Abstract

Introduction: Bacteriophages are an abundant component of the mucosal microbiota in humans and some animal species. Intestinal epithelial cells (IECs) are the key element responsible for the induction and regulation of immune responses in the gut mucosa. The objective of this study was to evaluate the effects of T4 and A5/80 bacteriophages on the expression of immunologically important genes in Caco-2, a model cell line for IECs. Materials & Method: Bacteriophages were added to cultures of differentiated Caco-2 cells for 12 hours, while control cultures were treated with phosphate-buffered saline (PBS). Expression of genes in Caco-2 cells was determined using custom-made RT2 Profiler PCR Arrays, which allow for the evaluation of gene expression with the sensitivity and specificity of real-time PCR. We evaluated the expression of 21 genes which are important for the immune functions of IECs, including IL1B, IL6, IL7, IL10, IL15, IL18, IL25, IL33, TGFB1, TNF, CXCL8, CCL2, TSLP, FCER2, PIGR, DEFB4A, CAMP, REG3G, TNFSF13, TNFSF13B, and MUC2. Results: Both examined phages significantly influenced the expression of a number of genes compared with control cultures. In particular, T4 significantly increased the expression of the CCL2 and DEFB4A genes, while A5/80 induced the expression of the PIGR gene. Discussion: Together with the findings from previous studies, our results suggest that by modulating the expression of some genes, bacteriophages may affect immune responses in the gut mucosa.

References

• 1. Aggarwala V., Liang G., Bushman F.D.: Viral communities of thehuman gut: metagenomic analysis of composition and dynamics.Mob. DNA, 2017; 8: 12
  Google Scholar
• 2. Boratyński J., Syper D., Weber-Dąbrowska B., Łusiak-Szelachowska M., Poźniak G., Górski A.: Preparation of endotoxinfreebacteriophages. Cell. Mol. Biol. Lett., 2004; 9: 253–259
  Google Scholar
• 3. Borysowski J., Przybylski M., Międzybrodzki R., Owczarek B.,Górski A.: The effects of bacteriophages on the expression of genesinvolved in antimicrobial immunity. Postępy Hig. Med. Dośw., 2019;73: 414–420
  Google Scholar
• 4. Clooney A.G., Sutton T.D., Shkoporov A.N., Holohan R.K., DalyK.M., O’Regan O., Ryan F.J., Draper L.A., Plevy S.E., Ross R.P., HillC.: Whole-virome analysis sheds light on viral dark matter in inflammatorybowel disease. Cell Host Microbe, 2019; 26: 764–778
  Google Scholar
• 5. Dalman M.R., Deeter A., Nimishakavi G., Duan Z.H.: Fold change and p-value cutoffs significantly alter microarray interpretations.BMC Bioinformatics, 2012; 13 (Suppl. 2): S11
  Google Scholar
• 6. Edwards R.A., McNair K., Faust K., Raes J., Dutilh B.E.: Computationalapproaches to predict bacteriophage-host relationships.FEMS Microbiol. Rev., 2016; 40: 258–272
  Google Scholar
• 7. Fusco A., Savio V., Cammarota M., Alfano A., Schiraldi C., DonnarummaG.: Beta-defensin-2 and beta-defensin-3 reduce intestinaldamage caused by Salmonella typhimurium modulating theexpression of cytokines and enhancing the probiotic activity ofEnterococcus faecium. J. Immunol. Res., 2017; 2017: 6976935
  Google Scholar
• 8. Gogokhia L., Buhrke K., Bell R., Hoffman B., Brown D.G., Hanke-Gogokhia C., Ajami N.J., Wong M.C., Ghazaryan A., Valentine J.F.,Porter N., Martens E., O’Connell R., Jacob V., Scherl E., et al.: Expansionof bacteriophages is linked to aggravated intestinal inflammationand colitis. Cell Host Microb., 2019; 25: 285–299.e8
