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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 TCR-mediated signaling.Human naïve CD4+ T cells were transfected with plasmids encoding either miR-20a or miR-control (miR-Ctrl) and cultered for 16 hours. (A) Cells were stimulated with microbeads coated with CD3 and CD28 mAbs (iAbs) for the indicated time periods. Subsequently, lysates were analyzed by immunoblotting using the indicated Abs. Bands in (A) were quantified using the ImageQuant software and values were normalized to the corresponding β-actin signal. Graphs in (B) show the phosphorylation levels of the indicated molecules as arbitrary units ± SEM of at least 4 independent experiments. (C) CD4+ T cells were incubated with Indo-1AM, stimulated with CD3 and CD28 mAbs, and Ca++ flux was measured by flow cytometry. Ionomycin is used to induce maximum Ca++ flux. Graph in (D) shows quantification of Ca++ flux expressed as arbitrary units ± SEM of at least 3 independent experiments. (E) CD4+ T cells were stimulated with iAbs for the indicated time periods. Subsequently, cell lysates were prepared and CD3ζ immunoprecipitations were analyzed by immunoblotting using the indicated Abs. Bands in (E) were quantified as described above. Graph in (F) shows the levels of CD3ζ-associated Zap-70 as arbitrary units ± SEM of 2 independent experiments. Significat 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.g002: miR-20a inhibits TCR-mediated signaling.Human naïve CD4+ T cells were transfected with plasmids encoding either miR-20a or miR-control (miR-Ctrl) and cultered for 16 hours. (A) Cells were stimulated with microbeads coated with CD3 and CD28 mAbs (iAbs) for the indicated time periods. Subsequently, lysates were analyzed by immunoblotting using the indicated Abs. Bands in (A) were quantified using the ImageQuant software and values were normalized to the corresponding β-actin signal. Graphs in (B) show the phosphorylation levels of the indicated molecules as arbitrary units ± SEM of at least 4 independent experiments. (C) CD4+ T cells were incubated with Indo-1AM, stimulated with CD3 and CD28 mAbs, and Ca++ flux was measured by flow cytometry. Ionomycin is used to induce maximum Ca++ flux. Graph in (D) shows quantification of Ca++ flux expressed as arbitrary units ± SEM of at least 3 independent experiments. (E) CD4+ T cells were stimulated with iAbs for the indicated time periods. Subsequently, cell lysates were prepared and CD3ζ immunoprecipitations were analyzed by immunoblotting using the indicated Abs. Bands in (E) were quantified as described above. Graph in (F) shows the levels of CD3ζ-associated Zap-70 as arbitrary units ± SEM of 2 independent experiments. Significat P values were done by using Student’s t Test. (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Mentions: Following TCR stimulation, two key tyrosine kinases, Lck and Zap-70, initiate signaling leading to gene transcription and T-cell responses [25]. Lck and Zap-70 activation is required for the formation of a signaling complex organized by the transmembrane adaptor protein LAT, which in turn will orchestrate the activation of PLC-γ1. PLC-γ1 generates second messengers mediating Ras-Erk1/2 signaling, PKCθ-CBM, and Ca++-Calcineurin pathways, which result in turn in the activation of AP-1, NF-κB, and NFAT, respectively. To investigate whether miR-20a regulates T-cell activation, we initially performed a biochemical analysis of TCR signaling. Freshly purified naïve CD4+ T cells were transfected with plasmid encoding either miR-20a or miR-control. Subsequently, cells were stimulated with CD3 and CD28 mAbs immobilized on microbeads (iAbs). As shown in Fig 2A and 2B, overexpression of miR-20a inhibits the TCR-mediated phosphorylation of Zap-70, LAT, PLC-γ, and Erk1/2. In addition, overexpression of miR-20a results in the constitutive activation of p38 mitogen- activated protein kinase (Fig 2A and 2B). Moreover, it appears that p38 phosphorylation cannot be further induced upon TCR stimulation in cells overexpressing miR-20a. Given that activated PLCγ-1 mediates Ca++ signaling by generating IP3, a decrease in the phosphorylation of PLCγ-1 upon miR-20a overexpression should also result in the reduction of the release of intracellular Ca++ in response to TCR stimulation. As expected, we have found that miR-20a overexpressing cells showed indeed a reduction in Ca++ flux compared to cells expressing miR-negative control (Fig 2C and 2D). We next assessed whether, miR-20a affects the activation of signaling molecules upstream of Zap-70. However, we did not observe any decrease in the phosphorylation of CD3ζ (Fig 2A and 2B). To test whether the inhibition of TCR-mediated signaling is due to a reduction in the recruitment of Zap-70 to CD3ζ, we performed CD3ζ immunoprecipitations. As shown in Fig 2E and 2F, we observed a slight dicrease in the recruitment of ZAP70 to CD3ζ upon miR-20a overexpression compared to miR-control. Collectively, these data indicate that miR-20a regulates TCR-mediated signaling at the level of Zap-70 activation.


