The Role of miR-155 in Modulating Gene Expression in CD4+ T Cells: Insights into Alternative Immune Pathways in Autoimmune Encephalomyelitis

Abstract

CD4+ T cells are considered the main orchestrators of autoimmune diseases. Their disruptive effect on CD4+ T cell differentiation and the imbalance between T helper cell populations can be most accurately determined using experimental autoimmune encephalomyelitis (EAE) as an animal model of multiple sclerosis (MS). One epigenetic factor known to promote autoimmune inflammation is miRNA-155 (miR-155), which is significantly upregulated in inflammatory T cells. The aim of the present study was to profile the transcriptome of immunized mice and determine their gene expression levels based on mRNA and miRNA sequencing. No statistically significant differences in miRNA profile were observed; however, substantial changes in gene expression between miRNA-155 knockout (KO) mice and WT were noted. In miR-155 KO mice, mRNA expression in CD4+ T cells changed in response to immunization with the myeloid antigen MOG_35-55. After restimulation with MOG_35-55, increased Ffar1 (free fatty acid receptor 1) and Scg2 (secretogranin-2) expression were noted in the CD4+ T cells of miR-155-deficient mice; this is an example of an alternative response to antigen stimulation.

Keywords: CD4+ T cells; KO mice; MOG; autoimmune disorders; experimental autoimmune encephalomyelitis (EAE); immunization; miR-155.

Figure 1

Scheme of the study.

Figure 2

Mutagenesis strategy for generating miRNA-155-deficient mice. (a) Diagram showing the miR-155 genomic locus (highlighted in yellow) and the entire region targeted for deletion (highlighted in red). (b) Representative PCR genotyping results using described primer pairs to distinguish WT (+/+), heterozygous (+/−), and miR-155 KO mice (−/−). (c) Comparison of positive droplet identification (shown in blue) using QuantaSoft Software, version 1.7. On the top left: Wells from A01 to C01 display the presence of miR-155-3p in splenocytes (A01: WT spleen; B01: heterozygous, C01: KO-miR-155) G01: NTC for miR-155-3p. Respectively, on the top right, wells from D01 to F01, include miR-155-5p results in the same order, with H01 as the NTC for miR-155-5p. The graphs below illustrate quantitative copy numbers of miRNA-155-3p and miRNA-155-5p in splenocytes from WT, heterozygous, and miR-155 KO mice and NTC.

Figure 2

Mutagenesis strategy for generating miRNA-155-deficient mice. (a) Diagram showing the miR-155 genomic locus (highlighted in yellow) and the entire region targeted for deletion (highlighted in red). (b) Representative PCR genotyping results using described primer pairs to distinguish WT (+/+), heterozygous (+/−), and miR-155 KO mice (−/−). (c) Comparison of positive droplet identification (shown in blue) using QuantaSoft Software, version 1.7. On the top left: Wells from A01 to C01 display the presence of miR-155-3p in splenocytes (A01: WT spleen; B01: heterozygous, C01: KO-miR-155) G01: NTC for miR-155-3p. Respectively, on the top right, wells from D01 to F01, include miR-155-5p results in the same order, with H01 as the NTC for miR-155-5p. The graphs below illustrate quantitative copy numbers of miRNA-155-3p and miRNA-155-5p in splenocytes from WT, heterozygous, and miR-155 KO mice and NTC.

Figure 3

(a) Confirmation of the successful deletion of miR-155 in KO mice. (b) The heat map illustrates gene expression patterns in CD4+ T cells from both miR-155-sufficient and -deficient mice (p = 0.05). CD4+ T cells from KO mice were re-stimulated in vitro with the MOG_35-55 antigen. Rows are centered; unit variance scaling is applied to rows. Both rows and columns are clustered using correlation distance and average linkage (39 rows, 4 columns). (c) Volcano plot highlights the differences in gene expression between MOG-stimulated miR-155-deficient CD4+ T cells and WT CD4+ T unstimulated cells.

Figure 3

(a) Confirmation of the successful deletion of miR-155 in KO mice. (b) The heat map illustrates gene expression patterns in CD4+ T cells from both miR-155-sufficient and -deficient mice (p = 0.05). CD4+ T cells from KO mice were re-stimulated in vitro with the MOG_35-55 antigen. Rows are centered; unit variance scaling is applied to rows. Both rows and columns are clustered using correlation distance and average linkage (39 rows, 4 columns). (c) Volcano plot highlights the differences in gene expression between MOG-stimulated miR-155-deficient CD4+ T cells and WT CD4+ T unstimulated cells.

Figure 4

Interactions between genes upregulated in CD4+ T cells from MOG-stimulated miR-155 KO mice and miRNAs. Network generated by miRNet.

Figure 5

Increased Ffar1 and Scg2 expression in MOG stimulated miR-155-deficient CD4+ T cells. Data represent the mean ± SD from three independent experiments, with each variant including 4–5 mice. * p = 0.014 or ** p < 0.0001.

Figure 6

GO analysis of gene expression in MOG restimulated miR-155-deficient CD4+ T cells compared to CD4+ T cells from control mice. Full description of processes: (1) non-membrane spanning protein tyrosine kinase activity; (2) oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NAD(P)H as one donor, and incorporation of one atom of oxygen; (3) G-protein coupled chemoattractant receptor activity.