Journal: PLoS Pathogens

Article Title: Interleukin-17A Mediates Acquired Immunity to Pneumococcal Colonization

doi: 10.1371/journal.ppat.1000159

Figure Lengend Snippet: A. Mice defective in IFN-γ, IL-4 or IL-17A receptor were immunized as described, then challenged with pneumococcal strain 0603. Mice with IFN-γ or IL-4 deficiency were significantly protected by WCV (P<0.001 vs. respective CT controls) whereas IL-17A receptor deficient mice were not protected (P>0.5 vs. CT). Dashed line represents the lower limit of detection of bacterial colonization. B. Expression of IL-17A from splenocytes of WCV-immunized mice. Cultured splenocytes from mice immunized with WCV (black columns) or CT alone (white columns) were stimulated for 72 hours with medium alone, Concanavalin A (5 µg/ml), WCA (10 µg dry weight) after which IL-17A production was measured by ELISA. Significantly more IL-17A was expressed following WCA stimulation of WCV-immunized vs. CT-immunized mice, although response to concanavalin A was similar. C. Effect of CD4+ T cell depletion upon IL-17A expression from splenocytes. Splenocytes (without or with CD4+ T cell depletion) from mice immunized with WCV were stimulated for 72 hours with medium alone or WCA after which IL-17A was measured by ELISA. IL-17A expression in splenocytes following WCA stimulation was significantly higher in the presence of CD4+ T cells compared to stimulation with medium alone or when CD4+ T cells were depleted. Repletion of CD4+ T cells restored the response. ** P<0.01 compared to cells stimulated with medium alone. D. IL-17A intracellular staining of splenocytes from WCV immunized mice. Splenocytes from WCV immunized mice were stimulated with WCA, blocked with monensin, harvested and stained for CD4+ and intracellular IL-17A as described. There is a statistically significant increase in CD4+ IL-17A positive cells following stimulation with WCA, which is not observed in the CD4- population. No increase in IL-17A positive cells could be detected in cells from unimmunized mice (data not shown). **P = 0.008 for comparison of frequency of IL-17A-positive cells in absence and presence of WCA stimulation among CD4+ cells. Data shown here are representative of three experiments, including at least 5 mice per experiment. E. Expression of IL-17A from NALT of WCV- vs. CT-immunized mice. Cultured splenocytes from mice immunized with WCV (black columns) or CT alone (white columns) were stimulated for 72 hours with medium alone or with WCA (10 µg dry weight) after which IL-17A production was measured by ELISA. Significantly more IL-17A was expressed following WCA stimulation of WCV-immunized vs. CT-immunized mice. **P<0.01 for comparison of IL-17A in WCV vs. CT-immunized mice following stimulation with WCA.

Article Snippet: Cells were cultured in 96-well culture plates (Corning Inc, Corning, NY), and cell culture supernatants were collected at predetermined times and stored at −70°C until assays for human IL-17A were performed by sandwich ELISA (R&D Biosystems).

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining

Journal: PLoS Pathogens

Article Title: Interleukin-17A Mediates Acquired Immunity to Pneumococcal Colonization

doi: 10.1371/journal.ppat.1000159

Figure Lengend Snippet: Three weeks after immunization of mice (n = 90) with CT with doses of WCA ranging from 1 to 100 µg, and one week before pneumococcal challenge, blood samples were obtained and stimulated with WCA (10 µg) for 6 days, after which supernatants were collected and assayed for IL-17A concentration. The correlation between density of colonization (cfu/nasal wash) 7 days after challenge and pre-challenge IL-17A expression was evaluated. IL-17A expression was significantly correlated with density of colonization.

Article Snippet: Cells were cultured in 96-well culture plates (Corning Inc, Corning, NY), and cell culture supernatants were collected at predetermined times and stored at −70°C until assays for human IL-17A were performed by sandwich ELISA (R&D Biosystems).

