Reversal of Pathogen-Induced Barrier Defects in Intestinal Epithelial Cells by Contra-pathogenicity Agents

Abstract

Background

Environmental enteropathy (EE) is associated with stunting, impairment of responses to oral vaccines, and other adverse health consequences in young children throughout the developing world. EE is characterized by chronic low-grade intestinal inflammation and disrupted epithelial barrier integrity, partly resulting from dysregulation of tight junction proteins, observed in other enteropathies such as celiac disease. During EE, this dysregulation of tight junction expression amplifies translocation of pathogenic bacteria across the intestinal mucosa.

Aims

The aim was to determine whether enteropathogen-mediated epithelial barrier failure can be ameliorated using contra-pathogenicity therapies.

Methods

Intestinal epithelial barrier damage was assessed in Caco-2 cells incubated with three important enteropathogens identified in EE patients: Enteropathogenic Escherichia coli (EPEC), Citrobacter rodentium (C. rodentium), and Cryptosporidium parvum (C. parvum). Potential therapeutic molecules were tested to detect effects on transepithelial resistance (TER), bacterial translocation (BT), claudin-4 expression, and regulation of the inflammatory cytokine response.

Results

All three enteropathogens compared to uninfected cells, reduced TER (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0007), reduced claudin-4 expression, and permitted BT (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0003) through the monolayer. Zinc, colostrum, epidermal growth factor, trefoil factor 3, resistin-like molecule-β, hydrocortisone, and the myosin light chain kinase inhibitor ML7 (Hexahydro-1-[(5-iodo-1-naphthalenyl)sulfonyl]-1H-1,4-diazepine hydrochloride); ML7) improved TER (up to 70%) and decreased BT (as much as 96%). Only zinc demonstrated modest antimicrobial activity.

Conclusion

The enteropathogens impaired intestinal–epithelial barrier integrity with dysregulation of claudin-4 and increased bacterial translocation. Enteropathogen-mediated damage was reduced using contra-pathogenicity agents which mitigated the effects of pathogens without direct antimicrobial activity.

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References

  1. 1.

    Turner JR. Intestinal mucosal barrier function in health and disease. Ann Rev Immunol. 2009;9:799–809.

    CAS  Google Scholar 

  2. 2.

    Salazar-Lindo E, Allen S, Brewster DR, Elliott EJ, et al. Intestinal infections and environmental enteropathy: working group report of the second world congress of pediatric gastroenterology, hepatology, and nutrition. J Pediatr Gastroenterol Nutr. 2004;39:S662–S669.

    PubMed  Article  PubMed Central  Google Scholar 

  3. 3.

    Platts-Mills JA, Babji S, Bodhidatta L, Gratz J, et al. MAL-ED network. Investigators pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (MAL-ED). Lancet Glob Health. 2015;3:e564–e575.

    PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Lee GO, McCormick BJJ, Seidman JC, Kosek MN, et al. For the MAL-ED network investigators. Infant nutritional status, feeding practices, enteropathogen exposure, socioeconomic status, and illness are associated with gut barrier function as assessed by the lactulose mannitol test in the MAL-ED birth cohort. Am J Trop Med Hyg. 2017;97:281–290.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. 5.

    Prendergast A, Kelly P. Enteropathies in the developing world; neglected effects on global health. Am J Trop Med Hyg. 2012;86:756–763.

    PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Naylor C, Lu M, Haque R, Mondal D, et al. Environmental Enteropathy, oral vaccine failure and growth faltering in infants in Bangladesh. EBioMedicine. 2015;2:1759–1766.

    PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Manary MJ, Abrams SA, Griffin IJ, Quimper MM, et al. Perturbed zinc homeostasis in rural 3-5-y-old Malawian children is associated with abnormalities in intestinal permeability attributed to tropical enteropathy. Pediatr Res. 2010;67:671–675.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  8. 8.

    Tostman A, Mtabho CM, Semvua HH, van den Boogaard J, et al. Pharmacokinetics of first-line tuberculosis drugs in Tanzanian patients. Antimicrob Agents Chemother. 2013;57:3208–3213.

    Article  CAS  Google Scholar 

  9. 9.

    Campbell DI, Elia M, Lunn PG. Growth faltering in rural Gambian infants is associated with impaired small intestinal barrier function, leading to endotoxemia and systemic inflammation. J Nutr. 2003;133:1332–1338.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  10. 10.

    Kelly P, Besa E, Zyambo K, Louis-Auguste J, et al. Endomicroscopic and transcriptomic analysis of impaired barrier function and malabsorption in environmental enteropathy. PLoS Negl Trop Dis. 2016;10:e0004600.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  11. 11.

