Inhibition of Rho-Kinase Downregulates Th17 Cells and Ameliorates Hepatic Fibrosis by Schistosoma japonicum Infection
Abstract
:1. Introduction
2. Materials and Methods
2.1. Mice and Parasites
2.2. Worm Burden and Hepatic Fibrosis
2.3. Histopathology
2.4. T Cell Activation and Differentiation
2.5. Flow Cytometry
2.6. Cytokine Assay
2.7. In Vivo Cytokine Capture Assay (IVCCA)
2.8. Real-Time PCR
2.9. Statistical Analysis
3. Results
3.1. RhoA-ROCK Inhibitor Fasudil Suppresses Hepatic Granuloma Formation and Fibrosis in Mice Infected with S. japonicum
3.2. Fasudil Inhibits Activation and Proliferation of CD4+ T Cells
3.3. Fasudil Impairs Th17 Differentiation and Cytokine Secretion In Vitro
3.4. Fasudil Upregulates iTreg Cells
3.5. Fasudil Therapy Downregulates Th2 and Th17 Cytokine Levels In Vivo in Infected Mice
3.6. Fasudil Induces Apoptosis of Hepatic Stellate Cells
4. Discussion
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
Col-I | collagen type I |
ELISA | enzyme-linked immunosorbent assay |
EPG | eggs per gram |
FACS | fluorescence-activated cell sorting |
GDP | guanosine diphosphate |
GTP | guanosine triphosphate |
HCC | hepatocellular carcinoma |
H&E | hematoxylin and eosin |
HSCs | hepatic stellate cells |
IL | interleukin |
i.p. | intraperitoneally |
IVCCA | in vivo cytokine capture assay |
KO | knock-out |
PBS | phosphate buffer saline |
PMA | phorbol 12-myristate 13-acetate |
RhoA | ras homolog family member A |
ROCK | Rho-associated kinase |
S. japonicum | Schistosoma japonicum |
Stat3 | signal transducer and activator of transcription 3 |
Th | T helper |
TGF-β1 | transforming growth factor-β1 |
Treg | regulatory T |
WT | wild-type |
Appendix A
References
- McManus, D.P.; Dunne, D.W.; Sacko, M.; Utzinger, J.; Vennervald, B.J.; Zhou, X.N. Schistosomiasis. Nat. Rev. Dis. Primers 2018, 4, 13. [Google Scholar] [CrossRef] [PubMed]
- Colley, D.G.; Bustinduy, A.L.; Secor, W.E.; King, C.H. Human schistosomiasis. Lancet 2014, 383, 2253–2264. [Google Scholar] [CrossRef]
- Lukacs, N.W.; Boros, D.L. Lymphokine regulation of granuloma formation in murine schistosomiasis mansoni. Clin. Immunol. Immunopathol. 1993, 68, 57–63. [Google Scholar] [CrossRef] [PubMed]
- Yang, J.Q.; Tasaka, K.; Chuang, C.K.; Yoshikawa, H.; Nakajima, Y. Dynamic analysis of T-lymphocyte function in relation to hepatopathologic changes and effect of interleukin-12 treatment in mice infected with Schistosoma japonicum. J. Parasitol. 1999, 85, 257–262. [Google Scholar] [CrossRef]
- Carson, J.P.; Ramm, G.A.; Robinson, M.W.; McManus, D.P.; Gobert, G.N. Schistosome-Induced Fibrotic Disease: The Role of Hepatic Stellate Cells. Trends Parasitol. 2018, 34, 524–540. [Google Scholar] [CrossRef]
- Grzych, J.M.; Pearce, E.; Cheever, A.; Caulada, Z.A.; Caspar, P.; Heiny, S.; Lewis, F.; Sher, A. Egg deposition is the major stimulus for the production of Th2 cytokines in murine schistosomiasis mansoni. J. Immunol. 1991, 146, 1322–1327. [Google Scholar]
- Wang, B.; Liang, S.; Wang, Y.; Zhu, X.Q.; Gong, W.; Zhang, H.Q.; Li, Y.; Xia, C.M. Th17 down-regulation is involved in reduced progression of schistosomiasis fibrosis in ICOSL KO mice. PLoS Negl. Trop. Dis. 2015, 9, e0003434. [Google Scholar] [CrossRef]
- Pellicoro, A.; Ramachandran, P.; Iredale, J.P.; Fallowfield, J.A. Liver fibrosis and repair: Immune regulation of wound healing in a solid organ. Nat. Rev. Immunol. 2014, 14, 181–194. [Google Scholar] [CrossRef]
