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miR-20a inhibits TCR-mediated signaling and cytokine production in human naïve CD4+ T cells.

Reddycherla AV, Meinert I, Reinhold A, Reinhold D, Schraven B, Simeoni L - PLoS ONE (2015)

Bottom Line: In this study, we have focused our investigation on miR-20a, a member of the miR-17-92 cluster, whose expression is decreased in patients suffering from multiple sclerosis.We have found that miR-20a is rapidly induced upon TCR-triggering in primary human naïve CD4+ T cells and that its transcription is regulated in a Erk-, NF-κB-, and Ca++-dependent manner.However, miR-20a overexpression strongly suppresses IL-10 secretion and moderately decreases IL-2, IL-6 and IL8 production, which are crucial regulators of inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: Otto-von-Guericke University, Institute of Molecular and Clinical Immunology, Leipziger Str. 44, Magdeburg, Germany.

ABSTRACT
Upon TCR stimulation by peptide-MHC complexes, CD4+ T cells undergo activation and proliferation. This process will ultimately culminate in T-cell differentiation and the acquisition of effector functions. The production of specific cytokines by differentiated CD4+ T cells is crucial for the generation of the appropriate immune response. Altered CD4+ T-cell activation and cytokine production result in chronic inflammatory conditions and autoimmune disorders. miRNAs have been shown to be important regulators of T-cell biology. In this study, we have focused our investigation on miR-20a, a member of the miR-17-92 cluster, whose expression is decreased in patients suffering from multiple sclerosis. We have found that miR-20a is rapidly induced upon TCR-triggering in primary human naïve CD4+ T cells and that its transcription is regulated in a Erk-, NF-κB-, and Ca++-dependent manner. We have further shown that overexpression of miR-20a inhibits TCR-mediated signaling but not the proliferation of primary human naïve CD4+ T cells. However, miR-20a overexpression strongly suppresses IL-10 secretion and moderately decreases IL-2, IL-6 and IL8 production, which are crucial regulators of inflammatory responses. Our study suggests that miR-20a is a new player in the regulation of TCR signaling strength and cytokine production.

No MeSH data available.


Related in: MedlinePlus

miR-20a inhibits early T-cell activation events.Human naïve CD4+ T cells were transfected with either miR-20a or miR- control expression plasmids. (A) 16h after transfection, cells were stimulated with immobilized CD3xCD28 mAbs for 6 hours and stained with an APC-conjugated CD69 mAb. Surface expression of CD69 was measured by flow cytometry. One representative experiment of 4 is shown. Graph in (B) represents relative mean fluorescent intensity (MFI) of CD69 expression of miR-20a overexpressing cells compared to miR-control. (C) Alexa Fluor 700 labelled cells were either left unstimulated or stimulated with platebound CD3xCD28 antibodies for 60 hours. Proliferation of cells was then analysed by flow cytometry by measuring the dilution of the fluorochrome by gating on GFP+ cells. Data represent arbitrary units ± SEM of at least 3 independent experiments. Significat P values were done by using Student’s t Test. (***, P < 0.001).
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pone.0125311.g005: miR-20a inhibits early T-cell activation events.Human naïve CD4+ T cells were transfected with either miR-20a or miR- control expression plasmids. (A) 16h after transfection, cells were stimulated with immobilized CD3xCD28 mAbs for 6 hours and stained with an APC-conjugated CD69 mAb. Surface expression of CD69 was measured by flow cytometry. One representative experiment of 4 is shown. Graph in (B) represents relative mean fluorescent intensity (MFI) of CD69 expression of miR-20a overexpressing cells compared to miR-control. (C) Alexa Fluor 700 labelled cells were either left unstimulated or stimulated with platebound CD3xCD28 antibodies for 60 hours. Proliferation of cells was then analysed by flow cytometry by measuring the dilution of the fluorochrome by gating on GFP+ cells. Data represent arbitrary units ± SEM of at least 3 independent experiments. Significat P values were done by using Student’s t Test. (***, P < 0.001).

Mentions: TCR triggering result in a rapid transcription of the CD69 gene, an early T-cell activation marker whose expression depends on the Ras-Erk cascade [26, 27]. As TCR-mediated signaling is defective upon overexpression of miR-20a, we next tested whether miR-20a also affects T-cell activation at a transcriptional level and we measured CD69 expression as a read-out system. As shown in Fig 5A and 5B, overexpression of miR-20a significantly decreased CD69 expression compared to cells transfected with miR-control.


miR-20a inhibits TCR-mediated signaling and cytokine production in human naïve CD4+ T cells.