  Google Scholar
• 9. Górski A., Międzybrodzki R., Borysowski J., Dąbrowska K., WierzbickiP., Ohams M., Korczak-Kowalska G., Olszowska-Zaremba N.,Łusiak-Szelachowska M., Kłak M., Jończyk E., Kaniuga E., Gołaś A.,Purchla S., Weber-Dąbrowska B., et al.: Phage as a modulator ofimmune responses: practical implications for phage therapy. Adv.Virus Res., 2012; 83: 41–71
  Google Scholar
• 10. Górski A., Wazna E., Dabrowska B.W., Dabrowska K., Switała-Jeleń K., Miedzybrodzki R.: Bacteriophage translocation. FEMSImmunol. Med. Microbiol., 2006; 46: 313–319
  Google Scholar
• 11. Greenhough J., Papadakis E.S., Cutress R.I., Townsend P.A.,Oreffo R.O., Tare R.S.: Regulation of osteoblast development byBcl-2-associated athanogene-1 (BAG-1). Sci. Rep., 2016; 6: 33504
  Google Scholar
• 12. Koeninger L., Armbruster N.S., Brinch K.S., Kjaerulf S., AndersenB., Langnau C.: Human β-defensin 2 mediated immunemodulation as treatment for experimental colitis. Front. Immunol.,2020; 11: 93
  Google Scholar
• 13. Kropinski A.M., Mazzocco A., Waddell T.E., Lingohr E., JohnsonR.P.: Enumeration of bacteriophages by double agar overlay plaqueassay. Methods Mol. Biol., 2009; 501: 69–76
  Google Scholar
• 14. Letarov A., Kulikov E.: The bacteriophages in human- and animalbody-associated microbial communities. J. Appl. Microbiol.,2009; 107: 1–13
  Google Scholar
• 15. Lu J.T., Xu A.T., Shen J., Ran Z.H.: Crosstalk between intestinalepithelial cell and adaptive immune cell in intestinal mucosal immunity.J. Gastroenterol. Hepatol., 2017; 32: 975–980
  Google Scholar
• 16. Łobocka M., Hejnowicz M.S., Dąbrowski K., Gozdek A., KosakowskiJ., Witkowska M., Ulatowska M.I., Weber-DąbrowskaB., Kwiatek M., Parasion S., Gawor J., Kosowska H., Głowacka A.:Genomics of staphylococcal Twort-like phages – potential therapeuticsof the post-antibiotic era. Adv. Virus Res., 2012; 83: 143–216
  Google Scholar
• 17. Łusiak-Szelachowska M., Żaczek M., Weber-Dąbrowska B.,Międzybrodzki R., Kłak M., Fortuna W., Letkiewicz S., Rogóż P.,Szufnarowski K., Jończyk-Matysiak E., Owczarek B., Górski A.: Phageneutralization by sera of patients receiving phage therapy. ViralImmunol., 2014; 27: 295–304
  Google Scholar
• 18. Manrique P., Bolduc B., Walk S.T., van der Oost J., de Vos W.M.,Young M.J.: Healthy human gut phageome. Proc. Natl. Acad. Sci.USA, 2016; 113: 10400–10405
  Google Scholar
• 19. Manrique P., Dills M., Young M.J.: The human gut phage communityand its implications for health and disease. Viruses, 2017;9: E141
  Google Scholar
• 20. Murie C., Woody O., Lee A.Y., Nadon R.: Comparison of small nstatistical tests of differential expression applied to microarrays.BMC Bioinformatics, 2009; 10: 45
  Google Scholar
• 21. Natoli M., Leoni B.D., D’Agnano I., Zucco F., Felsani A.: GoodCaco-2 cell culture practices. Toxicol. In Vitro, 2012; 26: 1243–1246
  Google Scholar
• 22. Norman J.M., Handley S.A., Baldridge M.T., Droit L., Liu C.Y.,Keller B.C., Kambal A., Monaco C.L., Zhao G., Fleshner P., Stappenbeck T.S., McGovern D.P., Keshavarzian A., Mutlu E.A., Sauk J., et al.:Disease-specific alterations in the enteric virome in inflammatorybowel disease. Cell, 2015; 160: 447–460
  Google Scholar
• 23. Peterson L.W., Artis D.: Intestinal epithelial cells: regulatorsof barrier function and immune homeostasis. Nat. Rev. Immunol.,2014; 14: 141–153
  Google Scholar
