.org/10.1038/s41577-018-0034-2
Cui J, Shin T, Kawano T et al (1997) Requirement for valpha14 NKT cells in IL-12-mediated rejection of tumors. Science 278(5343):1623–1626. https://doi.org/10.1126/science.278.5343.1623
Cui K, Yan G, Zheng X et al (2017) Suppression of natural killer cell activity by regulatory NKT10 cells aggravates alcoholic hepatosteatosis. Front Immunol 8:1414. https://doi.org/10.3389/fimmu.2017.01414
De Giorgi L, Sorini C, Cosorich I, Ferrarese R, Canducci F, Falcone M (2018) Increased iNKT17 Cell frequency in the intestine of non-obese diabetic mice correlates with high bacterioidales and low clostridiales abundance. Front Immunol 9:1752. https://doi.org/10.3389/fimmu.2018.01752
de Graaf KL, Lapeyre G, Guilhot F et al (2018) NI-0801, an anti-chemokine (C-X-C motif) ligand 10 antibody, in patients with primary biliary cholangitis and an incomplete response to ursodeoxycholic acid. Hepatol Commun 2(5):492–503. https://doi.org/10.1002/hep4.1170
Doisne JM, Soulard V, Becourt C et al (2011) Cutting edge: crucial role of IL-1 and IL-23 in the innate IL-17 response of peripheral lymph node NK1.1- invariant NKT cells to bacteria. J Immunol. https://doi.org/10.4049/jimmunol.1002725
Exley MA, Dellabona P, Casorati G (2021) Exploiting CD1-restricted T cells for clinical benefit. Mol Immunol 132:126–131. https://doi.org/10.1016/j.molimm.2020.12.015
Floreani A, Mangini C (2018) Primary biliary cholangitis: Old and novel therapy. Eur J Intern Med 47:1–5. https://doi.org/10.1016/j.ejim.2017.06.020
Hang S, Paik D, Yao L et al (2019) Bile acid metabolites control TH17 and treg cell differentiation. Nature 576(7785):143–148. https://doi.org/10.1038/s41586-019-1785-z
Hao H, Cao L, Jiang C et al (2017) Farnesoid X Receptor regulation of the NLRP3 inflammasome underlies cholestasis-associated sepsis. Cell Metab 25(4):856–867
Huh JR, Leung MW, Huang P et al (2011) Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing rorgammat activity. Nature 472(7344):486–490. https://doi.org/10.1038/nature09978
Hydes TJ, Blunt MD, Naftel J et al (2019) Constitutive activation of natural killer cells in primary biliary cholangitis. Front Immunol 10:2633. https://doi.org/10.3389/fimmu.2019.02633
Jia H, Chen J, Zhang X et al (2022) IL-17A produced by invariant natural killer T cells and CD3(+) CD56(+) alphaGalcer-CD1d tetramer(-) T cells promote liver fibrosis in patients with primary biliary cholangitis. J Leukoc Biol. https://doi.org/10.1002/JLB.2A0622-586RRRR
Jin F, Cheng D, Tao JY et al (2013) Anti-inflammatory and anti-oxidative effects of corilagin in a rat model of acute cholestasis. BMC Gastroenterol 13:79. https://doi.org/10.1186/1471-230X-13-79
Johnston B, Kim CH, Soler D, Emoto M, Butcher EC (2003) Differential chemokine responses and homing patterns of murine TCR alpha beta NKT cell subsets. J Immunol 171(6):2960–2969. https://doi.org/10.4049/jimmunol.171.6.2960
Jordan-Williams KL, Poston S, Taparowsky EJ (2013) BATF regulates the development and function of IL-17 producing iNKT cells. BMC Immunol 14:16. https://doi.org/10.1186/1471-2172-14-16
Karin N, Razon H (2018) Chemokines beyond chemo-attraction: CXCL10 and its significant role in cancer and autoimmunity. Cytokine 109:24–28. https://doi.org/10.1016/j.cyto.2018.02.012
Kawaguchi M, Kokubu F, Huang SK et al (2007) The IL-17F signaling pathway is involved in the induction of IFN-gamma-inducible protein 10 in bronchial epithelial cells. J Allergy Clin Immunol 119(6):1408–1414. https://doi.org/10.1016/j.jaci.2007.02.036
Khader SA, Bell GK, Pearl JE et al (2007) IL-23 and IL-17 in the establishment of protective pulmonary CD4+ T cell responses after vaccination and during mycobacterium tuberculosis challenge. Nat Immunol 8(4):369–377. https://doi.org/10.1038/ni1449
Kim CH, Johnston B, Butcher EC (2002) Trafficking machinery of NKT cells: shared and differential chemokine receptor expression among V alpha 24(+)V beta 11(+) NKT cell subsets with distinct cytokine-producing capacity. Blood 100(1):11–16. https://doi.org/10.1182/blood-2001-12-0196
