Crossover Subsets of CD4+ T Lymphocytes in the Intestinal Lamina Propria of Patients with Crohn’s Disease and Ulcerative Colitis

Abstract

Background

Hovhannisyan et al. first showed evidence of plasticity between Treg and Th17 in the inflamed intestine of Crohn’s disease (CD) patients. Our previous report suggests that the inflammatory cytokine milieu generates IL-17+ Foxp3+ CD4+ T lymphocytes which is a crossover population converting Treg subset to Th17 in the peripheral blood of IBD patients. This is considered as an evidence of Treg/Th17 plasticity.

Aim

The aim of this study was to characterize a variety of helper T cell crossover population, not limited to IL-17+ Foxp3+ CD4+ T lymphocytes, in the lamina propria (LP) of IBD patients.

Methods

Fresh colonoscopic biopsies were obtained from patients with CD (n = 50) and ulcerative colitis (UC, n = 32) and from healthy controls (HC, n = 25). LP mononuclear cells were assessed for intracellular cytokines and transcription factors such as IFNγ, IL-13, IL-17, IL-22, T-bet, Gata-3, RORγt, and Foxp3 using multicolor flow cytometry to detect subsets of LP CD4+ T lymphocytes.

Results

Patients with IBD demonstrated increased crossover populations in IL-17+ Foxp3+, T-bet+ Foxp3+, Gata3+ Foxp3+, RORγt+ Foxp3+ populations compared to HC. There was an inverse correlation of Harvey–Bradshaw index with Gata3+ Foxp3+ population in CD patients, while IL-13+ Foxp3+ population was directly correlated with Mayo clinical scores in UC patients. Furthermore, total IL-22 expressing cells as well as Th22 and IL-22+ Th1 populations were decreased in UC compared to CD and HC.

Conclusion

IBD patients exhibit the increased crossover populations in LP Treg cells toward Th2 and Th17 compared to HC. The prevalence of Treg/Th2 crossover populations is associated with clinical disease score of IBD.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Abbreviations

CD:

Crohn’s disease

HC:

Healthy control

IBD:

Inflammatory bowel diseases

LP:

Lamina propria

Th1:

T helper type 1 cells

Th2:

T helper type 2 cells

Th17:

T helper type 17 cells

Th22:

T helper type 22 cells

TNF:

Tumor necrosis factor

Treg:

Regulatory T helper cells

UC:

Ulcerative colitis

References

  1. 1.

    Funderburg NT, Stubblefield Park SR, Sung HC, et al. Circulating CD4(+) and CD8(+) T cells are activated in inflammatory bowel disease and are associated with plasma markers of inflammation. Immunology. 2013;140:87–97.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Baumgart DC, Sandborn WJ. Crohn’s disease. Lancet. 2012;380:1590–1605.

    Article  PubMed  Google Scholar 

  3. 3.

    Ordas I, Eckmann L, Talamini M, et al. Ulcerative colitis. Lancet. 2012;380:1606–1619.

    Article  PubMed  Google Scholar 

  4. 4.

    Bai A, Lu N, Guo Y, et al. All-trans retinoic acid down-regulates inflammatory responses by shifting the Treg/Th17 profile in human ulcerative and murine colitis. J Leukoc Biol. 2009;86:959–969.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Malmhall C, Bossios A, Radinger M, et al. Immunophenotyping of circulating T helper cells argues for multiple functions and plasticity of T cells in vivo in humans-possible role in asthma. PLoS ONE. 2012;7:e40012.

    Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Li L, Boussiotis VA. The role of IL-17-producing Foxp3+ CD4+ T cells in inflammatory bowel disease and colon cancer. Clin Immunol. 2013;148:246–253.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Li J, Ueno A, Fort Gasia M, et al. Distinctive Th17 lymphocyte plasticity in intestinal lamina propria of IBD patients compared with healthy controls. Gastoroenterology. 2015;148:S-323.

    Article  Google Scholar 

  8. 8.

    Jiang W, Su J, Zhang X, et al. Elevated levels of Th17 cells and Th17-related cytokines are associated with disease activity in patients with inflammatory bowel disease. Inflamm Res. 2014;63:943–950.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Fujino S, Andoh A, Bamba S, et al. Increased expression of interleukin 17 in inflammatory bowel disease. Gut. 2003;52:65–70.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Rovedatti L, Kudo T, Biancheri P, et al. Differential regulation of interleukin 17 and interferon gamma production in inflammatory bowel disease. Gut. 2009;58:1629–1636.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Lord JD, Valliant-Saunders K, Hahn H, et al. Paradoxically increased Foxp3+ T cells in IBD do not preferentially express the isoform of Foxp3 lacking exon 2. Dig Dis Sci. 2012;57:2846–2855. doi:10.1007/s10620-012-2292-3.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Ban H, Andoh A, Shioya M, et al. Increased number of FoxP3+ CD4+ regulatory T cells in inflammatory bowel disease. Mol Med Rep. 2008;1:647–650.

