Abstract
Background
Forkhead box P3 (FOXP3)+ regulatory T cells (Tregs) are critical for controlling inflammation in the gastrointestinal tract. There is a paradoxical increase of mucosal FOXP3+ T cells in patients with inflammatory bowel disease (IBD). These FOXP3+ cells were recently shown to include interleukin (IL)-17A-producing cells in Crohn’s disease, resembling Th17 cells implicated in autoimmune diseases. FOXP3 inhibits IL-17A production, but a naturally occurring splice variant of FOXP3 lacking exon 2 (Δexon2) cannot.
Aims
We hypothesized that IBD patients preferentially express the Δexon2 variant of FOXP3 so the paradoxically increased mucosal Tregs in IBD could represent cells expressing only Δexon2.
Methods
We used antibodies and primers that can distinguish between the full-length and Δexon2 splice variant of FOXP3 to evaluate expression of these isoforms in human intestinal tissue by immunohistochemistry and quantitative polymerase chain reaction (PCR), respectively.
Results
No difference in the expression pattern of Δexon2 relative to full-length FOXP3 was seen in ulcerative colitis or Crohn’s disease versus non-IBD controls. By immunofluorescence microscopy and flow cytometry, we also did not find individual cells which expressed FOXP3 protein exclusively in the Δexon2 isoform in either IBD or control tissue. FOXP3+ mucosal CD4+ T cells from both IBD and control specimens were able to make IL-17A in vitro after phorbol myristate acetate (PMA) and ionomycin stimulation, but these cells did not preferentially express Δexon2.
Conclusions
Our data do not support the hypothesis that selective expression of FOXP3 in the Δexon2 isoform accounts for the inability of copious FOXP3+ T cells to inhibit inflammation or IL-17 expression in IBD.
Similar content being viewed by others
References
Rudensky AY. Regulatory T cells and Foxp3. Immunol Rev. 2011;241:260–268.
Bennett CL, Christie J, Ramsdell F, et al. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet. 2001;27:20–21.
Brunkow ME, Jeffery EW, Hjerrild KA, et al. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet. 2001;27:68–73.
Patel DD. Escape from tolerance in the human X-linked autoimmunity-allergic disregulation syndrome and the Scurfy mouse. J Clin Invest. 2001;107:155–157.
Wildin RS, Ramsdell F, Peake J, et al. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet. 2001;27:18–20.
Makita S, Kanai T, Oshima S, Uraushihara K, et al. CD4+ CD25bright T cells in human intestinal lamina propria as regulatory cells. J Immunol. 2004;173:3119–3130.
Maul J, Loddenkemper C, Mundt P, et al. Peripheral and intestinal regulatory CD4+ CD25(high) T cells in inflammatory bowel disease. Gastroenterology. 2005;128:1868–1878.
Saruta M, Yu QT, Fleshner PR, et al. Characterization of FOXP3+ CD4+ regulatory T cells in Crohn’s disease. Clin Immunol. 2007;125:281–290.
Uhlig HH, Coombes J, Mottet C, et al. Characterization of Foxp3+ CD4+ CD25+ and IL-10-secreting CD4+ CD25+ T cells during cure of colitis. J Immunol. 2006;177:5852–5860.
Allan SE, Passerini L, Bacchetta R, et al. The role of 2 FOXP3 isoforms in the generation of human CD4+ Tregs. J Clin Invest. 2005;115:3276–3284.
Mailer RK, Falk K, Rotzschke O. Absence of leucine zipper in the natural FOXP3Delta2Delta7 isoform does not affect dimerization but abrogates suppressive capacity. PLoS One. 2009;4:e6104.
Kobayashi I, Shiari R, Yamada M, et al. Novel mutations of FOXP3 in two Japanese patients with immune dysregulation, polyendocrinopathy, enteropathy, X linked syndrome (IPEX). J Med Genet. 2001;38:874–876.
