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miR-155 inhibition sensitizes CD4+ Th cells for TREG mediated suppression.

Stahl HF, Fauti T, Ullrich N, Bopp T, Kubach J, Rust W, Labhart P, Alexiadis V, Becker C, Hafner M, Weith A, Lenter MC, Jonuleit H, Schmitt E, Mennerich D - PLoS ONE (2009)

Bottom Line: DNA-Microarray analyses of human as well as murine conventional CD4(+) Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations.Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3.Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4(+) Th cells to nTreg cell-mediated suppression.

View Article: PubMed Central - PubMed

Affiliation: Boehringer Ingelheim Pharma GmbH & Co. KG, Respiratory Diseases Research, Genomics Group, Biberach an der Riss, Germany.

ABSTRACT

Background: In humans and mice naturally occurring CD4(+)CD25(+) regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance.

Principal findings: DNA-Microarray analyses of human as well as murine conventional CD4(+) Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4(+) Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4(+) Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression.

Conclusion: Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4(+) Th cells to nTreg cell-mediated suppression.

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BIC/miR-155 expression is not necessarily regulated by FoxP3.(A) Schematic overview of the ChIP-Seq analysis workflow. Genomic loci of all significantly and reproducible FoxP3-bound micro-RNAs are shown: intragenic micro-RNA (B) and intergenic located micro-RNAs (C). Each genomic capture shows in light red the micro-RNA(s) and in blue the overlapping annotated gene(s) including the intro/exon structure(s). In dark red (CD25+/nTREG) and in gray (CD4+ Th cell) the FoxP3-bound genomic regions of both donors are symbolized. In addition, every capture contains the underlying chromosome including the basepair coordinates. The visualisations were generated using the UCSC genome browser (human genome assembly of March 2006). (D) Using Taqman RT-PCR, the expression analysis of T lymphocytes for mature miR-155 showed no significant difference between wild type C57/BL6 and FoxP3 mutated Scurfy mice. Post activation (19 h) an increased expression of matured miR-155 was detectable, in both CD4+ Th cells of wild type and Scurfy mice. All values were calculated as relative fold changes using the ddCT method. As normalizer 5S was used (n = 3).
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pone-0007158-g003: BIC/miR-155 expression is not necessarily regulated by FoxP3.(A) Schematic overview of the ChIP-Seq analysis workflow. Genomic loci of all significantly and reproducible FoxP3-bound micro-RNAs are shown: intragenic micro-RNA (B) and intergenic located micro-RNAs (C). Each genomic capture shows in light red the micro-RNA(s) and in blue the overlapping annotated gene(s) including the intro/exon structure(s). In dark red (CD25+/nTREG) and in gray (CD4+ Th cell) the FoxP3-bound genomic regions of both donors are symbolized. In addition, every capture contains the underlying chromosome including the basepair coordinates. The visualisations were generated using the UCSC genome browser (human genome assembly of March 2006). (D) Using Taqman RT-PCR, the expression analysis of T lymphocytes for mature miR-155 showed no significant difference between wild type C57/BL6 and FoxP3 mutated Scurfy mice. Post activation (19 h) an increased expression of matured miR-155 was detectable, in both CD4+ Th cells of wild type and Scurfy mice. All values were calculated as relative fold changes using the ddCT method. As normalizer 5S was used (n = 3).

Mentions: In 2007, Zheng et al. [16] and Marson et al. [17] published mouse FoxP3 ChIP-on-CHIP analyses studies. They affirmed FoxP3 regulates the expression of BIC/miR-155 and that BIC/miR-155 is a direct target of FoxP3 in mice. To prove this hypothesis, we performed human FoxP3 ChIP-Seq experiments using activated (16 h) human CD4+ Th cells and nTregs of two independent donors (Fig. 3A). A miRNA-focussed bioinformatic analysis revealed at least 24 different miRNA loci, which are significantly and reproducibly detected to be bound by the transcription factor FoxP3. Due to their genomic localization 18 miRNAs were categorized as intergenic (Fig. 3B) und 6 miRNAs as intrageneic (Fig. 3C). A comprehensive list of FoxP3-bound micro-RNA-associated genomic loci can be found as a data file (table S1). The table also contains the miRNAs which are located nearby or directly within a promoter region of an annotated gene. The binding of FoxP3 to these regions can't be differentiated between specific gene and/or miRNA regulation. The group of intergenic FoxP3 bound miRNA targets (Fig. 3B) confirms the finding of Zheng et al. [16] and Marson et al. [17], that FoxP3 binds the genomic BIC/miR-155 locus in human regulatory T cells. Surprisingly, no binding of FoxP3 to BIC/miR-155 was detectable in CD4+ Th cells, which also express FoxP3 after activation.


miR-155 inhibition sensitizes CD4+ Th cells for TREG mediated suppression.