Techniques: Concentration Assay, Expressing

Journal: PLoS Pathogens

Article Title: Interleukin-17A Mediates Acquired Immunity to Pneumococcal Colonization

doi: 10.1371/journal.ppat.1000159

Figure Lengend Snippet: A. Expression of IL-17A from tonsillar mononuclear cells from children. Tonsillar cells (n = 8) were cultured as described and stimulated with WCA or WCA derived from an isogenic, pneumolysin-negative strain (WCAply-). Stimulation with WCA was associated with significantly increased IL-17A expression compared to exposure to medium alone (P = 0.008 by Wilcoxon signed rank test), whereas stimulation with WCAply- did not increase IL-17A production. B. Expression of IL-17A from peripheral blood of adults and umbilical cord blood. Peripheral blood samples from adults (healthy adult volunteers (n = 7), parturient women (n = 11) and umbilical cord blood (n = 11) were stimulated with WCA for 6 days after which IL-17A concentration was assayed by ELISA. IL-17A production was significantly greater in adults than cord blood (P<0.001 by Mann-Whitney U test).

Article Snippet: Cells were cultured in 96-well culture plates (Corning Inc, Corning, NY), and cell culture supernatants were collected at predetermined times and stored at −70°C until assays for human IL-17A were performed by sandwich ELISA (R&D Biosystems).

Techniques: Expressing, Cell Culture, Derivative Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Interleukin-17A Mediates Acquired Immunity to Pneumococcal Colonization

doi: 10.1371/journal.ppat.1000159

Figure Lengend Snippet: A and B. Effect of human IL-17A on surface phagocytic killing of S. pneumoniae . A. Isolated neutrophils from healthy adult volunteers were incubated with recombinant human IL-17A at the indicated concentrations and evaluated in a surface phagocytic killing assay with pneumococcal strain 0603; colonies were counted after overnight incubation at 37°C with 5% CO 2 . IL-17A induces a dose-dependent enhancement of neutrophil killing of S. pneumoniae (P = 0.01 for 1 or 10 µg of IL-17A vs. no added IL-17A). B. Supernatant obtained from neutrophils after incubation with IL-17A did not have any demonstrable antipneumococcal effect, whereas washed neutrophils after incubation with IL-17A demonstrated enhanced killing. C. Effect of human IL-17A on opsonophagocytic killing of S. pneumoniae . Neutrophils purified from the peripheral blood of healthy adult volunteers were incubated with pneumococci anticapsular antibodies, complement, and a range of concentrations of IL-17A as indicated for 90 minutes, following which viable counts were obtained by plating on blood agar plates. Each line represents a different volunteer. IL-17A enhanced killing of pneumococci in a dose-dependent fashion in 6/6 subjects. *P = 0.016 by Wilcoxon matched pairs test.

Article Snippet: Cells were cultured in 96-well culture plates (Corning Inc, Corning, NY), and cell culture supernatants were collected at predetermined times and stored at −70°C until assays for human IL-17A were performed by sandwich ELISA (R&D Biosystems).

Techniques: Isolation, Incubation, Recombinant, Purification

Journal: Scientific Reports

Article Title: Function-specific IL-17A and dexamethasone interactions in primary human airway epithelial cells

