Limits...
In vivo microRNA-155 expression influences antigen-specific T cell-mediated immune responses generated by DNA vaccination.

Mao CP, He L, Tsai YC, Peng S, Kang TH, Pang X, Monie A, Hung CF, Wu TC - Cell Biosci (2011)

Bottom Line: Biolistic epidermal transfection with DNA encoding miR-155 suppressed the induction of antigen-specific T cell-mediated immunity, whereas reduction of endogenous miR-155 by a partially complementary antisense sequence reversed this effect.Because DCs represent a significant component of epidermal tissue and are among the most potent of antigen-presenting cells, the inhibitory actions of miR-155 could be mediated through this subset of cells.These results suggest that miR-155 may repress the expression of key molecules involved in lymph node migration, antigen presentation, or T cell activation in DCs, and thus forms part of a negative regulatory pathway that dampens the generation of T cell-mediated immune responses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA. wutc@jhmi.edu.

ABSTRACT

Background: MicroRNA (miRNA) molecules are potent mediators of post-transcriptional gene silencing that are emerging to be critical in the regulation of innate and adaptive immunity.

Results: Here we report that miR-155--an oncogenic miRNA with important function in the mammalian immune system--is induced in dendritic cells (DCs) upon maturation and potentially attenuates their ability to activate T cells. Biolistic epidermal transfection with DNA encoding miR-155 suppressed the induction of antigen-specific T cell-mediated immunity, whereas reduction of endogenous miR-155 by a partially complementary antisense sequence reversed this effect. Because DCs represent a significant component of epidermal tissue and are among the most potent of antigen-presenting cells, the inhibitory actions of miR-155 could be mediated through this subset of cells.

Conclusions: These results suggest that miR-155 may repress the expression of key molecules involved in lymph node migration, antigen presentation, or T cell activation in DCs, and thus forms part of a negative regulatory pathway that dampens the generation of T cell-mediated immune responses. Modulation of miR-155 expression in epidermis therefore represents a potentially promising form of gene therapy for the control of diseases ranging from autoimmunity to cancer and viral infection.

No MeSH data available.


Related in: MedlinePlus

Generation and characterization of the ability of I155, a mammalian expression vector encoding sequences partially antisense to miR-155, to restore GFP expression in a DC line cotransfected with bic155and GFP/miR-155as. A, Schematic diagram to depict I155, a DNA plasmid construct that contains 6 tandemly repeated sequences partially antisense to miR-155 cloned into the 3' UTR of red fluorescent protein. The partially complementary sequences of I155 shown in the diagram bind imperfectly to miR-155 to form a 4-nucleotide mismatched bulged site and may serve as a competitive inhibitor of miR-155. B, Fluorescence microscope image at 24 hrs of DC-1 cells transfected with GFP/miR-155as in combination with a control construct encoding non-specific miRNA (panel I), bic155 (panel II) or both bic155 and I155 (panels III and IV). The empty pcDNA3 vector was used to standardize the total amount of transfected DNA. GFP/miR-155as and bic155 transfection complexes were formulated in Lipofectamine 2000 in a 1:9 mass ratio. The transfection efficiency of I155 was measured by the red fluorescent intensity (panel V) and the degree to which I155 restored GFP expression in bic155- and GFP/miR-155as-cotransfected cells (panel IV). To further characterize the I155 construct, DC-1 cells were transfected with 0.1 μg of either RFP/control (panel V) or I155 (panel VI).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3116247&req=5

Figure 4: Generation and characterization of the ability of I155, a mammalian expression vector encoding sequences partially antisense to miR-155, to restore GFP expression in a DC line cotransfected with bic155and GFP/miR-155as. A, Schematic diagram to depict I155, a DNA plasmid construct that contains 6 tandemly repeated sequences partially antisense to miR-155 cloned into the 3' UTR of red fluorescent protein. The partially complementary sequences of I155 shown in the diagram bind imperfectly to miR-155 to form a 4-nucleotide mismatched bulged site and may serve as a competitive inhibitor of miR-155. B, Fluorescence microscope image at 24 hrs of DC-1 cells transfected with GFP/miR-155as in combination with a control construct encoding non-specific miRNA (panel I), bic155 (panel II) or both bic155 and I155 (panels III and IV). The empty pcDNA3 vector was used to standardize the total amount of transfected DNA. GFP/miR-155as and bic155 transfection complexes were formulated in Lipofectamine 2000 in a 1:9 mass ratio. The transfection efficiency of I155 was measured by the red fluorescent intensity (panel V) and the degree to which I155 restored GFP expression in bic155- and GFP/miR-155as-cotransfected cells (panel IV). To further characterize the I155 construct, DC-1 cells were transfected with 0.1 μg of either RFP/control (panel V) or I155 (panel VI).

Mentions: Ebert et al. have recently demonstrated that partially complementary antisense RNA sequences may serve as competitive inhibitors of miRNA [13]. Based on this finding, we designed a mammalian expression vector which contains 6 tandemly repeated miR-155 partially antisense regions cloned into the 3' UTR of the red fluorescence protein (RFP) gene to generate I155. Figure 4A shows a schematic diagram of I155. The miR-155 partially antisense regions have been shown to bind stably to miR-155-RISC and form bulges at the site normally cleaved by Argonaute-2, the catalytic component of RISC, effectively protecting I155 from degradation by the cellular RNA interference machinery [13]. Furthermore, the presence of multiple partially antisense repeats has been shown to enhance the avidity between I155 and miR-155-RISC and enables this miRNA inhibitor to successfully outcompete endogenous miR-155 target genes [13]. I155 contains the RFP gene to facilitate verification of its transfection efficiency as well as the intracellular stability of its encoded RNA.


