Article | Published:

Microbial dysbiosis associated with impaired intestinal Na+/H+ exchange accelerates and exacerbates colitis in ex-germ free mice

Mucosal Immunologyvolume 11pages13291341 (2018) | Download Citation



Intestinal epithelial Na+/H+ exchange facilitated by the apical NHE3 (Slc9a3) is a highly regulated process inhibited by intestinal pathogens and in inflammatory bowel diseases. NHE3−/− mice develop spontaneous, bacterially mediated colitis, and IBD-like dysbiosis. Disruption of epithelial Na+/H+ exchange in IBD may thus represent a host response contributing to the altered gut microbial ecology, and may play a pivotal role in modulating the severity of inflammation in a microbiome-dependent manner. To test whether microbiome fostered in an NHE3-deficient environment is able to drive mucosal immune responses affecting the onset or severity of colitis, we performed a series of cohousing experiments and fecal microbiome transplants into germ-free Rag-deficient or IL-10−/− mice. We determined that in the settings where the microbiome of NHE3-deficient mice was stably engrafted in the recipient host, it was able accelerate the onset and amplify severity of experimental colitis. NHE3-deficiency was characterized by the reduction in pH-sensitive butyrate-producing Firmicutes families Lachnospiraceae and Ruminococcaceae (Clostridia clusters IV and XIVa), with an expansion of inflammation-associated Bacteroidaceae. We conclude that the microbiome fostered by impaired epithelial Na+/H+ exchange enhances the onset and severity of colitis through disruption of the gut microbial ecology.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Additional information

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Coskun, M. Intestinal epithelium in inflammatory bowel disease. Front. Med. 1, 24 (2014).

  2. 2.

    Ghishan, F. K. & Kiela, P. R. Epithelial transport in inflammatory bowel diseases. Inflamm. Bowel Dis. 20, 1099–1109 (2014).

  3. 3.

    Kiela, P. R., Xu, H. & Ghishan, F. K. Apical NA+/H+exchangers in the mammalian gastrointestinal tract. J. Physiol. Pharmacol. 57(Suppl 7), 51–79 (2006).

  4. 4.

    Gurney, M. A., Laubitz, D., Ghishan, F. K. & Kiela, P. R. Pathophysiology of Intestinal Na+/H+exchange. Cell Mol. Gastroenterol. Hepatol. 3, 27–40 (2017).

  5. 5.

    Schultheis, P. J., Clarke, L. L., Meneton, P., Miller, M. L., Soleimani, M. & Gawenis, L. R. et al. Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+exchanger. Nat. Genet 19, 282–285 (1998).

  6. 6.

    Clayburgh, D. R., Musch, M. W., Leitges, M., Fu, Y. X. & Turner, J. R. Coordinated epithelial NHE3 inhibition and barrier dysfunction are required for TNF-mediated diarrhea in vivo. J. Clin. Invest 116, 2682–2694 (2006).

  7. 7.

    Amin, M. R., Malakooti, J., Sandoval, R., Dudeja, P. K. & Ramaswamy, K. IFN-gamma and TNF-alpha regulate human NHE3 gene expression by modulating the Sp family transcription factors in human intestinal epithelial cell line C2BBe1. Am. J. Physiol. Cell Physiol. 291, C887–C896 (2006).

  8. 8.

    Rocha, F., Musch, M. W., Lishanskiy, L., Bookstein, C., Sugi, K. & Xie, Y. et al. IFN-gamma downregulates expression of Na(+)/H(+) exchangers NHE2 and NHE3 in rat intestine and human Caco-2/bbe cells. Am. J. Physiol. Cell Physiol. 280, C1224–C1232 (2001).

  9. 9.

    Barmeyer, C., Harren, M., Schmitz, H., Heinzel-Pleines, U., Mankertz, J. & Seidler, U. et al. Mechanisms of diarrhea in the interleukin-2-deficient mouse model of colonic inflammation. Am. J. Physiol. Gastrointest. Liver Physiol. 286, G244–G252 (2004).

  10. 10.

    Lenzen, H., Lunnemann, M., Bleich, A., Manns, M. P., Seidler, U. & Jorns, A. Downregulation of the NHE3-binding PDZ-adaptor protein PDZK1 expression during cytokine-induced inflammation in interleukin-10-deficient mice. PLoS One 7, e40657 (2012).

  11. 11.

    Sullivan, S., Alex, P., Dassopoulos, T., Zachos, N. C., Iacobuzio-Donahue, C. & Donowitz, M. et al. Downregulation of sodium transporters and NHERF proteins in IBD patients and mouse colitis models: potential contributors to IBD-associated diarrhea. Inflamm. Bowel Dis. 15, 261–274 (2009).

