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microRNA-142 guards against autoimmunity by controlling Treg cell homeostasis and function

['Wei-Le Wang', 'Irell', 'Manella Graduate School Of Biological Sciences', 'Beckman Research Institute', 'City Of Hope', 'Duarte', 'California', 'United States Of America', 'Department Of Systems Biology', 'Ching Ouyang']

Date: 2022-02 

Abstract Regulatory T (T reg ) cells are critical in preventing aberrant immune responses. Posttranscriptional control of gene expression by microRNA (miRNA) has recently emerged as an essential genetic element for T reg cell function. Here, we report that mice with T reg cell–specific ablation of miR-142 (hereafter Foxp3CremiR-142fl/fl mice) developed a fatal systemic autoimmune disorder due to a breakdown in peripheral T-cell tolerance. Foxp3CremiR-142fl/fl mice displayed a significant decrease in the abundance and suppressive capacity of T reg cells. Expression profiling of miR-142–deficient T reg cells revealed an up-regulation of multiple genes in the interferon gamma (IFNγ) signaling network. We identified several of these IFNγ-associated genes as direct miR-142-3p targets and observed excessive IFNγ production and signaling in miR-142–deficient T reg cells. Ifng ablation rescued the T reg cell homeostatic defect and alleviated development of autoimmunity in Foxp3CremiR-142fl/fl mice. Thus, our findings implicate miR-142 as an indispensable regulator of T reg cell homeostasis that exerts its function by attenuating IFNγ responses.

Citation: Wang W-L, Ouyang C, Graham NM, Zhang Y, Cassady K, Reyes EY, et al. (2022) microRNA-142 guards against autoimmunity by controlling T reg cell homeostasis and function. PLoS Biol 20(2): e3001552. https://doi.org/10.1371/journal.pbio.3001552 Academic Editor: Paula M. Oliver, Children’s Hospital of Philadelphia and The University of Pennsylvania School of Medicine, UNITED STATES Received: November 10, 2021; Accepted: January 21, 2022; Published: February 18, 2022 Copyright: © 2022 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The underlying raw data can be found within the paper and its Supporting Information files. The RNA-seq data used in the paper are publicly available from the GEO database (accession number GSE190192). The underlying flow cytometry raw data can be found at the Figshare data repository (https://figshare.com/projects/microRNA-142_guards_against_autoimmunity_by_controlling_Treg_cell_homeostasis_and_function/128960). Funding: This study was funded in part by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health R01 AI125615 (to M. P. B.) and R01 AI146313 (to M. P. B.) grants and the American Association of Immunologists Careers in Immunology Fellowship (to M. P. B. and W. L. W.). The work in the Zeng lab was supported by the National Cancer Institute of the National Institutes of Health R01 CA228465 grant (to D. Z.). Research reported in this publication includes work performed in the Analytical Cytometry, Integrative Genomics and Veterinary Pathology Cores supported by the National Cancer Institute of the National Institutes of Health under P30 CA033572 award (to City of Hope Comprehensive Cancer Center). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: Ab, antibody; aGVHD, acute graft-versus-host disease; BMT, bone marrow transplantation; BrdU, 5-bromo-2-deoxyuridine; CPM, counts per million; CTLA4, cytotoxic T-lymphocyte associated protein 4; CTV, CellTrace Violet; DC, dendritic cell; DP, double positive; FACS, fluorescence activated cell sorting; FDR, false discovery rate; GITR, glucocorticoid-induced tumor necrosis factor receptor family-related gene; GLM, generalized linear model; GSEA, Gene Set Enrichment Analysis; Hif1a, hypoxia-induced factor 1 alpha; HITS-CLIP, high-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation; IACUC, Institutional Animal Care and Use Committee; ICOS, inducible T-cell co-stimulator; IFNγ, interferon gamma; IL, interleukin; KO, knockout; LR, likelihood ratio; miRNA, microRNA; PD-1, programmed death-1; PMA, phorbol 12-myristate 13-acetate; pStat1, phosphorylated Stat1; pT reg , peripheral Treg; qCML, quantile-adjusted conditional maximum likelihood; QLF, quasi-likelihood F; RBC, red blood cell; RIN, RNA integrity number; RNA-seq, RNA sequencing; SP, single positive; T conv , conventional T; TCR, T cell receptor; T eff , T effector; Th1, T helper cell 1; T reg , regulatory T; TMM, trimmed mean of M values; VHL, von Hippel–Lindau; WT, wild-type; YFP, yellow fluorescent protein

Introduction Regulatory T (T reg ) cells are vital in maintaining immune self-tolerance and restraining aberrant immune responses against infections [1,2]. Foxp3, an X chromosome–linked member of the forkhead box/winged helix family of transcription factors, is a master regulator of the genetic program that governs development and suppressive activity of T reg cells. Humans and mice that carry loss-of-function Foxp3 mutations develop a fatal autoimmune disease due to impaired T reg cell activity [3–7]. The majority of T reg cells are generated in the thymus (tT reg cells) through a selection process that favors cells with a strong functional T cell receptor (TCR) avidity toward self-antigens. In contrast, peripheral T reg (pT reg ) cells arise from naive CD4+ T cells upon encounter of non–self-antigens in the context of appropriate cytokine stimulation [8–10]. Harnessing the power of T reg cells to control immunological responses has a great potential for human therapy because, on one hand, T reg cells can promote transplantation