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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

Inhibition of miR-20a slightly increases TCR-mediated signaling.Human naïve CD4+ T cells were transfected with miR-20a or scramble decoys. (A) Quantification of miR-20a expression in resting and in stimulated cells upon stimulation with plate-bound CD3 and CD28 mAbs for 45 min was performed using RT qPCR. (B) 16 hours after transfection, cells were stimulated with microbeads coated with CD3 and CD28 mAbs for the indicated time periods. Subsequently, lysates were analyzed by immunoblotting using the indicated Abs. Bands in (B) were quantified using the ImageQuant software and values were normalized to the corresponding β-actin signal. Graphs in (C) show the phosphorylation levels of the indicated molecules as arbitrary units ± SEM of 3 independent experiments. Significat P values were calculated by using Student’s t Test. (*, P < 0.05).
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pone.0125311.g004: Inhibition of miR-20a slightly increases TCR-mediated signaling.Human naïve CD4+ T cells were transfected with miR-20a or scramble decoys. (A) Quantification of miR-20a expression in resting and in stimulated cells upon stimulation with plate-bound CD3 and CD28 mAbs for 45 min was performed using RT qPCR. (B) 16 hours after transfection, cells were stimulated with microbeads coated with CD3 and CD28 mAbs for the indicated time periods. Subsequently, lysates were analyzed by immunoblotting using the indicated Abs. Bands in (B) were quantified using the ImageQuant software and values were normalized to the corresponding β-actin signal. Graphs in (C) show the phosphorylation levels of the indicated molecules as arbitrary units ± SEM of 3 independent experiments. Significat P values were calculated by using Student’s t Test. (*, P < 0.05).

Mentions: In order to shed more light onto the function of miR-20a in TCR-mediated signaling, we performed inhibition experiments using a miR-20a decoy. As shown in Fig 4A, we were able to achieve an efficient suppression (about 80%) of miR-20a in resting CD4+ naïve T cells. However, 45 minutes after TCR stimulation inhibition was less efficient, as decoy-treated CD4+ T cells displayed only 35% reduction of miR-20a expression compared to scrambled controls (Fig 4A). When we assessed TCR-mediated signaling, we observed an increase in the phosphorylation of different signaling molecules upon treatment with miR-20a decoy (Fig 4B and 4C). These data are in agreement with the overexpression data and support the hypothesis that miR-20a is a negative regulator of TCR-mediated signaling. However, the difference between decoy- and scrambled-treated T cells is only minor. Based upon these data, we used the overexpression system for further analyses.


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)

Inhibition of miR-20a slightly increases TCR-mediated signaling.Human naïve CD4+ T cells were transfected with miR-20a or scramble decoys. (A) Quantification of miR-20a expression in resting and in stimulated cells upon stimulation with plate-bound CD3 and CD28 mAbs for 45 min was performed using RT qPCR. (B) 16 hours after transfection, cells were stimulated with microbeads coated with CD3 and CD28 mAbs for the indicated time periods. Subsequently, lysates were analyzed by immunoblotting using the indicated Abs. Bands in (B) were quantified using the ImageQuant software and values were normalized to the corresponding β-actin signal. Graphs in (C) show the phosphorylation levels of the indicated molecules as arbitrary units ± SEM of 3 independent experiments. Significat P values were calculated by using Student’s t Test. (*, P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401545&req=5

pone.0125311.g004: Inhibition of miR-20a slightly increases TCR-mediated signaling.Human naïve CD4+ T cells were transfected with miR-20a or scramble decoys. (A) Quantification of miR-20a expression in resting and in stimulated cells upon stimulation with plate-bound CD3 and CD28 mAbs for 45 min was performed using RT qPCR. (B) 16 hours after transfection, cells were stimulated with microbeads coated with CD3 and CD28 mAbs for the indicated time periods. Subsequently, lysates were analyzed by immunoblotting using the indicated Abs. Bands in (B) were quantified using the ImageQuant software and values were normalized to the corresponding β-actin signal. Graphs in (C) show the phosphorylation levels of the indicated molecules as arbitrary units ± SEM of 3 independent experiments. Significat P values were calculated by using Student’s t Test. (*, P < 0.05).
Mentions: In order to shed more light onto the function of miR-20a in TCR-mediated signaling, we performed inhibition experiments using a miR-20a decoy. As shown in Fig 4A, we were able to achieve an efficient suppression (about 80%) of miR-20a in resting CD4+ naïve T cells. However, 45 minutes after TCR stimulation inhibition was less efficient, as decoy-treated CD4+ T cells displayed only 35% reduction of miR-20a expression compared to scrambled controls (Fig 4A). When we assessed TCR-mediated signaling, we observed an increase in the phosphorylation of different signaling molecules upon treatment with miR-20a decoy (Fig 4B and 4C). These data are in agreement with the overexpression data and support the hypothesis that miR-20a is a negative regulator of TCR-mediated signaling. However, the difference between decoy- and scrambled-treated T cells is only minor. Based upon these data, we used the overexpression system for further analyses.

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