- Chen, D.; Luo, X.; Xie, H.; Gao, Z.; Fang, H.; Huang, J. Characteristics of IL-17 induction by Schistosoma japonicum infection in C57BL/6 mouse liver. Immunology 2013, 139, 523–532. [Google Scholar] [CrossRef]
- Qiu, S.; Fan, X.; Yang, Y.; Dong, P.; Zhou, W.; Xu, Y.; Zhou, Y.; Guo, F.; Zheng, Y.; Yang, J.Q. Schistosoma japonicum infection downregulates house dust mite-induced allergic airway inflammation in mice. PLoS ONE 2017, 12, e0179565. [Google Scholar] [CrossRef]
- Zhong, W.; Gao, L.; Zhou, Z.; Lin, H.; Chen, C.; Huang, P.; Huang, W.; Zhou, C.; Huang, S.; Nie, L.; et al. Indoleamine 2,3-dioxygenase 1 deficiency attenuates CCl4-induced fibrosis through Th17 cells down-regulation and tryptophan 2,3-dioxygenase compensation. Oncotarget 2017, 8, 40486–40500. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Meng, F.; Wang, K.; Aoyama, T.; Grivennikov, S.I.; Paik, Y.; Scholten, D.; Cong, M.; Iwaisako, K.; Liu, X.; Zhang, M.; et al. Interleukin-17 signaling in inflammatory, Kupffer cells, and hepatic stellate cells exacerbates liver fibrosis in mice. Gastroenterology 2012, 143, 765.e763–776.e763. [Google Scholar] [CrossRef] [PubMed]
- Mulloy, J.C.; Cancelas, J.A.; Filippi, M.D.; Kalfa, T.A.; Guo, F.; Zheng, Y. Rho GTPases in hematopoiesis and hemopathies. Blood 2010, 115, 936–947. [Google Scholar] [CrossRef] [PubMed]
- Stengel, K.; Zheng, Y. Cdc42 in oncogenic transformation, invasion, and tumorigenesis. Cell. Signal. 2011, 23, 1415–1423. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Zhou, X.; Florian, M.C.; Arumugam, P.; Chen, X.; Cancelas, J.A.; Lang, R.; Malik, P.; Geiger, H.; Zheng, Y. RhoA GTPase controls cytokinesis and programmed necrosis of hematopoietic progenitors. J. Exp. Med. 2013, 210, 2371–2385. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Loirand, G. Rho Kinases in Health and Disease: From Basic Science to Translational Research. Pharmacol. Rev. 2015, 67, 1074–1095. [Google Scholar] [CrossRef] [Green Version]
- Li, H.; Peyrollier, K.; Kilic, G.; Brakebusch, C. Rho GTPases and cancer. Biofactors 2014, 40, 226–235. [Google Scholar] [CrossRef]
- Feng, Y.; LoGrasso, P.V.; Defert, O.; Li, R. Rho Kinase (ROCK) Inhibitors and Their Therapeutic Potential. J. Med. Chem. 2016, 59, 2269–2300. [Google Scholar] [CrossRef]
- Zhang, Y.; Wu, S. Effects of fasudil on pulmonary hypertension in clinical practice. Pulm. Pharmacol. Ther. 2017, 46, 54–63. [Google Scholar] [CrossRef]
- Guo, R.; Su, Y.; Yan, J.; Sun, H.; Wu, J.; Liu, W.; Xu, Y. Fasudil improves short-term echocardiographic parameters of diastolic function in patients with type 2 diabetes with preserved left ventricular ejection fraction: A pilot study. Heart Vessel. 2015, 30, 89–97. [Google Scholar] [CrossRef]
- Wang, Q.; Yang, X.; Xu, Y.; Shen, Z.; Cheng, H.; Cheng, F.; Liu, X.; Wang, R. RhoA/Rho-kinase triggers epithelial-mesenchymal transition in mesothelial cells and contributes to the pathogenesis of dialysis-related peritoneal fibrosis. Oncotarget 2018, 9, 14397–14412. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Xie, Y.; Song, T.; Huo, M.; Zhang, Y.; Zhang, Y.Y.; Ma, Z.H.; Wang, N.; Zhang, J.P.; Chu, L. Fasudil alleviates hepatic fibrosis in type 1 diabetic rats: Involvement of the inflammation and RhoA/ROCK pathway. Eur. Rev. Med. Pharmacol. Sci. 2018, 22, 5665–5677. [Google Scholar] [CrossRef] [PubMed]
