Article | Published:

A screen of Crohn’s disease-associated microbial metabolites identifies ascorbate as a novel metabolic inhibitor of activated human T cells

Mucosal Immunologyvolume 12pages457467 (2019) | Download Citation

Subjects

Abstract

Microbial metabolites are an emerging class of mediators influencing CD4+ T-cell function. To advance the understanding of direct causal microbial factors contributing to Crohn’s disease, we screened 139 predicted Crohn’s disease-associated microbial metabolites for their bioactivity on human CD4+ T-cell functions induced by disease-associated T helper 17 (Th17) polarizing conditions. We observed 15 metabolites with CD4+ T-cell bioactivity, 3 previously reported, and 12 unprecedented. A deeper investigation of the microbe-derived metabolite, ascorbate, revealed its selective inhibition on activated human CD4+ effector T cells, including IL-17A-, IL-4-, and IFNγ-producing cells. Mechanistic assessment suggested the apoptosis of activated human CD4+ T cells associated with selective inhibition of energy metabolism. These findings suggest a substantial rate of relevant T-cell bioactivity among Crohn’s disease-associated microbial metabolites, and evidence for novel modes of bioactivity, including targeting of T-cell energy metabolism.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

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.

References

  1. 1.

    Integrative HMPRNC. The Integrative Human Microbiome Project: dynamic analysis of microbiome-host omics profiles during periods of human health and disease. Cell Host Microbe 16, 276–289 (2014).

  2. 2.

    Huttenhower, C., Kostic, A. D. & Xavier, R. J. Inflammatory bowel disease as a model for translating the microbiome. Immunity 40, 843–854 (2014).

  3. 3.

    Sartor, R. B. & Wu, G. D. Roles for intestinal bacteria, viruses, and fungi in pathogenesis of inflammatory bowel diseases and therapeutic approaches. Gastroenterology 152, 327–339.e324 (2017).

  4. 4.

    Brestoff, J. R. & Artis, D. Commensal bacteria at the interface of host metabolism and the immune system. Nat. Immunol. 14, 676–684 (2013).

  5. 5.

    Dorrestein, P. C., Mazmanian, S. K. & Knight, R. Finding the missing links among metabolites, microbes, and the host. Immunity 40, 824–832 (2014).

  6. 6.

    Thorburn, A. N., Macia, L. & Mackay, C. R. Diet, metabolites, and “western-lifestyle” inflammatory diseases. Immunity 40, 833–842 (2014).

  7. 7.

    Kaiko, G. E. et al. The colonic crypt protects stem cells from microbiota-derived metabolites. Cell 167, 1137 (2016).

  8. 8.

    Furusawa, Y. et al. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. Nature 504, 446–450 (2013).

  9. 9.

    Gevers, D. et al. The treatment-naive microbiome in new-onset Crohn’s disease. Cell Host Microbe 15, 382–392 (2014).

  10. 10.

    Tong, M. et al. Reprograming of gut microbiome energy metabolism by the FUT2 Crohn’s disease risk polymorphism. ISME J. 8, 2193–2206 (2014).

  11. 11.

    Lewis, J. D. et al. Inflammation, antibiotics, and diet as environmental stressors of the gut microbiome in pediatric Crohn’s disease. Cell Host Microbe 18, 489–500 (2015).

  12. 12.

    Gao, Z. et al. Butyrate improves insulin sensitivity and increases energy expenditure in mice. Diabetes 58, 1509–1517 (2009).

  13. 13.

    Smith, P. M. et al. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science 341, 569–573 (2013).

  14. 14.

    Chang, P. V., Hao, L., Offermanns, S. & Medzhitov, R. The microbial metabolite butyrate regulates intestinal macrophage function via histone deacetylase inhibition. Proc. Natl Acad. Sci. USA 111, 2247–2252 (2014).

  15. 15.

    Mascanfroni, I. D. et al. Metabolic control of type 1 regulatory T cell differentiation by AHR and HIF1-alpha. Nat. Med. 21, 638–646 (2015).

  16. 16.

    Hooper, L. V., Littman, D. R. & Macpherson, A. J. Interactions between the microbiota and the immune system. Science 336, 1268–1273 (2012).

  17. 17.

    Knights, D. et al. Complex host genetics influence the microbiome in inflammatory bowel disease. Genome Med. 6, 107 (2014).

  18. 18.

    Khor, B., Gardet, A. & Xavier, R. J. Genetics and pathogenesis of inflammatory bowel disease. Nature 474, 307–317 (2011).

  19. 19.

    Jostins, L. et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature 491, 119–124 (2012).

  20. 20.

    Yen, D. et al. IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6. J. Clin. Invest. 116, 1310–1316 (2006).

  21. 21.

