Journal: Immunity

Article Title: microRNA-142 is critical for the homeostasis and function of type-1 innate lymphoid cells

doi: 10.1016/j.immuni.2019.06.016

Figure Lengend Snippet: BM, SP, and blood (BL) of Mir142+/+ (control, open green) and Mir142−/− (filled blue) were harvested and assessed for type-1 ILCs (CD45+CD3−NK1.1+NKp46+). (A) Representative flow plot of NK1.1+ NKp46+ cells (gated on CD3−) from the SP and BM. (B) Summary data showing the total NK1.1+ NKp46+ cells, see Fig. S1 (C-D) Schema of the BM chimera assay. (D) Summary of 2 independent experiments with >8 mice per group. (E) Non-competitive BM chimera experiment where control or Mir142−/− BM were transferred into congenic, irradiated recipients. Summary data showing percent CD45.2+CD3−NK1.1+NKp46+ cells from 3 independent experiments with 6 mice per group. (F) Summary data showing the NK cell precursor population numbers: pre-NK precursor (pre-NKp) and restricted NK precursor (rNKp). Data are from >3 independent experiments with >12 mice per group. Data were compared using Student’s T test or Mann-Whitney test. (G) Experimental schema for trafficking experiments. Briefly, control and miR142−/− BM cells were differently labeled with cell trace violet and cell trace yellow. The cells were mixed, injected iv. into WT recipients, and tissues examined for the presence of labeled NK1.1+NKp46+ cells. Flow plots of pre-infusion NK1.1+ cells present at 1:1 ratio. (H) Representative flow cytometry showing NK1.1+ cells. (I) Summary data from (H) showing the ratio of control and miR142−/− NK1.1+ cells. N=10 mice from 2 independent experiments. Significance was determined by Wilcoxon signed rank test against the theoretical value of one (no trafficking alteration, dashed line). Please also see Figure S3.

Article Snippet: APC-CY7 mouse anti-mouse NK1.1 mAb (PK136) , BD Biosciences , Cat #560618.

Techniques: Irradiation, MANN-WHITNEY, Labeling, Injection, Flow Cytometry

Journal: Immunity

Article Title: microRNA-142 is critical for the homeostasis and function of type-1 innate lymphoid cells

doi: 10.1016/j.immuni.2019.06.016

Figure Lengend Snippet: (A-B) Splenocytes from Mir142+/+ and Mir142−/− mice were stimulated for 6 hours. (A) Representative flow plots showing intracellular IFN-γ staining in CD45+CD3−NK1.1+ cells. (B) Summary data from (A). (C-D) Splenocytes were isolated from Ncr1-cre+ Mir142+/+ (control, open green bars) and Ncr1-cre+ Mir142f/f (filled blue bars) mice and stimulated for 6 hours. (C) Representative flow plots showing intracellular IFN-γ staining in CD45+CD3−NK1.1+YFP+ cells. (D) Summary data from (C). (E-F) 36 hours after MCMV infection, SP NK1.1+ cells were assessed immediately for intracellular IFN-γ protein by flow cytometry. (E) Representative flow plots showing IFN-γ in CD45+CD3−NK1.1+ cells. (F) Summary from (E). (G) Viral copy numbers in the SP of MCMV-naïve control, Mir142+/+ (WT) and Mir142−/− (KO) MCMV-infected mice, were assessed using qPCR 4 days after infection (units: IE1×1000/B-Actin). (H) Mice were infected with 1e5 PFU MCMV and survival assessed. Pooled data from 2-3 independent experiments with >4 mice per group per experiment. Comparisons were made using Student’s T test or Mann-Whitney test, and log-rank (H). See also Figure S7.

Article Snippet: APC-CY7 mouse anti-mouse NK1.1 mAb (PK136) , BD Biosciences , Cat #560618.

Techniques: Staining, Isolation, Infection, Flow Cytometry, MANN-WHITNEY

Journal: Immunity

Article Title: microRNA-142 is critical for the homeostasis and function of type-1 innate lymphoid cells