doi: 10.1038/s41598-022-15393-2

Figure Lengend Snippet: IL-17A stimulates IL-17 signature genes in basal airway epithelial cells (hAECs). hAECs were cultured submerged and exposed to IL-17A (10 ng/ml) with or without dexamethasone (Dex; 10 nM) for 24 h. Expression of ( a ) IL-6 , ( b ) CXCL3 , ( c ) CXCL8 , ( d ) CSF3 , ( e ) CXCL5 , ( f ) SAA2 were analyzed by real-time qPCR. Data represent means ± SEM. N = 4 donors, [IL-17A] = IL-17A concentration. Statistical analyses were performed with two-way ANOVA, *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: Experiments in submerged cells were conducted by exposing cells to 10 nM of Dexamethasone (Sigma-aldrich D4902), recombinant human IL-17A (R&D 7955-IL-025/CF) in three different concentrations (1, 10, and 100 ng/ml), or to combinations of IL-17A and Dexamethasone for 24 h. For experiments in ALI cultured cells, cells from passage 1–2 were grown submerged on collagen-coated Transwell inserts until confluence, after which they were cultured at an air–liquid interface (ALI) and differentiated for 2 weeks in B/D medium (1:1), which is a mixture of DMEM [Gibco] and bronchial epithelial growth medium (BEGM) [Lonza], supplemented with 0.4% [w/v] bovine pituitary extract [BPE], 0.5 ng/ml epidermal growth factor [EGF], 5 µg/ml insulin, 10 µg/ml transferrin, 1 µM hydrocortisone, 6.5 ng/ml T3, 0.5 µg/ml epinephrine [all from Lonza], 15 ng/ml retinoic acid [Sigma chemical] 1.5 µg/ml bovine serum albumin [Sigma chemical] 0.5 mM sodium pyruvate [Gibco], 20 U/ml penicillin and 20 µg/ml streptomycin [Gibco] .

Techniques: Cell Culture, Expressing, Concentration Assay

Journal: Scientific Reports

Article Title: Function-specific IL-17A and dexamethasone interactions in primary human airway epithelial cells

doi: 10.1038/s41598-022-15393-2

Figure Lengend Snippet: IL-17A induces gene expression that is insensitive to dexamethasone (Dex) in human airway epithelial cells (hAECs). hAECs were cultured and differentiated at an air liquid interface (ALI) and exposed to IL-17A (10 ng/ml) with or without dexamethasone (10 nM) for 14 days. Gene expression was determined by bulk RNA sequencing and differentially expressed genes were analyzed and presented as heatmap of the top 10 most regulated genes, with each column in the groups representing an individual donor ( a ) and by a volcano plot ( b ). Pathway analysis (GSEA) of the statistically significant genes was done and presented as the top 10 most regulated pathways ( c ). The IL-17A gene signature of the top 10 differentially expressed genes was calculated as Z-score ratio ( d ). Data represent means ± SEM. N = 4 donors.

Article Snippet: Experiments in submerged cells were conducted by exposing cells to 10 nM of Dexamethasone (Sigma-aldrich D4902), recombinant human IL-17A (R&D 7955-IL-025/CF) in three different concentrations (1, 10, and 100 ng/ml), or to combinations of IL-17A and Dexamethasone for 24 h. For experiments in ALI cultured cells, cells from passage 1–2 were grown submerged on collagen-coated Transwell inserts until confluence, after which they were cultured at an air–liquid interface (ALI) and differentiated for 2 weeks in B/D medium (1:1), which is a mixture of DMEM [Gibco] and bronchial epithelial growth medium (BEGM) [Lonza], supplemented with 0.4% [w/v] bovine pituitary extract [BPE], 0.5 ng/ml epidermal growth factor [EGF], 5 µg/ml insulin, 10 µg/ml transferrin, 1 µM hydrocortisone, 6.5 ng/ml T3, 0.5 µg/ml epinephrine [all from Lonza], 15 ng/ml retinoic acid [Sigma chemical] 1.5 µg/ml bovine serum albumin [Sigma chemical] 0.5 mM sodium pyruvate [Gibco], 20 U/ml penicillin and 20 µg/ml streptomycin [Gibco] .

Techniques: Expressing, Cell Culture, RNA Sequencing Assay

Journal: Scientific Reports

Article Title: Function-specific IL-17A and dexamethasone interactions in primary human airway epithelial cells

doi: 10.1038/s41598-022-15393-2

Figure Lengend Snippet: Dexamethasone (Dex)-inducible genes are dampened by the presence of IL-17A in human airway epithelial cells (hAECs). hAECs were cultured and differentiated at an air liquid interface (ALI) and exposed to IL-17A (10 ng/ml) with or dexamethasone (10 nM) for 14 days. Gene expression was determined by bulk RNA sequencing and differentially expressed genes were analyzed and presented as a heatmap of the top 10 most regulated genes by dexamethasone, with each column in the groups representing an individual donor ( a ) and by a volcano plot ( b ). Pathway analysis (GSEA) of the differentially expressed genes was done subsequently ( c ). The dexamethasone gene signature was calculated as Z-score ratio of the most regulated genes ( d ). Canonical corticosteroid gene expression, HSD11B2 ( e ) and FKBP5 ( f ), were analyzed from normalized gene counts. Data represent means ± SEM. N = 4 donors. Statistical analyses were performed with paired two-way ANOVA and Šidák’s multiple comparison test, *p < 0.05, ***p < 0.001.

