Journal: The Journal of Biological Chemistry

Article Title: Innate lymphoid cell type 3–derived interleukin-22 boosts lipocalin-2 production in intestinal epithelial cells via synergy between STAT3 and NF-κB

doi: 10.1074/jbc.RA118.007290

Figure Lengend Snippet: Cytokine-mediated antimicrobial gene and protein expression in human IECs. A, HT-29 cells were stimulated for 24 h with IL-17A (50 ng/ml), IL-22 (50 ng/ml), TNF (20 ng/ml), IFN-γ (10 ng/ml), or a combination of these cytokines after which gene expression for the indicated genes was determined by qPCR. Subsequent hierarchical clustering was performed based on the calculated Pearson distance between genes. Colors represent average fold change on log scale compared with untreated controls based on three independent experiments. B, HT-29 cells and primary human CD45-depleted intestinal cells were stimulated for 24 h with IL-17A (50 ng/ml), IL-22 (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) after which gene expression for 19 antimicrobial genes was determined by qPCR. For HT-29 cells, values represent average fold change compared with no stimulation based on three independent experiments. For primary IECs, values indicate average fold change compared with no stimulation for three individual patient samples. C, HT-29, SW480, Caco-2, T84, and HCT116 cells were stimulated for 24 h with IL-17A (50 ng/ml), IL-22 (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) after which gene expression for the indicated genes was determined by qPCR. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Dunnett's post hoc test as compared with the unstimulated cells (expression value of 1). D–F, HT-29 and T84 cells were stimulated for 24 h with IL-17A (50 ng/ml), IL-22 (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) after which hBD-2 and LCN-2 concentrations in supernatant were determined by ELISA, and nitrite concentrations were determined by Griess assay. Values indicate the mean with S.E. of three independent experiments. Statistical analysis performed with two-way ANOVA followed by Šidák's post hoc test. p < 0.005 = **, and p < 0.0005 = ***.

Article Snippet: Reagents The following cytokines were obtained from BioTechne (Minneapolis, MN) and used at the following concentrations unless stated otherwise: 50 ng/ml human IL-22 (782-IL-010); 50 ng/ml human IL-17A (317-ILB-050); 20 ng/ml human TNF (210-TA-005); 10 ng/ml human IFN-γ (285-IF-100); 50 ng/ml human IL-1β (201-LB-005); 50 ng/ml human IL-23; and 50 ng/ml human IL-6 (206-IL-010).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Griess Assay

Journal: The Journal of Biological Chemistry

Article Title: Innate lymphoid cell type 3–derived interleukin-22 boosts lipocalin-2 production in intestinal epithelial cells via synergy between STAT3 and NF-κB

doi: 10.1074/jbc.RA118.007290

Figure Lengend Snippet: Specific cytokine synergy regulates the expression and release of LCN-2, NO, and hBD-2. A–F, HT-29 cells were stimulated for 24 h with all possible combinations of IL-22 (50 ng/ml), IL-17A (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) after which qPCR was performed to determine LCN2 (A) DEFB4 (B), and NOS2 (C) expression. Protein levels for LCN-2 (D) and hBD-2 (E) were determined in supernatants by ELISA, whereas nitrite levels (F) were determined by Griess assay. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with one-way ANOVA followed by Dunnett's post hoc test as compared with unstimulated cells. G, Venn diagrams representing gene expression levels (left) and protein/nitrite levels (right) for different cytokine combinations. Color intensity corresponds to the values in A–F redistributed on a gray scale from 0 to 255. p < 0.05 = *, p < 0.005 = **, and p < 0.0005 = ***.

Article Snippet: Reagents The following cytokines were obtained from BioTechne (Minneapolis, MN) and used at the following concentrations unless stated otherwise: 50 ng/ml human IL-22 (782-IL-010); 50 ng/ml human IL-17A (317-ILB-050); 20 ng/ml human TNF (210-TA-005); 10 ng/ml human IFN-γ (285-IF-100); 50 ng/ml human IL-1β (201-LB-005); 50 ng/ml human IL-23; and 50 ng/ml human IL-6 (206-IL-010).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Griess Assay

Journal: The Journal of Biological Chemistry

Article Title: Innate lymphoid cell type 3–derived interleukin-22 boosts lipocalin-2 production in intestinal epithelial cells via synergy between STAT3 and NF-κB

doi: 10.1074/jbc.RA118.007290

Figure Lengend Snippet: Involvement of STAT3 in IL-22, TNF, and IL-17A–mediated LCN-2 production. A and B, HT-29 cells were stimulated for 30 min with the indicated cytokines after which protein lysate was used for Western blot analysis to determine levels of pSTAT3–Tyr-705, pSTAT3–Ser-727, ac-STAT3–Lys-685, STAT3, and total protein. A, images of Western blottings representative of three independent experiments. B, band intensity quantification. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. C and D, HT-29 cells were stimulated for 24 h with IL-22 (50 ng/ml), IL-17A (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) in combination with indicated concentrations of STATTIC or DMSO control after which LCN-2 concentrations were determined by ELISA (C) and HT-29 viability was determined by WST-1 assay (D). Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Dunnett's post hoc test. E, HT-29 cells were stimulated with indicated cytokine combinations for 30 min after which ChIP-qPCR was performed using anti-STAT3 or control IgG. Fold-increase in enrichment is shown compared with unstimulated cells. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. p < 0.05 = *, p < 0.005 = **, and p < 0.0005 = ***. Molecular masses indicate the location of the closest protein ladder marker on the blot.

