IL-1β promotes Th17 differentiation by inducing alternative splicing of FOXP3

Abstract

CD4(+)FOXP3(+) regulatory T (Treg) cells are essential for maintaining immunological self-tolerance. Treg cell development and function depend on the transcription factor FOXP3, which is present in several distinct isoforms due to alternative splicing. Despite the importance of FOXP3 in the proper maintenance of Treg cells, the regulation and functional consequences of FOXP3 isoform expression remains poorly understood. Here, we show that in human Treg cells IL-1β promotes excision of FOXP3 exon 7. FOXP3 is not only expressed by Treg cells but is also transiently expressed when naïve T cells differentiate into Th17 cells. Forced splicing of FOXP3 into FOXP3Δ2Δ7 strongly favored Th17 differentiation in vitro. We also found that patients with Crohn's disease express increased levels of FOXP3 transcripts lacking exon 7, which correlate with disease severity and IL-17 production. Our results demonstrate that alternative splicing of FOXP3 modulates T cell differentiation. These results highlight the importance of characterizing FOXP3 expression on an isoform basis and suggest that immune responses may be manipulated by modulating the expression of FOXP3 isoforms, which has broad implications for the treatment of autoimmune diseases.

Figure 1. Increased alternative splicing of FOXP3 exon 7 in Crohn’s disease.

(a) Schematic overview of FOXP3 depicting alternatively spliced exons, epitopes of antibody clones and binding sites for primers used for detection of each splice variant. (b) Real-time PCR quantification of FOXP3 transcripts expressing exon 2 (FOXP3ex1/2), lacking exon 2 (FOXPex1/3), and lacking exon 7 (FOXP3ex6/8) in PBMCs obtained from Crohn’s disease patients (n = 8) or healthy donors (n = 10). (c) Percentage of FOXP3ex6/8 transcripts in relation to total FOXP3 transcripts in PBMCs (n = 11) of Crohn’s disease patients and healthy donors (n = 10). (d) Crohn’s disease colon biopsies (n = 29) stratified into 50% lowest and highest FOXP3ex6/8 expression samples plotted versus clinical score. (e) Percentage of FOXP3ex6/8 transcripts in relation to total FOXP3 in intestinal biopsies obtained from Crohn’s disease patients (n = 7) before and after successful anti-TNF-α treatment. (b–e) Data represent one pooled experiment with n biological replicates and are presented as (b,d,e) mean ± SD, (c) median ± IQR. P < 0.05 was considered significant ((b,d) two-tailed unpaired Student’s t test, (c) Kruskal-Wallis ANOVA and Dunn’s post hoc test, (e) two-tailed paired Student’s t test).

Figure 2. Activation and IL-1β regulate alternative splicing of FOXP3.

Quantitative PCR was used to analyze: (a) fold induction of FOXP3 transcripts in CD4⁺CD25^hiCD127^low cells activated with plate bound α-CD3 and soluble α-CD28 and IL-2 for 18 hours relative to freshly isolated cells. (b-d) Fold induction of FOXP3 transcripts in CD4⁺CD25^hiCD127^low cells activated as above in the presence of 10 ng/ml IL-1β, IL-6 or TNF-α relative to cells activated without cytokines. (e) FOXP3ex1/2 (dark gray), FOXP3ex1/3 (gray), and FOXP3ex6/8 (white) transcripts in CD4⁺ T cells sorted for low, intermediate (int), and high (hi) expression of CD25. (a,b) Data are representative of four independent experiments and presented as mean ± SD. P < 0.05 was considered significant (two-tailed unpaired Student’s t test). (c) Data (n = 3 technical replicates) are presented as mean ± SD.

Figure 3. FOXP3Δ2Δ7 promote IL-17A production in naïve T cells.

(a) Density plots of FOXP3 expression (total and exon 2) in enriched CD25⁺CD4⁺ T cells (n = 10), that had been transfected with control MAO (control), or with splice-redirecting MAO specific for FOXP3 exon 2 (MAO Δ2) and/or MAO specific for FOXP3 exon 7 (MAO Δ7). (b) Real-time PCR quantification of FOXP3ex1/2 (black), FOXP3ex1/3 (gray) and FOXP3ex6/8 (white) transcripts of naïve T cells transfected with control MAO or MAO Δ2 and MAO Δ7 (n = 10). Expression was normalized to HPRT-1 and transcription ratio was calculated relative to total FOXP3. (c,d) MAO transfected CD4⁺ Naïve T cells were differentiated towards the Th17 lineage for 5 days and (c) IL-2 (n = 7) and (d) IL-17A (n = 10) cytokine expression were assessed by flow cytometry. Data are presented as mean ± SD, P<0.05 was considered significant,two-tailed paired Student’s t test.

Figure 4. Alternative splicing of FOXP3 exon 7 correlates with IL-17A expression.

Quantitative PCR was used to measure FOXP3 and IL-17A transcript levels in colon biopsies from patients suffering from Crohn’s disease and normalized to GAPDH expression (n = 26). Data are presented as mean of n = 3 technical replicates. P < 0.05 was considered significant, Spearman rank correlation test; r = correlation coefficient).