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MicroRNA-146a controls functional plasticity in γδ T cells by targeting NOD1

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Science Immunology  04 May 2018:
Vol. 3, Issue 23, eaao1392
DOI: 10.1126/sciimmunol.aao1392
  • Fig. 1 miR-146a is highly expressed selectively on γδ27 T cells.

    (A) Microarray heat map of differentially expressed miRNAs in duplicate samples of γδ27+ (n = 4 mice per sample) and γδ27CCR6+ T cells (n = 8 mice per sample) isolated from pooled lymph nodes and spleen of C57BL/6 mice (more than twofold enrichment). (B) RT-qPCR analysis of miR-146a and miR-146b expression in sorted γδ27+ and γδ27 T cells from pooled peripheral organs (lymph node and spleen) of C57BL/6 mice. NS, not significant. (C) RT-qPCR analysis of miR-146a expression in sorted DN2 (CD4CD8CD44+CD25+), DN3 (CD4CD8CD44CD25+), γδ25+ (CD25+CD27+), γδ27+, and γδ27 thymocytes of C57BL/6 mice. Results are presented relative to miR-423-3p or RNU5G (reference small RNA) expression. Each symbol in (B) and (C) represents an individual mouse. *P < 0.05 and **P < 0.01 (Mann-Whitney two-tailed test).

  • Fig. 2 miR-146a inhibits IFN-γ production in γδ T cells.

    Workflow and results of (A to D) retroviral (RV) overexpression of miR-146a and (E to G) electroporation of miR-146a mimics in sorted peripheral γδ T cells. (B) Flow cytometry analysis of intracellular IFN-γ and IL-17 expression; and frequency of (C) IFN-γ+ and (D) Ki-67+ cells in sorted GFP+ retrovirally transduced γδ T cells expressing either an unrelated miR (RV-unrelated miR), a control vector (RV-control), or miR-146a (RV-146a). (F) Flow cytometry analysis of intracellular IFN-γ and IL-17 expression and (G) frequency of IFN-γ+ in electroporated γδ T cells with either RNA control or miR-146a mimic. Numbers in quadrants of flow cytometry plots indicate percentages of cells. Data are representative of five independent experiments. *P < 0.05 and **P < 0.01 (Mann-Whitney two-tailed test).

  • Fig. 3 miR-146a limits IFN-γ production and the functional plasticity of γδ27 T cells.

    (A) Flow cytometry analysis of intracellular IFN-γ and IL-17 and frequency of IFN-γ+ and IL-17+ IFN-γ+ in γδ27 T cells isolated from peripheral organs and stimulated in vitro overnight with αCD3/28 or for 72 hours in the presence of IL-1β plus IL-23. (B) Schematic for 1:1 mixed miR-146a+/+ and miR-146a−/− adult BM and neonatal thymocyte chimeras in Rag2−/− hosts. (C) Flow cytometry analysis of intracellular IFN-γ and IL-17, mean fluorescence intensity (MFI) of IFN-γ+, and fold difference of IFN-γ+ IL-17+ in γδ27 T cells isolated from peripheral organs of above-introduced chimeras after in vitro stimulation for 72 hours with IL-1β plus IL-23. Numbers in quadrants of flow cytometry plots indicate percentages of cells. Each symbol in (A) and (C) represents an individual experiment. *P < 0.05 (Mann-Whitney two-tailed test).

  • Fig. 4 miR-146a restricts functional plasticity of γδ27 T cells in Listeria infection.

