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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

Expression of miR-20a and miR-20a precursors in human naïve CD4+ T cells.Human naïve CD4+ T cells were stimulated with plate-bound CD3 and CD28 mAbs for the indicated time periods. Quantification of (A) miR-20a and (B) miR-20a precursors (both pri- and pre-miRNAs) was performed using RT qPCR. (C) Naïve CD4+ T cells were also preincubated with DMSO, U0126 (10 μM), IKK VII (200 nM) and EGTA (10 mM) for 30 minutes. Subsequently, samples were either left unstimulated or stimulated with plate-bound CD3 for 4 hours at 37°C. Expression of miR-20a precursors was quantified using RT qPCR. (D) Quantification of miR-20a level upon overexpression. Human naïve CD4+ T cells were transfected with plasmids encoding either miR-20a or miR-control (miR-Ctrl). Cells were either left unstimulated or stimulated for 2 hours with CD3xCD28 mAbs. Quantification of miR-20a in both resting and activated cells was performed using real-time PCR. Unless otherwise mentioned, all the data was normalized to either resting cells or unstimulated control transfected cells. Data represent arbitrary units ± SEM of at least 3 (A, C, D) and 2 (B) independent experiments. Significant P values were done by using Student’s t Test. (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
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pone.0125311.g001: Expression of miR-20a and miR-20a precursors in human naïve CD4+ T cells.Human naïve CD4+ T cells were stimulated with plate-bound CD3 and CD28 mAbs for the indicated time periods. Quantification of (A) miR-20a and (B) miR-20a precursors (both pri- and pre-miRNAs) was performed using RT qPCR. (C) Naïve CD4+ T cells were also preincubated with DMSO, U0126 (10 μM), IKK VII (200 nM) and EGTA (10 mM) for 30 minutes. Subsequently, samples were either left unstimulated or stimulated with plate-bound CD3 for 4 hours at 37°C. Expression of miR-20a precursors was quantified using RT qPCR. (D) Quantification of miR-20a level upon overexpression. Human naïve CD4+ T cells were transfected with plasmids encoding either miR-20a or miR-control (miR-Ctrl). Cells were either left unstimulated or stimulated for 2 hours with CD3xCD28 mAbs. Quantification of miR-20a in both resting and activated cells was performed using real-time PCR. Unless otherwise mentioned, all the data was normalized to either resting cells or unstimulated control transfected cells. Data represent arbitrary units ± SEM of at least 3 (A, C, D) and 2 (B) independent experiments. Significant P values were done by using Student’s t Test. (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Mentions: To investigate whether miR-20a is involved in human naïve CD4+ T-cell activation, we first determined the expression pattern of miR-20a upon TCR triggering. Freshly purified naïve CD4+ T cells from healthy donors were stimulated with plate-bound CD3 and CD28 monoclonal antibodies (mAbs). As shown in Fig 1A, the expression of miR-20a was rapidly (within 2 h) and significantly upregulated after T-cell stimulation. In addition to a rapid induction, the expression of miR-20a was also sustained up to 48 h and decreased thereafter to the basal level (Fig 1A).


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)

Expression of miR-20a and miR-20a precursors in human naïve CD4+ T cells.Human naïve CD4+ T cells were stimulated with plate-bound CD3 and CD28 mAbs for the indicated time periods. Quantification of (A) miR-20a and (B) miR-20a precursors (both pri- and pre-miRNAs) was performed using RT qPCR. (C) Naïve CD4+ T cells were also preincubated with DMSO, U0126 (10 μM), IKK VII (200 nM) and EGTA (10 mM) for 30 minutes. Subsequently, samples were either left unstimulated or stimulated with plate-bound CD3 for 4 hours at 37°C. Expression of miR-20a precursors was quantified using RT qPCR. (D) Quantification of miR-20a level upon overexpression. Human naïve CD4+ T cells were transfected with plasmids encoding either miR-20a or miR-control (miR-Ctrl). Cells were either left unstimulated or stimulated for 2 hours with CD3xCD28 mAbs. Quantification of miR-20a in both resting and activated cells was performed using real-time PCR. Unless otherwise mentioned, all the data was normalized to either resting cells or unstimulated control transfected cells. Data represent arbitrary units ± SEM of at least 3 (A, C, D) and 2 (B) independent experiments. Significant P values were done by using Student’s t Test. (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
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Related In: Results  -  Collection

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pone.0125311.g001: Expression of miR-20a and miR-20a precursors in human naïve CD4+ T cells.Human naïve CD4+ T cells were stimulated with plate-bound CD3 and CD28 mAbs for the indicated time periods. Quantification of (A) miR-20a and (B) miR-20a precursors (both pri- and pre-miRNAs) was performed using RT qPCR. (C) Naïve CD4+ T cells were also preincubated with DMSO, U0126 (10 μM), IKK VII (200 nM) and EGTA (10 mM) for 30 minutes. Subsequently, samples were either left unstimulated or stimulated with plate-bound CD3 for 4 hours at 37°C. Expression of miR-20a precursors was quantified using RT qPCR. (D) Quantification of miR-20a level upon overexpression. Human naïve CD4+ T cells were transfected with plasmids encoding either miR-20a or miR-control (miR-Ctrl). Cells were either left unstimulated or stimulated for 2 hours with CD3xCD28 mAbs. Quantification of miR-20a in both resting and activated cells was performed using real-time PCR. Unless otherwise mentioned, all the data was normalized to either resting cells or unstimulated control transfected cells. Data represent arbitrary units ± SEM of at least 3 (A, C, D) and 2 (B) independent experiments. Significant P values were done by using Student’s t Test. (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
Mentions: To investigate whether miR-20a is involved in human naïve CD4+ T-cell activation, we first determined the expression pattern of miR-20a upon TCR triggering. Freshly purified naïve CD4+ T cells from healthy donors were stimulated with plate-bound CD3 and CD28 monoclonal antibodies (mAbs). As shown in Fig 1A, the expression of miR-20a was rapidly (within 2 h) and significantly upregulated after T-cell stimulation. In addition to a rapid induction, the expression of miR-20a was also sustained up to 48 h and decreased thereafter to the basal level (Fig 1A).

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