Article Snippet: Experiments in submerged cells were conducted by exposing cells to 10 nM of Dexamethasone (Sigma-aldrich D4902), recombinant human IL-17A (R&D 7955-IL-025/CF) in three different concentrations (1, 10, and 100 ng/ml), or to combinations of IL-17A and Dexamethasone for 24 h. For experiments in ALI cultured cells, cells from passage 1–2 were grown submerged on collagen-coated Transwell inserts until confluence, after which they were cultured at an air–liquid interface (ALI) and differentiated for 2 weeks in B/D medium (1:1), which is a mixture of DMEM [Gibco] and bronchial epithelial growth medium (BEGM) [Lonza], supplemented with 0.4% [w/v] bovine pituitary extract [BPE], 0.5 ng/ml epidermal growth factor [EGF], 5 µg/ml insulin, 10 µg/ml transferrin, 1 µM hydrocortisone, 6.5 ng/ml T3, 0.5 µg/ml epinephrine [all from Lonza], 15 ng/ml retinoic acid [Sigma chemical] 1.5 µg/ml bovine serum albumin [Sigma chemical] 0.5 mM sodium pyruvate [Gibco], 20 U/ml penicillin and 20 µg/ml streptomycin [Gibco] .

Techniques: Cell Culture, Expressing, RNA Sequencing Assay

Journal: Scientific Reports

Article Title: Function-specific IL-17A and dexamethasone interactions in primary human airway epithelial cells

doi: 10.1038/s41598-022-15393-2

Figure Lengend Snippet: IL-17A and Dexamethasone (Dex) interaction in human airway epithelial cells (hAECs). hAECs were cultured and differentiated at an air liquid interface (ALI) and exposed to IL-17A (10 ng/ml) with or without dexamethasone (10 nM) for 14 days. Gene expression was determined by bulk RNA sequencing and differentially expressed genes were analyzed. 9 genes showed significantly interaction by IL-17A and dexamethasone and are presented in a heatmap, with each column in the groups representing an individual donor ( a ) and by a volcano plot ( b ). Genes that were differentially expressed by at least 1.5-fold response to IL-17A stimulation were included in the analysis and for these genes, the ability of dexamethasone to reverse this gene expression was assessed. The resulting score was normalized to 100%, yielding genes with a score of 100% as being completely dexamethasone resistant and genes with a score of 0% as being completely dexamethasone sensitive ( c ). N = 4 donors.

Article Snippet: Experiments in submerged cells were conducted by exposing cells to 10 nM of Dexamethasone (Sigma-aldrich D4902), recombinant human IL-17A (R&D 7955-IL-025/CF) in three different concentrations (1, 10, and 100 ng/ml), or to combinations of IL-17A and Dexamethasone for 24 h. For experiments in ALI cultured cells, cells from passage 1–2 were grown submerged on collagen-coated Transwell inserts until confluence, after which they were cultured at an air–liquid interface (ALI) and differentiated for 2 weeks in B/D medium (1:1), which is a mixture of DMEM [Gibco] and bronchial epithelial growth medium (BEGM) [Lonza], supplemented with 0.4% [w/v] bovine pituitary extract [BPE], 0.5 ng/ml epidermal growth factor [EGF], 5 µg/ml insulin, 10 µg/ml transferrin, 1 µM hydrocortisone, 6.5 ng/ml T3, 0.5 µg/ml epinephrine [all from Lonza], 15 ng/ml retinoic acid [Sigma chemical] 1.5 µg/ml bovine serum albumin [Sigma chemical] 0.5 mM sodium pyruvate [Gibco], 20 U/ml penicillin and 20 µg/ml streptomycin [Gibco] .

