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Cytokine response after stimulation of culture cells by zinc and probiotic strain

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Abstract

Intestinal porcine epithelial cells were used for an in vitro analysis of mRNA expression levels of inflammatory cytokines (IL-8, IL-18) and transcriptional factors (MyD88 and NF-κβ). Cells were exposed to inorganic and organic zinc sources (in two different concentrations—50 μmol/L and 100 μmol/L) alone or combined with Lactobacillus reuteri B6/1, which was also applied individually. The total exposure time was 4 h. Quantitative reverse transcriptase PCR was used to determine expression levels of the aforementioned parameters. In general, upregulation was observed; however, a decrease of some mRNA’s abundance was also determined. Differences in expression were analysed statistically using ANOVA and Tukey analyses. High relative expression was shown for IL-8, IL-18 and MyD88 in groups treated with 100 μmol/L of inorganic sources of zinc (ZnSO4) (p < 0.05), while groups treated with the organic form did not exhibit significant changes in expression. Also, 50 μmol/L of either zinc source did not significantly modify the transcriptional profile of the cytokines and transcription factors, showing that even inorganic sources, at lower concentrations, do not elicit a significant inflammatory reaction. In summary, supplementation of organic zinc source (Gly-Zn chelate) ensures that IL-8, IL-18, MyD88 and NF-κβ expression levels are not positively regulated. In contrast, inorganic sources of zinc (ZnSO4) could induce an inflammatory reaction. However, this response could be dampened if L. reuteri B6/1 is administered, showing the helpful aspect of using probiotics to modulate an inflammatory response. Conclusively, the use Gly-Zn chelate appears as an optimal alternative for Zn administration that does not compromise normal intestinal homeostasis.

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References

  • Bao YM, Choct M, Iji PA, Bruerton K (2010) The digestibility of organic trace minerals along the small intestine in broiler chickens. Asian Australas J Anim Sci 23:90–97

    Article  CAS  Google Scholar 

  • Bonaventura P, Benedetti G, Albarède F, Miossec P (2015) Zinc and its role in immunity and inflammation. Autoimmun Rev 14:277–285

    Article  CAS  PubMed  Google Scholar 

  • Brown SL, Riehl TE, Walker MR, Geske MJ, Doherty JM, Stenson WF, Stappenbeck TS (2007) MyD88-dependent positioning of Ptgs2-expressing stromal cells maintains colonic epithelial proliferation during injury. J Clin Invest 117:258–269

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chia SL, Tay CY, Setyawati MI, Leong DT (2014) Biomimicry 3D gastrointestinal spheroid platform for the assessment of toxicity and inflammatory effects of zinc oxide nanoparticles. Small 11:702–712

    Article  PubMed  CAS  Google Scholar 

  • Chon H, Choi B, Jeong G, Lee E, Lee S (2010) Suppression of proinflammatory cytokine production by specific metabolites of Lactobacillus plantarum 10hk2 via inhibiting NF-kappaB and p38 MAPK expressions. Comp Immunol Microbiol Infect Dis 33:e41–e49

    Article  PubMed  Google Scholar 

  • Chytilová M, Nemcová R, Gancarčíková S, Mudroňová D, Tkáčiková L (2014) Flax-seed oil and Lactobacillus plantarum supplementation modulate TLR and NF-κβ gene expression in enterotoxigenic Escherichia coli challenged gnotobiotic pigs. Acta Vet Hung 62:463–472

    Article  PubMed  CAS  Google Scholar 

  • De Boever S, Vangestel C, De Vacker P, Croubels S, Sys SU (2008) Identification and validation of housekeeping genes as internal control for gene expression in an intravenous LPS inflammation model in chickens. Vet Immunol Immunopathol 122:312–317

    Article  PubMed  CAS  Google Scholar 

  • Deguine J, Barton GM (2014) MyD88: a central player in innate immune signaling. F1000Prime rep 6:97

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Elliott CL, Allport VC, Loudon JA, Wu GD, Bennett PR (2001) Nuclear factor-kappa B is essential for up-regulation of interleukin-8 expression in human amnion and cervical epithelial cells. Mol Hum Reprod 7:787–790

