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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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RT-PCR expression analysis of pre-mature BIC transcript and its processed microRNA miR-155 in mice and men.The BIC transcript is strongly up-regulated upon activation using anti-CD3/anti-CD28 mAb in human donors (A) and in C57/BL6 mice (B). Whereas, in C57/BL6 mice the BIC transcript as well as the matured miR-155 was found to be higher expressed in nTregs than in CD4+ Th cells (B) and (D) (n = 3). In human cells, BIC and the matured form miR-155 were not present in resting cells, but strongly elevated levels were found in Th cells as well as in nTreg cells after TCR activation (A) and (C) (n = 4). Analyzing the kinetic of BIC/miR-155 expression RNA was collected using an activation time course experiment. (E) The activation of human T cell populations showed a temporary activation of the BIC transcription. Whereas, the CD4+ Th cells reached their maximum after 4 h upon activation, the peak for nTreg cells is shifted to the 16 h time point. (F) The levels of matured miR-155 were found to permanently increase within time (until 120 h) in activated CD4+ Th cells, whereas in nTregs a plateau was reached after 24 h. All values were calculated as relative fold changes using the ddCT method. As normalizer RNA Pol II (human & mouse BIC) as well as U18 (human miR-155) and 5S (mouse miR-155) were used.
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pone-0007158-g002: RT-PCR expression analysis of pre-mature BIC transcript and its processed microRNA miR-155 in mice and men.The BIC transcript is strongly up-regulated upon activation using anti-CD3/anti-CD28 mAb in human donors (A) and in C57/BL6 mice (B). Whereas, in C57/BL6 mice the BIC transcript as well as the matured miR-155 was found to be higher expressed in nTregs than in CD4+ Th cells (B) and (D) (n = 3). In human cells, BIC and the matured form miR-155 were not present in resting cells, but strongly elevated levels were found in Th cells as well as in nTreg cells after TCR activation (A) and (C) (n = 4). Analyzing the kinetic of BIC/miR-155 expression RNA was collected using an activation time course experiment. (E) The activation of human T cell populations showed a temporary activation of the BIC transcription. Whereas, the CD4+ Th cells reached their maximum after 4 h upon activation, the peak for nTreg cells is shifted to the 16 h time point. (F) The levels of matured miR-155 were found to permanently increase within time (until 120 h) in activated CD4+ Th cells, whereas in nTregs a plateau was reached after 24 h. All values were calculated as relative fold changes using the ddCT method. As normalizer RNA Pol II (human & mouse BIC) as well as U18 (human miR-155) and 5S (mouse miR-155) were used.

Mentions: Validation of BIC expression was conducted by real time PCR (RT-PCR) using miRNA/RNA preparations from donors which were independent of the original 10 healthy human volunteers used for the array-based expression profiling. Real-time PCR analysis revealed a 14-fold up-regulation of human BIC upon activation compared resting CD4+ Th cells and resting nTregs (Fig. 2A). This tendency could be validated in mice as well (Fig. 2B). In accordance with results published by Haasch et al 2002 [6], our data show that BIC is hardly detectable in resting human T cells, but is strongly up-regulated rather early upon activation. To determine, if BIC is processed into mature microRNA, murine and human miR-155-specific cDNA was generated and analyzed by Taqman RT-PCR. As depicted in Fig. 2C (human) and Fig. 2D (mouse), BIC was processed into mature miR-155 in both species. Interestingly, resting murine nTregs showed an elevated basal level of the BIC transcript and of mature miR-155 when compared to human resting nTregs. This could be confirmed by the recently published paper of Lu et al. [10]. They also showed a 6-fold higher expression of miR-155 in mouse peripheral FoxP3 positive T cells compared to FoxP3 negative T cells. Assuming that miR-155 will be regulated in mice by the transcription factor FoxP3.


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)

RT-PCR expression analysis of pre-mature BIC transcript and its processed microRNA miR-155 in mice and men.The BIC transcript is strongly up-regulated upon activation using anti-CD3/anti-CD28 mAb in human donors (A) and in C57/BL6 mice (B). Whereas, in C57/BL6 mice the BIC transcript as well as the matured miR-155 was found to be higher expressed in nTregs than in CD4+ Th cells (B) and (D) (n = 3). In human cells, BIC and the matured form miR-155 were not present in resting cells, but strongly elevated levels were found in Th cells as well as in nTreg cells after TCR activation (A) and (C) (n = 4). Analyzing the kinetic of BIC/miR-155 expression RNA was collected using an activation time course experiment. (E) The activation of human T cell populations showed a temporary activation of the BIC transcription. Whereas, the CD4+ Th cells reached their maximum after 4 h upon activation, the peak for nTreg cells is shifted to the 16 h time point. (F) The levels of matured miR-155 were found to permanently increase within time (until 120 h) in activated CD4+ Th cells, whereas in nTregs a plateau was reached after 24 h. All values were calculated as relative fold changes using the ddCT method. As normalizer RNA Pol II (human & mouse BIC) as well as U18 (human miR-155) and 5S (mouse miR-155) were used.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2743997&req=5

pone-0007158-g002: RT-PCR expression analysis of pre-mature BIC transcript and its processed microRNA miR-155 in mice and men.The BIC transcript is strongly up-regulated upon activation using anti-CD3/anti-CD28 mAb in human donors (A) and in C57/BL6 mice (B). Whereas, in C57/BL6 mice the BIC transcript as well as the matured miR-155 was found to be higher expressed in nTregs than in CD4+ Th cells (B) and (D) (n = 3). In human cells, BIC and the matured form miR-155 were not present in resting cells, but strongly elevated levels were found in Th cells as well as in nTreg cells after TCR activation (A) and (C) (n = 4). Analyzing the kinetic of BIC/miR-155 expression RNA was collected using an activation time course experiment. (E) The activation of human T cell populations showed a temporary activation of the BIC transcription. Whereas, the CD4+ Th cells reached their maximum after 4 h upon activation, the peak for nTreg cells is shifted to the 16 h time point. (F) The levels of matured miR-155 were found to permanently increase within time (until 120 h) in activated CD4+ Th cells, whereas in nTregs a plateau was reached after 24 h. All values were calculated as relative fold changes using the ddCT method. As normalizer RNA Pol II (human & mouse BIC) as well as U18 (human miR-155) and 5S (mouse miR-155) were used.
Mentions: Validation of BIC expression was conducted by real time PCR (RT-PCR) using miRNA/RNA preparations from donors which were independent of the original 10 healthy human volunteers used for the array-based expression profiling. Real-time PCR analysis revealed a 14-fold up-regulation of human BIC upon activation compared resting CD4+ Th cells and resting nTregs (Fig. 2A). This tendency could be validated in mice as well (Fig. 2B). In accordance with results published by Haasch et al 2002 [6], our data show that BIC is hardly detectable in resting human T cells, but is strongly up-regulated rather early upon activation. To determine, if BIC is processed into mature microRNA, murine and human miR-155-specific cDNA was generated and analyzed by Taqman RT-PCR. As depicted in Fig. 2C (human) and Fig. 2D (mouse), BIC was processed into mature miR-155 in both species. Interestingly, resting murine nTregs showed an elevated basal level of the BIC transcript and of mature miR-155 when compared to human resting nTregs. This could be confirmed by the recently published paper of Lu et al. [10]. They also showed a 6-fold higher expression of miR-155 in mouse peripheral FoxP3 positive T cells compared to FoxP3 negative T cells. Assuming that miR-155 will be regulated in mice by the transcription factor FoxP3.

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