Open Access
Research (Published online: 03-04-2019)
4. Recombinant horse interleukin-4 and interleukin-10 induced a mixed inflammatory cytokine response in horse peripheral blood mononuclear cells
Sheetal Saini, Harisankar Singha, Priyanka Siwach and B. N. Tripathi
Veterinary World, 12(4): 496-503

Sheetal Saini: ICAR-National Research Centre on Equines, Hisar, Haryana, India; Department of Biotechnology, Chaudhary Devi Lal University, Sirsa, Haryana, India.
Harisankar Singha: ICAR-National Research Centre on Equines, Hisar, Haryana, India.
Priyanka Siwach: Department of Biotechnology, Chaudhary Devi Lal University, Sirsa, Haryana, India.
B. N. Tripathi: ICAR-National Research Centre on Equines, Hisar, Haryana, India.

doi: 10.14202/vetworld.2019.496-503

Share this article on [Facebook] [LinkedIn]

Article history: Received: 29-11-2018, Accepted: 20-02-2019, Published online: 03-04-2019

Corresponding author: Harisankar Singha


Citation: Saini S, Singha H, Siwach P, Tripathi BN (2019) Recombinant horse interleukin-4 and interleukin-10 induced a mixed inflammatory cytokine response in horse peripheral blood mononuclear cells, Veterinary World, 12(4): 496-503.

Background and Aim: Interleukin (IL)-4 and IL-10 activate plethora of immune cells and induce the humoral immune response. However, recombinant version of horse IL-4 and IL-10 has not been investigated to understand their immunomodulating activities. This study aimed to produce recombinant horse mature IL-4 and IL-10 in Escherichia coli. Immune-modulating activities of recombinant horse IL-4 and IL-10 were investigated in peripheral blood mononuclear cells (PBMCs).

Materials and Methods: Equine PBMCs were stimulated with recombinant IL-4 and IL-10. A proliferation of PBMCs was measured by XTT assay and cytokines induction was measured by enzyme-linked immunosorbent assay and real-time polymerase chain reaction.

Results: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis displayed a molecular weight of 15 kDa for IL-4 and 19 kDa for IL-10. Recombinant IL-4 and IL-10 significantly induced cell proliferation at 250 ng/ml. The results demonstrated that IL-4 enhanced expression of interferon-gamma (IFN-γ), IL-6, tumor necrosis factor-alpha (TNF-α), and IL-10, while recombinant horse IL-10 induced expression of IL-6, IFN-γ, and TNF-α.

Conclusion: The present study demonstrated that biologically active horse IL-4 and IL-10 could be produced in E. coli.

Keywords: cell proliferation, cytokines, enzyme-linked immunosorbent assay, real-time polymerase chain reaction.


1. Mitchell, R.E., Hassan, M., Burton, B.R., Britton, G., Hill, E.V., Verhagen, J. and Wraith, D.C. (2017) IL-4 enhances IL-10 production in Th1 cells: Implications for Th1 and Th2 regulation. Sci. Rep., 7(1): 11315. [Crossref]

2. Wynn, T.A. (2015) Type 2 cytokines: Mechanisms and therapeutic strategies. Nat. Rev. Immunol., 15(5): 271-282. [Crossref] [PubMed]

3. Hsieh, C.S., Heimberger, A.B., Gold, J.S., O'Garra, A. and Murphy, K.M. (1992) Differential regulation of T helper phenotype development by interleukins 4 and 10 in an alpha-beta T-cell-receptor transgenic system. Proc. Natl. Acad. Sci. U. S. A., 89(13): 6065-6069. [Crossref]

4. McLeod, J.J., Baker, B. and Ryan, J.J. (2015) Mast cell production and response to IL-4 and IL-13. Cytokine, 75(1): 57-61. [Crossref] [PubMed] [PMC]

5. Junttila, I.S. (2018) Tuning the cytokine responses: An update on interleukin (IL)-4 and IL-13 receptor complexes. Front. Immunol., 9(888): 1-6. [Crossref]