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 TCR-mediated signaling.Human naïve CD4+ T cells were transfected with plasmids encoding either miR-20a or miR-control (miR-Ctrl) and cultered for 16 hours. (A) Cells were stimulated with microbeads coated with CD3 and CD28 mAbs (iAbs) for the indicated time periods. Subsequently, lysates were analyzed by immunoblotting using the indicated Abs. Bands in (A) were quantified using the ImageQuant software and values were normalized to the corresponding β-actin signal. Graphs in (B) show the phosphorylation levels of the indicated molecules as arbitrary units ± SEM of at least 4 independent experiments. (C) CD4+ T cells were incubated with Indo-1AM, stimulated with CD3 and CD28 mAbs, and Ca++ flux was measured by flow cytometry. Ionomycin is used to induce maximum Ca++ flux. Graph in (D) shows quantification of Ca++ flux expressed as arbitrary units ± SEM of at least 3 independent experiments. (E) CD4+ T cells were stimulated with iAbs for the indicated time periods. Subsequently, cell lysates were prepared and CD3ζ immunoprecipitations were analyzed by immunoblotting using the indicated Abs. Bands in (E) were quantified as described above. Graph in (F) shows the levels of CD3ζ-associated Zap-70 as arbitrary units ± SEM of 2 independent experiments. Significat P values were done by using Student’s t Test. (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
© Copyright Policy
Related In: Results  -  Collection

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pone.0125311.g002: miR-20a inhibits TCR-mediated signaling.Human naïve CD4+ T cells were transfected with plasmids encoding either miR-20a or miR-control (miR-Ctrl) and cultered for 16 hours. (A) Cells were stimulated with microbeads coated with CD3 and CD28 mAbs (iAbs) for the indicated time periods. Subsequently, lysates were analyzed by immunoblotting using the indicated Abs. Bands in (A) were quantified using the ImageQuant software and values were normalized to the corresponding β-actin signal. Graphs in (B) show the phosphorylation levels of the indicated molecules as arbitrary units ± SEM of at least 4 independent experiments. (C) CD4+ T cells were incubated with Indo-1AM, stimulated with CD3 and CD28 mAbs, and Ca++ flux was measured by flow cytometry. Ionomycin is used to induce maximum Ca++ flux. Graph in (D) shows quantification of Ca++ flux expressed as arbitrary units ± SEM of at least 3 independent experiments. (E) CD4+ T cells were stimulated with iAbs for the indicated time periods. Subsequently, cell lysates were prepared and CD3ζ immunoprecipitations were analyzed by immunoblotting using the indicated Abs. Bands in (E) were quantified as described above. Graph in (F) shows the levels of CD3ζ-associated Zap-70 as arbitrary units ± SEM of 2 independent experiments. Significat P values were done by using Student’s t Test. (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
Mentions: Following TCR stimulation, two key tyrosine kinases, Lck and Zap-70, initiate signaling leading to gene transcription and T-cell responses [25]. Lck and Zap-70 activation is required for the formation of a signaling complex organized by the transmembrane adaptor protein LAT, which in turn will orchestrate the activation of PLC-γ1. PLC-γ1 generates second messengers mediating Ras-Erk1/2 signaling, PKCθ-CBM, and Ca++-Calcineurin pathways, which result in turn in the activation of AP-1, NF-κB, and NFAT, respectively. To investigate whether miR-20a regulates T-cell activation, we initially performed a biochemical analysis of TCR signaling. Freshly purified naïve CD4+ T cells were transfected with plasmid encoding either miR-20a or miR-control. Subsequently, cells were stimulated with CD3 and CD28 mAbs immobilized on microbeads (iAbs). As shown in Fig 2A and 2B, overexpression of miR-20a inhibits the TCR-mediated phosphorylation of Zap-70, LAT, PLC-γ, and Erk1/2. In addition, overexpression of miR-20a results in the constitutive activation of p38 mitogen- activated protein kinase (Fig 2A and 2B). Moreover, it appears that p38 phosphorylation cannot be further induced upon TCR stimulation in cells overexpressing miR-20a. Given that activated PLCγ-1 mediates Ca++ signaling by generating IP3, a decrease in the phosphorylation of PLCγ-1 upon miR-20a overexpression should also result in the reduction of the release of intracellular Ca++ in response to TCR stimulation. As expected, we have found that miR-20a overexpressing cells showed indeed a reduction in Ca++ flux compared to cells expressing miR-negative control (Fig 2C and 2D). We next assessed whether, miR-20a affects the activation of signaling molecules upstream of Zap-70. However, we did not observe any decrease in the phosphorylation of CD3ζ (Fig 2A and 2B). To test whether the inhibition of TCR-mediated signaling is due to a reduction in the recruitment of Zap-70 to CD3ζ, we performed CD3ζ immunoprecipitations. As shown in Fig 2E and 2F, we observed a slight dicrease in the recruitment of ZAP70 to CD3ζ upon miR-20a overexpression compared to miR-control. Collectively, these data indicate that miR-20a regulates TCR-mediated signaling at the level of Zap-70 activation.

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