tolerance, but on the other, can hinder antitumor immunity. Posttranscriptional regulation of gene expression by microRNA (miRNA), a class of small (approximately 22 nucleotides) noncoding RNA, recently emerged as a critical genetic element that is essential for T reg cell function. T reg cell–specific knockouts (KOs) of either Drosha or Dicer genes, encoding 2 endonucleases required for mature miRNA generation from precursor transcripts, phenocopy mice with Foxp3 ablation, and develop severe systemic autoimmunity because of a defect in T reg cell activity [11–13]. Furthermore, deletion of Dicer at the double positive (DP) thymocyte stage in mice significantly diminished the frequency of tT reg cells, suggesting that miRNA-dependent gene control is also required for normal development of T reg cells [11]. Thus, the current pressing challenge in the field is to determine how specific miRNA gene(s) exert control of the T reg cell genetic program. The present report addresses this goal by examining the role of miR-142 in T reg cell development and function. miR-142 is predominantly expressed in cells of hematopoietic origin and encodes 2 abundant mature miRNA molecules—miR-142-5p and miR-142-3p—which arise from the opposite strands of the hairpin-like miR-142 precursor. Using genetic loss-of-function studies, miR-142 was previously implicated in the regulation of ontogenesis and function of several immune cell types. Our earlier report determined that deletion of this miRNA gene in mice results in aberrant B lymphopoiesis and impaired humoral immunity [14]. In addition, miR-142–deficient mice develop thrombocytopenia stemming from defective megakaryocyte maturation [15] and exhibit dysregulation of dendritic cell (DC) function [16]. Disruption of 2 miR-142 paralog genes in zebrafish using zinc-finger nucleases was reported to cause aberrant neutrophil differentiation [17]. In the T-cell compartment, miR-142 is required for the homeostasis of peripheral T effector (T eff ) cells, but is apparently dispensable for conventional T (T conv ) cell development in the thymus [14,18,19]. A recent study by Anandagoda and colleagues has demonstrated that miR-142 is essential for the immunosuppressive activity of T reg cells, but failed to reveal a significant role fot this miRNA in T reg cell development and homeostasis [20]. The authors suggest that posttranscriptional repression of the cAMP-hydrolyzing enzyme Pde3b by miR-142-5p isoform plays a key role in the regulation of the T reg cell suppressive function. Here, we show that mice with a conditional deletion of miR-142 in T reg cells develop severe autoimmune disease due to a profound defect in T reg cell homeostasis and function. In addition, our findings suggest that miR-142 plays an important role in tT reg cell development. We have determined that miR-142-3p isoform and its capacity to silence multiple interferon gamma (IFNγ)-associated genes play a critical role in mediating the regulatory activity of miR-142 in T reg cells. Global ablation of IFNγ rescues the T reg cell defect and autoimmunity in Foxp3CremiR-142fl/fl mice, thus providing further evidence for the essential role of the miR-142-IFNγ signaling pathway in the regulation of T reg cell homeostasis and function.

Discussion Although miRNA-mediated posttranscriptional control of gene expression is recognized as crucial for T reg cell development and function [11–13], our understanding of how specific miRNA genes govern T reg cell responses is incomplete. Here, we report that mice with T reg cell–specific miR-142 deletion develop a fatal systemic autoimmune disease due to a severe defect in T reg cell homeostasis and suppressive activity. Furthermore, we found that constitutive miR-142 ablation results in aberrant thymic T reg cell development. Thus, our findings have uncovered an indispensable role for miR-142 in the control of T reg cell–mediated immunological tolerance. We propose that miR-142 plays a dominant role among miRNAs involved in the regulation of T reg cell activity because the phenotype of Foxp3CremiR-142fl/fl mice closely resembles the autoimmune pathology observed in mice with global disruption of miRNA biogenesis in T reg cells [11–13]. This notion should be confirmed by further conditional KO studies of several miRNA genes that were previously implicated in the regulation of T reg cell function, including miR-146a, miR-155, and miR-27 [22,36,37]. The role of miR-142 in T reg cell function was previously examined by Anandagoda and colleagues using a conditional KO mouse model [20]. In contrast with the findings from this report, we determined that deletion of miR-142-3p and not miR-142-5p (as proposed by Anandagoda and colleagues) is the major driver of the T reg cell defect and subsequent systemic autoimmunity in Foxp3CremiR-142fl/fl mice. This conclusion is strongly supported by the observed global derepression of miR-142-3p target genes in miR-142–deficient T reg cells, whereas the levels of the majority of miR-142-5p targets did not significantly change. Our inference of a critical role for miR-142-3p in T reg cells is well aligned with the fact that miR-142-3p is more abundantly expressed in T reg cells than miR-142-5p and a large body of literature that assigns the main regulatory role in immune cells to miR-142-3p [14–18,28–30]. Our data revealing that miR-142-3p can directly bind and regulate the phosphodiesterase Pde3b gene, through which, as Anandagoda and colleagues suggest [20], miR-142 controls T reg cell immunosuppressive activity, further validates miR-142-3p