  12. 12.

    Siddique, I., Hasan, F. & Khan, I. Suppression of Na+/H+exchanger isoform-3 in human inflammatory bowel disease: lack of reversal by 5’-aminosalicylate treatment. Scand. J. Gastroenterol. 44, 56–64 (2009).

  13. 13.

    Yeruva, S., Farkas, K., Hubricht, J., Rode, K., Riederer, B. & Bachmann, O. et al. Preserved Na(+)/H(+) exchanger isoform 3 expression and localization, but decreased NHE3 function indicate regulatory sodium transport defect in ulcerative colitis. Inflamm. Bowel Dis. 16, 1149–1161 (2010).

  14. 14.

    Farkas, K., Yeruva, S., Rakonczay, Z. Jr., Ludolph, L., Molnar, T. & Nagy, F. et al. New therapeutic targets in ulcerative colitis: the importance of ion transporters in the human colon. Inflamm. Bowel Dis. 17, 884–898 (2011).

  15. 15.

    Subramanya, S. B., Rajendran, V. M., Srinivasan, P., Nanda Kumar, N. S., Ramakrishna, B. S. & Binder, H. J. Differential regulation of cholera toxin-inhibited Na-H exchange isoforms by butyrate in rat ileum. Am. J. Physiol. Gastrointest. Liver Physiol. 293, G857–G863 (2007).

  16. 16.

    Hayashi, H., Szaszi, K., Coady-Osberg, N., Furuya, W., Bretscher, A. P. & Orlowski, J. et al. Inhibition and redistribution of NHE3, the apical Na+/H+exchanger, by Clostridium difficile toxin B. J. Gen. Physiol. 123, 491–504 (2004).

  17. 17.

    Hodges, K., Alto, N. M., Ramaswamy, K., Dudeja, P. K. & Hecht, G. The enteropathogenic Escherichia coli effector protein EspF decreases sodium hydrogen exchanger 3 activity. Cell Microbiol 10, 1735–1745 (2008).

  18. 18.

    Ambort, D., Johansson, M. E., Gustafsson, J. K., Nilsson, H. E., Ermund, A. & Johansson, B. R. et al. Calcium and pH-dependent packing and release of the gel-forming MUC2 mucin. Proc. Natl Acad. Sci. USA 109, 5645–5650 (2012).

  19. 19.

    Johansson, M. E., Gustafsson, J. K., Holmen-Larsson, J., Jabbar, K. S., Xia, L. & Xu, H. et al. Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis. Gut 63, 281–291 (2014).

  20. 20.

    Laubitz, D., Larmonier, C. B., Bai, A., Midura-Kiela, M. T., Lipko, M. A. & Thurston, R. D. et al. Colonic gene expression profile in NHE3-deficient mice: evidence for spontaneous distal colitis. Am. J. Physiol. Gastrointest. Liver Physiol. 295, G63–G77 (2008).

  21. 21.

    Kiela, P. R., Laubitz, D., Larmonier, C. B., Midura-Kiela, M. T., Lipko, M. A. & Janikashvili, N. et al. Changes in mucosal homeostasis predispose NHE3 knockout mice to increased susceptibility to DSS-induced epithelial injury. Gastroenterology 137, 965–975 (2009). 975 e961-910.

  22. 22.

    Larmonier, C. B., Laubitz, D., Hill, F. M., Shehab, K. W., Lipinski, L. & Midura-Kiela, M. T. et al. Reduced colonic microbial diversity is associated with colitis in NHE3-deficient mice. Am. J. Physiol. Gastrointest. Liver Physiol. 305, G667–G677 (2013).

  23. 23.

    Engevik, M. A., Aihara, E., Montrose, M. H., Shull, G. E., Hassett, D. J. & Worrell, R. T. Loss of NHE3 alters gut microbiota composition and influences Bacteroides thetaiotaomicron growth. Am. J. Physiol. Gastrointest. Liver Physiol. 305, G697–G711 (2013).

  24. 24.

    Bloom, S. M., Bijanki, V. N., Nava, G. M., Sun, L., Malvin, N. P. & Donermeyer, D. L. et al. Commensal Bacteroides species induce colitis in host-genotype-specific fashion in a mouse model of inflammatory bowel disease. Cell Host Microbe 9, 390–403 (2011).

  25. 25.

    Hansen, J. J., Huang, Y., Peterson, D. A., Goeser, L., Fan, T. J. & Chang, E. B. et al. The colitis-associated transcriptional profile of commensal Bacteroides thetaiotaomicron enhances adaptive immune responses to a bacterial antigen. PLoS One 7, e42645 (2012).