• 24. Przybylski M., Dzieciątkowski T., Borysowski J., MiędzybrodzkiR., Górski A.: Inhibitory effects of bacteriophage preparations onadenoviral replication. Intervirology, 2019; 62: 37–44
  Google Scholar
• 25. Putra R.D., Lyrawati D.: Interactions between bacteriophagesand eukaryotic cells. Scientifica, 2020; 2020: 3589316
  Google Scholar
• 26. Reyes A., Haynes M., Hanson N., Angly F.E., Heath A.C., RohwerF., Gordon J.I.: Viruses in the faecal microbiota of monozygotictwins and their mothers. Nature, 2010; 466: 334–338
  Google Scholar
• 27. Ruder B., Atreya R., Becker C.: Tumour necrosis factor alphain intestinal homeostasis and gut related diseases. Int. J. Mol. Sci.,2019; 20: E1887
  Google Scholar
• 28. Salem A., Almahmoudi R., Hagström J., Stark H., NordströmD., Salo T., Eklund K.K.: Human β-defensin 2 expression in oral epithelium:Potential therapeutic targets in oral lichen planus. Int. J.Mol. Sci., 2019; 20: E1780
  Google Scholar
• 29. Sausset R., Petit M.A., Gaboriau-Routhiau V., De Paepe M.:New insights into intestinal phages. Mucosal Immunol., 2020; 13:205–215
  Google Scholar
• 30. Schreiner P., Neurath M.F., Ng S.C., El-Omar E.M., Sharara A.I.,Kobayashi T., Hisamatsu T., Hibi T., Rogler G.: Mechanism-basedtreatment strategies for IBD: Cytokines, cell adhesion molecules,JAK inhibitors, gut flora, and more. Inflamm. Intest. Dis., 2019;4: 79–96
  Google Scholar
• 31. Shkoporov A.N., Clooney A.G., Sutton T.D., Ryan F.J., Daly K.M.,Nolan J.A., McDonnell S.A., Khokhlova E.V., Draper L.A., Forde A.,Guerin E., Velayudhan V., Ross R.P., Hill C.: The human gut viromeis highly diverse, stable, and individual specific. Cell Host Microbe,2019; 26: 527–541.e5
  Google Scholar
• 32. Sutton T.D., Hill C.: Gut bacteriophage: Current understandingand challenges. Front. Endocrinol., 2019; 10: 784
  Google Scholar
• 33. Ślopek S., Durlakowa I., Weber-Dąbrowska B., Kucharewicz-Krukowska A., Dąbrowski M., Bisikiewicz R.: Results of bacteriophagetreatment of suppurative bacterial infections. I. Generalevaluation of the results. Arch. Immunol. Ther. Exp., 1983; 31:267–291
  Google Scholar
• 34. Tosiek M.J., Fiette L., El Daker S., Eberl G., Freitas A.A.: IL-15-dependent balance between Foxp3 and RORγt expression impactsinflammatory bowel disease. Nat. Commun., 2016; 7: 10888
  Google Scholar
• 35. Turula H., Wobus C.E.: The role of the polymeric immunoglobulinreceptor and secretory immunoglobulins during mucosalinfection and immunity. Viruses, 2018; 10: E237
  Google Scholar
• 36. Wang W., Jovel J., Halloran B., Wine E., Patterson J., Ford G.,OʼKeefe S., Meng B., Song D., Zhang Y., Tian Z., Wasilenko S.T.,Rahbari M., Reza S., Mitchell T., et al.: Metagenomic analysis ofmicrobiome in colon tissue from subjects with inflammatory boweldiseases reveals interplay of viruses and bacteria. Inflamm. BowelDis., 2015; 21: 1419–1427
  Google Scholar
• 37. Yang J.Y., Kim M.S., Kim E., Cheon J.H., Lee Y.S., Kim Y., LeeS.H., Seo S.U., Shin S.H., Choi S.S., Kim B., Chang S.Y., Ko H.J., BaeJ.W., Kweon M.N.: Enteric viruses ameliorate gut inflammation viaToll-like receptor 3 and Toll-like receptor 7-mediated interferon-βproduction. Immunity, 2016; 44: 889–900
  Google Scholar
• 38. Yap M.L., Rossmann M.G.: Structure and function of bacteriophageT4. Future Microbiol., 2014; 9: 1319–1327
  Google Scholar

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