    Article  CAS  PubMed  Google Scholar 

  • Heller KJ (2001) Probiotic bacteria in fermented foods: product characteristics and starter organisms. Am J Clin Nutr 73:374–379

    Article  Google Scholar 

  • Huang D, Zhuo Z, Fang S, Yue M, Feng J (2016) Different zinc sources have diverse impacts on gene expression of zinc absorption related transporters in intestinal porcine epithelial cells. Biol Trace Elem Res 176:325–332

    Article  CAS  Google Scholar 

  • Husáková E, Bobíková K, Stašová D (2015) Total IgA in spleen, bursa and intestine of chickens pretreated with E. faecium AL41 and challenged with Salmonella Enteritidis PT4. Food Agric Immunol 26:366–370

    Article  CAS  Google Scholar 

  • Jarosz M, Olbert M, Wyszogrodzka G, Młyniec K, Librowski T (2017) Antioxidant and anti-inflammatory effects of zinc. Zinc-dependent NF-κβ signaling. Inflammopharmacology 25:11–24

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jiang Y, Lu X, Man C, Han L, Shan Y, Qu X, Liu Y, Yang S, Xue Y, Zhanga Y (2012) Lactobacillus acidophilus induces cytokine and chemokine production via NF-kappaB and p38 mitogen-activated protein kinase signaling pathways in intestinal epithelial cells. Clin Vaccine Immunol 19:603–608

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Karaffová V, Husáková E, Bobíková K, Herich R, Revajová V, Stašová D, Kavuľová A, Levkutová M, Levkut M Jr, Lauková A, Ševčíková Z, Levkut M Sr (2017) TLR4 and TLR21 expression, MIF, IFN-β, MD-2, CD14 activation, and sIgA production in chickens administered with EFAL41 strain challenged with Campylobacter jejuni. Folia Microbiol 62:89–97

    Article  CAS  Google Scholar 

  • Kumar A, Collier-Hyams LS, Kwon YM, Hanson JM, Neish AS (2009) The bacterial fermentation product butyrate influences epithelial signaling via reactive oxygen species-mediated changes in cullin-1 neddylation. J Immunol 182:538–546

    Article  CAS  PubMed  Google Scholar 

  • Lan JG (2005) Different cytokine responses of primary colonic epithelial cells to commensal bacteria. World J Gastroenterol 11:3375–3384

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu T, Zhang L, Joo D, Sun SC (2017) NF-κβ signaling in inflammation. Signal Transduct Target Ther 2:e17023

    Article  Google Scholar 

  • Lodemann U, Einspanier R, Scharfen F, Martens H, Bondzio A (2013) Effects of zinc on epithelial barrier properties and viability in a human and a porcine intestinal cell culture model. Toxicol in Vitro 27:834–843

    Article  CAS  PubMed  Google Scholar 

  • Lügering N, Kucharzik T, Gockel H, Sorg C, Stoll R, Domschke W (1998) Human intestinal epithelial cells down-regulate IL-8 expression in human intestinal microvascular endothelial cells; role of transforming growth factor-beta 1 (TGF-beta1). Clin Exp Immunol 114:377–384

    Article  PubMed  PubMed Central  Google Scholar 

  • Mariani V, Palermo S, Fiorentini S, Lanubile A, Giuffra E (2009) Gene expression study of two widely used pig intestinal epithelial cell lines: IPEC-J2 and IPI-2I. Vet Immunol Immunopathol 131:278–284

    Article  CAS  PubMed  Google Scholar 

  • Mohamadzadeh M, Olson S, Kalina WV, Ruthel G, Demmin GL, Warfield KL, Klaenhammer TR (2005) Lactobacilli activate human dendritic cells that skew T cells toward T helper 1 polarization. Proc Natl Acad Sci 102:2880–2885

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Morita H, He F, Fuse T, Ouwehand AC, Hashimoto H, Hosoda M, Mizumachi K, Kurisaki J (2002) Adhesion of lactic acid bacteria to caco-2 cells and their effect on cytokine secretion. Microbiol Immunol 46:293–297