In vivo microRNA-155 expression influences antigen-specific T cell-mediated immune responses generated by DNA vaccination.

Mao CP, He L, Tsai YC, Peng S, Kang TH, Pang X, Monie A, Hung CF, Wu TC - Cell Biosci (2011)

Generation and characterization of the ability of I155, a mammalian expression vector encoding sequences partially antisense to miR-155, to restore GFP expression in a DC line cotransfected with bic155and GFP/miR-155as. A, Schematic diagram to depict I155, a DNA plasmid construct that contains 6 tandemly repeated sequences partially antisense to miR-155 cloned into the 3' UTR of red fluorescent protein. The partially complementary sequences of I155 shown in the diagram bind imperfectly to miR-155 to form a 4-nucleotide mismatched bulged site and may serve as a competitive inhibitor of miR-155. B, Fluorescence microscope image at 24 hrs of DC-1 cells transfected with GFP/miR-155as in combination with a control construct encoding non-specific miRNA (panel I), bic155 (panel II) or both bic155 and I155 (panels III and IV). The empty pcDNA3 vector was used to standardize the total amount of transfected DNA. GFP/miR-155as and bic155 transfection complexes were formulated in Lipofectamine 2000 in a 1:9 mass ratio. The transfection efficiency of I155 was measured by the red fluorescent intensity (panel V) and the degree to which I155 restored GFP expression in bic155- and GFP/miR-155as-cotransfected cells (panel IV). To further characterize the I155 construct, DC-1 cells were transfected with 0.1 μg of either RFP/control (panel V) or I155 (panel VI).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3116247&req=5

Figure 4: Generation and characterization of the ability of I155, a mammalian expression vector encoding sequences partially antisense to miR-155, to restore GFP expression in a DC line cotransfected with bic155and GFP/miR-155as. A, Schematic diagram to depict I155, a DNA plasmid construct that contains 6 tandemly repeated sequences partially antisense to miR-155 cloned into the 3' UTR of red fluorescent protein. The partially complementary sequences of I155 shown in the diagram bind imperfectly to miR-155 to form a 4-nucleotide mismatched bulged site and may serve as a competitive inhibitor of miR-155. B, Fluorescence microscope image at 24 hrs of DC-1 cells transfected with GFP/miR-155as in combination with a control construct encoding non-specific miRNA (panel I), bic155 (panel II) or both bic155 and I155 (panels III and IV). The empty pcDNA3 vector was used to standardize the total amount of transfected DNA. GFP/miR-155as and bic155 transfection complexes were formulated in Lipofectamine 2000 in a 1:9 mass ratio. The transfection efficiency of I155 was measured by the red fluorescent intensity (panel V) and the degree to which I155 restored GFP expression in bic155- and GFP/miR-155as-cotransfected cells (panel IV). To further characterize the I155 construct, DC-1 cells were transfected with 0.1 μg of either RFP/control (panel V) or I155 (panel VI).
Mentions: Ebert et al. have recently demonstrated that partially complementary antisense RNA sequences may serve as competitive inhibitors of miRNA [13]. Based on this finding, we designed a mammalian expression vector which contains 6 tandemly repeated miR-155 partially antisense regions cloned into the 3' UTR of the red fluorescence protein (RFP) gene to generate I155. Figure 4A shows a schematic diagram of I155. The miR-155 partially antisense regions have been shown to bind stably to miR-155-RISC and form bulges at the site normally cleaved by Argonaute-2, the catalytic component of RISC, effectively protecting I155 from degradation by the cellular RNA interference machinery [13]. Furthermore, the presence of multiple partially antisense repeats has been shown to enhance the avidity between I155 and miR-155-RISC and enables this miRNA inhibitor to successfully outcompete endogenous miR-155 target genes [13]. I155 contains the RFP gene to facilitate verification of its transfection efficiency as well as the intracellular stability of its encoded RNA.

Bottom Line: Biolistic epidermal transfection with DNA encoding miR-155 suppressed the induction of antigen-specific T cell-mediated immunity, whereas reduction of endogenous miR-155 by a partially complementary antisense sequence reversed this effect.Because DCs represent a significant component of epidermal tissue and are among the most potent of antigen-presenting cells, the inhibitory actions of miR-155 could be mediated through this subset of cells.These results suggest that miR-155 may repress the expression of key molecules involved in lymph node migration, antigen presentation, or T cell activation in DCs, and thus forms part of a negative regulatory pathway that dampens the generation of T cell-mediated immune responses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA. wutc@jhmi.edu.

ABSTRACT

Background: MicroRNA (miRNA) molecules are potent mediators of post-transcriptional gene silencing that are emerging to be critical in the regulation of innate and adaptive immunity.

Results: Here we report that miR-155--an oncogenic miRNA with important function in the mammalian immune system--is induced in dendritic cells (DCs) upon maturation and potentially attenuates their ability to activate T cells. Biolistic epidermal transfection with DNA encoding miR-155 suppressed the induction of antigen-specific T cell-mediated immunity, whereas reduction of endogenous miR-155 by a partially complementary antisense sequence reversed this effect. Because DCs represent a significant component of epidermal tissue and are among the most potent of antigen-presenting cells, the inhibitory actions of miR-155 could be mediated through this subset of cells.

Conclusions: These results suggest that miR-155 may repress the expression of key molecules involved in lymph node migration, antigen presentation, or T cell activation in DCs, and thus forms part of a negative regulatory pathway that dampens the generation of T cell-mediated immune responses. Modulation of miR-155 expression in epidermis therefore represents a potentially promising form of gene therapy for the control of diseases ranging from autoimmunity to cancer and viral infection.

No MeSH data available.


Related in: MedlinePlus