Techniques: Cell Culture, Expressing, RNA Sequencing Assay

Journal: Scientific Reports

Article Title: Function-specific IL-17A and dexamethasone interactions in primary human airway epithelial cells

doi: 10.1038/s41598-022-15393-2

Figure Lengend Snippet: IL-17A stimulates IL-17A gene signature and inflammatory marker expression in human airway epithelial cells (hAECs), and these effects were not prevented by dexamethasone (Dex). hAECs were cultured and differentiated at an air liquid interface (ALI) and exposed to IL-17A (10 ng/ml) with or without dexamethasone (10 nM) for 14 days. CXCL8 and CCL20 protein release was measured by ELISA ( a , b ). CXCL8, CCL20, SLC26A4, SAA1, SAA2, IL1β, ALPL, CXCL3, CXCL6, CXCL1 , and CXCL2 gene expression was determined by total RNA sequencing and normalized gene counts were analyzed. Data represent means ± SEM. N = 10 donors for protein and N = 4 donors for gene expressions analysis. Statistical analyses were performed with paired one-way ANOVA and Tukey’s multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Experiments in submerged cells were conducted by exposing cells to 10 nM of Dexamethasone (Sigma-aldrich D4902), recombinant human IL-17A (R&D 7955-IL-025/CF) in three different concentrations (1, 10, and 100 ng/ml), or to combinations of IL-17A and Dexamethasone for 24 h. For experiments in ALI cultured cells, cells from passage 1–2 were grown submerged on collagen-coated Transwell inserts until confluence, after which they were cultured at an air–liquid interface (ALI) and differentiated for 2 weeks in B/D medium (1:1), which is a mixture of DMEM [Gibco] and bronchial epithelial growth medium (BEGM) [Lonza], supplemented with 0.4% [w/v] bovine pituitary extract [BPE], 0.5 ng/ml epidermal growth factor [EGF], 5 µg/ml insulin, 10 µg/ml transferrin, 1 µM hydrocortisone, 6.5 ng/ml T3, 0.5 µg/ml epinephrine [all from Lonza], 15 ng/ml retinoic acid [Sigma chemical] 1.5 µg/ml bovine serum albumin [Sigma chemical] 0.5 mM sodium pyruvate [Gibco], 20 U/ml penicillin and 20 µg/ml streptomycin [Gibco] .

Techniques: Marker, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, RNA Sequencing Assay

Journal: Scientific Reports

Article Title: Function-specific IL-17A and dexamethasone interactions in primary human airway epithelial cells

doi: 10.1038/s41598-022-15393-2

Figure Lengend Snippet: IL-17A induces goblet cell metaplasia and mucus production in human airway epithelial cells (hAECs), and these effects were not inhibited by dexamethasone (Dex). hAECs were cultured and differentiated at an air liquid interface (ALI) and exposed to IL-17A (10 ng/ml) with or without dexamethasone (10 nM) for 14 days. Mucus was collected and weighed at Day-11 ( a ). Representative images of ALI-cultured hAECs that were fluorescently labeled with MUC5AC (stained green (Alexa fluor 488); scale bar 100 µm; 20 X magnification) ( b ). Gene expression was determined by bulk RNA sequencing and normalized gene counts were analyzed ( c – e ). Data represent means ± SEM. N = 7 donors for mucus production measurement and N = 4 donors for gene expression analysis. Statistical analyses were performed with paired one-way ANOVA and Tukey’s multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Experiments in submerged cells were conducted by exposing cells to 10 nM of Dexamethasone (Sigma-aldrich D4902), recombinant human IL-17A (R&D 7955-IL-025/CF) in three different concentrations (1, 10, and 100 ng/ml), or to combinations of IL-17A and Dexamethasone for 24 h. For experiments in ALI cultured cells, cells from passage 1–2 were grown submerged on collagen-coated Transwell inserts until confluence, after which they were cultured at an air–liquid interface (ALI) and differentiated for 2 weeks in B/D medium (1:1), which is a mixture of DMEM [Gibco] and bronchial epithelial growth medium (BEGM) [Lonza], supplemented with 0.4% [w/v] bovine pituitary extract [BPE], 0.5 ng/ml epidermal growth factor [EGF], 5 µg/ml insulin, 10 µg/ml transferrin, 1 µM hydrocortisone, 6.5 ng/ml T3, 0.5 µg/ml epinephrine [all from Lonza], 15 ng/ml retinoic acid [Sigma chemical] 1.5 µg/ml bovine serum albumin [Sigma chemical] 0.5 mM sodium pyruvate [Gibco], 20 U/ml penicillin and 20 µg/ml streptomycin [Gibco] .

