Il 17a

Lymphocytes (106) from blood and jejunum were stimulated in vitro with 0.1 µM phorbol 12-myristate-13-acetate (PMA) and 0.5 µg/ml ionomycin (Sigma-Aldrich, St.Louis, MO), Cells were cultured for 4 hours in the presence of 5 µg/ml Brefeldin A (Sigma-Aldrich) then stained for cell surface markers, fixed in 2% paraformaldehyde, permeabilized in Cytofix/Cytoperm solution (BD Biosciences), and intracellularly co-stained with fluorochrome-labelled antibodies for the cytokines. For examining the effects of IL-22/IL-17A on maintenance of intestinal epithelial cells, total cell suspensions containing epithelial cells and lymphocytes isolated from the intestine were stimulated with IL-22 (10 ng/ml, BioLegend), IL-17A (10 ng/ml, BioLegend) or both and survival in vitro was compared with controls without cytokine stimulation. After 24 hours, cells were stained with Dead/lived cell staining kit and cell surface markers, and percentages of live epithelial cells were analyzed. Cells were acquired with a LSR II cytometer (Becton Dickinson). Data was analyzed with Flowjo software (Tree star, Ashland, OR).

Health mice were anaesthetized using 2% isoflurane inhalation and killed by cervical dislocation. The bronchus was isolated from lobes of lung, minced to small pieces and digested by 0.05% pronase (Sigma, MA, USA) in DMEM/F12 media (Invitrogen, CA, USA) at 4 °C overnight. Digestion was stopped by adding FBS (Gibco, CA, USA). The bronchial epithelial cells were identified by CK-18 immunofluorescence staining.
Murine bronchial epithelial cells were cultured for 24 h, and then were cocultured with cigarette smoke extract (CSE) or/and IL-17A. CSE was prepared as previously reported [27 (link)]. Briefly, filtered cigarettes (Nanning Jiatianxia unfiltered cigarettes, Guangxi, China) were smoked using a peristaltic pump (VWR International) after cutting the filters. Each cigarette was smoked with a 1.5 cm butt remaining. Four cigarettes were bubbled through 40 ml of cell growth medium, and this solution, regarded as 100% strength CSE. In CSE group, bronchial epithelial cells were induced by 20% CSE. In IL-17A group, the cells were induced by 50 ng/ml IL-17A (Biolegend, CA, USA). In CSE + IL-17A group, the cells were cocultured with 20% CSE and 50 ng/ml IL-17A (Biolegend, CA, USA). Cells in all groups were cultured at 37 °C with 5%CO₂ for 72 h. The cells without administration of CSE or IL-17A were control group.

Data represented in Figures 5A–5L: Cytokines were added for 12 h overnight at increasing doses (0, 0.1, 0.5, 1, 2, 5, 10 ng/mL) of IL-4 (human: Biolegend 574002), IL-17A (human: Biolegend 570502), IFNγ (human: Biolegend 570202; mouse: Peprotech 315-05), IFNα (human: Biolegend 592702; mouse: Biolegend 752802), or IFNβ (mouse: R&D Systems 8234-MB-010). Each condition was run as a biological triplicate. Data represented in Figure S3C-K: cytokines were added for 12 h overnight at increasing doses (0, 0.1, 0.5, 1, 5, 10 ng/mL) of human IL-4 (Biolegend 574004), IL-13 (Biolegend 571104), IFNα (Biolegend 592704), IFNγ (Biolegend 570204), IL-17A (Biolegend 570504), or IL-1β (Biolegend 579404) (each condition run as a biological quadruplicate). All populations were lysed in 50 μL lysis buffer (RLT + 1% BME, QIAGEN and Sigma, respectively) and snap frozen on dry ice. Bulk RNA-seq was performed as described previously and summarized above (Ordovas-Montanes et al., 2018 (link)). Populations were sequenced to an average ± SEM read depth of 3.95 ± 0.11 million reads per sample, with an average ± SEM alignment percentage to either hg19 or mm10 reference transcriptomes of 71 ± 0.3%. All samples met quality thresholds regarding genomic and transcriptomic alignment.

Paw tissue culture supernatants were examined for inflammatory/pain mediators using the following commercial ELISA kits: IL-17A (BioLegend, San Diego, CA, USA), IL-1β (Thermo Scientific, Pierce Biotechnology, Rockford, IL, USA), PGE2, IL-10 and TGF-β (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions. Standard curve was calculated for each assay with limits of detection for IL-17 = 8 pg/ml, IL-1β = 6.5 pg/mL, TNF-α = 2 pg/mL, IL-10 < 10 pg/mL, TGF-β = 4.6 pg/mL and PGE2 < 39 pg/mL. Cytokines and PGE2 concentrations were normalized to paw weight.
The concentration of nitrite, as the end-product of NO production, was measured in the paw tissue culture supernatants using a method based on the Griess reaction^{54 (link)}. The nitrite concentration was calculated using a NaNO₂ standard curve with a range from 1 to 40 μM and normalized to paw weight.

The cytokine levels in the supernatant were measured by enzyme-linked immunosorbent assay (ELISA) using commercially available IFN-γ, IL-4, IL-17A, and IL-10 ELISA kits (Biolegend, San Diego, CA, USA) according to the manufacturer’s instructions.

Antibodies employed in flow cytometric analysis were obtained from various commercial sources: anti-CD1d (Biolegend, 1B1), anti-αGalcer:CD1d (eBioscience, L363), CD86 (BD Bioscience, GL1), TCRαβ (eBioscience,IP26), F4/80 (eBioscience, BM8), IL17-A (Biolegend,TC11-18H10.1), NK1.1 (Biolegend, PK136), and CD3 (Biolegend, 17A2). Cells were blocked with anti-CD16/32 antibody (Biolegend) for 15 min before incubation with fluorescently labeled antibodies at a concentration of 2 μg/ml for 45 min on ice. Stained cells were washed once with FACS buffer and analyzed by FACSan (Becton Dickinson). For internal staining, cells stained with surface makers were fixed in 2% PFA Fixation buffer (eBioscience) at 4°C overnight, followed by 3 washes with permeabilization buffer (R&D) and incubation with permeabilization buffer for 20 min on ice before staining with internal antibody. Stained cells were washed once with permeabilization buffer and suspended in PBS for analysis. Flow cytometry data were processed with FlowJo software (Tree Star, inc.,).