    Korpe PS, Petri WA. Environmental enteropathy: critical implications of a poorly understood condition. Trends Mol Med. 2012;18:328–336.

    PubMed  PubMed Central  Article  Google Scholar 

  12. 12.

    Prendergast AJ, Kelly P. Interactions between intestinal pathogens, enteropathy and malnutrition in developing countries. Curr Opin Infect Dis. 2016;29:229–236.

    PubMed  PubMed Central  Article  Google Scholar 

  13. 13.

    Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet. 2013;382:209–222.

    PubMed  Article  PubMed Central  Google Scholar 

  14. 14.

    Liu J, Platts-Mills JA, Juma J, Kabir F, et al. Use of quantitative molecular diagnostic methods to identify causes of diarrhoea in children: a reanalysis of the GEMS case-control study. Lancet. 2016;388:1291–1301.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. 15.

    Lima AAM, Leite ÁM, Di Moura A, Lima NL, et al. Determinant variables, enteric pathogen burden, gut function and immune-related inflammatory biomarkers associated with childhood malnutrition: a prospective case-control study in Northeastern Brazil. Pediatr Infect Dis J. 2017;36:1177–1185.

    PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Amour C, Gratz J, Mduma E, Svensen E, et al. Etiology, Risk factors, and interactions of enteric infections and malnutrition and the consequences for child health and development project (MAL-ED) network investigators. Epidemiology and impact of campylobacter infection in children in 8 low-resource settings: results from the MAL-ED study. Clin Infect Dis. 2016;63:1171–1179.

    PubMed  PubMed Central  Google Scholar 

  17. 17.

    Kosek MN, the MAL-ED Network Investigators. Causal pathways from enteropathogens to environmental enteropathy: findings from the MAL-ED birth cohort study. e-Biomedicine 2017; in press.

  18. 18.

    Marchiando AM, Shen L, Graham WV, Edelblum KL, et al. The epithelial barrier is maintained by in vivo tight junction expansion during pathologic intestinal epithelial shedding. Gastroenterology. 2011;140:1208–1218.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Flynn AN, Buret AG. Caspases-3, -8, and -9 are required for induction of epithelial cell apoptosis by enteropathogenic E. coli but are dispensable for increased paracellular permeability. Microb Pathog. 2008 Apr;44:311-9. Epub. 10/24/2007.

  20. 20.

    Tapia R, Kralicek SE, Hecht GA. Modulation of epithelial cell polarity by bacterial pathogens. Ann N Y Acad Sci. 2017;1405:16–24.

    PubMed  PubMed Central  Article  Google Scholar 

  21. 21.

    Shifflett DE, Clayburgh DR, Koutsouris A, Turner JR, Hecht GA. Enteropathogenic E. coli disrupts tight junction barrier function and structure in vivo. Lab Invest. 2005;85:1308–1324.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  22. 22.

    Kelly P, Menzies I, Crane R, Zulu I, et al. Responses of small intestinal architecture and function over time to environmental factors in a tropical population. Am J Trop Med Hyg. 2004;70:412–419.

    PubMed  Article  PubMed Central  Google Scholar 

  23. 23.

    Amadi B, Mwiya M, Musuku J, Watuka A, et al. Effect of nitazoxanide on morbidity and mortality in Zambian children with cryptosporidiosis: a randomised controlled trial. Lancet. 2002;360:1375–1380.

    PubMed  Article  PubMed Central  Google Scholar 

  24. 24.

    Deng W, Li Y, Vallance BA, Finlay BB. Locus of enterocyte effacement from Citrobacter rodentium: sequence analysis and evidence for horizontal transfer among attaching and effacing pathogens. Infect Immun.. 2001;69:6323–6335.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. 25.

    Lai Y, Rosenshine I, Leong JM, Frankel G. Intimate host attachment: enteropathogenic and enterohaemorrhagic Escherichia coli. Cell Microbiol. 2013;15:1796–1808.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Yang J, Tauschek M, Hart E, Hartland EL, Robins-Browne RM. Virulence regulation in Citrobacter rodentium: the art of timing. Microb Biotechnol. 2010;3:259–268.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. 27.

    Liu J, Bolick DT, Kolling GL, Fu Z, Guerrant RL. Protein malnutrition impairs intestinal epithelial cell turnover, a potential mechanism of increased cryptosporidiosis in a murine model. Infect Immun. 2016;84:3542–3549.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28.

    Butler M, Ng CY, van Heel DA, Lombardi G, et al. Modulation of dendritic cell phenotype and function in an in vitro model of the intestinal epithelium. Eur J Immunol. 2006;36:864–874.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. 29.