Article Snippet: Reagents The following cytokines were obtained from BioTechne (Minneapolis, MN) and used at the following concentrations unless stated otherwise: 50 ng/ml human IL-22 (782-IL-010); 50 ng/ml human IL-17A (317-ILB-050); 20 ng/ml human TNF (210-TA-005); 10 ng/ml human IFN-γ (285-IF-100); 50 ng/ml human IL-1β (201-LB-005); 50 ng/ml human IL-23; and 50 ng/ml human IL-6 (206-IL-010).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, WST-1 Assay, Marker

Journal: The Journal of Biological Chemistry

Article Title: Innate lymphoid cell type 3–derived interleukin-22 boosts lipocalin-2 production in intestinal epithelial cells via synergy between STAT3 and NF-κB

doi: 10.1074/jbc.RA118.007290

Figure Lengend Snippet: Involvement of NF-κB in IL-22, TNF, and IL-17A-mediated LCN-2 production. A and B, HT-29 cells were stimulated for 30 min with the indicated cytokines after which protein lysate was used for Western blot analysis to determine levels of pNF-κB–Ser-536, ac-NF-κB–Lys-310, IκB, NF-κB, and total protein. A, images of Western blottings representative of three independent experiments. B, band intensity quantification. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. C and D, HT-29 cells were stimulated for 24 h with IL-22 (50 ng/ml), IL-17A (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) in combination with indicated concentrations of BMS-345541 or DMSO control after which LCN-2 concentrations were determined by ELISA (C) and HT-29 viability was determined by WST-1 assay (D). Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Dunnett's post hoc test. E, HT-29 cells were stimulated with indicated cytokine combinations for 30 min after which ChIP-qPCR was performed using anti-NF-κB or control IgG. Fold-increase in enrichment was compared with unstimulated cells. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. p < 0.05 = *, p < 0.005 = **, p < 0.0005 = ***. Molecular masses indicate the location of the closest protein ladder marker on the blot.

Article Snippet: Reagents The following cytokines were obtained from BioTechne (Minneapolis, MN) and used at the following concentrations unless stated otherwise: 50 ng/ml human IL-22 (782-IL-010); 50 ng/ml human IL-17A (317-ILB-050); 20 ng/ml human TNF (210-TA-005); 10 ng/ml human IFN-γ (285-IF-100); 50 ng/ml human IL-1β (201-LB-005); 50 ng/ml human IL-23; and 50 ng/ml human IL-6 (206-IL-010).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, WST-1 Assay, Marker

Journal: Cell host & microbe

Article Title: Gestational stage and IFN-λ signaling regulate ZIKV infection in utero

doi: 10.1016/j.chom.2017.08.012

Figure Lengend Snippet: A. Schematic of the intrauterine compartment during human pregnancy denoting chorionic villi, fetal membranes (amnion and chorion), and decidua basalis. B. Confocal micrographs of human chorionic villi, fetal membrane, or decidua stained for cytokeratin-19 (green, left panel; red, middle panel), HLA-G (green, right panel), or β-actin (red, left of right panels; green, middle panel). DAPI-stained nuclei are shown in blue. Scale bar, 50 μm. C. IFN-λ induction of OAS1 in midgestation chorionic villi (**, P < 0.01; n = 6), fetal membranes (P = 0.07; n = 6), or decidua (**, P < 0.01; n = 3) after treatment with 100 ng/mL of IFN-λ (λ1 or λ3) for 16 h as assessed by RT-qPCR. Experiments with recombinant IFN-β (100 ng/ml) were performed in parallel (***, P < 0.001). Data are normalized to mock-treated controls and are shown as mean ± standard deviation. D. Infectious ZIKV-Brazil titers from midgestation chorionic villi (n = 7), fetal membranes (n = 6), or decidua (n = 3) preparations pre-treated with medium (Mock) or 100 ng/ml of IFN-β, IFN-λ1, or IFN-λ3 overnight (~16 h) and then infected with ZIKV-Brazil for 72 h. Data are shown as mean ± standard deviations. Statistical analyses in C and D were peformed using a Kruskal-Wallis ANOVA (*, P < 0.05). See also Figures S3, S4, and S5.

Article Snippet: Human placental explants For infections, placental, decidual, or fetal membrane explants were infected immediately with 5 x 10 5 FFU/ml of ZIKV for 72 h following isolation and treatment (~16 h) with 100 ng/mL of recombinant IFN-β, IFN-λ1 or IFN-λ3 (R&D Systems; 1598-IL-025, 5259-IL-025, 8499-IF-010).

Techniques: Membrane, Staining, Quantitative RT-PCR, Recombinant, Standard Deviation, Infection