as the key miR-142 isoform in T reg cells. Our findings indicate that miR-142-3p controls T reg cell homeostasis and function by attenuating IFNγ production and signaling. Interestingly, dysregulation of IFNγ responses is increasingly recognized as a critical factor that negatively impacts T reg cell activity. For example, excessive IFNγ production was reported to drive functional “fragility” of T reg cells in the context of antitumor immunity [38]. Additionally, conditional deletion of the E3 ubiquitin ligase von Hippel–Lindau (Vhl) gene was shown to impair T reg cell function through IFNγ dysregulation [35]. Moreover, unrestrained Stat1 activation and subsequent IFNγ production by Socs1-deficient T reg cells was linked to a severe failure of immunological tolerance [22]. Finally, a loss of functional activity by Dicer-deficient T reg cells was associated with excessive IFNγ production [13]. miRNAs, despite eliciting a moderate effect on the expression of their target genes, often have a significant impact on cellular physiology through coordinated and coherent targeting of multiple key molecules in a signaling cascade [39]. In agreement with this notion, we found that miR-142 is predicted to control expression of several IFNγ-associated genes, including Ifngr2, Gbp3, Stat1, Irf1, and Hif1a and validated some of these as bona fide miR-142-3p targets. We propose that coordinated derepression of these target genes in miR-142–deficient T reg cells drives the observed dysregulation of IFNγ responses. In support of this hypothesis, we found that genetic blockade of IFNγ production rescues the homeostatic defect in miR-142–deficient T reg cells and prevents development of systemic lymphoproliferative and autoimmune disorder in Foxp3CremiR-142fl/fl mice. Despite a complete restoration of normal T reg cell frequency in Foxp3CremiR-142fl/flIfng−/− mice, the partial rescue of peripheral T eff cell hyperactivation in these mice suggests a possibility that miR-142–mediated control of T reg cell function is not limited to the attenuation of IFNγ signaling and likely involves additional molecular targets. Another caveat of our genetic epistasis experiments using Foxp3CremiR-142fl/flIfng−/− mice is a potential non–cell-autonomous effect of IFNγ deletion on miR-142–deficient T reg cells. Because we found a significant IFNγ up-regulation in both miR-142–deficient T reg cells and miR-142–sufficient T eff cells in Foxp3CremiR-142fl/fl mice, the global IFNγ ablation might potentially impact miR-142–deficient T reg cell function in cell-extrinsic manner via changes in their IFNγ-rich inflammatory environment. Future analysis of miR-142–sufficient and miR-142–deficient T reg cells from animals that lack IFNγ expression such as Foxp3CremiR-142fl/flIfng−/− and female Foxp3Cr/+miR-142fl/fl mice will be required to corroborate the intrinsic effect of IFNγ on miR-142-deficient T reg cells and uncover the additional signaling pathways through which miR-142 mediates its regulatory functions in T reg cells. Our investigation of Hif1a, a validated miR-142-3p target, revealed an important role for the miR-142-Hif1a axis in the regulation of T reg cell homeostasis. We observed that lowering the Hif1a gene dose in miR-142–deficient T reg cells partially rescued the T reg homeostatic defect and modestly reduced the hyperactivation of peripheral T eff cells in Foxp3CremiR-142fl/fl mice. The role suggested by our findings for Hif1a as a negative regulator of T reg cell homeostasis is consistent with the conclusions of 2 previous reports [35,38]. However, Hif1a haploinsufficiency had little impact on the dysregulated IFNγ production in miR-142–deficient T reg cells and failed to prevent development of fatal autoimmunity in Foxp3CremiR-142fl/fl mice. This outcome is not surprising given the fact that the T reg cell number in Foxp3CremiR-142fl/flHif1a+/fl mice is only partially restored. The failure of Hif1a haploinsufficiency to fully rescue the T reg cell defect in Foxp3CremiR-142fl/fl mice is probably linked to the abundance of miR-142-3p target genes in the IFNγ signaling pathway. The existence of multiple miR-142-3p targets likely makes the dysregulated state of IFNγ signaling in miR-142–deficient T reg cells refractory to changes in the expression of a single target. This conclusion is supported by studies in miR-142-3p-deficient zebrafish, which display impaired myelopoiesis due to an aberrant activation of the IFNγ signaling pathway [17]. This developmental defect in miR-142-3p-deficient zebrafish could be rescued by a compound knockdown of stat1a and irf1b genes, whereas silencing of either factor alone was insufficient to restore normal neutrophil differentiation. Of note, based on the observations of dysregulated IFNγ signaling in miR-142–deficient immune cells from zebrafish and rodents, the role of miR-142 in suppressing IFNγ signaling appears to be evolutionary conserved. In contrast, the function of miR-142-Pde3b signaling axis is unlikely to be evolutionary preserved, because miR-142-3p and miR-142-5p binding sites in the mouse Pde3b 3′ UTR are poorly conserved. In summary, our results have established miR-142 as a central regulator of T reg cell development, homeostasis, and suppressive activity that mediates its function in T reg cells in part by limiting IFNγ production and responsiveness. Besides advancing our understanding of the T reg cell biology, these novel insights may open a new avenue for targeted pharmacological manipulation of T reg cell activity in cancer immunotherapy and autoimmune disease settings.

