The characteristics of CD4+T-helper cell subset differentiation in experimental Clonorchis sinensis-infected FVB mice

Document Type : Original Article


Jiangsu Key Laboratory of Immunity and Metabolism, Department of Pathogenic Biology and Immunology, Laboratory of Infection and Immunity, Xuzhou Medical University, Xuzhou, 221004, Jiangsu Province, People’s Republic of China


Objective(s): Immune responses are tightly regulated in the development of clonorchiasis. However, the adaptive immune regulatory pathway contributing to the pathological processes of clonorchis sinensis (C. sinensis) infection is still not clear. In this study, we assessed the dynamic changes of CD4+T cell subsets and the related cytokines as well as transcription factors during C. sinensis infection.
Materials and Methods: We used female FVB mice to establish the infection model. H&E and Masson’s stain were performed in 2 or 8 weeks post-infection (PI) liver of C. sinensis. The percentages of splenic Th1, Th2, and Treg in CD4+T cells were detected by flow cytometry. The transcription factors T-bet, GATA3, Foxp3, and RORγt gene expression were detected by qPCR. The protein expression of IL-10, IL-17, IL-4, IL-2, and Tumor Necrosis Factor-α (TNF-α) were examined using ELISA.
Results: The percentages of splenic Th1, Th2, and Treg in CD4+T cells were significantly increased in both 2 and 8 weeks PI of C. sinensis, while the ratio of Treg/Th17 as well as the percentage of Treg in serum was gradually increased during the development of infection. The expressions of T-bet, GATA3, Foxp3, and RORγt were increased in 8 weeks PI. Serum levels of IL-10, IL-17, IL-4, and IL-2 were profoundly increased in infected mice, while the concentrations of TNF-α increased to peak two weeks PI.
Conclusion: Our results suggested that the imbalance of CD4+T cell subsets may regulate and contribute to an appropriate compromise between pathology, tissue repair, and elimination in a susceptible murine host.