- Santos, G.L.; Hartmann, S.; Zimmermann, W.H.; Ridley, A.; Lutz, S. Inhibition of Rho-associated kinases suppresses cardiac myofibroblast function in engineered connective and heart muscle tissues. J. Mol. Cell. Cardiol. 2019, 134, 13–28. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dai, K.; Wang, Y.; Tai, S.; Ni, H.; Lian, H.; Yu, Y.; Liao, W.; Zheng, C.; Chen, Q.; Kuver, A.; et al. Fasudil exerts a cardio-protective effect on mice with coxsackievirus B3-induced acute viral myocarditis. Cardiovasc. Ther. 2018, e12477. [Google Scholar] [CrossRef] [PubMed]
- Dos Santos, T.M.; Righetti, R.F.; Camargo, L.D.N.; Saraiva-Romanholo, B.M.; Aristoteles, L.; de Souza, F.C.R.; Fukuzaki, S.; Alonso-Vale, M.I.C.; Cruz, M.M.; Prado, C.M.; et al. Effect of Anti-IL17 Antibody Treatment Alone and in Combination With Rho-Kinase Inhibitor in a Murine Model of Asthma. Front. Physiol. 2018, 9, 1183. [Google Scholar] [CrossRef]
- Rozo, C.; Chinenov, Y.; Maharaj, R.K.; Gupta, S.; Leuenberger, L.; Kirou, K.A.; Bykerk, V.P.; Goodman, S.M.; Salmon, J.E.; Pernis, A.B. Targeting the RhoA-ROCK pathway to reverse T-cell dysfunction in SLE. Ann. Rheum. Dis. 2017, 76, 740–747. [Google Scholar] [CrossRef]
- Yang, J.Q.; Kalim, K.W.; Li, Y.; Zhang, S.; Hinge, A.; Filippi, M.D.; Zheng, Y.; Guo, F. RhoA orchestrates glycolysis for TH2 cell differentiation and allergic airway inflammation. J. Allergy Clin. Immunol. 2016, 137, 231–245. [Google Scholar] [CrossRef]
- Yang, J.Q.; Kalim, K.W.; Li, Y.; Zheng, Y.; Guo, F. Ablation of RhoA impairs Th17 cell differentiation and alleviates house dust mite-triggered allergic airway inflammation. J. Leukoc. Biol. 2019. [Google Scholar] [CrossRef]
- Yang, Y.; Dong, P.; Zhao, J.; Zhou, W.; Zhou, Y.; Xu, Y.; Mei, C.; Guo, F.; Zheng, Y.; Yang, J.Q. PKClambda/iota regulates Th17 differentiation and house dust mite-induced allergic airway inflammation. Biochim. Biophys. Acta Mol. Basis Dis. 2018, 1864, 934–941. [Google Scholar] [CrossRef]
- Yang, J.Q.; Wen, X.; Kim, P.J.; Singh, R.R. Invariant NKT cells inhibit autoreactive B cells in a contact- and CD1d-dependent manner. J. Immunol. 2011, 186, 1512–1520. [Google Scholar] [CrossRef]
- Yang, J.Q.; Kalim, K.W.; Li, Y.; Duan, X.; Nguyen, P.; Khurana Hershey, G.K.; Kroner, J.; Ruff, B.; Zhang, L.; Salomonis, N.; et al. Rational targeting Cdc42 restrains Th2 cell differentiation and prevents allergic airway inflammation. Clin. Exp. Allergy 2019, 49, 92–107. [Google Scholar] [CrossRef] [PubMed]
- Finkelman, F.D.; Morris, S.C. Development of an assay to measure in vivo cytokine production in the mouse. Int. Immunol. 1999, 11, 1811–1818. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Stoilov, I.; Starcher, B.C.; Mecham, R.P.; Broekelmann, T.J. Measurement of elastin, collagen, and total protein levels in tissues. Methods Cell Biol. 2018, 143, 133–146. [Google Scholar] [CrossRef] [PubMed]
- Gagliano, N.; Arosio, B.; Santambrogio, D.; Balestrieri, M.R.; Padoani, G.; Tagliabue, J.; Masson, S.; Vergani, C.; Annoni, G. Age-dependent expression of fibrosis-related genes and collagen deposition in rat kidney cortex. J. Gerontol. A Biol. Sci. Med. Sci. 2000, 55, B365–B372. [Google Scholar] [CrossRef] [PubMed]
- Sondel, P.M.; Buhtoiarov, I.N.; DeSantes, K. Pleasant memories: Remembering immune protection while forgetting about graft-versus-host disease. J. Clin. Investig. 2003, 112, 25–27. [Google Scholar] [CrossRef] [PubMed]
- Boyman, O.; Letourneau, S.; Krieg, C.; Sprent, J. Homeostatic proliferation and survival of naive and memory T cells. Eur. J. Immunol. 2009, 39, 2088–2094. [Google Scholar] [CrossRef]
