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. 2015 Jul 21;43(1):52-64.
doi: 10.1016/j.immuni.2015.04.022. Epub 2015 Jul 7.

A Single miRNA-mRNA Interaction Affects the Immune Response in a Context- and Cell-Type-Specific Manner

Li-Fan Lu  1 , Georg Gasteiger  2 , I-Shing Yu  3 , Ashutosh Chaudhry  4 , Jing-Ping Hsin  4 , Yuheng Lu  5 , Paula D Bos  4 , Ling-Li Lin  1 , Carolyn L Zawislak  4 , Sunglim Cho  6 , Joseph C Sun  4 , Christina S Leslie  5 , Shu-Wha Lin  7 , Alexander Y Rudensky  8
Affiliations

A Single miRNA-mRNA Interaction Affects the Immune Response in a Context- and Cell-Type-Specific Manner

Li-Fan Lu et al. Immunity. .

Abstract

MicroRNA (miRNA)-dependent regulation of gene expression confers robustness to cellular phenotypes and controls responses to extracellular stimuli. Although a single miRNA can regulate expression of hundreds of target genes, it is unclear whether any of its distinct biological functions can be due to the regulation of a single target. To explore in vivo the function of a single miRNA-mRNA interaction, we mutated the 3' UTR of a major miR-155 target (SOCS1) to specifically disrupt its regulation by miR-155. We found that under physiologic conditions and during autoimmune inflammation or viral infection, some immunological functions of miR-155 were fully or largely attributable to the regulation of SOCS1, whereas others could be accounted only partially or not at all by this interaction. Our data suggest that the role of a single miRNA-mRNA interaction is dependent on cell type and biological context.