    Fujino, S. et al. Increased expression of interleukin 17 in inflammatory bowel disease. Gut 52, 65–70 (2003).

  22. 22.

    Kleinschek, M. A. et al. Circulating and gut-resident human Th17 cells express CD161 and promote intestinal inflammation. J. Exp. Med. 206, 525–534 (2009).

  23. 23.

    Bogaert, S. et al. Differential mucosal expression of Th17-related genes between the inflamed colon and ileum of patients with inflammatory bowel disease. BMC Immunol. 11, 61 (2010).

  24. 24.

    Kobayashi, T. et al. IL23 differentially regulates the Th1/Th17 balance in ulcerative colitis and Crohn’s disease. Gut 57, 1682–1689 (2008).

  25. 25.

    Neurath, M. F. New targets for mucosal healing and therapy in inflammatory bowel diseases. Mucosal Immunol. 7, 6–19 (2014).

  26. 26.

    Hueber, W. et al. Secukinumab, a human anti-IL-17A monoclonal antibody, for moderate to severe Crohn’s disease: unexpected results of a randomised, double-blind placebo-controlled trial. Gut 61, 1693–1700 (2012).

  27. 27.

    Sandborn, W. J. et al. Ustekinumab induction and maintenance therapy in refractory Crohn’s disease. N. Engl. J. Med. 367, 1519–1528 (2012).

  28. 28.

    Sands, B. E. et al. OP025: a randomized, double-blind placebo-controlled phase 2a induction study of MEDI2070 (anti-p19 antibody) in patients with active Crohn’s disease who have failed anti-TNF antibody therapy. J. Crohn’s Colitis 9, S15–S16 (2015).

  29. 29.

    Targan, S. R. et al. Mo2083: a randomized, double-blind, placebo-controlled study to evaluate the safety, tolerability, and efficacy of AMG 827 in subjects with moderate to severe Crohn’s disease. Gastroenterology 143, e26 (2012).

  30. 30.

    Colombel, J. F., Sendid, B., Jouault, T. & Poulain, D. Secukinumab failure in Crohn’s disease: the yeast connection? Gut 62, 800–801 (2013).

  31. 31.

    Maxwell, J. R. et al. Differential roles for interleukin-23 and interleukin-17 in intestinal immunoregulation. Immunity 43, 739–750 (2015).

  32. 32.

    Morgan, X. C. et al. Dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment. Genome Biol. 13, R79 (2012).

  33. 33.

    Wilson, N. J. et al. Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nat. Immunol. 8, 950–957 (2007).

  34. 34.

    Wang, C. et al. CD5L/AIM regulates lipid biosynthesis and restrains Th17 cell pathogenicity. Cell 163, 1413–1427 (2015).

  35. 35.

    Pacheco, R. et al. Glutamate released by dendritic cells as a novel modulator of T cell activation. J. Immunol. 177, 6695–6704 (2006).

  36. 36.

    Wishart, D. S. et al. HMDB 3.0--The Human Metabolome Database in 2013. Nucleic Acids Res. 41, D801–D807 (2013).

  37. 37.

    Yun, J. et al. Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH. Science 350, 1391–1396 (2015).

  38. 38.

    Macintyre, A. N. et al. The glucose transporter Glut1 is selectively essential for CD4 T cell activation and effector function. Cell Metab. 20, 61–72 (2014).

  39. 39.

    Buck, M. D., O’Sullivan, D. & Pearce, E. L. T cell metabolism drives immunity. J. Exp. Med. 212, 1345–1360 (2015).

  40. 40.

    Slack, M., Wang, T. & Wang, R. T cell metabolic reprogramming and plasticity. Mol. Immunol. 68, 507–512 (2015).

  41. 41.

    Hancock, R. D. & Viola, R. Biotechnological approaches for L-ascorbic acid production. Trends Biotechnol. 20, 299–305 (2002).

  42. 42.

    DuPage, M. & Bluestone, J. A. Harnessing the plasticity of CD4(+) T cells to treat immune-mediated disease. Nat. Rev. Immunol. 16, 149–163 (2016).

  43. 43.

    Sallusto, F. Heterogeneity of human CD4(+) T cells against microbes. Annu. Rev. Immunol. 34, 317–334 (2016).

  44. 44.

    Hong, J. M., Kim, J. H., Kang, J. S., Lee, W. J. & Hwang, Y. I. Vitamin C is taken up by human T cells via sodium-dependent vitamin C transporter 2 (SVCT2) and exerts inhibitory effects on the activation of these cells in vitro. Anat. Cell Biol. 49, 88–98 (2016).

  45. 45.

    Maratou, E. et al. Glucose transporter expression on the plasma membrane of resting and activated white blood cells. Eur. J. Clin. Invest. 37, 282–290 (2007).