Materials and methods Mice C57BL/6J (stock#000664), B6/CD45.1 (stock#002014), Hif1afl/fl (stock#007561), and Foxp3YFP-Cre (stock#016959) mice were purchased from the Jackson Laboratory. BALB/c mice were obtained from Charles River Laboratories. miR-142fl/fl mice were described previously [14]. Foxp3CremiR-142fl/fl mice were generated by crossing miR-142fl/fl mice with Foxp3YFP-Cre deleter/reporter mice [21]. Mice were kept in a specific pathogen-free facility at the City of Hope Animal Resource Center, and all animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the City of Hope. Our approved animal protocols that are relevant for this work are the following: IACUC#13021, IACUC#13020, and IACUC#03008. Flow cytometry For surface marker analysis, single cell suspensions from thymus, spleen, and peripheral lymph nodes (axillary, brachial, inguinal, and cervical) were treated with red blood cell (RBC) lysis buffer (BioLegend) to eliminate mature erythrocytes and then blocked with anti-CD16/CD32 antibody (Ab) to prevent nonspecific binding. Cells were stained with monoclonal fluorophore-conjugated antibodies against specific cell surface markers, such as anti-CD4 (clone RM4-5), anti-CD8α (clone 53–6.7), anti-CD25 (clone PC61), anti-CD44 (clone IM7), anti-CD62L (clone MEL-14), anti-CD45.1 (clone A20), anti-ICOS (clone 7E.17G9), anti-GITR (clone DTA-1), anti-CTLA-4 (clone UC10-4B9), anti-PD-1 (clone 29F.1A12), and anti-CD103 (clone 2E7) antibodies (all from BioLegend). Intracellular staining with anti-Foxp3 (clone FJK-16s; eBioscience) and anti-Hif1α (clone 241812; R&D Systems) antibodies was performed using fixed and permeabilized cells following the manufacturer’s protocol. For detection of phosphorylated Stat1 (pStat1), purified CD4+ T cells were first stimulated with IFNγ (100 ng/mL) for 24 hours, fixed with Cytofix buffer and permeabilized with Phosflow Perm Buffer III, and subsequently stained with anti-pStat1 (Y701; clone 4a; BD Biosciences) antibodies. For intracellular cytokine staining, T cells derived from spleen and lymph nodes or purified T reg cells were first stimulated with phorbol 12-myristate 13-acetate (PMA; 50 ng/mL) and ionomycin (500 ng/mL) for 4 hours in the presence of monensin (2 μM) and then fixed, permeabilized, and stained following the manufacturer’s protocol (BioLegend). Data were acquired on Accuri C6 flow cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar). Cell sorting for the analysis of miR-142 expression in T-cell lineage was performed using FACSAriaIII (BD Biosciences) instrument. All flow cytometry raw data generated in this study can be found at the Figshare data repository (https://figshare.com/projects/microRNA-142_guards_against_autoimmunity_by_controlling_Treg_cell_homeostasis_and_function/128960). Global transcriptome profiling by RNA-seq CD4+YFP+ T reg cells were sorted using BD FACSAria II machine from single-cell splenocyte suspensions derived from 8- to 11-week-old Foxp3Cre and Foxp3CremiR-142fl/fl female mice (n = 3 per group). Total RNA from purified WT and miR-142–deficient T reg cells was isolated using miRNeasy kit (QIAGEN) and subjected to RNA-seq. RNA quality was determined with an Agilent Bioanalyzer (RNA integrity number (RIN) > 7.5 for all samples). Library was prepared according to the manufacturer’s protocol using Illumina TruSeq RNA Library Prep Kit v2 (San Diego, Califonia, USA) and subsequently loaded on an Illumina HiSeq 2500 for parallel sequencing. The 51 base pair single-ended sequence reads were mapped to the mouse reference genome (mm10) using the alignment program HISAT (https://daehwankimlab.github.io/hisat2). Gene expression was measured from alignment bam files by read counting function featureCounts in the Bioconductor package Rsubread (http://bioconductor.org/packages/Rsubread). The unstranded raw counts were then normalized using a trimmed mean of M values (TMM) method implemented in the Bioconductor package edgeR (https://bioconductor.org/packages/edgeR). A total of 11,658 genes having counts per million (CPM) values higher than 1 in at least 3 samples were included in the downstream differential expression analysis. Differentially expressed genes were tested using 3 statistical methods in edgeR, including the generalized linear model (GLM) quasi-likelihood F (QLF) test, the likelihood ratio (LR) test, and the exact test based on quantile-adjusted conditional maximum likelihood (qCML) methods. A complete list of genes and the statistical test results (miR-142–deficient (KO) versus miR-142–sufficient (WT) T reg cells) are shown in S1 Table. Statistical P values were adjusted by Benjamini–Hochberg method for false discovery rate (FDR) controls. The volcano plot visualizing the distribution of differentially expressed genes was based on QLF test results. The RNA-seq raw data were deposited in the Gene Expression Omnibus under the accession number GSE190192. Pathway and GSEA Differentially expressed genes passing the criterion of FDR lower than 0.05 for all 3 statistical methods mentioned above were subjected for pathway and GSEA using Enrichr (http://amp.pharm.mssm.edu/Enrichr) software algorithm [31]. Top ranked pathways across major databases, including Panther (http://www.pantherdb.org), Reactome (https://reactome.org), KEGG (https://www.genome.jp/kegg), and WikiPathways (https://www.wikipathways.org) were identified and are listed in S2 Table. In addition, GSEA analysis (http://software.broadinstitute.org/gsea/index.jsp)) [32] was performed with the CPM values from the 11,658 expressed genes rank-ordered by Signal2Noise