Techniques: Cell Culture, Labeling, Staining, Expressing, RNA Sequencing Assay

Journal: Scientific Reports

Article Title: Function-specific IL-17A and dexamethasone interactions in primary human airway epithelial cells

doi: 10.1038/s41598-022-15393-2

Figure Lengend Snippet: Dexamethasone (Dex) reverses IL-17A-induced epithelial barrier disruption in human airway epithelial cells (hAECs). hAECs were cultured and differentiated at an air liquid interface (ALI) and exposed to IL-17A (10 ng/ml) with or without dexamethasone (10 nM) for 14 days. Epithelial barrier function was assessed by permeability to fluorescein isothiocyanate-dextran (FITC-dextran) after 2 weeks incubation with IL-17A or dexamethasone of combination of both. FITC concentration was measured and calculated from fluorescence intensity. Data are from N = 7 donors ( a ). Gene expression was determined by bulk RNA sequencing and differentially expressed genes were analyzed. Statistically significant genes were used for pathway analysis (GSEA) and presented as top 10 most regulated pathways ( b ). String analysis ( https://string-db.org/ ) was done to the genes that are related to cilia function and development ( c ). Data represent means ± SEM. N = 4 donors. Statistical analyses were performed with paired one-way ANOVA and Tukey’s multiple comparison test, *p < 0.05.

Article Snippet: Experiments in submerged cells were conducted by exposing cells to 10 nM of Dexamethasone (Sigma-aldrich D4902), recombinant human IL-17A (R&D 7955-IL-025/CF) in three different concentrations (1, 10, and 100 ng/ml), or to combinations of IL-17A and Dexamethasone for 24 h. For experiments in ALI cultured cells, cells from passage 1–2 were grown submerged on collagen-coated Transwell inserts until confluence, after which they were cultured at an air–liquid interface (ALI) and differentiated for 2 weeks in B/D medium (1:1), which is a mixture of DMEM [Gibco] and bronchial epithelial growth medium (BEGM) [Lonza], supplemented with 0.4% [w/v] bovine pituitary extract [BPE], 0.5 ng/ml epidermal growth factor [EGF], 5 µg/ml insulin, 10 µg/ml transferrin, 1 µM hydrocortisone, 6.5 ng/ml T3, 0.5 µg/ml epinephrine [all from Lonza], 15 ng/ml retinoic acid [Sigma chemical] 1.5 µg/ml bovine serum albumin [Sigma chemical] 0.5 mM sodium pyruvate [Gibco], 20 U/ml penicillin and 20 µg/ml streptomycin [Gibco] .

Techniques: Cell Culture, Permeability, Incubation, Concentration Assay, Fluorescence, Expressing, RNA Sequencing Assay