Kim ND, Moon JO, Slitt AL, Copple BL (2006) Early growth response factor-1 is critical for cholestatic liver injury. Toxicol Sci 90(2):586–595. https://doi.org/10.1093/toxsci/kfj111
Kirbas A, Biberoglu E, Ersoy AO et al (2016) The role of interleukin-17 in intrahepatic cholestasis of pregnancy. J Matern Fetal Neonatal Med 29(6):977–981. https://doi.org/10.3109/14767058.2015.1028354
Kobayashi E, Kobayashi M, Tsuneyama K, Fukami T, Nakajima M, Yokoi T (2009) Halothane-induced liver injury is mediated by interleukin-17 in mice. Toxicol Sci 111(2):302–310. https://doi.org/10.1093/toxsci/kfp165
Kobayashi M, Higuchi S, Mizuno K et al (2010) Interleukin-17 is involved in alpha-naphthylisothiocyanate-induced liver injury in mice. Toxicology 275(1–3):50–57. https://doi.org/10.1016/j.tox.2010.05.011
Kong X, Kong Y, Zhang F, Wang T, Zhu X (2018) Expression and significance of dendritic cells and Th17/Treg in serum and placental tissues of patients with intrahepatic cholestasis of pregnancy. J Matern Fetal Neonatal Med 31(7):901–906. https://doi.org/10.1080/14767058.2017.1300652
Kontturi M, Sotaniemi E (1969) Effect of oestrogen on liver function of prostatic cancer patients. Br Med J 4(5677):204–205. https://doi.org/10.1136/bmj.4.5677.204
Lee YJ, Wang H, Starrett GJ, Phuong V, Jameson SC, Hogquist KA (2015) Tissue-specific distribution of iNKT cells impacts their cytokine response. Immunity 43(3):566–578. https://doi.org/10.1016/j.immuni.2015.06.025
Li X, Liu R, Luo L et al (2017a) Role of AMP-activated protein kinase alpha1 in 17alpha-ethinylestradiol-induced cholestasis in rats. Arch Toxicol 91(1):481–494. https://doi.org/10.1007/s00204-016-1697-8
Li X, Liu R, Zhang L, Jiang Z (2017b) The emerging role of AMP-activated protein kinase in cholestatic liver diseases. Pharmacol Res 125(Pt B):105–113. https://doi.org/10.1016/j.phrs.2017.09.002
Manousou P, Kolios G, Drygiannakis I et al (2013) CXCR3 axis in patients with primary biliary cirrhosis: a possible novel mechanism of the effect of ursodeoxycholic acid. Clin Exp Immunol 172(1):9–15. https://doi.org/10.1111/cei.12032
McGinley AM, Sutton CE, Edwards SC et al (2020) Interleukin-17A serves a priming role in autoimmunity by recruiting IL-1beta-producing myeloid cells that promote pathogenic T cells. Immunity 52(2):342–356
Mencarelli A, Renga B, Migliorati M et al (2009) The bile acid sensor farnesoid X receptor is a modulator of liver immunity in a rodent model of acute hepatitis. J Immunol 183(10):6657–6666. https://doi.org/10.4049/jimmunol.0901347
Monteiro M, Almeida CF, Agua-Doce A, Graca L (2013) Induced IL-17-producing invariant NKT cells require activation in presence of TGF-beta and IL-1beta. J Immunol 190(2):805–811. https://doi.org/10.4049/jimmunol.1201010
Nishioji K, Okanoue T, Itoh Y et al (2001) Increase of chemokine interferon-inducible protein-10 (IP-10) in the serum of patients with autoimmune liver diseases and increase of its mRNA expression in hepatocytes. Clin Exp Immunol 123(2):271–279. https://doi.org/10.1046/j.1365-2249.2001.01391.x
Nong C, Zou M, Xue R et al (2020) The role of invariant natural killer T cells in experimental xenobiotic-induced cholestatic hepatotoxicity. Biomed Pharmacother 122:109579. https://doi.org/10.1016/j.biopha.2019.109579
O’Brien KM, Allen KM, Rockwell CE, Towery K, Luyendyk JP, Copple BL (2013) IL-17A synergistically enhances bile acid-induced inflammation during obstructive cholestasis. Am J Pathol 183(5):1498–1507. https://doi.org/10.1016/j.ajpath.2013.07.019
Oh SJ, Chung DH (2011) Invariant NKT cells producing IL-4 or IL-10, but not IFN-gamma, inhibit the Th1 response in experimental autoimmune encephalomyelitis, whereas none of these cells inhibits the Th17 response. J Immunol 186(12):6815–6821. https://doi.org/10.4049/jimmunol.1003916
Ouyang X, Han SN, Zhang JY et al (2018) Digoxin suppresses pyruvate kinase M2-promoted HIF-1alpha transactivation in steatohepatitis. Cell Metab 27(2):339–350