  46. 46.

    Rumsey, S. C. et al. Glucose transporter isoforms GLUT1 and GLUT3 transport dehydroascorbic acid. J. Biol. Chem. 272, 18982–18989 (1997).

  47. 47.

    Vera, J. C., Rivas, C. I., Fischbarg, J. & Golde, D. W. Mammalian facilitative hexose transporters mediate the transport of dehydroascorbic acid. Nature 364, 79–82 (1993).

  48. 48.

    May, J. M., Mendiratta, S., Hill, K. E. & Burk, R. F. Reduction of dehydroascorbate to ascorbate by the selenoenzyme thioredoxin reductase. J. Biol. Chem. 272, 22607–22610 (1997).

  49. 49.

    Winkler, B. S., Orselli, S. M. & Rex, T. S. The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective. Free Radic. Biol. Med. 17, 333–349 (1994).

  50. 50.

    Hwang, N. R. et al. Oxidative modifications of glyceraldehyde-3-phosphate dehydrogenase play a key role in its multiple cellular functions. Biochem. J. 423, 253–264 (2009).

  51. 51.

    Shenton, D. & Grant, C. M. Protein S-thiolation targets glycolysis and protein synthesis in response to oxidative stress in the yeast Saccharomyces cerevisiae. Biochem. J. 374, 513–519 (2003).

  52. 52.

    Ravichandran, V., Seres, T., Moriguchi, T., Thomas, J. A. & Johnston, R. B. Jr. S-thiolation of glyceraldehyde-3-phosphate dehydrogenase induced by the phagocytosis-associated respiratory burst in blood monocytes. J. Biol. Chem. 269, 25010–25015 (1994).

  53. 53.

    Ullah, M. F., Khan, H. Y., Zubair, H., Shamim, U. & Hadi, S. M. The antioxidant ascorbic acid mobilizes nuclear copper leading to a prooxidant breakage of cellular DNA: implications for chemotherapeutic action against cancer. Cancer Chemother. Pharmacol. 67, 103–110 (2011).

  54. 54.

    Franchi, L. et al. Inhibiting oxidative phosphorylation in vivo restrains Th17 effector responses and ameliorates murine colitis. J. Immunol. 198, 2735–2746 (2017).

  55. 55.

    Fiorani, M. et al. The mitochondrial transporter of ascorbic acid functions with high affinity in the presence of low millimolar concentrations of sodium and in the absence of calcium and magnesium. Biochim. Biophys. Acta 1848, 1393–1401 (2015).

  56. 56.

    Tsukaguchi, H. et al. A family of mammalian Na+-dependent L-ascorbic acid transporters. Nature 399, 70–75 (1999).

  57. 57.

    Savini, I., Rossi, A., Pierro, C., Avigliano, L. & Catani, M. V. SVCT1 and SVCT2: key proteins for vitamin C uptake. Amino Acids 34, 347–355 (2008).

  58. 58.

    Li, X., Cobb, C. E. & May, J. M. Mitochondrial recycling of ascorbic acid from dehydroascorbic acid: dependence on the electron transport chain. Arch. Biochem. Biophys. 403, 103–110 (2002).

  59. 59.

    Sasidharan Nair, V., Song, M. H. & Oh, K. I. Vitamin C facilitates demethylation of the Foxp3 enhancer in a Tet-dependent manner. J. Immunol. 196, 2119–2131 (2016).

  60. 60.

    Yue, X. et al. Control of Foxp3 stability through modulation of TET activity. J. Exp. Med. 213, 377–397 (2016).

  61. 61.

    Nishikimi, M., Fukuyama, R., Minoshima, S., Shimizu, N. & Yagi, K. Cloning and chromosomal mapping of the human nonfunctional gene for L-gulono-gamma-lactone oxidase, the enzyme for L-ascorbic acid biosynthesis missing in man. J. Biol. Chem. 269, 13685–13688 (1994).

  62. 62.

    Naidu, K. A. Vitamin C in human health and disease is still a mystery? An overview. Nutr. J. 2, 7 (2003).

  63. 63.

    Costa, K. C., Glasser, N. R., Conway, S. J. & Newman, D. K. Pyocyanin degradation by a tautomerizing demethylase inhibits Pseudomonas aeruginosa biofilms. Science 355, 170–173 (2017).

  64. 64.

    Vera, J. C. et al. Resolution of the facilitated transport of dehydroascorbic acid from its intracellular accumulation as ascorbic acid. J. Biol. Chem. 270, 23706–23712 (1995).

  65. 65.

    Amir Shaghaghi, M., Bernstein, C. N., Serrano Leon, A., El-Gabalawy, H. & Eck, P. Polymorphisms in the sodium-dependent ascorbate transporter gene SLC23A1 are associated with susceptibility to Crohn disease. Am. J. Clin. Nutr. 99, 378–383 (2014).