    Moura RA, Sircili MP, Leomil L, Matté MH, et al. Clonal relationship among atypical enteropathogenic Escherichia coli strains isolated from different animal species and humans. Appl Environ Microbiol. 2009;75:7399–7408.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Moon HW, Whipp SC, Argenzio RA, Levine MM, Giannella RA. Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect Immun. 1983;41:1340–1351.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Amadi B, Besa E, Zyambo K, Kaonga P, et al. Impaired barrier function and autoantibody generation in malnutrition enteropathy in Zambia. EBioMedicine. 2017;22:191–199.

    PubMed  PubMed Central  Article  Google Scholar 

  32. 32.

    Flynn AN, Buret AG. Tight junctional disruption and apoptosis in an in vitro model of Citrobacter rodentium infection. Microb Pathog. 2008;45:98–104.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  33. 33.

    de Sablet T, Potiron L, Marquis M, Bussière FI, Lacroix-Lamandé S, Laurent F. Cryptosporidium parvum increases intestinal permeability through interaction with epithelial cells and IL-1β and TNFα released by inflammatory monocytes. Cell Microbiol. 2016;18:1871–1880.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  34. 34.

    De Rycke J, Comtet E, Chalareng C, Boury M, Tasca C, Milon A. Escherichia coli O103 from rabbit elicits actin stress fibers and focal adhesions in HeLa epithelial cells, cytopathic effects that are linked to an analog of the locus of enterocyte effacement. Infect Immun. 1997;65:2555–2563.

    PubMed  PubMed Central  Article  Google Scholar 

  35. 35.

    Higgins LM, Frankel G, Douce G, Dougan G, MacDonald TT. Citrobacter rodentium infection in mice elicits a mucosal Th1 cytokine response and lesions similar to those in murine inflammatory bowel disease. Infect Immun. 1999;67:3031–3039.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36.

    Kumar A, Chatterjee I, Anbazhagan AN, Jayawardena D, et al. Cryptosporidium parvum disrupts intestinal epithelial barrier function via altering expression of key tight junction and adherens junction proteins. Cell Microbiol. 2018;20:e12830.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  37. 37.

    McNamara BP, Koutsouris A, O’Connell CB, Nougayréde JP, Donnenberg MS, Hecht G. Translocated EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function. J Clin Invest. 2001;107:621–629.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Glotfelty LG, Zahs A, Hodges K, Shan K, Alto NM, Hecht GA. Enteropathogenic E. coli effectors EspG1/G2 disrupt microtubules, contribute to tight junction perturbation and inhibit restoration. Cell Microbiol. 2014;16:1767–1783.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. 39.

    Imdad A, Yakoob MY, Sudfeld C, Haider BA, Black RE, Bhutta ZA. Impact of vitamin A supplementation on infant and childhood mortality. BMC Public Health. 2011;11:S20.

    PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Lazzerini M, Wanzira H. Oral zinc for treating diarrhoea in children. Cochrane Database Syst Rev. 2016;2016:CD005436.

    PubMed Central  Google Scholar 

  41. 41.

    Turner JR, Buschmann MM, Sailer A, Calvo IR, Shen L. The role of molecular remodeling in differential regulation of tight junction permeability. Semin Cell Dev Biol. 2014;36:204–212.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  42. 42.

    Marie CS, Verkerke HP, Paul SN, Mackey AJ, Petri WA Jr. Leptin protects host cells from entamoeba histolytica cytotoxicity by a STAT3-dependent mechanism. Infect Immun. 2012;80:1934–1943.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. 43.

    Reeves PG, Briske-Anderson M, Johnson L. Pre-treatment of Caco-2 cells with zinc during the differentiation phase alters the kinetics of zinc uptake and transport (2). J Nutr Biochem. 2001;12:674–684.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  44. 44.

    Metzler-Zebeli BU, Caine WR, McFall M, Miller B, et al. Supplementation of diets for lactating sows with zinc amino acid complex and gastric nutriment-intubation of suckling pigs with zinc methionine on mineral status, intestinal morphology and bacterial translocation in lipopolysaccharide-challenged weaned pigs. J Anim Physiol Anim Nutr (Berl). 2010;94:237–249.

    CAS  Article  Google Scholar 

  45. 45.

    Hogan SP, Seidu L, Blanchard C, Groschwitz K, et al. Resistin-like molecule beta regulates innate colonic function: barrier integrity and inflammation susceptibility. J Allergy Clin Immunol. 2006;118:257–268.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. 46.

