Journal: Frontiers in Immunology

Article Title: Neuronal-Activated ILC2s Promote IL-17A Production in Lung γδ T Cells During Sepsis

doi: 10.3389/fimmu.2021.670676

Figure Lengend Snippet: Sepsis induces IL-17A-producing γδ T cell expansion and IL-17A expression in the lungs. Wild type (WT, C57BL/6J) mice were subjected to cecal ligation and puncture (CLP) to induce sepsis or sham surgery, plasma and lung tissue were then collected at different time points as indicated. (A) ELISA analysis of plasma IL-17A from CLP or sham mice (n = 4). (B) Real-time PCR detection of lung Il17a mRNA from mice at 24h after CLP or sham surgery (n = 4). Data were normalized by S18. (C) ELISA analysis of IL-17A protein in lung homogenates from CLP or sham mice (n = 4). Data were normalized by protein concentrations. (D) Representative flow cytometry plots for IL-17A expression within lung live CD45 + populations at 24h after CLP or sham surgery. The relative contribution of different cells (Lineage - ILCs, CD4 + T cells, CD8 + T cells, and γδ T cells) to lung IL-17A + cells was determined. (E) The percentages of the IL-17A + cell population within lung live CD45 + populations at 24h after CLP or sham surgery (n = 5). (F) Representative flow cytometry plots for ILC2 population within lung live CD45 + Lineage - populations and IL-17A + ILC2 population within ILC2 population at 24h after CLP or sham surgery. (G) The percentages of IL-17A + ILC2 population within lung ILC2 population at 24h after CLP or sham surgery (n = 6). (H) Representative flow cytometry plots for ILC2 population within lung live CD45 + IL-17A + populations at 24h after CLP or sham surgery. (I–K) Representative flow cytometry plots (I) , percentages (J) , and numbers (K) of γδ T cells within lung live CD45 + populations at 24h after CLP or sham surgery (n = 3). All data are mean ± SEM, with symbols representing the values of individual mice. * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001, n.s., not significant. One-way ANOVA in (A, C) ; two-tailed Student’s t-test in (B, E, G, J, K) .

Article Snippet: For determination of mouse IL-1β, IL-9, IL-17A and IL-23, Quantikine ELISA Kits from R&D Systems were used according to manufacturers’ instructions.

Techniques: Expressing, Ligation, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Flow Cytometry, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Neuronal-Activated ILC2s Promote IL-17A Production in Lung γδ T Cells During Sepsis

doi: 10.3389/fimmu.2021.670676

Figure Lengend Snippet: ILC2s mediate NMU-indued increase in lung γδ T cells. (A–D) Representative flow cytometry plots (A) , numbers (B) , percentages (C) of γδ T cell population, and ELISA analysis (D) of supernatant IL-17A in different groups. ILC2s and γδ T cells were co-cultured for 48h with or without NMU (10 μg/ml). IL-1β (100 ng/ml) and IL-23 (100 ng/ml) were added to polarize IL-17A-producing γδ T cells, LPS (1 μg/ml) plus TNF-α (20 ng/ml) were added to mimic sepsis stimulation (n = 4). (E, F) Numbers (E) of γδ T cell population and ELISA analysis (F) of supernatant IL-17A in groups co-cultured with different numbers of ILC2s. ILC2s and γδ T cells were co-cultured for 48 hours with NMU (10 μg/ml) (n = 4). (G, H) Representative flow cytometry plots (G) of γδ T cell population and ELISA analysis (H) of supernatant IL-17A in co-culture group with different concentrations of NMU (1 or 10 μg/ml). ILC2s and γδ T cells were co-cultured for 48h (n = 4). (I) Real-time PCR detection of nmur1 mRNA in ILC2s after nmur1 sgRNA transfection using CRISPR/Cas9 approach for 48h (n = 3). (J–M) Representative flow cytometry plots (J) , numbers (K) , percentages (L) of γδ T cell population, and ELISA analysis (M) of supernatant IL-17A in control and nmur1 knockdown groups. ILC2s and γδ T cells were co-cultured for 48h (n = 4). All data are mean ± SEM, with symbols representing the values of individual mice. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant. One-way ANOVA in (B–D) ; two-tailed Student’s t-test in (E, F, H, I, K–M) .

Article Snippet: For determination of mouse IL-1β, IL-9, IL-17A and IL-23, Quantikine ELISA Kits from R&D Systems were used according to manufacturers’ instructions.

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Co-Culture Assay, Real-time Polymerase Chain Reaction, Transfection, CRISPR, Control, Knockdown, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Neuronal-Activated ILC2s Promote IL-17A Production in Lung γδ T Cells During Sepsis

doi: 10.3389/fimmu.2021.670676

Figure Lengend Snippet: IL-9 mediates ILC2 regulation of γδ T cell expansion and IL-17A production. (A) ELISA analysis of supernatant IL-9 in different groups (n = 3). ILC2s and γδ T cells were co-cultured for 48h with or without NMU (10 μg/ml). (B) ELISA analysis of supernatant IL-9 in groups after nmur1 sgRNA transfection using CRISPR/Cas9 approach for 48h and then co-cultured with γδ T cells for 48h (n = 4). (C) ELISA analysis of supernatant IL-9 in groups after Il9 sgRNA transfection using CRISPR/Cas9 approach for 48h (n = 4). (D–G) Representative flow cytometry plots (D) , numbers (E) , percentages (F) of γδ T cell population, and ELISA analysis (G) of supernatant IL-17A in control and Il9 knockdown groups. ILC2s and γδ T cells were co-cultured for 48h (n = 4). All data are mean ± SEM, with symbols representing the values of individual mice. * P < 0.05, ** P < 0.01, *** P < 0.001. One-way ANOVA in (A) ; two-tailed Student’s t-test in (B, C, E, F, G) .

Article Snippet: For determination of mouse IL-1β, IL-9, IL-17A and IL-23, Quantikine ELISA Kits from R&D Systems were used according to manufacturers’ instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Transfection, CRISPR, Flow Cytometry, Control, Knockdown, Two Tailed Test