6. Rojas, J.M., Avia, M., Martin, V. and Sevilla, N. (2017) IL-10: A multifunctional cytokine in viral infections. J. Immunol. Res., 2017(4): 1-14. [Crossref]

7. MacKenzie, K.F., Pattison, M.J. and Arthur, J.S. (2014) Transcriptional regulation of IL-10 and its cell-specific role in vivo. Crit. Rev. Immunol., 34(4): 315-345. [Crossref]

8. Cordeau, M.E., Joubert, P., Dewachi, O., Hamid, Q. and Lavoie, J.P. (2004) IL-4, IL-5 and IFN-gamma mRNA expression in pulmonary lymphocytes in equine heaves. Vet. Immunol. Immunopathol., 97(1-2): 87-96. [Crossref]

9. Curto, E., Messenger, K.M., Salmon, J.H. and Gilger, B.C. (2016) Cytokine and chemokine profiles of aqueous humor and serum in horses with uveitis measured using multiplex bead immunoassay analysis. Vet. Immunol. Immunopathol., 182(12): 43-51. [Crossref] [PubMed]

10. Pacholewska, A., Jagannathan, V., Drogemuller, M., Klukowska-Rotzler, J., Lanz, S., Hamza, E., Dermitzakis, E.T., Marti, E., Leeb, T. and Gerber, V. (2015) Impaired cell cycle regulation in a natural equine model of asthma. PLoS One, 10(8): e0136103. [Crossref]

11. Steinbach, F., Stark, R., Ibrahim, S., Gawad, E.A., Ludwig, H., Walter, J., Commandeur, U. and Mauel, S. (2005) Molecular cloning and characterization of markers and cytokines for equid myeloid cells. Vet. Immunol. Immunopathol., 108(1-2): 227-236. [Crossref] [PubMed]

12. Dohmann, K., Wagner, B., Horohov, D.W. and Leibold, W. (2000) Expression and characterization of equine interleukin 2 and interleukin 4. Vet. Immunol. Immunopathol., 77(3-4): 243-256. [Crossref]

13. Wagner, B., Robeson, J., McCracken, M., Wattrang, E. and Antczak, D.F. (2005) Horse cytokine/IgG fusion proteins--mammalian expression of biologically active cytokines and a system to verify antibody specificity to equine cytokines. Vet. Immunol. Immunopathol., 105(1-2): 1-14. [Crossref]

14. Berghaus, L.J., Giguere, S., Bordin, A.I. and Cohen, N.D. (2018) Effects of priming with cytokines on intracellular survival and replication of Rhodococcus equi in equine macrophages. Cytokine, 102(2): 7-11. [Crossref] [PubMed]

15. Wagner, B., Perkins, G., Babasyan, S., Freer, H., Keggan, A., Goodman, L.B., Glaser, A., Torsteinsdottir, S., Svansson, V. and Bjornsdottir, S. (2017) Neonatal immunization with a single IL-4/antigen dose induces increased antibody responses after challenge infection with equine herpesvirus Type 1 (EHV-1) at weanling age. PLoS One, 12(1): e0169072. [Crossref]

16. Singha, H., Goyal, S.K. and Malik, P. (2015) Cloning and sequence analysis of interleukin (IL)-2, IL-4,IL-10, and IL-18 of Marwari horse. Indian J. Anim. Sci., 85(6): 621-627.

17. Sambrook, J.J. and Green, M.R. (2012) Molecular Cloning: A Laboratory Manual. 4. Cold Spring Harbor Laboratory Press, New York. p1599-1609.