Reddycherla AV, Meinert I, Reinhold A, Reinhold D, Schraven B, Simeoni L - PLoS ONE (2015)

miR-20a inhibits early T-cell activation events.Human naïve CD4+ T cells were transfected with either miR-20a or miR- control expression plasmids. (A) 16h after transfection, cells were stimulated with immobilized CD3xCD28 mAbs for 6 hours and stained with an APC-conjugated CD69 mAb. Surface expression of CD69 was measured by flow cytometry. One representative experiment of 4 is shown. Graph in (B) represents relative mean fluorescent intensity (MFI) of CD69 expression of miR-20a overexpressing cells compared to miR-control. (C) Alexa Fluor 700 labelled cells were either left unstimulated or stimulated with platebound CD3xCD28 antibodies for 60 hours. Proliferation of cells was then analysed by flow cytometry by measuring the dilution of the fluorochrome by gating on GFP+ cells. Data represent arbitrary units ± SEM of at least 3 independent experiments. Significat P values were done by using Student’s t Test. (***, P < 0.001).
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4401545&req=5

pone.0125311.g005: miR-20a inhibits early T-cell activation events.Human naïve CD4+ T cells were transfected with either miR-20a or miR- control expression plasmids. (A) 16h after transfection, cells were stimulated with immobilized CD3xCD28 mAbs for 6 hours and stained with an APC-conjugated CD69 mAb. Surface expression of CD69 was measured by flow cytometry. One representative experiment of 4 is shown. Graph in (B) represents relative mean fluorescent intensity (MFI) of CD69 expression of miR-20a overexpressing cells compared to miR-control. (C) Alexa Fluor 700 labelled cells were either left unstimulated or stimulated with platebound CD3xCD28 antibodies for 60 hours. Proliferation of cells was then analysed by flow cytometry by measuring the dilution of the fluorochrome by gating on GFP+ cells. Data represent arbitrary units ± SEM of at least 3 independent experiments. Significat P values were done by using Student’s t Test. (***, P < 0.001).
Mentions: TCR triggering result in a rapid transcription of the CD69 gene, an early T-cell activation marker whose expression depends on the Ras-Erk cascade [26, 27]. As TCR-mediated signaling is defective upon overexpression of miR-20a, we next tested whether miR-20a also affects T-cell activation at a transcriptional level and we measured CD69 expression as a read-out system. As shown in Fig 5A and 5B, overexpression of miR-20a significantly decreased CD69 expression compared to cells transfected with miR-control.

Bottom Line: In this study, we have focused our investigation on miR-20a, a member of the miR-17-92 cluster, whose expression is decreased in patients suffering from multiple sclerosis.We have found that miR-20a is rapidly induced upon TCR-triggering in primary human naïve CD4+ T cells and that its transcription is regulated in a Erk-, NF-κB-, and Ca++-dependent manner.However, miR-20a overexpression strongly suppresses IL-10 secretion and moderately decreases IL-2, IL-6 and IL8 production, which are crucial regulators of inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: Otto-von-Guericke University, Institute of Molecular and Clinical Immunology, Leipziger Str. 44, Magdeburg, Germany.

ABSTRACT
Upon TCR stimulation by peptide-MHC complexes, CD4+ T cells undergo activation and proliferation. This process will ultimately culminate in T-cell differentiation and the acquisition of effector functions. The production of specific cytokines by differentiated CD4+ T cells is crucial for the generation of the appropriate immune response. Altered CD4+ T-cell activation and cytokine production result in chronic inflammatory conditions and autoimmune disorders. miRNAs have been shown to be important regulators of T-cell biology. In this study, we have focused our investigation on miR-20a, a member of the miR-17-92 cluster, whose expression is decreased in patients suffering from multiple sclerosis. We have found that miR-20a is rapidly induced upon TCR-triggering in primary human naïve CD4+ T cells and that its transcription is regulated in a Erk-, NF-κB-, and Ca++-dependent manner. We have further shown that overexpression of miR-20a inhibits TCR-mediated signaling but not the proliferation of primary human naïve CD4+ T cells. However, miR-20a overexpression strongly suppresses IL-10 secretion and moderately decreases IL-2, IL-6 and IL8 production, which are crucial regulators of inflammatory responses. Our study suggests that miR-20a is a new player in the regulation of TCR signaling strength and cytokine production.

No MeSH data available.


Related in: MedlinePlus