    CAS  PubMed  Google Scholar 

  13. 13.

    Annunziato F, Romagnani S. Do studies in humans better depict Th17 cells? Blood. 2009;114:2213–2219.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Eastaff-Leung N, Mabarrack N, Barbour A, et al. Foxp3+ regulatory T cells, Th17 effector cells, and cytokine environment in inflammatory bowel disease. J Clin Immunol. 2010;30:80–89.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

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

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Himmel ME, Yao Y, Orban PC, et al. Regulatory T-cell therapy for inflammatory bowel disease: more questions than answers. Immunology. 2012;136:115–122.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Ueno A, Jijon H, Chan R, et al. Increased prevalence of circulating novel IL-17 secreting Foxp3 expressing CD4+ T cells and defective suppressive function of circulating Foxp3+ regulatory cells support plasticity between Th17 and regulatory T cells in inflammatory bowel disease patients. Inflamm Bowel Dis. 2013;19:2522–2534.

    Article  PubMed  Google Scholar 

  18. 18.

    Hovhannisyan Z, Treatman J, Littman DR, et al. Characterization of interleukin-17-producing regulatory T cells in inflamed intestinal mucosa from patients with inflammatory bowel diseases. Gastroenterology. 2011;140:957–965.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Trifari S, Kaplan CD, Tran EH, et al. Identification of a human helper T cell population that has abundant production of interleukin 22 and is distinct from T(H)-17, T(H)1 and T(H)2 cells. Nat Immunol. 2009;10:864–871.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Eyerich S, Eyerich K, Pennino D, et al. Th22 cells represent a distinct human T cell subset involved in epidermal immunity and remodeling. J Clin Invest. 2009;119:3573–3585.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Duhen T, Geiger R, Jarrossay D, et al. Production of interleukin 22 but not interleukin 17 by a subset of human skin-homing memory T cells. Nat Immunol. 2009;10:857–863.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Immunology Leslie M. Cross-over immune cells blur the boundaries. Science. 2012;336:1228–1229.

    Article  Google Scholar 

  23. 23.

    Hueber W, Sands BE, Lewitzky S, 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. 2012;61:1693–1700.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Nikolaus S, Schreiber S. Diagnostics of inflammatory bowel disease. Gastroenterology. 2007;133:1670–1689.

    Article  PubMed  Google Scholar 

  25. 25.

    Colombel JF, Rutgeerts P, Reinisch W, et al. Early mucosal healing with infliximab is associated with improved long-term clinical outcomes in ulcerative colitis. Gastroenterology. 2011;141:1194–1201.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Daperno M, D’Haens G, Van Assche G, et al. Development and validation of a new, simplified endoscopic activity score for Crohn’s disease: the SES-CD. Gastrointest Endosc. 2004;60:505–512.

    Article  PubMed  Google Scholar 

  27. 27.

    Harvey RF, Bradshaw JM. A simple index of Crohn’s-disease activity. Lancet. 1980;1:514.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Yurchenko E, Shio MT, Huang TC, et al. Inflammation-driven reprogramming of CD4+ Foxp3+ regulatory T cells into pathogenic Th1/Th17 T effectors is abrogated by mTOR inhibition in vivo. PLoS ONE. 2012;7:e35572.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Leung JM, Davenport M, Wolff MJ, et al. IL-22-producing CD4+ cells are depleted in actively inflamed colitis tissue. Mucosal Immunol. 2014;7:124–133.

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Davenport M, Poles J, Leung JM, et al. Metabolic alterations to the mucosal microbiota in inflammatory bowel disease. Inflamm Bowel Dis. 2014;20:723–731.

    Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Ylikoski E, Lund R, Kylaniemi M, et al. IL-12 up-regulates T-bet independently of IFN-gamma in human CD4+ T cells. Eur J Immunol. 2005;35:3297–3306.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Koch MA, Thomas KR, Perdue NR, et al. T-bet(+) Treg cells undergo abortive Th1 cell differentiation due to impaired expression of IL-12 receptor beta2. Immunity. 2012;37:501–510.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Mann ER, Bernardo D, English NR, et al. Compartment-specific immunity in the human gut: properties and functions of dendritic cells in the colon versus the ileum. Gut. 2016;65:256–270.

    Article  PubMed  Google Scholar 

  34. 34.

    Belkaid Y, Hand TW. Role of the microbiota in immunity and inflammation. Cell. 2014;157:121–141.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Donaldson GP, Lee SM, Mazmanian SK. Gut biogeography of the bacterial microbiota. Nat Rev Microbiol. 2016;14:20–32.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Esposito M, Ruffini F, Bergami A, et al. IL-17- and IFN-gamma-secreting Foxp3+ T cells infiltrate the target tissue in experimental autoimmunity. J Immunol. 2010;185:7467–7473.

