miR-182 and miR-10a Are Key Regulators of Treg Specialisation and Stability during Schistosome and Leishmania-associated Inflammation

A diverse suite of effector immune responses provide protection against various pathogens. However, the array of effector responses must be immunologically regulated to limit pathogen- and immune-associated damage. CD4+Foxp3+ regulatory T cells (Treg) calibrate immune responses; however, how Treg cells adapt to control different effector responses is unclear. To investigate the molecular mechanism of Treg diversity we used whole genome expression profiling and next generation small RNA sequencing of Treg cells isolated from type-1 or type-2 inflamed tissue following Leishmania major or Schistosoma mansoni infection, respectively. In-silico analyses identified two miRNA “regulatory hubs” miR-10a and miR-182 as critical miRNAs in Th1- or Th2-associated Treg cells, respectively. Functionally and mechanistically, in-vitro and in-vivo systems identified that an IL-12/IFNγ axis regulated miR-10a and its putative transcription factor, Creb. Importantly, reduced miR-10a in Th1-associated Treg cells was critical for Treg function and controlled a suite of genes preventing IFNγ production. In contrast, IL-4 regulated miR-182 and cMaf in Th2-associed Treg cells, which mitigated IL-2 secretion, in part through repression of IL2-promoting genes. Together, this study indicates that CD4+Foxp3+ cells can be shaped by local environmental factors, which orchestrate distinct miRNA pathways preserving Treg stability and suppressor function

miR-182 and miR-10a Are Key Regulators of Treg Specialisation and Stability during <i>Schistosome</i> and <i>Leishmania</i>-associated Inflammation

<div><p>A diverse suite of effector immune responses provide protection against various pathogens. However, the array of effector responses must be immunologically regulated to limit pathogen- and immune-associated damage. CD4⁺Foxp3⁺ regulatory T cells (Treg) calibrate immune responses; however, how Treg cells adapt to control different effector responses is unclear. To investigate the molecular mechanism of Treg diversity we used whole genome expression profiling and next generation small RNA sequencing of Treg cells isolated from type-1 or type-2 inflamed tissue following <i>Leishmania major</i> or <i>Schistosoma mansoni</i> infection, respectively. <i>In-silico</i> analyses identified two miRNA “regulatory hubs” miR-10a and miR-182 as critical miRNAs in Th1- or Th2-associated Treg cells, respectively. Functionally and mechanistically, in-vitro and in-vivo systems identified that an IL-12/IFNγ axis regulated miR-10a and its putative transcription factor, Creb. Importantly, reduced miR-10a in Th1-associated Treg cells was critical for Treg function and controlled a suite of genes preventing IFNγ production. In contrast, IL-4 regulated miR-182 and cMaf in Th2-associed Treg cells, which mitigated IL-2 secretion, in part through repression of IL2-promoting genes. Together, this study indicates that CD4⁺Foxp3⁺ cells can be shaped by local environmental factors, which orchestrate distinct miRNA pathways preserving Treg stability and suppressor function.</p></div

Differentially expressed miRNAs and candidate miRNA regulatory hubs in Th1- and Th2-Treg cells.

<p>miRNAs with significantly altered expression following (A) <i>Schistosoma mansoni</i> (Sm) or (C) <i>Leishmania major</i> (Lm) infection (Student's t-test p-value<0.05); y-axis: log (fold-change) of miRNA expression level. Representation of predicted targets for up-regulated miRNAs among down-regulated genes following <i>S. mansoni</i> (Sm) infection (B). Representation of predicted targets for down-regulated miRNAs among up-regulated genes following <i>L. major</i> (Lm) infection (D). Y-axis: −log of the empirical p-value of predicted target site enrichment over background expectation. Orange: miRNAs predicted to target differentially expressed genes significantly more than expected by chance, full details in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003451#ppat.1003451.s012" target="_blank">Table S4</a>. Dashed line: p-value = 0.05.</p

miR-182 and miR-10a target a significant number of <i>in-silico</i> predicted targets in Foxp3⁺ Treg cells.

<p>CD4⁺Foxp3⁺ cells isolated from naïve mice and transfected with miR-182 mimics or hairpin inhibitors (A) or miR-10a mimics or hairpin inhibitors (B) to identify predicted target gene regulation. RNA was extracted 24 hours post transfection for analysis. One of 3 individual experiments shown, with 3 biological replicates in each experiment. p-value = 0.05. with data expressed as mean ±SEM.</p

Differential gene expression in CD4⁺Foxp3⁺ cells isolated from chronic <i>S. Mansoni</i> or chronic <i>L. Major</i> infected tissue.

<p>(A) Isolation and FACS sorting of CD4⁺Foxp3⁺ cells from the liver of <i>S. Mansoni</i> or ear of <i>L. Major infected</i> mice. (B) Heat map of differential gene expression showing biological replicates <i>(naïve Treg = 7, S. m. Treg = 4, L. m. Treg = 3 biological replicates)</i> for the isolated Foxp3⁺ populations. 3944 array probes were differentially expressed at a false discovery rate (FDR) less than 0.05. The list of 3944 array probes is provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003451#ppat.1003451.s009" target="_blank">Table S1</a>. (C) Number of common (overlap) and unique genes that were differentially regulated between the Sm-Foxp3⁺ cells and Lm Foxp3⁺ cells, relative to ‘Naïve’ Foxp3+ cells (FDR<0.1 and fold change >1.5). (D) Immunity-associated genes up-regulated in Lm Foxp3⁺ cells. (E) Immunity-associated genes up-regulated in and Sm Foxp3⁺ cells.</p