doi: 10.1016/j.immuni.2019.06.016

Figure Lengend Snippet: The CD3−NK1.1+NKp46+ compartment of Mir142+/+ (open green) and Mir142−/− (filled blue) mice was assessed for CD49a and CD49b by flow cytometry. (A) Representative flow plots of CD3−NK 1.1 +NKp46+ cells. (B) Total number of NK cells (NK, CD49a−CD49b+), ILC1-like (CD49a+CD49b+), and ILC1s (CD49a+CD49b−) within the indicated tissues. Summary from >3 independent experiments with > 10 mice per group. (C) Total numbers of NK, ILC1-like, and ILC1 cells within control (Ncr1-cre+ Mir142+/+) and ILC-specific miR-142-deficient (Ncr1-cre+ Mir142f/f) mice, cells gated on CD3−NK1.1+NKp46+ YFP+. (D) Experimental Schema. BM from control and Ncr1-cre+ Mir142f/f mice were transferred into irradiated recipients and spleens assessed 6 weeks later. (E) Summary data showing the percent of ILC1-like cells from CD3-NK1.1+NKp46+ CD45.1+ (WT) or YFP+ (Mir142f/f) within the indicated recipient mice. Data from 2-3 independent experiments, N=7-10 mice, compared using T-test or Mann-Whitney corrected for multiple comparisons, when appropriate. See also Figure S2.

Article Snippet: APC-CY7 mouse anti-mouse NK1.1 mAb (PK136) , BD Biosciences , Cat #560618.

Techniques: Flow Cytometry, Irradiation, MANN-WHITNEY

Journal: Immunity

Article Title: microRNA-142 is critical for the homeostasis and function of type-1 innate lymphoid cells

doi: 10.1016/j.immuni.2019.06.016

Figure Lengend Snippet: (A) Summary data of integrin αVβ3 and CD49a on CD3−NK1.1+NKp46+CD49b+ cells from the SP of Il15−/− or control mice. (B) Representative histogram showing integrin αV expression in the indicated tissues of Global and Ncr1-cre+ mice. (C) Summary showing percent integrin αV+ NK1.1+NKp46+ cells in the indicated tissues from Mir142−/− (left), Ncr1-cre+ Mir142f/f (YFP+, right), and controls. (D) Predicted miR-142-3p binding site in 3’UTR of Itgav (top). Luciferase reporter assay for Itgav 3’UTR (bottom). Data summarize 3 independent experiments and were compared using an ANOVA. (E) Mir142−/− mice were treated for 2 weeks with an αV inhibitor or vehicle. Summary data of total type-1 ILCs in the SP from 4 independent experiments, n=11 mice per group. (C, E) Data were compared using Student’s T test. See also Figure S3.

Article Snippet: APC-CY7 mouse anti-mouse NK1.1 mAb (PK136) , BD Biosciences , Cat #560618.

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay

Journal: Immunity

Article Title: microRNA-142 is critical for the homeostasis and function of type-1 innate lymphoid cells

doi: 10.1016/j.immuni.2019.06.016

Figure Lengend Snippet: (A) Representative histograms depict CD43 expression on NK1.1+NKp46+ cells from Mir142−/− (blue) mice compared to controls (green). Gray filled histograms represent CD43− lymphocytes. (B) Summary from (A). (C) Summary CD73 and TIGIT median expression on NK1.1+NKp46+ cells. (D) Transcription factor median expression on Mir142−/− and control NK1.1+NKp46+ cells. Data are from 2-4 independent experiments with N=5-10 mice per group. Data were compared using Student’s T or Mann Whitney tests.

Article Snippet: APC-CY7 mouse anti-mouse NK1.1 mAb (PK136) , BD Biosciences , Cat #560618.

Techniques: Expressing, MANN-WHITNEY

Journal: Immunity

Article Title: microRNA-142 is critical for the homeostasis and function of type-1 innate lymphoid cells

doi: 10.1016/j.immuni.2019.06.016

Figure Lengend Snippet: (A-B) CD3−NK1.1+NKp46+ cells were flow-sorted from Mir142+/+ (WT) or Mir142−/− (KO) BM or SP and microarray performed. (A) GSEA of the TGF-β Hallmark Signaling Pathway gene set in Mir142−/− versus WT from SP and BM. NES, Normalized enrichment score. (B) Heat map of differentially regulated genes between Mir142+/+, Smad4Δ/Δ, and Mir142−/− NK1.1+ cells. Genes associated with the ILC1-like signature (black), miR-142 targets (blue), and NK cell development and effector functions (green) are indicated. Data are from 3 biological replicates. (C) Predicted miR-142-3p binding site in the 3’UTR of Tgfbr1. (D) Luciferase reporter assay for miR-142 overexpression (OE) without the Tgfbr1 3’UTR (empty), with Tgfbr1 3’ UTR (WT) or with Tgfbr1-mutated at the miR-142-3p binding site in the 3’ UTR (Δ). Data summarize 3 independent experiments and were compared using an ANOVA. (E) Summary TGFBR1 expression on type-1 ILCs from SP of indicated mice (relative expression: median minus FMO); data from > 3 independent experiments were compared with T test (n=6-8 mice per group). (F-G) SP cells were stimulated with 10 ng/mL IL-15 plus 10 ng/mL TGF-β1 or left unstimulated (0 minutes) and assessed for phosphorylated (p) SMAD-2 and/or −3. (F) Representative flow plot showing pSMAD-2 and/or −3 in unstimulated (0m, top) or stimulated (60m, bottom) CD3−NK1.1+ cells. (G) Summary from (F). Data are from 2 independent experiments with 6-7 mice per group. Significance was determined by 2-Way ANOVA. Please also see Figure S4.