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank to the Alberta IBD consortium for financial support; Ms. Gurmeet Bindra in the inflammatory intestinal tissue bank (IITB) and the staff in IBD clinic for assistance with research sample collection; Dr. Karen Poon (Nicole Perkins Microbe Core Facility, Calvin, Phoebe and Joan Snyder Institute for Chronic Disease) and Mr. Ronald Chan for assistance with data acquisition; Drs. Tie Wang, Donald Buie, Anthony MacLean, and Iwona Auer-Grzesiak for assistance with optimizing study design; and all patients and healthy individuals who participated into this study.

Author information

Affiliations

Authors

Contributions

AU, JLi, JLu, XG, SG were involved in study concept; JLi, AU, MFG were involved in data acquisition and analysis; MFG, MI, GK, PB, RP, SG were involved in patient recruitment; JQ, HB, SG were involved in project supervision; AU, HJ, MI, SG, HB, JLi were involved in manuscript preparation.

Corresponding author

Correspondence to Aito Ueno.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Ji Li and Aito Ueno share the first authorship.

Xianyong Gui and Subrata Ghosh share the senior authorship.

Study Sponsor Alberta IBD Consortium/Alberta Innovates—Health Solutions.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Figure 1

Flow cytometry plots. The gating strategy is shown in (A). First, the acquired data were gated lymphocytes in FSC-A vs. SSC-A display, followed by doublets discrimination in FSC-A vs. FSC-H display, and then live/dead discrimination. Live CD4+ T cells are further analyzed. Representative data of CD, UC, and HC are shown in B-D. The upper panels display Foxp3 (X-axis) vs. IFNγ (Y-axis) (B), the middle panels Foxp3 (X-axis) vs. IL-17 (Y-axis) display (C) and the lower panels IL-17 (X-axis) versus IFNγ (Y-axis) (D). (PPTX 113 kb)

Supplementary Figure 2

Prevalence of Th subsets in lamina propria mononuclear cells. The prevalence of expressing a Th1 signature cytokine, IFNγ (A), a Th1 transcription factor, T-bet (B), a Th2 signature cytokine, IL-13 (C), a Th2 transcription factor, Gata3 (D), a Th17 signature cytokine, IL-17A (E), a Th17 transcription factor RORγt (F), a Treg transcription factor Foxp3 (G), and a combination of CD25 and Foxp3 (H) in CD4 + T cells are shown in this figure. * p < 0.05, **p < 0.01, *** p < 0.001, **** P < 0.0001. (PPTX 99 kb)

Supplementary Figure 3

Concordance between endoscopic scores (SES-CD scores) and Foxp3+ crossover populations in lamina propria mononuclear cells of CD patients. The prevalence of IFNγ+ Foxp3+ (A), IL-13+ Foxp3+ (B), IL-17+ Foxp3+ (C), T-bet+ Foxp3+ (D), Gata3+ Foxp3+ (E), RORγt+ Foxp3+ (F), and IFNγ+ IL-17+ (G) is shown in this figure. (PPTX 146 kb)

Supplementary Figure 4

Concordance between Mayo endoscopic subscores and CD4+ T cell crossover populations in lamina propria mononuclear cells of UC patients. The prevalence of IFNγ+ Foxp3+ (A), IL-13+ Foxp3+ (B), IL-17+ Foxp3+ (C), T-bet+ Foxp3+ (D), Gata3+ Foxp3+ (E), RORγt+ Foxp3+ (F), and IFNγ+ IL-17+ (G) is shown in this figure. * p < 0.05 (PPTX 102 kb)

Supplementary Figure 5

Association between IL-22+ T helper cell subsets and clinical disease activity scores evaluated by Harvey–Bradshaw index in CD (A) and Mayo clinical index scores in UC cohort (B). Total IL-22+, IL-22+ IL-17+ (IL-22+ Th17), IL-22+ IFN+ (IL-22+ Th1) and IL-22+ IL-17-IFNγ-(Th22) cells are shown in this figure. (PPTX 81 kb)

Supplementary Figure 6

Association between IL-22+ T helper cell subsets and endoscopic disease activity evaluated by SES-CD scores in CD cohort (A) and Mayo endoscopic subscores in UC cohort (B). Total IL-22+, IL-22+ IL-17+ (IL-22+ Th17), IL-22+ IFN+ (IL-22+ Th1) and IL-22+ IL-17-IFNγ-(Th22) cells are shown in this figure. (PPTX 80 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Li, J., Ueno, A., Iacucci, M. et al. Crossover Subsets of CD4+ T Lymphocytes in the Intestinal Lamina Propria of Patients with Crohn’s Disease and Ulcerative Colitis. Dig Dis Sci 62, 2357–2368 (2017). https://doi.org/10.1007/s10620-017-4596-9

Download citation

Keywords

  • T cell crossover population
  • Crohn’s disease
  • Ulcerative colitis
  • Regulatory T cells
  • Th17
  • Th22