    (A) Flow cytometry analysis of intracellular IFN-γ and IL-17 expression in γδ T cells isolated from spleens of miR-146a+/+ or miR-146a−/− mice, 7 days after infection with L. monocytogenes. (B) Frequency and total cell numbers of IL-17+ IFN-γ+ in γδ T cells isolated from spleens of miR-146a+/+ and miR-146a−/− mice, 7 days after infection with L. monocytogenes. (C) Representative flow cytometry analysis of Vγ6 chain usage (left), frequency of IFN-γ+ IL-17+ (middle), and total cell numbers of IFN-γ+ IL-17+ (right) of Vγ1Vγ4 γδ T cells isolated from spleen of miR-146a+/+ or miR-146a−/− mice, 7 days after infection with L. monocytogenes. (D) Representative flow cytometry analysis of IL-17+ IFN-γ+ γδ T cells isolated from the chimeras established in Fig. 3B. (Right) frequency within IFN-γ+ IL-17+ γδ T cells of either miR-146a+/+ or miR-146a−/− origin. Numbers in quadrants of flow cytometry plots indicate percentages of cells. Each symbol in (B) to (D) represents an individual mouse. *P < 0.05 and **P < 0.01 (Mann-Whitney two-tailed test). WT, wild-type; KO, knockout.

  • Fig. 5 miR-146a targets Nod1 mRNA that is depleted in γδ27 T cells.

    (A) Putative binding sites of miR-146a-5p in the 3′UTR region of Nod1 and Atf2. The line represents canonical Watson-Crick base pairing, whereas the dot represents noncanonical base pairing between miR-146a and Nod1 or Atf2 mRNAs. The red bases above illustrated the mutated sites in the region of Nod1 and Atf2 3′UTRs. (B) Dual luciferase reporter assay was performed to verify binding between miR-146a and Nod1 or Atf2 mRNAs. HEK293 T cells were cotransfected with a pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) containing either the WT or mutated 3′UTR target sites plus miR-146a, an unrelated miRNA, or GFP control expression vector. A negative construct (without miR-146a binding sites) and a positive construct (3′UTR of Notch1) were included. Data are from three to four independent experiments with technical replicates. *P < 0.05, **P < 0.005, ***P < 0.001, Student’s t test. RT-qPCR analysis of Nod1 and Atf2 and expression in (C) sorted γδ27+, γδ27CCR6, and γδ27CCR6+ T cells from pooled peripheral organs (lymph node and spleen) of C57BL/6 mice; (D) sorted Vγ1+, Vγ4+, and Vγ1γ4 γδ T cells and CD4+ T cells from pooled peripheral organs (lymph node and spleen) of C57BL/6 mice; and (E) sorted Vγ4+CD27 and Vγ1γ4 CD27-γδ T cells from pooled lymph nodes of either miR-146a+/+ or miR-146a−/− mice. Results are presented relative to β-actin and HPRT expression. Each symbol in (C) to (E) represents an individual mouse. *P < 0.05 and **P < 0.01 (Mann-Whitney two-tailed test).

  • Fig. 6 Nod1 is required for IFN-γ production and functional plasticity in γδ T cells.

    (A) Flow cytometry analysis of intracellular IFN-γ and IL-17 expression and frequency of IFN-γ+ IL-17+ (left), IL-17+ (middle), and Ki-67+ (right) in γδ27 T cells isolated from peripheral organs (spleen and lymph nodes) of Nod1+/+ and Nod−/− littermates as well as Atf2+/+ and Atf2−/− littermates after overnight stimulation with IL-1β plus IL-23. (B) Flow cytometry analysis of intracellular IFN-γ and IL-17 expression and frequency of IFN-γ+ IL-17+ (left), IL-17+ (up) and Ki-67+ (bottom) in γδ27 T cells isolated from peripheral organs (spleen and lymph nodes) of miR-146a+/+Nod1+/+, miR-146a−/−Nod1+/+, and miR-146a−/−Nod1+/− littermates stimulated for 72 hours in the presence of IL-1β plus IL-23. (C) Bacterial CFUs of L. monocytogenes bacterial burden was enumerated in the spleen 4 days after infection (left), weight loss (middle), and survival (right) of Nod1+/+, Nod+/−, Nod−/−, and TCRd−/− mice infected with Listeria (n = 7 to 18 in three independent experiments). (D) Flow cytometry analysis of intracellular IFN-γ and IL-17 expression in γδ T cells isolated from spleen of Nod1+/+, Nod+/−, and Nod−/− mice, 7 days after infection with Listeria. Numbers in quadrants of flow cytometry plots indicate percentages of cells. (E) Frequency of IFN-γ+ IL-17+ within γδ T cells (left) and Vγ1Vγ4 γδ T cells (right) isolated from spleen of Nod1+/+, Nod+/−, and Nod−/−, 7 days after infection with Listeria (n = 7 to 18 in three independent experiments). Each symbol in (A) and (D) represents an individual mouse. *P < 0.05 and **P < 0.01 (Mann-Whitney two-tailed test).