metric. Gene ontology gene sets from Molecular Signatures Database (MSigDB) v7.1 were evaluated for the enrichment. Sylamer analysis Analysis of miRNA seed enrichment in the 3′ UTRs of genes that are differentially expressed in miR-142 KO T reg cells was performed by the Web-based SylArray software algorithm (http://www.ebi.ac.uk/enright-srv/sylarray)) [26]. In vitro T reg cell suppression assay CD4+ T cells were isolated from Foxp3Cre, Foxp3CremiR-142fl/fl, and Foxp3CremiR-142fl/flHif1afl/fl spleens using anti-CD4-biotin (clone GK1.5), anti-Biotin MicroBeads (Miltenyi), LS columns (Miltenyi), and a MiniMACS Separator (Miltenyi). Enriched CD4+ T cells were sorted for YFP+(T reg ) and YFP−(T con ) cells using BD FACS Fusion. Moreover, 105 Foxp3Cre CD4+YFP− T con cells were labeled with 4μM CellTrace Violet (CTV) (Thermo Fisher) at 37°C for 7.5 minutes and cultured with either Foxp3CremiR-142fl/fl or Foxp3CremiR-142fl/flHif1afl/fl CD4+YFP+ T reg cells at ratios of 1:0.5 and 1:0.125 in the presence of CD3ɛ/CD28-conjugated MACSiBead Particles (mouse T Cell Activation/Expansion Kit, Miltenyi) at a bead-to-cell ratio of 2:1. After 3 days of culturing, proliferation of Foxp3Cre CD4+YFP− T con cells was measured by flow cytometry as CTV dilution. In vitro T reg survival assay CD4+ T cells isolated from Foxp3Cre and Foxp3CremiR-142fl/fl spleens by EasySep Mouse CD4+ T Cell Isolation Kit (STEMCELL Technologies) were sorted for YFP expressing cells using BD FACSAria II instrument. CD4+YFP+ T reg cells were stimulated with anti-CD3 (1 μg/mL) specific antibodies for 48 hours, stained with Annexin V-PE (BioLegend) and analyzed by flow cytometry. For the IFNγ-induced apoptosis of T reg cells assay, splenocytes were cultured for 24 hours in the presence or absence of IFNγ (10 ng/mL) and then harvested and stained with Annexin V-PE for fluorescence activated cell sorting (FACS) analysis. In vivo labeling of T reg cells with BrdU Foxp3Cre and Foxp3CremiR-142fl/fl mice were injected intraperitoneally with 1 mg of BrdU and splenic BrdU+ T reg cells (CD4+YFP+) were quantified by flow cytometry 16 hours postinjection. Intracellular staining with anti-BrdU specific antibodies was performed using BrdU Flow Kit (BD Biosciences). aGVHD mouse model BALB/c recipient mice were lethally irradiated (850 cGy) 8 to 10 hours prior to bone marrow transplantation (BMT) and subsequently transplanted via intravenous injection with T-cell–depleted bone marrow from C57BL/6J mice (TCD-BM, 2.5 × 106 cells), Thy1.2+CD4+ T cells from C57BL/6J spleens (0.5 to 0.6 × 106 cells), and CD4+YFP+ T reg cells derived from either Foxp3Cre or Foxp3CremiR-142fl/fl spleens (0.1 to 0.12 × 106 cells). Body weight and severity of diarrhea in host mice were monitored and recorded for 15 days after BMT. Donor T reg cell frequency in the host spleen was assessed by FACS on day 17 after transplantation. miRNA qRT-PCR Total RNA was isolated using miRNeasy kit (QIAGEN) and reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies). Mature miR-142-3p expression was assessed by TaqMan Real-Time miRNA assay (Life Technologies) and normalized to snoRNA234 levels. 3′ UTR luciferase reporter assays DNA fragments encompassing WT and miR-142-3p seed mutated 3′ UTRs of mouse Hif1a, Ifngr2, and Pde3b genes were synthesized and cloned into pMIR-Report vector (Ambion). The sequence complementary to the miR-142-3p.2 seed sequence (8-mer) in the Ifngr2 3′ UTR was mutated from 5′-AACACTAA-3′ to 5′-ACGTACCA-3′. The sequence complementary to the miR-142-3p.2 seed sequence (8-mer) in the Hif1a 3′ UTR was mutated from 5′- AACACTAA-3′ to 5′- ACGTACCA-3′. The sequence complementary to the miR-142-3p.1 seed sequence (6-mer) in the Pde3b 3′ UTR was mutated from 5′- CACTAC-3′ to 5′- CCAGCC-3′. To perform the 3′ UTR reporter assays, 105 293T cells in 24-well plates were transiently transfected using calcium phosphate with 10 ng of pMIR-Report-3′ UTR firefly luciferase plasmid, 20 ng of Renilla luciferase reporter plasmid (pRL-SV40; Promega), and 450 ng of either pMDH1-miR-142 or control pMDH1 vector (Addgene). Cells were lysed 48 hours posttransfection, and the luciferase activities were assessed using Dual Luciferase Reporter assay (Promega). IFNγ ELISA CD4+ T cells purified from Foxp3Cre and Foxp3CremiR-142fl/fl splenocytes with the help of EasySep Mouse CD4+ T Cell Isolation Kit (STEMCELL Technologies) were sorted for YFP expressing cells using BD FACSAria II instrument. CD4+YFP+ T reg cells (106/mL) were stimulated with anti-CD3 (5 μg/mL) and anti-CD28 (2 μg/mL) specific antibodies for 48 hours in the presence of IL-2 (50 ng/mL) and cell culture supernatants were assessed for IFNγ production by sandwich ELISA. Briefly, 96-well flat-bottom Maxisorp (Nunc) plates were coated with anti-mouse IFNγ-specific capture antibodies (eBioscience, clone XMG1.2), and the captured mouse IFNγ was detected with biotin labeled anti-mouse IFNγ-specific antibodies (eBioscience, clone R4-6A2). Mouse recombinant IFNγ (eBioscience) was used as a standard to quantify the results. Histopathology For histological sectioning, mouse tissues were collected and placed into 10% formalin, fixed for 24 hours, washed, and transferred to 70% ethanol before standard paraffin embedding. Tissue sections were stained with hematoxylin and eosin and examined by an experienced veterinary pathologist. Statistical analysis All statistical analyses were performed using Prism 6 (GraphPad) software. Statistical analyses were performed using 2-tailed Student t test or ANOVA. Results were considered significant when P ≤ 0.05.