    Herbert DR, Yang JQ, Hogan SP, Groschwitz K, et al. Intestinal epithelial cell secretion of RELM-beta protects against gastrointestinal worm infection. J Exp Med. 2009;206:2947–2957.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Bergstrom KS, Morampudi V, Chan JM, Bhinder G, et al. Goblet cell derived RELM-β recruits CD4 + T cells during infectious colitis to promote protective intestinal epithelial cell proliferation. PLoS Pathog. 2015;11:e1005108.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  48. 48.

    Ting F, Znalesniak EB, Kalinski T, et al. TFF peptides play a role in the immune response following oral infection of mice with Toxoplasma gondii. Eur J Microbiol Immunol (Bp). 2015;5:221–231.

    Article  CAS  Google Scholar 

  49. 49.

    Sansonetti PJ, Arondel J, Huerre M, Harada A, Matsusima K. Interleukin-8 controls bacterial transepithelial translocation at the cost of epithelial destruction in experimental Shigellosis. Infect Immun. 1999;67:1471–1480.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50.

    Playford RJ, Macdonald CE, Johnson WS. Colostrum and milk-derived peptide growth factors for the treatment of gastrointestinal disorders. Am J Clin Nutr. 2000;72:5–14.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  51. 51.

    Playford RJ, Hanby AM, Gschmeissner S, Peiffer LP, et al. The epidermal growth factor receptor (EGF-R) is present on the basolateral, but not the apical, surface of enterocytes in the human gastrointestinal tract. Gut. 1996;39:262–266.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. 52.

    Okuyama H, Urao M, Lee D, Drongowski RA, Coran AG. The effect of epidermal growth factor on bacterial translocation in newborn rabbits. J Pediatr Surg. 1998;33:225–228.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  53. 53.

    Lu L, Li T, Williams G, Petit E, Borowsky M, Walker WA. Hydrocortisone induces changes in gene expression and differentiation in immature human enterocytes. Am J Physiol Gastrointest Liver Physiol. 2011;300:G425–G432.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  54. 54.

    Tian JQ, Göke M, Podolsky DK. Glucocorticoids have pleiotropic effects on small intestinal crypt cells. Am J Physiol. 1999;277:G1027–G1040.

    PubMed  PubMed Central  Google Scholar 

  55. 55.

    Friedman J. The long road to leptin. J Clin Invest. 2016;126:4727–4734.

    PubMed  PubMed Central  Article  Google Scholar 

  56. 56.

    Cunningham KE, Turner JR. Myosin light chain kinase: pulling the strings of epithelial tight junction function. Ann N Y Acad Sci. 2012;1258:34–42.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. 57.

    Barrera GJ, Tortolero GS. Trefoil factor 3 (TFF3) from human breast milk activates PAR-2 receptors, of the intestinal epithelial cells HT-29, regulating cytokines and defensins. Bratisl Lek Listy. Infect Immun. 2008;76:796–811.

    Article  CAS  Google Scholar 

  58. 58.

    Kozakova H, Hanson LA, Stepankova R, Kahu H, Dahlgren UI, Wiedermann U. Vitamin A deficiency leads to severe functional disturbance of the intestinal epithelium enzymes associated with diarrhoea and increased bacterial translocation in gnotobiotic rats. Microbes Infect. 2003;5:405–411.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  59. 59.

    Wang X, Valenzano MC, Mercado JM, Zurbach EP, Mullin JM. Zinc supplementation modifies tight junctions and alters barrier function of CACO-2 human intestinal epithelial layers. Dig Dis Sci. 2013;58:77–87.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

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Acknowledgments

We gratefully acknowledge funding from Barts and The London Charity, and David Wareham (Blizard Institute, Barts and The London School of Medicine, UK) for a gift of E. coli K12.

Funding

Funding for this study was from Bart’s Charity and provided support in the form of salaries for authors [NC] but did not have any additional role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript. ME is an employee of Takeda Pharmaceuticals—This funder provided support in the form of salaries for authors [ME] but did not have any additional role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript. GJS is currently in receipt of funding from Takeda Pharmaceuticals, BBSRC together with 31, Benevolent and the Dunhill Foundation. He acts as a scientific advisor to 3 Takeda Pharmaceuticals and Zealand Pharma. “These funders provided support in the form of salaries for authors [GJS] but did not have any additional role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

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Correspondence to Naheed Choudhry.

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Choudhry, N., Scott, F., Edgar, M. et al. Reversal of Pathogen-Induced Barrier Defects in Intestinal Epithelial Cells by Contra-pathogenicity Agents. Dig Dis Sci 66, 88–104 (2021). https://doi.org/10.1007/s10620-020-06121-9

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Keywords

  • Intestinal barrier
  • Claudin-4
  • Microbial translocation
  • Enteropathogenic Escherichia coli
  • Citrobacter rodentium
  • Cryptosporidium parvum