  26. 26.

    Laubitz, D., Harrison, C. A., Midura-Kiela, M. T., Ramalingam, R., Larmonier, C. B. & Chase, J. H. et al. Reduced Epithelial Na+/H+Exchange Drives Gut Microbial Dysbiosis and Promotes Inflammatory Response in T Cell-Mediated Murine Colitis. PLoS One 11, e0152044 (2016).

  27. 27.

    Du, Z., Hudcovic, T., Mrazek, J., Kozakova, H., Srutkova, D. & Schwarzer, M. et al. Development of gut inflammation in mice colonized with mucosa-associated bacteria from patients with ulcerative colitis. Gut Pathog. 7, 32 (2015).

  28. 28.

    Garrett, W. S., Lord, G. M., Punit, S., Lugo-Villarino, G., Mazmanian, S. K. & Ito, S. et al. Communicable ulcerative colitis induced by T-bet deficiency in the innate immune system. Cell 131, 33–45 (2007).

  29. 29.

    Soave, O. & Brand, C. D. Coprophagy in animals: a review. Cornell Vet. 81, 357–364 (1991).

  30. 30.

    Laukens, D., Brinkman, B. M., Raes, J., De Vos, M. & Vandenabeele, P. Heterogeneity of the gut microbiome in mice: guidelines for optimizing experimental design. FEMS Microbiol Rev. 40, 117–132 (2016).

  31. 31.

    Sellon, R. K., Tonkonogy, S., Schultz, M., Dieleman, L. A., Grenther, W. & Balish, E. et al. Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice. Infect. Immun. 66, 5224–5231 (1998).

  32. 32.

    Chassaing, B., Srinivasan, G., Delgado, M. A., Young, A. N., Gewirtz, A. T. & Vijay-Kumar, M. Fecal lipocalin 2, a sensitive and broadly dynamic non-invasive biomarker for intestinal inflammation. PLoS One 7, e44328 (2012).

  33. 33.

    Buisson A., Vazeille E., Minet-Quinard R., Goutte M., Bouvier D., Goutorbe F. et al. Fecal Matrix Metalloprotease-9 and Lipocalin-2 as Biomarkers in Detecting Endoscopic Activity in Patients With Inflammatory Bowel Diseases. J. Clin. Gastroenterol. 2017.

  34. 34.

    Janecke, A. R., Heinz-Erian, P. & Muller, T. Congenital sodium diarrhea: a form of intractable diarrhea, with a link to inflammatory bowel disease. J. Pediatr. Gastroenterol. Nutr. 63, 170–176 (2016).

  35. 35.

    Pryde, S. E., Duncan, S. H., Hold, G. L., Stewart, C. S. & Flint, H. J. The microbiology of butyrate formation in the human colon. FEMS Microbiol. Lett. 217, 133–139 (2002).

  36. 36.

    Van den Abbeele, P., Belzer, C., Goossens, M., Kleerebezem, M., De Vos, W. M. & Thas, O. et al. Butyrate-producing Clostridium cluster XIVa species specifically colonize mucins in an in vitro gut model. ISME J. 7, 949–961 (2013).

  37. 37.

    Johansson, M. E., Larsson, J. M. & Hansson, G. C. The two mucus layers of colon are organized by the MUC2 mucin, whereas the outer layer is a legislator of host-microbial interactions. Proc. Natl Acad. Sci. USA 108(Suppl 1), 4659–4665 (2011).

  38. 38.

    Johansson, M. E., Phillipson, M., Petersson, J., Velcich, A., Holm, L. & Hansson, G. C. The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Proc. Natl Acad. Sci. USA 105, 15064–15069 (2008).

  39. 39.

    Walker, A. W., Duncan, S. H., McWilliam Leitch, E. C., Child, M. W. & Flint, H. J. pH and peptide supply can radically alter bacterial populations and short-chain fatty acid ratios within microbial communities from the human colon. Appl. Environ. Microbiol 71, 3692–3700 (2005).

  40. 40.

    Kiela, P. R., Kuscuoglu, N., Midura, A. J., Midura-Kiela, M. T., Larmonier, C. B. & Lipko, M. et al. Molecular mechanism of rat NHE3 gene promoter regulation by sodium butyrate. Am. J. Physiol. Cell Physiol. 293, C64–C74 (2007).

  41. 41.