Supporting information S1 Fig. miR-142 expression in T-cell lineage and analysis of germline (miR-142−/−) and T reg cell–specific (Foxp3CremiR-142fl/fl) miR-142 KO mice. (A) Impaired T reg cell development in miR-142−/− mice. Left panel, FACS analysis of WT and miR-142−/− thymocytes with anti-CD4 and anti-Foxp3 antibodies. Foxp3+CD4+ T reg cells are gated and numbers indicate the percentage of cells in the gate. Right panel, frequency of CD4+Foxp3+ T reg cells in WT and miR-142−/− thymi (n = 3 per group). (B, C) T reg cell defect in the periphery of miR-142−/− mice. FACS analysis of T reg cells in spleen (B) and MLNs (C) from WT and miR-142−/− mice. Right panel, frequency of CD4+Foxp3+ T reg cells in WT and miR-142−/− spleens and MLNs (n = 4 per group). (D) qRT-PCR analysis of mature miR-142-3p and miR-142-5p expression in different T-cell subsets purified from Foxp3Cre mice (n = 2). DN, double negative CD4-CD8- thymocytes; DP, double positive CD4+CD8+ thymocytes; SP, single positive CD4+YFP− thymocytes; T reg , CD4+YFP+ T reg cells from thymus and spleen, respectively; T Naive , naive CD4+YFP−CD62L+CD44− splenic T cells; T Activated , activated CD4+YFP−CD62L−CD44+ splenic T cells. Expression level of miR-142-3p in DN population was arbitrarily set to 1. snoRNA234 levels were used for normalization. (E) qRT-PCR analysis of mature miR-142-3p expression in CD4+YFP+ T reg and CD4+YFP− T eff cells purified from Foxp3Cre and Foxp3CremiR-142fl/fl spleens. Expression level of miR-142-3p in CD4+YFP+ T reg cells isolated from Foxp3CremiR-142fl/fl spleen was arbitrarily set to 1. snoRNA234 levels were used for normalization. Spleen (F) and thymus (G) weights in 8- to 11-week-old male Foxp3Cre and Foxp3CremiR-142fl/fl mice (n ≥ 7 per group). Absolute cell counts in spleen (H), thymus (I), and peripheral lymph nodes (J) from Foxp3Cre and Foxp3CremiR-142fl/fl mice (n = 6 per group). Results are shown as mean ± SD. P values were calculated using 2-tailed Student t test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, not significant. The underlying numerical raw data can be found in S1 Data file. The underlying flow cytometry raw data can be found at the Figshare repository. FACS, fluorescence activated cell sorting; KO, knockout; MLN, mesenteric lymph node; PLN, peripheral lymph node; SD, standard deviation; SP, spleen; T reg , regulatory T; WT, wild-type; YFP, yellow fluorescent protein. https://doi.org/10.1371/journal.pbio.3001552.s001 (PDF) S2 Fig. Characterization of T reg and T eff cell defects in Foxp3CremiR-142fl/fl mice. (A) FACS analysis of lymphocytes from PLNs (left panel) and thymus (right panel) of 12-week-old Foxp3Creand Foxp3CremiR-142fl/fl mice with anti-CD4 and anti-Foxp3 specific antibodies. Foxp3+CD4+ T reg cells are gated and numbers indicate the percentage of cells in the gate. (B) Frequency (left panel) and total number (right panel) of T reg cells in PLNs and thymi of 12-week-old Foxp3Cre (red bars) and Foxp3CremiR-142fl/fl (blue bars) mice (n = 3 per group). (C) FACS analysis of CD44 and CD62L expression on CD4+ (upper panel) and CD8+ (bottom panel) T cells from Foxp3Cre and Foxp3CremiR-142fl/fl PLNs. Numbers indicate percentage of cells in the quadrants. (D) Frequency of CD44−CD62L+ (naive) and CD44+CD62L− (activated) CD4+ (left panel) and CD8+ (right panel) T cells in Foxp3Cre and Foxp3CremiR-142fl/fl PLNs (n = 6 per group). (E) Intracellular FACS analysis of IFNγ production by CD4+ (upper panel) and CD8+ (bottom panel) T cells from Foxp3Cre and Foxp3CremiR-142fl/fl PLNs. IFNγ+ T cells are gated and numbers indicate percentage of cells in the gate. (F) Frequency of IFNγ-expressing CD4+ and CD8+ T cells in Foxp3Cre (filled bars) and Foxp3CremiR-142fl/fl (open bars) PLNs (n = 4 per group). (G) Total CD4+ and CD8+ T cell counts in Foxp3Cre (filled bars) and Foxp3CremiR-142fl/fl (open bars) PLNs (n = 6 per group). (H) Frequencies of IFNγ-, IL-4-, and IL-17-expressing CD4+ T cells isolated from Foxp3Cre (filled bars) and Foxp3CremiR-142fl/fl (open bars) spleens (n ≥ 3 per group). Results are shown as mean ± SD. P values were calculated using 2-tailed Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, not