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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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Overexpression of mature miR-155 rendered murine CD4+ Th cells unresponsive to nTreg-mediated suppression.Suppression assay performed with naïve murine CD4+ Th cells and murine nTregs: Th cells transfected with pre miR-155 showed up to 40% decreased susceptibility for nTreg cell-mediated suppression compared to the control-transfected CD4+ Th cells. Shown is the 3H-thymidine uptake after 18 h. The bar plot diagrams are indicating the grade of suppression levels. The added numbers are showing the differences (%) for the susceptibility of nTreg-mediated suppression of proliferation of the activated CD4+ Th cells. One representative experiment out of at least three experiments is shown.
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pone-0007158-g005: Overexpression of mature miR-155 rendered murine CD4+ Th cells unresponsive to nTreg-mediated suppression.Suppression assay performed with naïve murine CD4+ Th cells and murine nTregs: Th cells transfected with pre miR-155 showed up to 40% decreased susceptibility for nTreg cell-mediated suppression compared to the control-transfected CD4+ Th cells. Shown is the 3H-thymidine uptake after 18 h. The bar plot diagrams are indicating the grade of suppression levels. The added numbers are showing the differences (%) for the susceptibility of nTreg-mediated suppression of proliferation of the activated CD4+ Th cells. One representative experiment out of at least three experiments is shown.

Mentions: To further corroborate these findings, human CD4+ Th cells were transfected with human mimic-miR-155 or its respective human pre-miR-155 (precursor) to analyze whether this evokes the opposite effect, namely rendering CD4+ Th cells unresponsive to nTreg cell-mediated suppression. To this end, miR-155, pre miR-155 as well as control-transfected CD4+ Th cells where co-cultured with nTregs at different ratios. Interestingly, overexpression of miR-155 resulted in a strongly decreased susceptibility of the CD4+ Th cells to nTreg cell-mediated suppression (Fig. 4B). Equivalent experiments were performed with naïve murine CD4+ Th cells and murine nTregs. Murine CD4+ Th cells transfected with murine pre-miR-155 showed an up to 40% decreased susceptibility to nTreg cell-mediated suppression compared to the control-transfected CD4+ Th cells, underscoring the results obtained with human T cells (Fig. 5). Again, increasing the levels of miR-155 in murine or human nTregs did not significantly influence their ability to suppress CD4+ Th cells (data not shown). Previous work has shown that a loss of miR-155 in Tregs did not impair their sensitivity to impair Treg cell suppressor function, whereas miR-155 is involved during thymic differentiation by promoting the fitness and the proliferative potential of differentiating nTregs [10].


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)

Overexpression of mature miR-155 rendered murine CD4+ Th cells unresponsive to nTreg-mediated suppression.Suppression assay performed with naïve murine CD4+ Th cells and murine nTregs: Th cells transfected with pre miR-155 showed up to 40% decreased susceptibility for nTreg cell-mediated suppression compared to the control-transfected CD4+ Th cells. Shown is the 3H-thymidine uptake after 18 h. The bar plot diagrams are indicating the grade of suppression levels. The added numbers are showing the differences (%) for the susceptibility of nTreg-mediated suppression of proliferation of the activated CD4+ Th cells. One representative experiment out of at least three experiments is shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2743997&req=5

pone-0007158-g005: Overexpression of mature miR-155 rendered murine CD4+ Th cells unresponsive to nTreg-mediated suppression.Suppression assay performed with naïve murine CD4+ Th cells and murine nTregs: Th cells transfected with pre miR-155 showed up to 40% decreased susceptibility for nTreg cell-mediated suppression compared to the control-transfected CD4+ Th cells. Shown is the 3H-thymidine uptake after 18 h. The bar plot diagrams are indicating the grade of suppression levels. The added numbers are showing the differences (%) for the susceptibility of nTreg-mediated suppression of proliferation of the activated CD4+ Th cells. One representative experiment out of at least three experiments is shown.
Mentions: To further corroborate these findings, human CD4+ Th cells were transfected with human mimic-miR-155 or its respective human pre-miR-155 (precursor) to analyze whether this evokes the opposite effect, namely rendering CD4+ Th cells unresponsive to nTreg cell-mediated suppression. To this end, miR-155, pre miR-155 as well as control-transfected CD4+ Th cells where co-cultured with nTregs at different ratios. Interestingly, overexpression of miR-155 resulted in a strongly decreased susceptibility of the CD4+ Th cells to nTreg cell-mediated suppression (Fig. 4B). Equivalent experiments were performed with naïve murine CD4+ Th cells and murine nTregs. Murine CD4+ Th cells transfected with murine pre-miR-155 showed an up to 40% decreased susceptibility to nTreg cell-mediated suppression compared to the control-transfected CD4+ Th cells, underscoring the results obtained with human T cells (Fig. 5). Again, increasing the levels of miR-155 in murine or human nTregs did not significantly influence their ability to suppress CD4+ Th cells (data not shown). Previous work has shown that a loss of miR-155 in Tregs did not impair their sensitivity to impair Treg cell suppressor function, whereas miR-155 is involved during thymic differentiation by promoting the fitness and the proliferative potential of differentiating nTregs [10].

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