Article Snippet: APC-CY7 mouse anti-mouse NK1.1 mAb (PK136) , BD Biosciences , Cat #560618.

Techniques: Microarray, Binding Assay, Luciferase, Reporter Assay, Over Expression, Expressing

Journal: Immunity

Article Title: microRNA-142 is critical for the homeostasis and function of type-1 innate lymphoid cells

doi: 10.1016/j.immuni.2019.06.016

Figure Lengend Snippet: (A) WT CD3−NK1.1+ cells were sorted from the SP and incubated with 10 ng/mL IL-15. Immediately after sorting (baseline) and after stimulation, RNA was extracted from the cells and miR-142-3p/5p expression were assessed by qPCR. Summary data (normalized to baseline) from 2 independent experiments with 3 biological replicates. (B) Splenocytes were incubated with IL-15 for 48 hours in vitro. Summary data depict the percent of type-1 ILCs (CD45+CD3−NK1.1+) positive for 7-AAD and/or Annexin V. (C) Representative histograms of surface expression of CD122 (IL-2 and −15Rβ) on Mir142+/+ and Mir142−/− CD45+CD3−NK1.1+ cells (Global, Top) and Ncr1-cre+ Mir142+/+ and Ncr1-cre+ Mir142f/f CD45+CD3−NK1.1+YFP+ cells (Ncr1-cre+, Bottom). Gray filled histograms depict CD122-negative lymphocytes. (D) Summary data from (C) showing the MFI. (E) Representative histograms showing intracellular pSTAT-5 (Y694) staining in global (left) and Ncrl-cre+ (right) NK1.1+ and/or YFP+ cells after 15-minute stimulation with IL-15. (F) Summary data from (E). (G) Representative histograms showing pSTAT-4 in global (Top) and Ncr1-cre+ (Bottom) CD3− NK1.1+ and/or YFP+ cells after stimulation with IL-12 for 15 minutes. (H) Summary data from (G). (I) Predicted miR-142-5p binding site in Socs1 3’ UTR (top). A luciferase reporter assay for Socs1 3’UTR. Data are compared using an ANOVA. Data summarize 3 independent experiments. (J-K) CD3−NK1.1+ SP cells from control and Mir142−/− SP were sorted and assessed for Socs1 and β-actin by western blot. (J) Representative blot from 3 independent experiments showing Socs1 (top) and β-Actin (bottom). (K) Summary data depicting Socs1 band intensity (normalized using β-actin). Comparisons were made using RM-ANOVA (A, I), Student’s T test/Mann-Whitney (B-D, H), RM-ANOVA (F), paired T test (K). Please also see Figure S5.

Article Snippet: APC-CY7 mouse anti-mouse NK1.1 mAb (PK136) , BD Biosciences , Cat #560618.

Techniques: Incubation, Expressing, In Vitro, Staining, Binding Assay, Luciferase, Reporter Assay, Western Blot, MANN-WHITNEY

Journal: Immunity

Article Title: microRNA-142 is critical for the homeostasis and function of type-1 innate lymphoid cells

doi: 10.1016/j.immuni.2019.06.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: APC-CY7 mouse anti-mouse NK1.1 mAb (PK136) , BD Biosciences , Cat #560618.

