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

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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.

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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

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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

Authors and Affiliations

  1. Gastrointestinal Research Group/Department of Medicine, HSC1838, University of Calgary, 2500 University Dr. NW, Calgary, AB, Canada

    Ji Li, Aito Ueno, Marietta Iacucci, Miriam Fort Gasia, Humberto B. Jijon, Remo Panaccione, Gilaad G. Kaplan, Paul L. Beck, Herman W. Barkema & Subrata Ghosh

  2. Department of Gastroenterology, Peking Union Medical College Hospital, Beijing, China

    Ji Li & Jiaming Qian

  3. Institute of Translational Medicine, University of Birmingham, Birmingham, UK

    Marietta Iacucci & Subrata Ghosh

  4. Calgary Laboratory Services, Calgary, AB, Canada

    Joanne Luider & Xianyong Gui

  5. Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, AB, Canada

    Xianyong Gui

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  1. Ji Li
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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.

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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)

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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

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