- Wei, L.; Laurence, A.; Elias, K.M.; O’Shea, J.J. IL-21 is produced by Th17 cells and drives IL-17 production in a STAT3-dependent manner. J. Biol. Chem. 2007, 282, 34605–34610. [Google Scholar] [CrossRef]
- Weiskirchen, R.; Tacke, F. Cellular and molecular functions of hepatic stellate cells in inflammatory responses and liver immunology. Hepatobiliary Surg. Nutr. 2014, 3, 344–363. [Google Scholar] [CrossRef]
- Wen, X.; He, L.; Chi, Y.; Zhou, S.; Hoellwarth, J.; Zhang, C.; Zhu, J.; Wu, C.; Dhesi, S.; Wang, X.; et al. Dynamics of Th17 cells and their role in Schistosoma japonicum infection in C57BL/6 mice. PLoS Negl. Trop. Dis. 2011, 5, e1399. [Google Scholar] [CrossRef]
- Liu, Y.; Li, L.; Liu, J.; She, W.M.; Shi, J.M.; Li, J.; Wang, J.Y.; Jiang, W. Activated hepatic stellate cells directly induce pathogenic Th17 cells in chronic hepatitis B virus infection. Exp. Cell Res. 2017. [Google Scholar] [CrossRef]
- Zhou, S.; Qi, Q.; Wang, X.; Zhang, L.; Xu, L.; Dong, L.; Zhu, J.; Li, Y.; Xu, Z.; Liu, F.; et al. SjHSP60 induces CD4(+) CD25(+) Foxp3(+) Tregs via TLR4-Mal-drived production of TGF-beta in macrophages. Immunol. Cell Biol. 2018, 96, 958–968. [Google Scholar] [CrossRef] [PubMed]
- Ikeda, H.; Kume, Y.; Tejima, K.; Tomiya, T.; Nishikawa, T.; Watanabe, N.; Ohtomo, N.; Arai, M.; Arai, C.; Omata, M.; et al. Rho-kinase inhibitor prevents hepatocyte damage in acute liver injury induced by carbon tetrachloride in rats. Am. J. Physiol. Gastrointest. Liver Physiol. 2007, 293, G911–G917. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kuroda, S.; Tashiro, H.; Igarashi, Y.; Tanimoto, Y.; Nambu, J.; Oshita, A.; Kobayashi, T.; Amano, H.; Tanaka, Y.; Ohdan, H. Rho inhibitor prevents ischemia-reperfusion injury in rat steatotic liver. J. Hepatol. 2012, 56, 146–152. [Google Scholar] [CrossRef] [PubMed]
- Wang, R.K.; Shao, X.M.; Yang, J.P.; Yan, H.L.; Shao, Y. MicroRNA-145 inhibits proliferation and promotes apoptosis of HepG2 cells by targeting ROCK1 through the ROCK1/NF-kappaB signaling pathway. Eur. Rev. Med. Pharmacol. Sci. 2019, 23, 2777–2785. [Google Scholar] [CrossRef]
- Takeba, Y.; Matsumoto, N.; Watanabe, M.; Takenoshita-Nakaya, S.; Ohta, Y.; Kumai, T.; Takagi, M.; Koizumi, S.; Asakura, T.; Otsubo, T. The Rho kinase inhibitor fasudil is involved in p53-mediated apoptosis in human hepatocellular carcinoma cells. Cancer Chemother. Pharmacol. 2012, 69, 1545–1555. [Google Scholar] [CrossRef]
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Zhou, W.; Yang, Y.; Mei, C.; Dong, P.; Mu, S.; Wu, H.; Zhou, Y.; Zheng, Y.; Guo, F.; Yang, J.-Q. Inhibition of Rho-Kinase Downregulates Th17 Cells and Ameliorates Hepatic Fibrosis by Schistosoma japonicum Infection. Cells 2019, 8, 1262. https://doi.org/10.3390/cells8101262
Zhou W, Yang Y, Mei C, Dong P, Mu S, Wu H, Zhou Y, Zheng Y, Guo F, Yang J-Q. Inhibition of Rho-Kinase Downregulates Th17 Cells and Ameliorates Hepatic Fibrosis by Schistosoma japonicum Infection. Cells. 2019; 8(10):1262. https://doi.org/10.3390/cells8101262
Chicago/Turabian StyleZhou, Wei, Yingying Yang, Congjin Mei, Panpan Dong, Shasha Mu, Hongchu Wu, Yonghua Zhou, Yi Zheng, Fukun Guo, and Jun-Qi Yang. 2019. "Inhibition of Rho-Kinase Downregulates Th17 Cells and Ameliorates Hepatic Fibrosis by Schistosoma japonicum Infection" Cells 8, no. 10: 1262. https://doi.org/10.3390/cells8101262