  66. 66.

    Hengstermann, S. et al. Altered status of antioxidant vitamins and fatty acids in patients with inactive inflammatory bowel disease. Clin. Nutr. 27, 571–578 (2008).

  67. 67.

    Langille, M. G. et al. Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nat. Biotechnol. 31, 814–821 (2013).

  68. 68.

    Abubucker, S. et al. Metabolic reconstruction for metagenomic data and its application to the human microbiome. PLoS Comput. Biol. 8, e1002358 (2012).

  69. 69.

    Sokol, P. A., Darling, P., Woods, D. E., Mahenthiralingam, E. & Kooi, C. Role of ornibactin biosynthesis in the virulence of Burkholderia cepacia: characterization of pvdA, the gene encoding L-ornithine N(5)-oxygenase. Infect. Immun. 67, 4443–4455 (1999).

  70. 70.

    Mc, F. J. The nephelometer:aN instrument for estimating the number of bacteria in suspensions used for calculating the opsonic index and for vaccines. J. Am. Med. Assoc. XLIX, 1176–1178 (1907).

Download references

Acknowledgements

This study was supported by the Crohn’s and Colitis Foundation 323814, and National Institutes of Health grants PO1-DK46763 (J.B., D.P.B.M.), P30-CA016042 (UCLA Jonsson Comprehensive Cancer Center), and NCATS UCLA CTSI UL1TR001881. We thank all the volunteers for their participation in this study. We particularly thank the Immune Assessment Core at University of California, Los Angeles, for their support in scaling up the screens and running Luminex experiments. We also thank members from Dr. Michael Teitell Laboratory, Dr. Laurent Vergnes, and Dr. Linsey Stiles from Cellular Bioenergetics Core at University of California, Los Angeles, for the help with the Seahorse experiments.

Author information

Affiliations

  1. Molecular Biology IDP, University of California, Los Angeles, CA, 90095, USA

    • Yu-Ling Chang
  2. Pathology and Laboratory Medicine, University of California, Los Angeles, CA, 90095, USA

    • Yu-Ling Chang
    • , Maura Rossetti
    • , David Casero
    • , Gemalene Sunga
    • , Nicholas Harre
    • , Shelley Miller
    • , Romney Humphries
    •  & Jonathan Braun
  3. The Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA

    • Hera Vlamakis
    • , Ramnik Xavier
    •  & Curtis Huttenhower
  4. Washington University School of Medicine, St. Louis, MO, 63110, USA

    • Thaddeus Stappenbeck
  5. College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA

    • Kenneth W. Simpson
  6. Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA

    • R. Balfour Sartor
  7. Department of Medicine, Division of Gastroenterology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

    • Gary Wu
  8. Center for Clinical Epidemiology and Biostatistics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

    • James Lewis
  9. Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

    • Frederic Bushman
  10. The F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA

    • Dermot P. B. McGovern
  11. Department of Pediatrics, Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

    • Nita Salzman
  12. Department of Plant Pathology and Microbiology, University of California, Riverside, CA, 92521, USA

    • James Borneman

Authors

  1. Search for Yu-Ling Chang in:

  2. Search for Maura Rossetti in:

  3. Search for Hera Vlamakis in:

  4. Search for David Casero in:

  5. Search for Gemalene Sunga in:

  6. Search for Nicholas Harre in:

  7. Search for Shelley Miller in:

  8. Search for Romney Humphries in:

  9. Search for Thaddeus Stappenbeck in:

  10. Search for Kenneth W. Simpson in:

  11. Search for R. Balfour Sartor in:

  12. Search for Gary Wu in:

  13. Search for James Lewis in:

  14. Search for Frederic Bushman in:

  15. Search for Dermot P. B. McGovern in:

  16. Search for Nita Salzman in:

  17. Search for James Borneman in:

  18. Search for Ramnik Xavier in:

  19. Search for Curtis Huttenhower in:

  20. Search for Jonathan Braun in:

Contributions

Y.-L.C., M.R. and J.B. were responsible for experimental design, data interpretation, and manuscript preparation. Y.-L.C., G.S., N.H., and S.M. performed the experiments. Y.-L.C. and D.C. contributed to data analysis, and C.H. and H.V. predicted metabolites from metagenomics analysis. R.H., K.W.S., R.B.S., G.W., J.L., F.B., D.P.B.M., N.S., and J.B. in the CCFA consortium contributed to study design. J.B., T.S., R.X., and C.H. contributed to information integration and analysis.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Jonathan Braun.

Electronic supplementary material

About this article

Publication history

Received

Revised

Accepted

Published

DOI

https://doi.org/10.1038/s41385-018-0022-7

Further reading