Torgerson TR, Ochs HD. Immune dysregulation, polyendocrinopathy, enteropathy, X-linked: forkhead box protein 3 mutations and lack of regulatory T cells. J Allergy Clin Immunol. 2007;120:744–750.
Oukka M. Th17 cells in immunity and autoimmunity. Ann Rheum Dis. 2008;67:iii26–iii29.
Annunziata F, Cosmi L, Santarlasci V, et al. Phenotypic and functional features of human Th17 cells. J Exp Med. 2007;204:1849–1861.
Witowski J, Ksiazek K, Jorres A. Interleukin-17: a mediator of inflammatory responses. Cell Mol Life Sci. 2004;61:567–579.
Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B. TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity. 2006;24:179–189.
Dubinsky MC, Wang D, Picornell Y, et al. IL-23 receptor (IL-23R) gene protects against pediatric Crohn’s disease. Inflamm Bowel Dis. 2007;13:511–515.
Duerr RH, Taylor KD, Brant SR, et al. A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science. 2006;314:1461–1463.
Franke A, McGovern DP, Barrett JC, et al. Genome-wide meta-analysis increases to 71 the number of confirmed Crohn’s disease susceptibility loci. Nat Genet. 2010;42:1118–1125.
Sandborn WJ, Feagan BG, Fedorak RN, et al. A randomized trial of Ustekinumab, a human interleukin-12/23 monoclonal antibody, in patients with moderate-to-severe Crohn’s disease. Gastroenterology. 2008;135:1130–1141.
Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature. 2006;441:235–238.
McCabe RP, Secrist H, Botney M, Egan M, Peters MG. Cytokine mRNA expression in intestine from normal and inflammatory bowel disease patients. Clin Immunol Immunopathol. 1993;66:52–58.
Hovhannisyan Z, Treatman J, Littman DR, Mayer L. Characterization of interleukin-17-producing regulatory T cells in inflamed intestinal mucosa from patients with inflammatory bowel diseases. Gastroenterology. 2011;140:957–965.
Ivanov II, McKenzie BS, Zhou L, et al. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell. 2006;126:1121–1133.
Ichiyama K, Yoshida H, Wakabayashi Y, et al. Foxp3 inhibits RORgammat-mediated IL-17A mRNA transcription through direct interaction with RORgammat. J Biol Chem. 2008;283:17003–17008.
Zhou L, Lopes JE, Chong MM, et al. TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function. Nature. 2008;453:236–240.
Ayyoub M, Deknuydt F, Raimbaud I, et al. Human memory FOXP3+ Tregs secrete IL-17 ex vivo and constitutively express the T(H)17 lineage-specific transcription factor RORgamma t. Proc Natl Acad Sci U S A. 2009;106:8635–8640.
Voo KS, Wang YH, Santori FR, et al. Identification of IL-17-producing FOXP3+ regulatory T cells in humans. Proc Natl Acad Sci U S A. 2009;106:4793–4798.
Yu QT, Saruta M, Avanesyan A, Fleshner PR, Banham AH, Papadakis KA. Expression and functional characterization of FOXP3+ CD4+ regulatory T cells in ulcerative colitis. Inflamm Bowel Dis. 2007;13:191–199.
Acknowledgments
We wish to thank Sandhya Mishra for assistance with patient recruitment, and thank Patti Stewart, Joyce Matsuoka-Hayashi, Joelle Averbuch, Rachael Williams, and all of the Anatomic Pathology attendings at Virginia Mason Medical Center for facilitating surgical specimen acquisition. We wish to thank Mary Beauchamps for assistance with histology preparation and K. Aru Arumuganathan for assistance with flow cytometry. Special thanks go to Jane Buckner and Elisa Boden for manuscript review. This work was funded through a grant from the NIDDK/NIH 1K08DK081659, and internal resources at the Benaroya Research Institute.
Conflict of interest
None.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Lord, J.D., 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 57, 2846–2855 (2012). https://doi.org/10.1007/s10620-012-2292-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10620-012-2292-3