  • Table 1 Putative miR-146a targets based on differential Ago2-RNA immunoprecipitation followed by deep sequencing in T cells.
    Gene symbolFold changeNo. of binding sites
    Receptor activity
    Nod14.211
    Tcp113.441
    Ghr3.071
    Paqr72.721
    Unc5a2.591
    Transcription factor activity
    Ccnh6.481
    Tef4.492
    Myf63.801
    Zfp7883.681
    Tfap2b3.672
    Grhl23.341
    Foxc13.211
    Sp12.901
    Mllt102.831
    Gtf2e22.833
    Atf22.532
    Yap12.502
    Phf21a2.323

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/23/eaao1392/DC1

    Fig. S1. miR-146a expression analysis.

    Fig. S2. Loss of miR-146a does not affect steady-state γδ T cell subsets.

    Fig. S3. Loss of miR-146a does not affect IFN-γ production by γδ27+ T cells or CD4+ TH1 cells upon in vitro polarization.

    Fig. S4. Mixed BM and neonatal thymocyte chimeras.

    Fig. S5. γδ T cell responses to L. monocytogenes infection.

    Fig. S6. Differential Argonaute 2 immunoprecipitation for identification of mRNA targets of miR-146a.

    Fig. S7. Nod1 is targeted by miR-146a in γδ T cells.

    Fig. S8. Nod1 restriction does not affect IFN-γ production by γδ27+ T cells.

    Table S1. Top 25 miscellaneous mRNAs differentially enriched upon Ago2-RNA immunoprecipitation in T cells overexpressing miR-146a.

    Table S2. Raw data sets.

  • Supplementary Materials

    Supplementary Material for:

    MicroRNA-146a controls functional plasticity in γδ T cells by targeting NOD1

    Nina Schmolka,* Pedro H. Papotto, Paula Vargas Romero, Tiago Amado, Francisco J. Enguita, Ana Amorim, Ana F. Rodrigues, Katrina E. Gordon, Ana S. Coroadinha, Mark Boldin, Karine Serre, Amy H. Buck, Anita Q. Gomes,* Bruno Silva-Santos*

    *Corresponding author. Email: nina.schmolka{at}uzh.ch (N.S.); anitagomes{at}medicina.ulisboa.pt (A.Q.G.); bssantos{at}medicina.ulisboa.pt (B.S.-S.)

    Published 4 May 2018, Sci. Immunol. 3, eaao1392 (2018)
    DOI: 10.1126/sciimmunol.aao1392

    This PDF file includes:

    • Fig. S1. miR-146a expression analysis.
    • Fig. S2. Loss of miR-146a does not affect steady-state γδ T cell subsets.
    • Fig. S3. Loss of miR-146a does not affect IFN-γ production by γδ27+ T cells or CD4+ TH1 cells upon in vitro polarization.
    • Fig. S4. Mixed BM and neonatal thymocyte chimeras.
    • Fig. S5. γδ T cell responses to L. monocytogenes infection.
    • Fig. S6. Differential Argonaute 2 immunoprecipitation for identification of mRNA targets of miR-146a.
    • Fig. S7. Nod1 is targeted by miR-146a in γδ T cells.
    • Fig. S8. Nod1 restriction does not affect IFN-γ production by γδ27+ T cells.
    • Table S1. Top 25 miscellaneous mRNAs differentially enriched upon Ago2-RNA immunoprecipitation in T cells overexpressing miR-146a.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Raw data sets.

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