Journal: ImmunoHorizons

Article Title: IL-17RA–Mediated Epithelial Cell Activity Prevents Severe Inflammatory Response to Helicobacter pylori Infection

doi: 10.4049/immunohorizons.2300078

Figure Lengend Snippet: Fibroblasts respond to IL-17, expressing PMN recruiting chemokines. ( A–C ) Primary fibroblasts were cultured from uninfected gastric tissue from C57BL/6, Il17ra ΔGI-Epi , and germline Il17ra −/− mice, expanded in culture, and then stimulated with 100 ng/ml rIL-17a for 5 h. Real-time RT-PCR was performed to measure relative expression of PMN recruiting chemokines, (A) Cxcl1 , (B) Cxcl2 , and (C) Cxcl5 . Data are expressed in relative units using Gapdh as the housekeeping gene and unstimulated fibroblasts as the calibrator sample. Individual values from each sample are shown with the SD for each group. One-way ANOVA was performed with a Dunnett multiple comparison test to assess statistical significance compared with unstimulated cells. *** p < 0.001, **** p < 0.0001.

Article Snippet: For stimulations, cells were serum starved overnight and then stimulated with 20–100 ng/ml rIL-17a (210-17, PeproTech, Rocky Hill, NJ) or 0.1% BSA in PBS for 6 h. RNA was isolated as described above, and real-time qPCR was performed using TaqMan gene expression assays for Cxcl1 (Mm04207460_m1), Cxcl2 (Mm00436450_m1), Cxcl5 (Mm00436451_g1), and Il17ra (Mm00493214_m1) (Applied Biosystems/Thermo Fisher Scientific).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Comparison

Journal: ImmunoHorizons

Article Title: IL-17RA–Mediated Epithelial Cell Activity Prevents Severe Inflammatory Response to Helicobacter pylori Infection

doi: 10.4049/immunohorizons.2300078

Figure Lengend Snippet: IL-17 signaling impacts expression of genes that contribute to innate barrier function and restricts chronic inflammation. ( A ) Differential abundance of genes in the stomach at 3 mo after H. pylori infection in Il17ra −/− mice compared with C57BL/6 mice using the nCounter Immunology NanoString panel. The table represents genes that are lower in expression in Il17ra −/− with a log 10 adjusted p value of >2 and a log 2 fold change of greater than −1.25. On the volcano plot, these genes are in purple. The multiplex RNA hybridization assay was performed on six mice per genotype. ( B ) Gastric murine organoids from C57BL/6 mice (gastroids) were stimulated with 50 ng/ml IL-17A, IL-17F, and the heterodimer IL-17A/F. RNA was then extracted from these gastroids and qPCR assays of genes associated with epithelial cell responses including Cxcl1 , Nox1 , and Pigr were measured. Gapdh was used as an endogenous control, and unstimulated gastroids were pooled and used as a reference sample. The data are representative of three independent experiments. Error bars represent ± SEM. One-way ANOVA with a Dunnett multiple comparison test was used to determine significance. * p < 0.05, **** p < 0.0001, compared with unstimulated.

Article Snippet: For stimulations, cells were serum starved overnight and then stimulated with 20–100 ng/ml rIL-17a (210-17, PeproTech, Rocky Hill, NJ) or 0.1% BSA in PBS for 6 h. RNA was isolated as described above, and real-time qPCR was performed using TaqMan gene expression assays for Cxcl1 (Mm04207460_m1), Cxcl2 (Mm00436450_m1), Cxcl5 (Mm00436451_g1), and Il17ra (Mm00493214_m1) (Applied Biosystems/Thermo Fisher Scientific).

Techniques: Expressing, Infection, Multiplex Assay, Hybridization, Control, Comparison