Stahl HF, Fauti T, Ullrich N, Bopp T, Kubach J, Rust W, Labhart P, Alexiadis V, Becker C, Hafner M, Weith A, Lenter MC, Jonuleit H, Schmitt E, Mennerich D - PLoS ONE (2009)

BIC/miR-155 expression is not necessarily regulated by FoxP3.(A) Schematic overview of the ChIP-Seq analysis workflow. Genomic loci of all significantly and reproducible FoxP3-bound micro-RNAs are shown: intragenic micro-RNA (B) and intergenic located micro-RNAs (C). Each genomic capture shows in light red the micro-RNA(s) and in blue the overlapping annotated gene(s) including the intro/exon structure(s). In dark red (CD25+/nTREG) and in gray (CD4+ Th cell) the FoxP3-bound genomic regions of both donors are symbolized. In addition, every capture contains the underlying chromosome including the basepair coordinates. The visualisations were generated using the UCSC genome browser (human genome assembly of March 2006). (D) Using Taqman RT-PCR, the expression analysis of T lymphocytes for mature miR-155 showed no significant difference between wild type C57/BL6 and FoxP3 mutated Scurfy mice. Post activation (19 h) an increased expression of matured miR-155 was detectable, in both CD4+ Th cells of wild type and Scurfy mice. All values were calculated as relative fold changes using the ddCT method. As normalizer 5S was used (n = 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2743997&req=5

pone-0007158-g003: BIC/miR-155 expression is not necessarily regulated by FoxP3.(A) Schematic overview of the ChIP-Seq analysis workflow. Genomic loci of all significantly and reproducible FoxP3-bound micro-RNAs are shown: intragenic micro-RNA (B) and intergenic located micro-RNAs (C). Each genomic capture shows in light red the micro-RNA(s) and in blue the overlapping annotated gene(s) including the intro/exon structure(s). In dark red (CD25+/nTREG) and in gray (CD4+ Th cell) the FoxP3-bound genomic regions of both donors are symbolized. In addition, every capture contains the underlying chromosome including the basepair coordinates. The visualisations were generated using the UCSC genome browser (human genome assembly of March 2006). (D) Using Taqman RT-PCR, the expression analysis of T lymphocytes for mature miR-155 showed no significant difference between wild type C57/BL6 and FoxP3 mutated Scurfy mice. Post activation (19 h) an increased expression of matured miR-155 was detectable, in both CD4+ Th cells of wild type and Scurfy mice. All values were calculated as relative fold changes using the ddCT method. As normalizer 5S was used (n = 3).
Mentions: In 2007, Zheng et al. [16] and Marson et al. [17] published mouse FoxP3 ChIP-on-CHIP analyses studies. They affirmed FoxP3 regulates the expression of BIC/miR-155 and that BIC/miR-155 is a direct target of FoxP3 in mice. To prove this hypothesis, we performed human FoxP3 ChIP-Seq experiments using activated (16 h) human CD4+ Th cells and nTregs of two independent donors (Fig. 3A). A miRNA-focussed bioinformatic analysis revealed at least 24 different miRNA loci, which are significantly and reproducibly detected to be bound by the transcription factor FoxP3. Due to their genomic localization 18 miRNAs were categorized as intergenic (Fig. 3B) und 6 miRNAs as intrageneic (Fig. 3C). A comprehensive list of FoxP3-bound micro-RNA-associated genomic loci can be found as a data file (table S1). The table also contains the miRNAs which are located nearby or directly within a promoter region of an annotated gene. The binding of FoxP3 to these regions can't be differentiated between specific gene and/or miRNA regulation. The group of intergenic FoxP3 bound miRNA targets (Fig. 3B) confirms the finding of Zheng et al. [16] and Marson et al. [17], that FoxP3 binds the genomic BIC/miR-155 locus in human regulatory T cells. Surprisingly, no binding of FoxP3 to BIC/miR-155 was detectable in CD4+ Th cells, which also express FoxP3 after activation.

Bottom Line: DNA-Microarray analyses of human as well as murine conventional CD4(+) Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations.Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3.Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4(+) Th cells to nTreg cell-mediated suppression.

View Article: PubMed Central - PubMed

Affiliation: Boehringer Ingelheim Pharma GmbH & Co. KG, Respiratory Diseases Research, Genomics Group, Biberach an der Riss, Germany.

ABSTRACT

Background: In humans and mice naturally occurring CD4(+)CD25(+) regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance.

Principal findings: DNA-Microarray analyses of human as well as murine conventional CD4(+) Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4(+) Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4(+) Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression.

Conclusion: Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4(+) Th cells to nTreg cell-mediated suppression.

Show MeSH
Related in: MedlinePlus