Techniques: Recombinant, Derivative Assay, Electron Microscopy, Cell Isolation, Flow Cytometry, Luciferase, Microarray, Control Assay, Software

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: Allergic phenotype in WT and Saa–/– C57BL/6 mice sensitized and challenged with HDM was analyzed seventy-two hours after the last challenge. Control mice received PBS at both sensitization and challenge. (a) Airway responses to cholinergic stimulation shown as airway pressure over time (APTI, *P = 0.0378, **P = 0.0019), (b) total serum IgE concentrations (**P = 0.0019), (c) bronchoalveolar lavage (BAL) eosinophilia and (d and e) histological examination of airway inflammation. Sections were stained for mucus production with periodic acid Schiff (PAS; Scale bars 100 μm) and bar graphs represent percentage of mucus positive cells in the airways (*P = 0.0241). Numbers of (f) CD11b+ DCs, (g) IL-13+ CD3+CD4+ and (h) IL-13+ILC2 cells (***P = 0.0003) in the lungs and (i and j) TH2 cytokine production from HDM-restimulated lung cells (**P = 0.0018). Data represent means ± SEM of pooled data from 2 independent experiments containing n=11 WT PBS, n=12 WT HDM, n=11 Saa–/– PBS and n=13 Saa–/– HDM animals per group (a, c, and f-j), n=2 WT PBS, n=3 WT HDM, n=2 Saa–/– PBS and n=4 Saa–/– HDM animals per group (d, e), or are representative of 2 experiments (b) with n=8 WT PBS, n=7 WT HDM, n=8 Saa–/– PBS and n=8 Saa–/– HDM animals per group. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Dunett’s post hoc (b, c, e-j) or Holm-Sidak’s (a) analysis that compares WT HDM to counterparts. ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: For ILC2 flow, cells were plated at 2 × 10 6 cells/ml, restimulated with PMA/ionomycin overnight as described above before staining with PerCP-Cy5.5 or APC-Cy7-conjugated lineage marker monoclonal antibodies (FcεRI, MAR-1, BioLegend; CD3ε, 145–2C11, BD Pharmingen; TCRβ, H57–597, BD Pharmingen; B220, RA3–682, BD Pharmingen; CD49b, DX5, eBioscience; CD11b, M1/70, BD Pharmingen; CD11c, N418; BioLegend; Gr1, RB6–85C, BD Pharmingen; NK1.1, PK136, BioLegend), Alexa Fluor700-conjugated CD45 (30-F11; BD Pharmingen), BV605-conjugated CD90.2 (53–2.1, BD), APC-e780-conjugated CD25 (PC615, eBioscience), PE-Cy7-conjugated ICOS (C398.4A; BioLegend), BV421-conjugated IL-33Rα (DIH9, BioLegend) and FITC-conjugated T1/ST2 (DJ8, MD Bioproducts).

Techniques: Staining, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: The parasitic worm Schistosoma mansoni (a Puerto Rican isolate) routinely maintained in the laboratory by cycling between the intermediate snail host, Biomphalaria glabrata, and outbred ICR mice as definitive hosts. (a) Numbers of CD11b+ DCs in the lungs of PBS or Schistosoma mansoni worm extract-treated WT and Saa–/– C57BL/6 mice (***P = 0.0002). (b) Frequency and (c) total numbers of CD3+CD4+ T cells (*P = 0.014). (d) TH2 (***P = 0.0003) and (e) TH17 (**P = 0.0014) cells in the lungs of these mice. (f-i) Cytokine production from worm extract-restimulated lung cells (IL-5: *P = 0.019, IL-10: *P = 0.011, IL-13: *P = 0.017, IL-17: *P = 0.017). Data represents means ± SEM of pooled data from 2 independent experiments containing a total of n= 8 animals in each group. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Dunett’s post hoc that compares WT worm to counterparts. ****P ≤ 0.0001.

Article Snippet: For ILC2 flow, cells were plated at 2 × 10 6 cells/ml, restimulated with PMA/ionomycin overnight as described above before staining with PerCP-Cy5.5 or APC-Cy7-conjugated lineage marker monoclonal antibodies (FcεRI, MAR-1, BioLegend; CD3ε, 145–2C11, BD Pharmingen; TCRβ, H57–597, BD Pharmingen; B220, RA3–682, BD Pharmingen; CD49b, DX5, eBioscience; CD11b, M1/70, BD Pharmingen; CD11c, N418; BioLegend; Gr1, RB6–85C, BD Pharmingen; NK1.1, PK136, BioLegend), Alexa Fluor700-conjugated CD45 (30-F11; BD Pharmingen), BV605-conjugated CD90.2 (53–2.1, BD), APC-e780-conjugated CD25 (PC615, eBioscience), PE-Cy7-conjugated ICOS (C398.4A; BioLegend), BV421-conjugated IL-33Rα (DIH9, BioLegend) and FITC-conjugated T1/ST2 (DJ8, MD Bioproducts).

Techniques: Two Tailed Test