    Article  CAS  PubMed  Google Scholar 

  • Muñoz M, Eidenschenk C, Ota N, Wong K, Lohmann U, Kühl AA, Wang X, Manzanillo P, Li Y, Rutz S, Zheng Y, Diehl L, Kayagaki N, van Lookeren-Campagne M, Liesenfeld O, Heimesaat M, Ouyang W (2015) Interleukin-22 induces interleukin-18 expression from epithelial cells during intestinal infection. Immunity 42:321–331

    Article  PubMed  CAS  Google Scholar 

  • Neish AS, Gewirtz AT, Zeng H, Young AN, Hobert ME, Karmali V, Rao AS, Madara JL (2000) Prokaryotic regulation of epithelial responses by inhibition of IkappaB-alpha ubiquitination. Science 289:1560–1563

    Article  CAS  PubMed  Google Scholar 

  • Nowarski R, Jackson R, Gagliani N, de Zoete MR, Palm NW, Bailis W, Low JS, Harman CC, Graham M, Elinav E, Flavell RA (2015) Epithelial IL-18 equilibrium controls barrier function in colitis. Cell 163:1444–1456

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Plaza-Diaz J, Gomez-Llorente C, Fontana L, Gil A (2014) Modulation of immunity and inflammatory gene expression in the gut, in inflammatory diseases of the gut and in the liver by probiotics. World J Gastroenterol 20:15632–15649

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Roselli M, Finamore A, Britti M, Bosi P, Oswald I, Mengheri E (2005) Alternatives to in-feed antibiotics in pigs: evaluation of probiotics, zinc or organic acids as protective agents for the intestinal mucosa. A comparison of in vitro and in vivo results. Anim Res 54:203–218

    Article  CAS  Google Scholar 

  • Sargeant HR, Miller HM, Shaw MA (2011) Inflammatory response of porcine epithelial IPEC J2 cells to enterotoxigenic E. coli infection is modulated by zinc supplementation. Mol Immunol 48:2113–2121

    Article  CAS  PubMed  Google Scholar 

  • Shao L, Serrano D, Mayer L (2001) The role of epithelial cells in immune regulation in the gut. Semin Immunol 13:163–176

    Article  CAS  PubMed  Google Scholar 

  • Shimazu T, Villena J, Tohno M, Fujie H, Hosoya S, Shimosato T, Kitazawa H (2012) Immunobiotic Lactobacillus jensenii elicits anti-inflammatory activity in porcine intestinal epithelial cells by modulating negative regulators of the toll-like receptor signaling pathway. Infect Immun 80:276–288

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Siegmund B (2010) Interleukin-18 in intestinal inflammation: friend and foe? Immunity 32:300–302

    Article  CAS  PubMed  Google Scholar 

  • Tien MT, Girardin SE, Regnault B, Le Bourhis L, Dillies MA, Coppée JY, Bourdet-Sicard R, Sansonetti PJ, Pédron T (2006) Anti-inflammatory effect of Lactobacillus casei on Shigella-infected human intestinal epithelial cells. J Immunol 176:1228–1237

    Article  CAS  PubMed  Google Scholar 

  • Vizoso Pinto MG, Rodriguez Gomez M, Seifert S, Watzl B, Holzapfel WH, Franz CM (2009) Lactobacilli stimulate the innate immune response and modulate the TLR expression of HT29 intestinal epithelial cells in vitro. Int J Food Microbiol 133:86–93

    Article  CAS  PubMed  Google Scholar 

  • Volf J, Stepanova H, Matiasovic J, Kyrova K, Sisak F, Havlickova H, Leva L, Faldyna M, Rychlik I (2012) Salmonella enterica serovar typhimurium and Enteritidis infection of pigs and cytokine signalling in palatine tonsils. Vet Microbiol 156:127–135

