Mouse monoclonal antibody against IL-33 (clone Nessy-1) (#ALX-804-840-C100) was purchased from Enzo. Rabbit polyclonal antibodies against KRT5 (#ab24647) and Ki-67 (#ab15580) were purchased from Abcam (Abcam, Cambridge, MA). Rabbit polyclonal antibody against KRT14 (#PRB-155P) was purchased from Covance (Covance, Princeton, NJ). Rabbit polyclonal antibody against KRT4 (#HPA034881) was purchased from Sigma (Sigma-Aldrich Corp, St. Louis, MO). Rabbit monoclonal antibodies against E-cadherin (#3195), p75 (#8238), and phospho-histone H3 (#3377) were purchased from Cell Signaling (Cell Signaling Technology, MA). Mouse monoclonal antibody against p63 (#sc-8431) was purchased from Santa Cruz (Santa Cruz Biotechnology, TX). Mouse monoclonal antibody against PCNA (#MAB424) was purchased from Millipore (Billerica, MA). Goat polyclonal antibody against IL-33 (#AF3625) was purchased from R&D (R&D Systems, Minneapolis, MN). Donkey anti-goat Alexa Fluor 488 (A11055), anti-rabbit Alexa Fluor 568 (A10042), and anti-mouse Alexa Fluor 647 (A31571) secondary antibodies were purchased from Life Technologies (Carlsbad, CA). Mouse anti-HSP90 (TA500494) and mouse anti-GAPDH (TA310153) primary antibodies were purchased from Origene (Rockville, MD).
Esophageal biopsy collection and processing
Esophageal biopsies were obtained and processed as previously described43. Briefly, this study was approved by the Institutional Review Board of Cincinnati Children’s Hospital Medical Center (CCHMC) before the start of the study. Active EoE was defined as having a physician-provided EoE diagnosis and ≥15 eosinophils per 400x high-power field in distal esophageal biopsies. Inactive EoE was defined as having a previous history of EoE but with 0 or 1 eosinophils per high-power field. Normal controls were defined as patients with any history of EoE nor any other eosinophilic gastrointestinal disorder (EGID) and 0 eosinophils per high-power field. After informed consent was received, distal esophageal biopsies were obtained and fixed with formalin and then embedded in paraffin (FFPE). All experimental methods utilizing processed esophageal biopsies were performed in accordance with all relevant guidelines and regulations.
Immunohistochemistry and immunofluorescence of esophageal biopsies
Before immunohistochemistry or immunofluorescence was performed, hematoxylin and eosin (H&E) stainings of esophageal biopsies were examined to confirm proper orientation and inclusion of all layers of the epithelium. H&E stainings and immunohistochemistry of distal esophageal biopsies using mouse anti-IL-33 antibody (Nessy-1) were performed by the Pathology Research Core at CCHMC. Images were obtained using an Apotome widefield microscope (Zeiss, Thornwood, NY). For immunofluorescence studies, slides with 4-µm sections of FFPE esophageal biopsies underwent deparaffinization (serial incubations with xylene, 100% ethanol, 95% ethanol, 70% ethanol, 50% ethanol), antigen retrieval using sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0), blocked with 10% donkey serum/phosphate-buffered saline (PBS), and then incubated with primary antibody diluted in 10% donkey serum/PBS overnight at 4 °C in a humidified chamber. The next day, slides were washed with PBS, incubated with secondary antibodies diluted in 10% donkey serum/PBS for 1 h at room temperature (RT) in a humidified chamber, and then washed in the presence of DAPI (0.5 µg/mL). Finally, a cover slip was added with ProLong Gold mounting reagent (Molecular Probes). The next day, slides were imaged using a Nikon A1R inverted confocal microscope. Analysis was performed with the Nikon Elements program.
Ex vivo culture of primary esophageal epithelial cells
One human distal esophageal biopsy obtained during routine endoscopy was collected for research purposes in 1 mL keratinocyte serum-free media (KSFM) (Invitrogen) containing human epidermal growth factor (EGF) (1 ng/mL), bovine pituitary extract (50 μg/mL), and 1X penicillin/streptomycin (Invitrogen) and subsequently placed in a 60-mm dish in 3 mL of Leibovitz’s L-15 media (Invitrogen) containing 115 U/mL collagenase, 1.2 U/mL dispase, and 1.25 mg/mL bovine serum albumin that had been filter sterilized (0.2 μm). The biopsy was mechanically dispersed using scissors into pieces less than 1 mm in size and then incubated at 37 °C for 1 h. The digested biopsy was collected and washed twice with 5 mL KSFM containing the same supplements as described above. Cells were then incubated in 1 mL of 0.05% trypsin/ethylenediaminetetraacetic acid (EDTA) (Invitrogen) (10 min, 37 °C, with agitation every 2 min). Soybean trypsin inhibitor (250 mg/L in 1X Dulbecco’s phosphate-buffered saline) was added (5 mL). Cells were pelleted and then resuspended in 1 mL KSFM (containing the same supplements as described above) and transferred to a 35-mm dish. Irradiated NIH 3T3 J2 fibroblasts (162,500 cells) were added to the dish. Media were changed at day 5 and every other day thereafter using KSFM containing the same supplements as describe above. After epithelial cells became 60–70% confluent, they were dispersed from the plate using 0.05% trypsin/EDTA, which was inactivated by soybean trypsin inhibitor; cells were then cultured in KSFM containing the same supplements as described above. In indicated experiments, cultures were stimulated with recombinant human IL-13 or OSM (both from Peprotech).
Immunofluorescence of primary esophageal epithelial cells
Primary esophageal epithelial cells were plated on Ibidi 8-well chambers (#80826). The next day, cells were fixed with 4% paraformaldehyde for 10 min and quenched with 50 mM ammonium chloride. Cells were blocked with 10% donkey serum/PBS for 30 min and incubated with primary antibody diluted in 10% donkey serum/PBS for 1 h at RT. Cells were washed with PBS, incubated with secondary antibodies diluted in 10% donkey serum/PBS for 1 h at RT, and washed in the presence of DAPI (0.5 µg/mL). Finally, cells were placed in fresh PBS and imaged using a Nikon A1R inverted confocal microscope. Analysis was performed with the Nikon Elements program.
Western blot analysis
Cells were lysed in RIPA buffer (50 mM Tris-HCl pH 8; 150 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate; 0.1% SDS, and 1 mM EGTA) supplemented with beta-mercaptoethanol and protease inhibitors (Roche), sonicated for three rounds of 10 seconds, boiled for 15 minutes, loaded onto a 4–12% SDS-PAGE gel (Invitrogen), and subjected to Western blot analysis. Membranes were probed with goat anti-IL-33, mouse anti-HSP90, or mouse anti-GAPDH antibodies. Secondary IRDye-conjugated antibodies were from LI-COR Biosciences (Lincoln, Nebraska). Quantification of signal was performed with Image Studio Lite software (http://www.licor.com/bio/products/software/image_studio_lite/).
Quantitative real-time polymerase chain reaction (RT-PCR)
RT-PCR was performed as previously described43. Briefly, total RNA was isolated from cells using the RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. RT-PCR was then performed using a 7900HT Fast Real-Time PCR system from Applied Biosystems (Life Technologies Grand Island, NY) with FastStart Universal SYBR Green Master mix (Roche Diagnostics Corporation Indianapolis, IN).
One-way ANOVA with Holm-Sidak correction for multiple testing was performed using GraphPad Prism 7.0 software.