significant. The underlying numerical raw data can be found in S1 Data file. The underlying flow cytometry raw data can be found at the Figshare repository. FACS, fluorescence activated cell sorting; IFNγ, interferon gamma; IL, interleukin; PLN, peripheral lymph node; SD, standard deviation; T reg , regulatory T. https://doi.org/10.1371/journal.pbio.3001552.s002 (PDF) S3 Fig. Immunophenotyping of miR-142–deficient T reg cells. (A) FACS analysis of T reg suppression activity in vitro. Purified CD4+ T conv cells were loaded with CTV dye and incubated with FACS-sorted T reg cells from Foxp3Cre (red line) and Foxp3CremiR-142fl/fl (blue line) spleens in the presence of beads coated with anti-CD3 and anti-CD28 specific antibodies. Several T reg to T conv cell ratios were shown as indicated. (B) Left panel, FACS analysis of Annexin V-stained CD4+YFP+ T reg cells from Foxp3Cre (WT) or Foxp3CremiR-142fl/fl (KO) spleens. Right panel, MFI of Annexin V staining on T reg cells from Foxp3Cre (WT) or Foxp3CremiR-142fl/fl (KO) spleens. (C) Frequency of splenic YFP+ T reg cells in female Foxp3Cre/WTmiR-142+/+ and Foxp3Cre/WTmiR-142fl/fl mice. (D) MFI of GITR, CD25, CD103, PD-1, CTLA-4, and ICOS expression on splenic CD4+YFP+ T reg cells from Foxp3Cre (WT) or Foxp3CremiR-142fl/fl (KO) mice. Results are shown as mean ± SD. P values were calculated using 2-tailed Student t test *, P < 0.05; **, P < 0.01; NS, not significant. The underlying numerical raw data can be found in S1 Data file. The underlying flow cytometry raw data can be found at the Figshare repository. CTV, cell trace violet; FACS, fluorescence activated cell sorting; KO, knockout; MFI, mean fluorescence intensity; T reg , regulatory T; WT, wild-type; YFP, yellow fluorescent protein. https://doi.org/10.1371/journal.pbio.3001552.s003 (PDF) S4 Fig. Gene expression signatures in miR-142–deficient T reg cells. Heatmap visualization of differentially expressed cytokine, chemokine, and immune receptor genes (A) and T reg cell signature genes (B). Genes highlighted in red are putative miR-142-3p targets. (C) Standard enrichment plot demonstrating the enrichment of up-regulated genes in miR-142–deficient T reg cells from gene ontology term “regulation of response to interferon gamma” using the GSEA tool. (D) ELISA analysis of IFNγ production by Foxp3Cre (WT) and Foxp3CremiR-142fl/fl (KO) T reg cells (n = 4 per group). Purified CD4+YFP+ T reg cells (106/mL) were stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml) antibodies in the presence of IL-2 (50 ng/ml) for 48 hours. (E) MFI of phospho-Stat1(Y701) levels in Foxp3Cre (WT; n = 6) and Foxp3CremiR-142fl/fl (KO; n = 5) T reg cells. P values were calculated using 2-tailed Student t test. *, P < 0.05; **, P < 0.01. The underlying raw data can be found in S1 Data file. GSEA, Gene Set Enrichment Analysis; IFNγ, interferon gamma; IL, interleukin; KO, knockout; MFI, mean fluorescence intensity; T reg , regulatory T; WT, wild-type; YFP, yellow fluorescent protein. https://doi.org/10.1371/journal.pbio.3001552.s004 (PDF) S5 Fig. Characterization of the molecular mechanism by which miR-142 controls T reg cell homeostasis and function. (A) Diagrams (top left) and sequence alignments (top right) of putative miR-142-3p binding sites in the 3′ UTRs of Stat1, Ifngr2, Irf1, Gbp3, and Pde3b genes. HITS-CLIP analysis of Ago2 binding to the 3′ UTRs of Ifngr2 (bottom-left) and Gbp3 (bottom-right) genes in WT (blue plot) and miR-155 KO (yellow plot) activated CD4+ T cells. Sequences corresponding to miR-142-3p binding sites are labeled by arrows. (B) Validation of Pde3b and Ifngr2 as direct miR-142-3p targets by the 3′ UTR luciferase reporter assay (n = 2). Relative expression of WT and miR-142-3p seed mutated Pde3b and Ifngr2 3′ UTR reporter constructs upon cotransfection with either miR-142 precursor expressing plasmid or empty vector control. Expression of WT Pde3b and Ifngr2 3′ UTR reporters in the presence of empty vector were set to 1. (C) MFI of Hif1α in Foxp3Cre (WT; red bars) and Foxp3CremiR-142fl/fl (KO; blue bars) CD4+Foxp3+ T reg and CD4+Foxp3- T conv cells (n = 6 per group). (D) Left panel, FACS analysis of IFNγR2 