    Musch, M. W., Bookstein, C., Xie, Y., Sellin, J. H. & Chang, E. B. SCFA increase intestinal Na absorption by induction of NHE3 in rat colon and human intestinal C2/bbe cells. Am. J. Physiol. Gastrointest. Liver Physiol. 280, G687–G693 (2001).

  42. 42.

    Li, X., Cai, L., Xu, H., Geng, C., Lu, J. & Tao, L. et al. Somatostatin regulates NHE8 protein expression via the ERK1/2 MAPK pathway in DSS-induced colitis mice. Am. J. Physiol. Gastrointest. Liver Physiol. 311, G954–G963 (2016).

  43. 43.

    Yeruva, S., Chodisetti, G., Luo, M., Chen, M., Cinar, A. & Ludolph, L. et al. Evidence for a causal link between adaptor protein PDZK1 downregulation and Na(+)/H(+) exchanger NHE3 dysfunction in human and murine colitis. Pflug. Arch. 467, 1795–1807 (2015).

  44. 44.

    Couturier-Maillard, A., Secher, T., Rehman, A., Normand, S., De Arcangelis, A. & Haesler, R. et al. NOD2-mediated dysbiosis predisposes mice to transmissible colitis and colorectal cancer. J. Clin. Invest 123, 700–711 (2013).

  45. 45.

    Berry, D., Kuzyk, O., Rauch, I., Heider, S., Schwab, C. & Hainzl, E. et al. Intestinal Microbiota Signatures Associated with Inflammation History in Mice Experiencing Recurring Colitis. Front Microbiol 6, 1408 (2015).

  46. 46.

    Engevik, M. A., Engevik, K. A., Yacyshyn, M. B., Wang, J., Hassett, D. J. & Darien, B. et al. Human Clostridium difficile infection: inhibition of NHE3 and microbiota profile. Am. J. Physiol. Gastrointest. Liver Physiol. 308, G497–G509 (2015).

  47. 47.

    Reeves, A. E., Koenigsknecht, M. J., Bergin, I. L. & Young, V. B. Suppression of Clostridium difficile in the gastrointestinal tracts of germfree mice inoculated with a murine isolate from the family Lachnospiraceae. Infect. Immun. 80, 3786–3794 (2012).

Download references


This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants 2R01DK041274 (to F.K.G. and P.K.R.) and 5R01DK073338 (to C.J.). The founders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Special thanks to Paula Campbell with the University of Arizona Flow Cytometry Core for her assistance flow sorting T cells for adoptive transfer.

Author information

Author notes

  1. These authors contributed equally: Christian Jobin, Fayez K. Ghishan, Pawel R. Kiela


  1. Department of Pediatrics, Steele Children’s Research Center, Tucson, AZ, USA

    • Christy A. Harrison
    • , Daniel Laubitz
    • , Monica T. Midura-Kiela
    • , Deepa R. Jamwal
    • , Fayez K. Ghishan
    •  & Pawel R. Kiela
  2. Department of Immunobiology, University of Arizona College of Medicine, Tucson, AZ, USA

    • Christy A. Harrison
    •  & Pawel R. Kiela
  3. University Animal Care, University of Arizona, Tucson, AZ, USA

    • Christina L. Ohland
    • , Karuna Patil
    •  & David G. Besselsen
  4. Division of Gastroenterology, Department of Medicine, University of Florida College of Medicine, Gainesville, FL, USA

    • Christian Jobin


  1. Search for Christy A. Harrison in:

  2. Search for Daniel Laubitz in:

  3. Search for Christina L. Ohland in:

  4. Search for Monica T. Midura-Kiela in:

  5. Search for Karuna Patil in:

  6. Search for David G. Besselsen in:

  7. Search for Deepa R. Jamwal in:

  8. Search for Christian Jobin in:

  9. Search for Fayez K. Ghishan in:

  10. Search for Pawel R. Kiela in:


C.A.H. designed and performed experiments, analyzed results, and wrote the paper; D.L. performed experiments, analyzed results, and edited the paper; C.L.O. designed and performed experiments with germ-free IL-10−/− mice; M.T.M-K., responsible for most experimental animals, performed experiments; K.P. designed and performed experiments with germ-free mice in Arizona; D.G.B. analyzed histology and scored samples; D.R.J. performed flow sorting of T cells; C.J. supervised experiments with germ-free IL-10−/− mice at the Univ. of Florida, provided funding, edited manuscript; F.K.G. supervised experiments at the University of Arizona, provided funding, edited manuscript; P.R.K. supervised experiments at the University of Arizona, designed the experiments, analyzed results provided funding, edited manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Pawel R. Kiela.

Electronic supplementary material

About this article

Publication history