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

Mailer RK, Joly AL, Liu S, Elias S, Tegner J, Andersson J - Sci Rep (2015)

Bottom Line: 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.Our results demonstrate that alternative splicing of FOXP3 modulates T cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Translational Immunology Unit, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f3: 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.

Mentions: Previous studies have demonstrated that FOXP3Δ2Δ7 is incapable of conferring suppressive ability to T cells19. However, FOXP3 is not only expressed by Treg cells but it is also transiently expressed during Th17 differentiation. Because IL-1β promotes both alternative splicing of FOXP3 and Th17 differentiation, we next assessed the ability of FOXP3 isoforms to modulate Th17 differentiation. We purposely did not use overexpression of FOXP3 isoforms as T cell activation induces relatively high levels of endogenous FOXP3fl and FOXP3Δ2. Instead, we altered the splicing pattern of FOXP3 using morpholino antisense oligonucleotides (MAO) that prevent splice-directing small nuclear ribonucleoproteins from binding the exon/exon boundaries of FOXP3 pre-mRNA21. We demonstrated that it is possible to remove FOXP3 exon 2 in Treg cells with great efficiency (Fig. 3a) but the MAO targeting exon 7 was less efficient and could only partially remove FOXP3 exon 7 in Treg cells as determined by qPCR (data not shown). On the other hand MAO targeting efficiently removed FOXP3 exon 7 in naïve T cells upon differentiating into Th17 cells, presumably because MAO transfection precedes the induction of FOXP3 mRNA expression and the total amount of FOXP3 mRNA is much lower in these cells (Fig 3b). MAO-mediated splice shifting was observed as early as 24 hours post-transfection and persisted for at least 1 week (data not shown). Importantly, in these cells we found that expression of IL-2 (Fig. 3c) and IL-17A (Fig. 3d), but not IFN-γ (data not shown), is modulated by alternative splicing of FOXP3. Indeed, the combined removal of exon 2 and exon 7 mediated by MAO treatment strongly enhanced expression of IL-2 and IL-17A (Fig. 3c,d).


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

Mailer RK, Joly AL, Liu S, Elias S, Tegner J, Andersson J - Sci Rep (2015)

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.
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Related In: Results  -  Collection

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Show All Figures
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f3: 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.
Mentions: Previous studies have demonstrated that FOXP3Δ2Δ7 is incapable of conferring suppressive ability to T cells19. However, FOXP3 is not only expressed by Treg cells but it is also transiently expressed during Th17 differentiation. Because IL-1β promotes both alternative splicing of FOXP3 and Th17 differentiation, we next assessed the ability of FOXP3 isoforms to modulate Th17 differentiation. We purposely did not use overexpression of FOXP3 isoforms as T cell activation induces relatively high levels of endogenous FOXP3fl and FOXP3Δ2. Instead, we altered the splicing pattern of FOXP3 using morpholino antisense oligonucleotides (MAO) that prevent splice-directing small nuclear ribonucleoproteins from binding the exon/exon boundaries of FOXP3 pre-mRNA21. We demonstrated that it is possible to remove FOXP3 exon 2 in Treg cells with great efficiency (Fig. 3a) but the MAO targeting exon 7 was less efficient and could only partially remove FOXP3 exon 7 in Treg cells as determined by qPCR (data not shown). On the other hand MAO targeting efficiently removed FOXP3 exon 7 in naïve T cells upon differentiating into Th17 cells, presumably because MAO transfection precedes the induction of FOXP3 mRNA expression and the total amount of FOXP3 mRNA is much lower in these cells (Fig 3b). MAO-mediated splice shifting was observed as early as 24 hours post-transfection and persisted for at least 1 week (data not shown). Importantly, in these cells we found that expression of IL-2 (Fig. 3c) and IL-17A (Fig. 3d), but not IFN-γ (data not shown), is modulated by alternative splicing of FOXP3. Indeed, the combined removal of exon 2 and exon 7 mediated by MAO treatment strongly enhanced expression of IL-2 and IL-17A (Fig. 3c,d).

Bottom Line: 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.Our results demonstrate that alternative splicing of FOXP3 modulates T cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Translational Immunology Unit, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden.

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.

No MeSH data available.


Related in: MedlinePlus