    Article  CAS  PubMed  Google Scholar 

  • Von Schillde MA, Hörmannsperger G, Weiher M, Alpert CA, Hahne H, Bäuerl C, Haller D (2012) Lactocepin secreted by Lactobacillus exerts anti-inflammatory effects by selectively degrading proinflammatory chemokines. Cell Host Microbe 11:387–396

    Article  CAS  Google Scholar 

  • Wu Y, Zhu C, Chen Z, Chen Z, Zhang W, Ma X, Jiang Z (2016) Protective effects of Lactobacillus plantarum on epithelial barrier disruption caused by enterotoxigenic Escherichia coli in intestinal porcine epithelial cells. Vet Immunol Immunopathol 172:55–63

    Article  CAS  PubMed  Google Scholar 

  • Yan M, Xu Q, Zhang P, Zhou X, Zhang Z, Chen W (2010) Correlation of NF-κB signal pathway with tumor metastasis of human head and neck squamous cell carcinoma. BMC Cancer 10:437

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Yang H, Xiong X, Li T, Yin Y (2016) Ethanolamine enhances the proliferation of intestinal epithelial cells via the mTOR signalling pathway and mitochondrial function. In vitro cell dev biol ˗. Animal 52:562–567

    CAS  Google Scholar 

  • Young L, Sung J, Stacey G, Masters JR (2010) Detection of mycoplasma in cell cultures. Nat

  • Yu Y, Lu L, Wang RL, Xi L, Luo XG, Liu B (2010) effects of source and phytate on zinc absorption by in situ ligated intestinal loops of broilers. Poult Sci 89:2157–2165

    Article  CAS  PubMed  Google Scholar 

  • Zeuthen LH, Fink LN, Metzdorff SB, Kristensen MB, Licht TR, Nellemann C, Frøkiaer H (2010) Lactobacillus acidophilus induces a slow but more sustained chemokine and cytokine response in naïve foetal enterocytes compared to commensal Escherichia coli. BMC Immunol 11:2

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Zhao S, Li B, Chen G, Hu Q, Zhao L (2017) Preparation, characterization, and anti-inflammatory effect of the chelate of Flammulina velutipes polysaccharide with Zn. Food Agric Immunol 28:162–177

    Article  CAS  Google Scholar 

  • Zhu C, Lv H, Chen Z, Wang L, Wu X, Chen Z, Jiang Z (2017) Dietary zinc oxide modulates antioxidant capacity, small intestine development, and jejunal gene expression in weaned piglets. Biol Trace Elem Res 175:331–338

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We are very grateful to Marco Larrea-Álvarez, PhD, and to César Larrea-Álvarez for the appropriate and constructive suggestions that help to improve the manuscript. This work was supported by the Grant Agency for Science of the Slovak Republik VEGA (1/0112/18, 1/0355/19); Slovak Research and Developmental Agency (APVV-15-0165).

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Authors and Affiliations

  1. Department of Pathological Anatomy and Pathological Physiology, University of Veterinary Medicine and Pharmacy, Komenského 73, 04181, Košice, Slovak Republic

    Miroslava Šefcová, Martin Levkut, Katarína Bobíková, Viera Karaffová, Viera Revajová, Zuzana Ševčíková, Róbert Herich & Mikuláš Levkut

  2. Department of Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, Komenského 73, 04181, Košice, Slovak Republic

    Ivana Cingeľová Maruščáková

  3. Department of Epizootiology and Parasitology, University of Veterinary Medicine and Pharmacy, Komenského 73, 04181, Košice, Slovak Republic

    Mária Levkutová

  4. Institute of Neuroimmunology, Slovak Academy of Science, Dúbravská cesta 9, 84510, Bratislava, Slovak Republic

    Mikuláš Levkut

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  1. Miroslava Šefcová
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Šefcová, M., Levkut, M., Bobíková, K. et al. Cytokine response after stimulation of culture cells by zinc and probiotic strain. In Vitro Cell.Dev.Biol.-Animal 55, 830–837 (2019). https://doi.org/10.1007/s11626-019-00401-z

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  • DOI: https://doi.org/10.1007/s11626-019-00401-z

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