Pichavant M, Goya S, Meyer EH et al (2008) Ozone exposure in a mouse model induces airway hyperreactivity that requires the presence of natural killer T cells and IL-17. J Exp Med 205(2):385–393. https://doi.org/10.1084/jem.20071507
Piechota J, Jelski W (2020) Intrahepatic cholestasis in pregnancy: review of the literature. J Clin Med. https://doi.org/10.3390/jcm9051361
Qian C, Jiang T, Zhang W et al (2013) Increased IL-23 and IL-17 expression by peripheral blood cells of patients with primary biliary cirrhosis. Cytokine 64(1):172–180. https://doi.org/10.1016/j.cyto.2013.07.005
Rachitskaya AV, Hansen AM, Horai R et al (2008) Cutting edge: NKT cells constitutively express IL-23 receptor and RORgammat and rapidly produce IL-17 upon receptor ligation in an IL-6-independent fashion. J Immunol 180(8):5167–5171. https://doi.org/10.4049/jimmunol.180.8.5167
Roma MG, Toledo FD, Boaglio AC, Basiglio CL, Crocenzi FA, Sanchez Pozzi EJ (2011) Ursodeoxycholic acid in cholestasis: linking action mechanisms to therapeutic applications. Clin Sci (lond) 121(12):523–544. https://doi.org/10.1042/CS20110184
Salomon I, Netzer N, Wildbaum G, Schif-Zuck S, Maor G, Karin N (2002) Targeting the function of IFN-gamma-inducible protein 10 suppresses ongoing adjuvant arthritis. J Immunol 169(5):2685–2693. https://doi.org/10.4049/jimmunol.169.5.2685
Schwandner R, Yamaguchi K, Cao Z (2000) Requirement of tumor necrosis factor receptor-associated factor (TRAF)6 in interleukin 17 signal transduction. J Exp Med 191(7):1233–1240. https://doi.org/10.1084/jem.191.7.1233
Shaker ME, Hamed MF, Shaaban AA (2022) Digoxin mitigates diethylnitrosamine-induced acute liver injury in mice via limiting production of inflammatory mediators. Saudi Pharm J 30(3):291–299. https://doi.org/10.1016/j.jsps.2022.01.007
Sidahmed AM, Leon AJ, Bosinger SE et al (2012) CXCL10 contributes to p38-mediated apoptosis in primary T lymphocytes in vitro. Cytokine 59(2):433–441. https://doi.org/10.1016/j.cyto.2012.05.002
Soria-Jasso LE, Carino-Cortes R, Munoz-Perez VM, Perez-Hernandez E, Perez-Hernandez N, Fernandez-Martinez E (2019) Beneficial and deleterious effects of female sex hormones, oral contraceptives, and phytoestrogens by immunomodulation on the liver. Int J Mol Sci. https://doi.org/10.3390/ijms20194694
Sun K, Ma S, Tian S et al (2021) An enhanced level of LAMP-2A participates in CD4(+)T cell hyperactivity in patients with primary biliary cholangitis. Ann Transl Med 9(2):101. https://doi.org/10.21037/atm-20-2427
Thomas SY, Hou R, Boyson JE et al (2003) CD1d-restricted NKT cells express a chemokine receptor profile indicative of Th1-type inflammatory homing cells. J Immunol 171(5):2571–2580. https://doi.org/10.4049/jimmunol.171.5.2571
Van Kaer L (2005) alpha-galactosylceramide therapy for autoimmune diseases: prospects and obstacles. Nat Rev Immunol 5(1):31–42. https://doi.org/10.1038/nri1531
Venken K, Jacques P, Mortier C et al (2019) RORgammat inhibition selectively targets IL-17 producing iNKT and gammadelta-T cells enriched in Spondyloarthritis patients. Nat Commun 10(1):9. https://doi.org/10.1038/s41467-018-07911-6
Wallace KL, Marshall MA, Ramos SI et al (2009) NKT cells mediate pulmonary inflammation and dysfunction in murine sickle cell disease through production of IFN-gamma and CXCR3 chemokines. Blood 114(3):667–676. https://doi.org/10.1182/blood-2009-02-205492
Wang X, Jiang Z, Xing M et al (2014) Interleukin-17 mediates triptolide-induced liver injury in mice. Food Chem Toxicol 71:33–41. https://doi.org/10.1016/j.fct.2014.06.004
Wang J, Fu T, Dong R et al (2019) Hepatoprotection of auraptene from the peels of citrus fruits against 17alpha-ethinylestradiol-induced cholestasis in mice by activating farnesoid X receptor. Food Funct 10(7):3839–3850. https://doi.org/10.1039/c9fo00318e
Wang L, You HM, Meng HW et al (2021) STING-mediated inflammation contributes to Gao binge ethanol feeding model. J Cell Physiol. https://doi.org/10.1002/jcp.30606