Journal: Immunity

Article Title: Lung γδ T cells mediate protective responses during neonatal influenza infection that are associated with Type-2 immunity

doi: 10.1016/j.immuni.2018.07.011

Figure Lengend Snippet: A. Representative flow cytometric plots (left, with CD27 and CD44 expression on x- and y-axis, respectively) and summary frequency plot (right) of γδ T cells from mock- (open, n=12) and influenza virus- (solid, n=11) infected mice at 2 days following infection. Data are combined from four independent experiments and presented as mean ± SEM.B. Representative flow cytometric histogram showing expression of CCR6 (left) and Sca-1 (right) gated on γδ T cells from mock- (open) and virus- (shaded) infected neonates (2 days after infection). Mean fluorescence intensity are shown in the upper right corners.C. Relative expression of Il17a by quantitative real-time PCR in sort-purified γδ T cells from mock (open, n=6) and virus-infected neonates at 1 (n=6) and 2 (n=6) days following infection. Samples are combined from two independent experiments and data are presented as mean ± SEM.D. Representative flow cytometric plots of IL-17A (top) and IFN-γ (bottom) expression in γδ T cells from mock- (left) and virus-infected (right) neonates at 1 day after infection.E. Scatter plot showing frequency and number of IL-17A- (top) and IFN-γ- (bottom) producing γδ T cells from (D). Samples from mock-infected (n=14) neonates were pooled from animals 1 (n=11) and 2 (n=10) days after mock infection.F. Representative flow cytometric plots (left) and summary frequency plots (right) of γδ TCR expression gated on total IL-17A-producing cells from mock- (n=14) and virus- (n=11) infected mice at 1 day following infection. (D-F) PMA/ionomycin was used for stimulation prior to intracellular staining. Data are combined from four independent experiments and presented as mean ± SEM. **p<0.01, ***p<0.001, ****p<0.0001, n.s., not significant.

Article Snippet: Cytokines were measured in supernatants as follows: IL-17A, IL-33, and amphiregulin were assayed by Human IL-17A, IL-33, and amphiregulin DuoSet ELISA kits (R&D), and the level of IFN-γ was detected by Human Milliplex assays (Millipore) according to manufacturer instructions.

Techniques: Expressing, Infection, Fluorescence, Real-time Polymerase Chain Reaction, Purification, Staining

Journal: Immunity

Article Title: Lung γδ T cells mediate protective responses during neonatal influenza infection that are associated with Type-2 immunity

doi: 10.1016/j.immuni.2018.07.011

Figure Lengend Snippet: A. Estimation of IL-17A in whole lung homogenates from mock- (open) or influenza virus-(solid)- infected wild-type (black) and TCRδ−/− (red) neonates by ELISA at 1 day after infection. Samples are pooled from at least two independent experiments and data are shown as mean ± SEM (Mann-Whitney test).B. Survival rate of influenza virus-infected TCRδ−/− neonates administered with low levels of recombinant mouse IL-17A (rmIL-17A, green, n=37, 100pg/mouse) or PBS control (red, n=19) at the time of infection. Data are combined from six independent trials, which individually showed the same trend, and data are presented as mean ± SEM.C. Schema outlining wild-type and Il17a−/− γδ T cell transfers to TCRδ−/− neonates and subsequent infection.D and E. Body weight profile (D) and survival rate (E) of influenza-infected TCRδ−/− neonates receiving wild-type (black, n=15) or Il17a−/− (red, n=13) γδ T cells intranasally or no cell transfer (grey, n=10). Data are combined from four independent trials, which individually showed the same trend. Weight profile data are presented as mean ± SEM.F. Protein levels of IL-33 assessed by ELISA in influenza virus-infected wild-type (black, n=15) and TCRδ−/− lungs (red, n=9) at 1 day following infection. Samples are pooled from three independent experiments, and data are shown as mean ± SEM.G. Protein levels of IL-33 detected by ELISA in the lungs of TCRδ−/− neonates that had been infected with influenza virus and simultaneously treated with either a low level of rmIL-17A (green, n=7, 100pg/mouse) or PBS control (red, n=6). Data were collected 1 day after infection/treatment. Samples are pooled from at least two independent experiments, and data are shown as mean ± SEM.H. Survival rate of influenza virus-infected wild-type (black, n=20) and Il33−/− neonates (blue, n=17) with intranasal influenza virus infection. Data are combined from ten independent experiments and shown as mean ± SEM in weight change.I. Survival rate of influenza virus-infected TCRδ−/− neonates administered with recombinant mouse IL-33 (rmIL-33, brown, n=18, 10ng/mouse) or PBS control (red, n=18) at the time of infection. Data are combined from six individual experiments.*p<0.05, **p<0.01, n.s., not significant.