expression in CD4+YFP+ T cells from Foxp3Cre (red line) and Foxp3CremiR-142fl/fl (blue line) spleens; right panel, MFI of IFNγR2 in Foxp3Cre (WT; red bars) and Foxp3CremiR-142fl/fl (KO; blue bars) CD4+YFP+ T reg cells. (E) Schematic diagram and sequence conservation of 2 miR-142-5p and one miR-142-3p binding sites in the 3′ UTR of mouse Pde3b gene as determined by the TargetScan algorithm. (F) Sequence alignment of miR-142-3p binding sites in mouse Pde3b-WT and Pde3b-MUT 3′ UTR reporter constructs. Results are shown as mean ± SD. P values were calculated using 2-tailed Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant. The underlying numerical raw data can be found in S1 Data file. The underlying flow cytometry raw data can be found at the Figshare repository. FACS, fluorescence activated cell sorting; IFNγ, interferon gamma; KO, knockout; MFI, mean fluorescence intensity; SD, standard deviation; T reg , regulatory T; WT, wild-type; YFP, yellow fluorescent protein. https://doi.org/10.1371/journal.pbio.3001552.s005 (PDF) S6 Fig. Phenotypic analysis of Foxp3CremiR-142fl/flHif1a+/fl and Foxp3CremiR-142fl/flHif1afl/fl mice. (A) Relative Hif1α protein expression in Foxp3Cre (WT; red bar), Foxp3CremiR-142fl/fl (KO; blue bar), and Foxp3CremiR-142fl/flHif1a+/fl (HET; green bar) T reg cells. The Hif1α levels in WT T reg cells were arbitrarily set to 1. (B) Frequency of CD4+Foxp3+ T reg cells in splenic lymphocytes from Foxp3Cre (WT; red bar; n = 7), Foxp3CremiR-142fl/fl (KO; blue bar; n = 5) and Foxp3CremiR-142fl/flHif1a+/fl (HET; green bar; n = 3) mice. (C) Kaplan–Meier survival curves for Foxp3Cre (red line; n = 27), Foxp3CremiR-142fl/fl (blue line; n = 27), and Foxp3CremiR-142fl/flHif1a+/fl (green line; n = 8) mice. Analysis of IFNγ production (D) and Stat1 activation (E) in T reg cells from Foxp3CremiR-142fl/flHif1a+/fl mice. Left panels, intracellular FACS analysis of splenic CD4+Foxp3+ T reg cells from Foxp3Cre (red line; WT), Foxp3CremiR-142fl/fl (blue line; KO) and Foxp3CremiR-142fl/flHif1a+/fl (green line; HET) mice with anti-IFNγ (D) and anti-pStat1 (Y701) (E) antibodies. Right panels, MFI of IFNγ and phospho-Stat1 (pSTAT1) in Foxp3Cre (WT; red bar), Foxp3CremiR-142fl/fl (KO; blue bar) and Foxp3CremiR-142fl/flHif1a+/fl (HET; green bar) T reg cells. (F, G) FACS analysis of immunosuppressive activity of T reg cells derived from Foxp3Cre (WT; red dot), Foxp3CremiR-142fl/fl (KO; blue dot) and Foxp3CremiR-142fl/flHif1afl/fl (DKO; green dot) mice (n = 3) in vitro. Several T reg to T conv cell ratios were analyzed as indicated in the graph. Unstimulated T conv cells were used as control. Representative FACS plot analysis is shown in G. (H) FACS analysis of CD44 and CD62L expression in splenic CD4+ T cells from Foxp3Cre, Foxp3CremiR-142fl/flHif1a+/fl, and Foxp3CremiR-142fl/flHif1afl/fl mice. Numbers indicate percentage of cells in the quadrants. Results are shown as mean ± SD. P values were calculated using 2-tailed Student t test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; NS, not significant. The underlying numerical raw data can be found in S1 Data file. The underlying flow cytometry raw data can be found at the Figshare repository. DKO, double knockout; FACS, fluorescence activated cell sorting; IFNγ, interferon gamma; KO, knockout; MFI, mean fluorescence intensity; pStat1, phosphorylated Stat1; SD, standard deviation; T reg , regulatory T; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001552.s006 (PDF) S1 Data. Data underlying figures. https://doi.org/10.1371/journal.pbio.3001552.s007 (XLSX) S1 Table. Differentially expressed genes in miR-142–deficient T reg cells. T reg , regulatory T. https://doi.org/10.1371/journal.pbio.3001552.s008 (XLSX) S2 Table. Pathway enrichment analysis by Enrichr. https://doi.org/10.1371/journal.pbio.3001552.s009 (XLSX)

Acknowledgments We would like to thank Dr. Zuoming Sun and members of the Boldin laboratory at the City of Hope for helpful discussions and suggestions regarding the manuscript. We thank the City of Hope Animal Research Center for their help and care in breeding and maintaining our mouse colonies.

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