Paradoxically Increased FOXP3+ T Cells in IBD Do Not Preferentially Express the Isoform of FOXP3 Lacking Exon 2

Abstract

Background FOXP3+ regulatory T cells (Tregs) are critical for controlling inflammation in the gastrointestinal (GI) 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 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 (IHC) and quantitative PCR, respectively. Results No difference in the expression pattern of Δexon2 relative to full length FOXP3 was seen in ulcerative colitis (UC) 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 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.

An expanded population of pathogenic regulatory T cells in giant cell arteritis is abrogated by IL-6 blockade therapy

“…Cells were labelled with Pacific Blue-conjugated anti-CD4, fluorescein isothiocyanate-conjugated anti-CD45RA, phycoerythrin (PE)-Cy7-conjugated anti-CCR4, PE-conjugated anti-CTLA4, allophycocyanin (APC)-Cy7-conjugated anti-IL-17A, PE-Cy7-conjugated anti-CD25, PE-conjugated anti-Ki67, APC-conjugated anti-CD161 (BioLegend); Alexa Fluor 488-conjugated anti-Foxp3 and Alexa Fluor 700-conjugated anti-Foxp3. Foxp3Δ2 was detected using clone PCH101 (eBiocience) that recognises the N-terminus portion of the protein and clone 150D (BioLegend) that recognises exon 2 24 25. Data were acquired on a LSRFortessa cell analyser (BD biosciences) and analysed with FlowJo software.…”

CD4 T Cells in IBD: Crossing the Line?

“…Although a splice variant of FOXP3 lacking exon 2 exists in humans, and therefore would not interact with and block RORct, this is not the sole form of FOXP3 present in IL-17-expressing FOXP3? T cells from the bowels of people with and without IBD [7]. Instead, another mechanism must be present to impair FOXP3's ability to suppress the pro-inflammatory cytokine production of T cells that express it in IBD.…”

The Role of T-Cell Subsets in Chronic Inflammation in Celiac Disease and Inflammatory Bowel Disease Patients: More Common Mechanisms or More Differences?

“…Treg accumulate in areas of active inflammation, including granulomas and retain potent regulatory activity ex vivo [100]. Linking these Treg to Th17 cells in CD, the Treg population includes IL-17-producing cells [101,102]. …”