18. Figueiredo, M.D., Salter, C.E., Andrietti, A.L., Vandenplas, M.L., Hurley, D.J. and Moore, J.N. (2009) Validation of a reliable set of primer pairs for measuring gene expression by real-time quantitative RT-PCR in equine leukocytes. Vet. Immunol. Immunopathol., 131(1-2): 65-72. [Crossref] [PubMed]

19. Sanchez-Matamoros, A., Kukielka, D., De las Heras, A.I. and Sanchez-Vizcaino, J.M. (2013) Development and evaluation of an SYBR Green real-time RT-PCR assay for evaluation of cytokine gene expression in horse. Cytokine, 61(1): 50-53. [Crossref] [PubMed]

20. Overbergh, L., Valckx, D., Waer, M. and Mathieu, C. (1999) Quantification of murine cytokine mRNAs using real-time quantitative reverse transcriptase PCR. Cytokine, 11(4): 305-312. [Crossref] [PubMed]

21. Cao, S., Liu, J., Song, L. and Ma, X. (2005) The protooncogene c-Maf is an essential transcription factor for IL-10 gene expression in macrophages. J. Immunol., 174(6): 3484-3492. [Crossref]

22. Morris, S.C., Orekhova, T., Meadows, M.J., Heidorn, S.M., Yang, J. and Finkelman, F.D. (2006) IL-4 induces in vivo production of IFN-gamma by NK and NKT cells. J. Immunol., 176(9): 5299-5305. [Crossref]

23. Oliver, J.A., Stolberg, V.R., Chensue, S.W. and King, P.D. (2012) IL-4 acts as a potent stimulator of IFN-gamma expression in CD8+ T cells through STAT6-dependent and independent induction of Eomesodermin and T-bet. Cytokine, 57(1): 191-199. [Crossref] [PubMed] [PMC]

24. Lavoie-Lamoureux, A., Moran, K., Beauchamp, G., Mauel, S., Steinbach, F., Lefebvre-Lavoie, J., Martin, J.G. and Lavoie, J.P. (2010) IL-4 activates equine neutrophils and induces a mixed inflammatory cytokine expression profile with enhanced neutrophil chemotactic mediator release ex vivo. Am. J. Physiol. Lung Cell Mol. Physiol., 299(4): L472-L482. [Crossref]

25. Hochrein, H., O'Keeffe, M., Luft, T., Vandenabeele, S., Grumont, R.J., Maraskovsky, E. and Shortman, K. (2000) Interleukin (IL)-4 is a major regulatory cytokine governing bioactive IL-12 production by mouse and human dendritic cells. J. Exp. Med., 192(6): 823-833. [Crossref]

26. Cush, S.S., Reynoso, G.V., Kamenyeva, O., Bennink, J.R., Yewdell, J.W. and Hickman, H.D. (2016) Locally produced IL-10 limits cutaneous vaccinia virus spread. PLoS Pathog., 12(3): e1005493. [Crossref]

27. Ma, X., Yan, W., Zheng, H., Du, Q., Zhang, L., Ban, Y., Li, N. and Wei, F. (2015) Regulation of IL-10 and IL-12 production and function in macrophages and dendritic cells. F1000Res. 4(F1000 Faculty Rev):1465. [Crossref]

28. Fiorentino, D.F., Zlotnik, A., Mosmann, T.R., Howard, M. and O'Garra, A. (1991) IL-10 inhibits cytokine production by activated macrophages. J. Immunol., 147(11): 3815-3822. [PubMed]

29. D'Andrea, A., Aste-Amezaga, M., Valiante, N.M., Ma, X., Kubin, M. and Trinchieri, G. (1993) Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J. Exp. Med., 178(3): 1041-1048. [Crossref]

30. Stordeur, P., Poulin, L.F., Craciun, L., Zhou, L., Schandene, L., de Lavareille, A., Goriely, S. and Goldman, M. (2002) Cytokine mRNA quantification by real-time PCR. J. Immunol. Methods, 259(1-2): 55-64. [Crossref]

31. Godornes, C., Leader, B.T., Molini, B.J., Centurion-Lara, A. and Lukehart, S.A. (2007) Quantitation of rabbit cytokine mRNA by real-time RT-PCR. Cytokine, 38(1): 1-7. [Crossref]

32. Seko, Y., Cole, S., Kasprzak, W., Shapiro, B.A. and Ragheb, J.A. (2006) The role of cytokine mRNA stability in the pathogenesis of autoimmune disease. Autoimmun. Rev., 5(5): 299-305. [Crossref] [PubMed]