Wildbaum G, Netzer N, Karin N (2002) Plasmid DNA encoding IFN-gamma-inducible protein 10 redirects antigen-specific T cell polarization and suppresses experimental autoimmune encephalomyelitis. J Immunol 168(11):5885–5892. https://doi.org/10.4049/jimmunol.168.11.5885
Yoshiga Y, Goto D, Segawa S et al (2008) Invariant NKT cells produce IL-17 through IL-23-dependent and -independent pathways with potential modulation of Th17 response in collagen-induced arthritis. Int J Mol Med 22(3):369–374
Yu L, Liu X, Li X et al (2016) Protective effects of SRT1720 via the HNF1alpha/FXR signalling pathway and anti-inflammatory mechanisms in mice with estrogen-induced cholestatic liver injury. Toxicol Lett 264:1–11. https://doi.org/10.1016/j.toxlet.2016.10.016
Zhang S, Huang D, Weng J et al (2016) Neutralization of interleukin-17 attenuates cholestatic liver fibrosis in mice. Scand J Immunol 83(2):102–108. https://doi.org/10.1111/sji.12395
Zhao P, Han SN, Arumugam S et al (2019) Digoxin improves steatohepatitis with differential involvement of liver cell subsets in mice through inhibition of PKM2 transactivation. Am J Physiol Gastrointest Liver Physiol 317(4):G387–G397. https://doi.org/10.1152/ajpgi.00054.2019
Zheng L, Chu J, Shi Y et al (2013) Bone marrow-derived stem cells ameliorate hepatic fibrosis by down-regulating interleukin-17. Cell Biosci 3(1):46. https://doi.org/10.1186/2045-3701-3-46
Zhu H, Zhang Q, Chen G (2019) CXCR6 deficiency ameliorates ischemia-reperfusion injury by reducing the recruitment and cytokine production of hepatic NKT cells in a mouse model of non-alcoholic fatty liver disease. Int Immunopharmacol 72:224–234. https://doi.org/10.1016/j.intimp.2019.04.021
Zhu X, Zhu J (2020) CD4 T helper cell subsets and related human immunological disorders. Int J Mol Sci. https://doi.org/10.3390/ijms21218011
Zohar Y, Wildbaum G, Novak R et al (2014) CXCL11-dependent induction of FOXP3-negative regulatory T cells suppresses autoimmune encephalomyelitis. J Clin Invest 124(5):2009–2022. https://doi.org/10.1172/JCI71951
Zou M, Nong C, Yu Z et al (2020) The role of invariant natural killer T cells and associated immunoregulatory factors in triptolide-induced cholestatic liver injury. Food Chem Toxicol 146:111777. https://doi.org/10.1016/j.fct.2020.111777
Zou M, Wang A, Wei J et al (2021) An insight into the mechanism and molecular basis of dysfunctional immune response involved in cholestasis. Int Immunopharmacol 92:107328. https://doi.org/10.1016/j.intimp.2020.107328
Acknowledgements
The present study was supported by the National Natural Science Foundation of China (No. 82073948, No. 81703626), National Innovation and Entrepreneurship Training Program for Undergraduate (No. 202210316040Z). The authors would like to thank Dr. Li Bai (University of Science and Technology of China, Hefei, China) for kindly giving us Jα18-/- mice and mouse iNKT cell hybridoma line DN32.D3, Dr. Pinghu Zhang (Yangzhou University, Yangzhou, China) for kindly giving us CXCL10-/- mice, NIH Tetramer Core Facility for kindly providing us tetramers, Jie Zhao, who works in the Pharmaceutical Animal Experimental Center of China Pharmaceutical University, for her kind help with in vivo experiments.
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WK, MZ and XL performed the experiments. MZ and XL collected data and analyzed the data. WK and XL completed the revision experiments. LZ and XW contributed to the guidance of experiments and the final manuscript. XW designed the study, wrote the manuscript, and answered the reviewers’ questions. XL drew the graphical abstract. YZ, XL and HC modified the language throughout the text. All the authors reviewed the manuscript.
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Kong, W., Li, X., Zou, M. et al. iNKT17 cells play a pathogenic role in ethinylestradiol-induced cholestatic hepatotoxicity. Arch Toxicol 97, 561–580 (2023). https://doi.org/10.1007/s00204-022-03403-1
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DOI: https://doi.org/10.1007/s00204-022-03403-1