1. Qian MB, Utzinger J, Keiser J, Zhou XN. Clonorchiasis. Lancet 2016; 387:800-810.
2. Bouchery T, Kyle R, Ronchese F, Le Gros G. The differentiation of CD4(+) T-helper cell subsets in the context of helminth parasite infection. Front Immunol 2014; 5:487-499.
3. Pletinckx K, Stijlemans B, Pavlovic V, Laube R, Brandl C, Kneitz S, et al. Similar inflammatory DC maturation signatures induced by TNF or Trypanosoma brucei antigens instruct default Th2-cell responses. Eur J Immunol 2011; 41:3479-3494.
4. Kara EE, Comerford I, Fenix KA, Bastow CR, Gregor CE, McKenzie DR, et al. Tailored immune responses: novel effector helper T cell subsets in protective immunity. PLoS Pathog 2014; 10:e1003905.
5. Tsai HC, Velichko S, Hung LY, Wu R. IL-17A and Th17 cells in lung inflammation: an update on the role of Th17 cell differentiation and IL-17R signaling in host defense against infection. Clin Dev Immunol 2013; 2013:267971- 267982.
6. Dekita M, Wu Z, Ni J, Zhang X, Liu Y, Yan X, et al. Cathepsin S is involved in Th17 differentiation through the upregulation of IL-6 by activating PAR-2 after systemic exposure to lipopolysaccharide from porphyromonas gingivalis. Front Pharmacol 2017; 8:470-482.
7.Geis AL, Fan H, Wu X, Wu S, Huso DL, Wolfe JL, et al. Regulatory T-cell response to enterotoxigenic bacteroides fragilis colonization triggers IL17-dependent colon carcinogenesis. Cancer Discov 2015; 5:1098-1109.
8. Silva JL, Rezende-Oliveira K, da Silva MV, Gomez-Hernandez C, Peghini BC, Silva NM, et al. IL-17-expressing CD4(+) and CD8(+) T lymphocytes in human toxoplasmosis. Mediators Inflamm 2014; 2014:573825-573831.
9. Nabavi NS, Pezeshkpoor F, Valizadeh N, Ahmadi Ghezeldasht S, Rezaee SA. Increased Th17 functions are accompanied by Tregs activities in lupoid leishmaniasis. Parasite Immunol 2018;40:e12507.
10. O’Quinn DB, Palmer MT, Lee YK, Weaver CT. Emergence of the Th17 pathway and its role in host defense. Adv Immunol 2008; 99:115-163.
11. D’Elia R, Behnke JM, Bradley JE, Else KJ. Regulatory T cells: a role in the control of helminth-driven intestinal pathology and worm survival. J Immunol 2009; 182:2340-2348.
12. Adalid-Peralta L, Arce-Sillas A, Fragoso G, Cardenas G, Rosetti M, Casanova-Hernandez D, et al. Cysticerci drive dendritic cells to promote in vitro and in vivo Tregs differentiation. Clin Dev Immunol 2013; 2013:981468-981476.
13. Kim EM, Bae YM, Choi MH, Hong ST. Cyst formation, increased anti-inflammatory cytokines and expression of chemokines support for Clonorchis sinensis infection in FVB mice. Parasitol Int 2012; 61:124-129.
14. Zhang BB, Yan C, Fang F, Du Y, Ma R, Li XY, et al. Increased hepatic Th2 and Treg subsets are associated with biliary fibrosis in different strains of mice caused by Clonorchis sinensis. PLoS One 2017; 12:e0171005.
15. Sher A, Pearce E, Kaye P. Shaping the immune response to parasites: role of dendritic cells. Curr Opin Immunol 2003; 15:421-429.
16. Choi BI, Han JK, Hong ST, Lee KH. Clonorchiasis and cholangiocarcinoma: etiologic relationship and imaging diagnosis. Clin Microbiol Rev 2004; 17:540-552.
17. Koyasu S, Moro K. Type 2 innate immune responses and the natural helper cell. Immunology 2011; 132:475-481.
18. Pleasance J, Wiedosari E, Raadsma HW, Meeusen E, Piedrafita D. Resistance to liver fluke infection in the natural sheep host is correlated with a type-1 cytokine response. Parasite Immunol 2011; 33:495-505.
19. Espino AM, Rivera F. Quantitation of cytokine mRNA by real-time RT-PCR during a vaccination trial in a rabbit model of fascioliasis. Vet Parasitol 2010; 169:82-92.
20. Dalton JP, Robinson MW, Mulcahy G, O’Neill SM, Donnelly S. Immunomodulatory molecules of Fasciola hepatica: candidates for both vaccine and immunotherapeutic development. Vet Parasitol 2013; 195:272-285.
21. Dessein A, Kouriba B, Eboumbou C, Dessein H, Argiro L, Marquet S, et al. Interleukin-13 in the skin and interferon-gamma in the liver are key players in immune protection in human schistosomiasis. Immunol Rev 2004; 201:180-190.
22. McManus DP, Loukas A. Current status of vaccines for schistosomiasis. Clin Microbiol Rev 2008; 21:225-242.
23. Chen YF, Zheng JJ, Qu C, Xiao Y, Li FF, Jin QX, et al. Inonotus obliquus polysaccharide ameliorates dextran sulphate sodium induced colitis involving modulation of Th1/Th2 and Th17/Treg balance. Artif Cells Nanomed Biotechnol 2019; 47:757-766.
24. Motran CC, Ambrosio LF, Volpini X, Celias DP, Cervi L. Dendritic cells and parasites: from recognition and activation to immune response instruction. Semin Immunopathol 2017; 39:199-213.
25. Yan C, Li XY, Li B, Zhang BB, Xu JT, Hua H, et al. Expression of toll-like receptor (TLR) 2 and TLR4 in the livers of mice infected by Clonorchis sinensis. J Infect Dev Ctries 2015; 9:1147-1155.
26. Hua H, Du Y, Ma R, Zhang BB, Yu Q, Li B, et al. The regulatory roles of toll-like receptor 4 in secretions of type 1/type 2 relative cytokines by splenocytes and dendritic cells exposed to clonorchis sinensis excretory/secretory products. Inflammation 2018; 41:213-220.
27. Yang XO, Panopoulos AD, Nurieva R, Chang SH, Wang D, Watowich SS, et al. STAT3 regulates cytokine-mediated generation of inflammatory helper T cells. J Biol Chem 2007; 282:9358-9363.
28. Galvez J. Role of Th17 cells in the pathogenesis of human IBD. ISRN Inflamm 2014; 2014:928461.
29. Walsh KP, Brady MT, Finlay CM, Boon L, Mills KH. Infection with a helminth parasite attenuates autoimmunity through TGF-beta-mediated suppression of Th17 and Th1 responses. J Immunol 2009; 183:1577-1586.
30. Jager A, Kuchroo VK. Effector and regulatory T-cell subsets in autoimmunity and tissue inflammation. Scand J Immunol 2010; 72:173-184.
31. Donnelly S, Stack CM, O’Neill SM, Sayed AA, Williams DL, Dalton JP. Helminth 2-Cys peroxiredoxin drives Th2 responses through a mechanism involving alternatively activated macrophages. FASEB J 2008; 22:4022-4032.
32. Dowling DJ, Hamilton CM, Donnelly S, La Course J, Brophy PM, Dalton J, et al. Major secretory antigens of the helminth Fasciola hepatica activate a suppressive dendritic cell phenotype that attenuates Th17 cells but fails to activate Th2 immune responses. Infect Immun 2010; 78:793-801.
33. Everts B, Smits HH, Hokke CH, Yazdanbakhsh M. Helminths and dendritic cells: sensing and regulating via pattern recognition receptors, Th2 and Treg responses. Eur J Immunol 2010; 40:1525-1537.
34. Moosbrugger-Martinz V, Tripp CH, Clausen BE, Schmuth M, Dubrac S. Atopic dermatitis induces the expansion of thymus-derived regulatory T cells exhibiting a Th2-like phenotype in mice. J Cell Mol Med 2016; 20:930-938.