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Figures

Figure 1
Figure 1. De-repression of SOCS1 in mice harboring mutations in the miR-155 target site in the 3′ UTR of the Socs1 gene
(A, B) Western blot analysis of SOCS1 amounts in different immune cell subsets isolated from miR-155KO and SOCS1KI mice. (n = 3–5) (C) Retroviral miR-155 or control miR-150 vectors equipped with a GFP reporter were expressed in T cells from miR-155KO and SOCS1KI mice. GFP+ cells were sorted 4 days after retroviral transduction and the amounts of SOCS1 and Myb proteins were assessed. (D) Immunoblot analysis of total and phospho-Stat5 (pStat5) in SOCS1KI Treg and Tconv cells. Densitometric expression values of SOCS1, Myb, total Stat5 or pStat5 normalized based on β-actin expression values and fold changes are shown below the corresponding lanes. The data are representative of two independent experiments (n = 2–4). See also Figure S1–S2.
Figure 2
Figure 2. SOCS1KI mice did not exhibit reduced Treg cell numbers
FACS analysis of (A) thymus and (B) spleen in 6~8 week old SOCS1KI mice and wild-type littermates. Percentages of different thymocyte and splenocyte subsets are shown. (C–F) Cellularity of the thymus and spleen and the proportion and absolute numbers of thymis and splenic Foxp3+CD4+ T cells in SOCS1KI and WT mice are shown. The data are shown as mean ± SD and are representative of four independent experiments (n = 4–8). See also Figure S3.
Figure 3
Figure 3. miR-155-mediated SOCS1 regulation is critical to maintain normal Treg and Tconv cell numbers in a competitive setting
(A)Ratios of frequencies of CD45.1−Foxp3+ and CD45.1+Foxp3+ Treg cells within each donor-derived CD4+ T cell population from peripheral blood lymphocytes 100 days after BM transfer. (B) Treg cell frequencies within each donor-derived T cell population from both the thymus and spleen 120 days after BM transfer. (C) Ratios of frequencies of thymic and splenic CD45.1-Foxp3+ and CD45.1+Foxp3+ Treg cells within each donor-derived CD4+ T cell population 120 days after BM transfer. Ratios of frequencies of CD45.1−Foxp3− and CD45.1+Foxp3−(D) CD4+ and (E) CD8+ Tconv cells within each donor-derived compartment from peripheral blood lymphocytes 100 days after BM transfer. Each symbol represents an individual mouse, and the data are shown as mean ± SD.
Figure 4
Figure 4. Intermediate EAE disease phenotype in SOCS1KI mice compared to WT and miR-155KO mice
(A)EAE was induced in SOCS1KI, miR-155KO and WT control mice. Their disease severity was scored regularly based upon clinical symptoms. The data are shown as mean clinical scores ± SD and are pooled from 2 independent experiments (n = 10). (B) Frequency of IL-17+ and IFNγ+ T cells in spleen and brain from WT, SOCS1KI and miR-155KO mice induced with EAE. (C) Ratios of frequencies of CD45.1−IL-17+ and CD45.1+IL-17+ cells within each donor-derived CD4+ T cell population in the brain. (D) Ratios of frequencies of CD45.1−Foxp3−CD4+ and CD45.1+Foxp3−CD4+ Teff cells within each donor-derived compartment in the brain. Each symbol represents an individual mouse, and the data are shown as mean ± SD. (E) In vitro proliferative responses of WT, SOCS1KI and miR-155KO splenic CD4+ T cells after restimulation with indicated concentrations of MOG35–55 peptide. T cells were isolated on day 25 after EAE induction and cultured for 72 hours. Their proliferation was measured by 3[H] thymidine incorporation. The data are shown as mean cpm ± SD and are representative of 4 independent experiments (n = 8–12). See also Figure S4–S5.
Figure 5
Figure 5. SOCS1 regulation by miR-155 is dispensable for acute antiviral T cell responses
Mixed BM chimeras were infected with LCMV Armstrong and splenic T cells were analyzed by flow cytometry on d7 of infection. (A) Expression of Ki67 in Foxp3− CD4+ Teff cells. Depicted are representative FACS plots and ratios of the proportions of Ki67+ cells among CD45.2+ CD4+ WT, SOCS1KI or miR155KO cells versus CD45.1+ CD4+ wt controls. (B) Percentage of activated CD44+ CD62L− Foxp3− CD4+ Teff cells. (C) Ratios of IFNγ producing CD4+ Teff cells upon stimulation with LCMV-GP61 peptide. (D, E) Percentage and ratios of LCMV-GP33 and –NP396 specific tetramer binding (D) and IFNγ production of splenic CD8+ T cells (E). (F) CD45.2+ miR-155KO or SOCS1KI and CD45.1+CD45.2+ naïve CD8+ P14 TCR-tg T cells (104 each) were co-transferred into CD45.1+ WT hosts prior to infection with LCMV Armstrong. Splenic T cells were analyzed on d8 pi. The data are shown as mean ± SD and are pooled from 2 independent experiments (n = 7).
Figure 6
Figure 6. Cell type-specific role of miR-155-mediated regulation of SOCS1 during acute viral infection
Mixed BM chimeras were infected with MCMV and splenic T cells were analyzed on d7 pi. (A) Percentage and ratios of MCMV-M45 specific tetramer binding of splenic CD8+ T cells. (B) CD45.2+ miR-155KO or SOCS1KI and CD45.1+ wt NK cells (2×105 each) were co-transferred into Ly49H-deficient hosts prior to infection with MCMV. Ratios of transferred Ly49H+ NK cells were analyzed in the spleen on d7 pi. The data are shown as mean ± SD and are representative of 3 independent experiments (n = 3–5). (C) Scatterplots depict log fold changes of gene expression in SOCS1KI and miR-155KO versus WT CD8+ T cells and Ly49H+ NK cells. Highlighted dots represent genes with significant log fold changes in SOCS1KI (red) or miR-155KO (blue) or both genotypes (black). (D) Bar graphs depict the percentage of genes similarly regulated in both SOCS1KI and miR-155KO from all the genes that were significantly changed in miR-155KO in indicated cell types. Sequencing data represent analysis of 3 biological replicates.
Figure 7
Figure 7. Context-dependent role of miR-155-SOCS1 interaction for antiviral CD8+ T cells
(A, B) CD45.2+ miR-155KO or SOCS1KI and CD45.1+ P14 TCR-tg T cells (104 each) were co-transferred into CD45.1+CD45.2+ WT hosts prior to infection with LCMV Armstrong (Arm) or clone 13. (A) Ratios of P14 T cells in the spleen on d8 pi. (B) Kinetic analysis of P14 T cells in peripheral blood of LCMV clone 13 infected mice. (C) Mixed BM chimeras were infected with MCMV and the percentage and ratios of MCMV-m38 and –m139 specific tetramer binding of splenic CD8+ T cells was analyzed on d40 pi. The data are shown as mean ± SD and are representative of 2–3 independent experiments (n = 4–7). See also Figure S6–S7.
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