Article Snippet: Cytokines were measured in supernatants as follows: IL-17A, IL-33, and amphiregulin were assayed by Human IL-17A, IL-33, and amphiregulin DuoSet ELISA kits (R&D), and the level of IFN-γ was detected by Human Milliplex assays (Millipore) according to manufacturer instructions.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Recombinant

Journal: Immunity

Article Title: Lung γδ T cells mediate protective responses during neonatal influenza infection that are associated with Type-2 immunity

doi: 10.1016/j.immuni.2018.07.011

Figure Lengend Snippet: A. Relative expression of Il33 mRNA, as assessed by quantitative real-time PCR, in mouse lung epithelial cells (LET1s) treated with medium (white), A/x31 influenza virus (blue, 3 MOI), rmIL-17A (green, 50ng/ml), or virus+rmIL-17A (red) at 24hrs and 48hrs after treatment. Data are shown as mean ± SEM and combined from three separate experiments that independently showed the same trend.B. Immunoblot assay of IL-33 protein in stimulated LET1s for the same conditions as in (A) at 48 hrs after stimulation.C. Quantitative real-time PCR analysis of Il33 mRNA in LET1 cells treated as in A and B with and without STAT3 phosphorylation inhibitor S3I-201 (100μM in 0.05% DMSO) at 48hrs after treatment. Data are shown as mean ± SEM and combined from two separate experiments that independently showed the same trend.D. Immunoblot analysis of IL-33 and phosphorylated STAT3 (pSTAT3) in the LET1 lysates treated as before (A, B, and C). Total STAT3 (tSTAT3) and β-actin are also shown. Immunoblots were repeated twice showing similar results.*p<0.05, ****p<0.0001

Article Snippet: Cytokines were measured in supernatants as follows: IL-17A, IL-33, and amphiregulin were assayed by Human IL-17A, IL-33, and amphiregulin DuoSet ELISA kits (R&D), and the level of IFN-γ was detected by Human Milliplex assays (Millipore) according to manufacturer instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Immunity

Article Title: Lung γδ T cells mediate protective responses during neonatal influenza infection that are associated with Type-2 immunity

doi: 10.1016/j.immuni.2018.07.011

Figure Lengend Snippet: A- C. Correlation between concentrations of human IL-17A and IL-33 (A), IFN-γ and IL-33 (B) and IL-33 and Areg protein levels (C) in the nasal aspirates of influenza-infected children (< 8 years old, n=51).D. Concentration of IL-17A at Day 0 after enrollment in the nasal aspirates of children with mild (black, n=11) and severe (red, n=12) influenza disease outcomes.E. Concentration of IFN-γ at Day 0 after enrollment in the nasal aspirates of children with mild (black, n=11) and severe (red, n=12) disease outcomes.Data are presented as mean ± SEM, and in each case cytokine values (pg/ml) were log10 transformed for visualization only. *p<0.05.

Article Snippet: Cytokines were measured in supernatants as follows: IL-17A, IL-33, and amphiregulin were assayed by Human IL-17A, IL-33, and amphiregulin DuoSet ELISA kits (R&D), and the level of IFN-γ was detected by Human Milliplex assays (Millipore) according to manufacturer instructions.

Techniques: Infection, Concentration Assay, Transformation Assay

Journal: Immunity

Article Title: Lung γδ T cells mediate protective responses during neonatal influenza infection that are associated with Type-2 immunity

doi: 10.1016/j.immuni.2018.07.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cytokines were measured in supernatants as follows: IL-17A, IL-33, and amphiregulin were assayed by Human IL-17A, IL-33, and amphiregulin DuoSet ELISA kits (R&D), and the level of IFN-γ was detected by Human Milliplex assays (Millipore) according to manufacturer instructions.

Techniques: Plasmid Preparation, Recombinant, Cell Stimulation, Protease Inhibitor, Blocking Assay, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Sequencing, Software