Subjects, Samples, and Clinical Information
Colonic mucosa was obtained from surgically resected colon specimens from patients with an established diagnosis of CD between February 2012 and Jan 2018. All samples were collected from disease-unaffected regions without macroscopic inflammation and were from ascending colon. Clinical information was collected from medical records up to 5 years after CD diagnosis.
Two Independent cohorts of adult CD and treatment-naïve pediatric CD samples were downloaded from GEO (accession numbers GSE57945 and GSE137344). Pediatric CD samples from GSE57945 were processed as described previously 12.
RNA isolation, sequencing, and processing
Adult samples from UNC hospitals were isolated and sequenced as previously described 8. Briefly, RNA was isolated using the Qiagen RNeasy Mini Kit following the manufacturer’s protocol, and RNA purity was assessed with Thermo Scientific NanoDrop 2000. RNA-seq libraries were prepared using the Illumina TruSeq polyA+ Sample Prep Kit. Paired-end (50 bp) sequencing was performed on the Illumina HiSeq 2500 and 4000 platforms.
Cutadapt v2.9 (https://doi.org/10.14806/ej.17.1.200) was used to remove sequencing adapters and filter low quality reads (-q 10). Quantification of sequencing reads was performed using Salmon v1.2. 9 to the hg38 genome with GC-bias and sequence-specific parameters enabled (--gcbias and –seqbias, respectively), and tximport v1.12.3 (10.12688/f1000research.7563.1) was used to summarize transcript-level to gene-level abundance estimates using R v3.6.0.
Data Availability Statement
The sequencing data underlying this article are available in public sequencing data from GEO, sratoolkit v2.10.1 (http://ncbi.github.io/sra-tools/). The remaining data underlying this article are available in the article and in its online supplementary material.
Raw sequencing counts from Salmon were DESeq2 normalized and VST transformed10. Box plots were generated using ggplot2, and PCA was performed using the prcomp function in R v3.6.0.
Human ileum and colon tissue biopsies from NIBD, colon-like Crohn’s disease (CL) and ileum-like Crohn’s disease (IL) were fixed in 10% (vol/vol) neutral buffered formalin, embedded in paraffin, and prepared as histological sections. After deparaffinization and epitope retrieval in 1X citrate buffer solution, sections were blocked for 1 h in 3% BSA before immunostaining was performed. Polyclonal goat anti-ACE2 antibody (R&D Systems #AF933) was applied overnight at 4oC, followed by a 1 h incubation with a secondary anti-goat (Alexa Fluor 594) antibody the next day. Slides were then incubated with DAPI (Invitrogen #D1306) for 5 min to stain nuclei and mounted using FluorSave Reagent (EMD Millipore #345789). Fluorescence was detected using an Olympus VS120 virtual slide microscope.
ACE2 Signal Intensity
ACE2 fluorescent signal intensity was measured using ImageJ software and normalized to background. To facilitate measurements, images of the stained tissue sections were converted to black and white images on the ACE2 channel, removing signal from DAPI. For each section, pixel intensity was measured in three different regions that were selected for optimal histological cut, showing intact villi (ileum) or colonocytes (colon). Five intensity measurements (e.g., yellow rectangles on supplemental data images) were analyzed per region (Supplemental Figure 1). N=4 patients per group. Intensity measurements were averaged per patient and normalized to disease-control group. Significance was determined via one-way ANOVA with multiple comparisons.
Reverse-Transcriptase qPCR Analysis
Total RNA was extracted from dissected colonic mucosa stored in RNAlater using TRIzol reagent and purified with the Total RNA Purification Plus Kit (48300; Norgen Biotek) according to the manufacturer’s instructions. Total RNA was extracted from isolated and cultured colonic IECs using the Single Cell RNA Purification Kit (51800; Norgen Biotek). Complementary DNA for mRNA was generated from 500 ng of RNA using the High-Capacity Complementary DNA Reverse Transcription Kit (4368814; Thermo Fisher Science). Comparative-Ct-TaqMan with a relative quantification qPCR for mRNAs was performed on the QuantStudio 3 RT-PCR system using TaqMan Fast Advanced Master Mix (4444557; Thermo Fisher Science) with individual TaqMan probes (TaqMan Gene Expression assays, assay ID: Hs01085333_m1 [ACE2], Hs02339424_g1 [RPS9]). Expression of ACE2 was normalized to RPS9.
All numeric data in the figures are expressed as means ± standard deviation (SD). Differences between the 2 groups were analyzed by a Mann–Whitney or Fisher exact test. Differences between the 3 groups were analyzed by a Kruskal-Wallis test followed by Dunn’s multiple comparison test (R v4.0.1). P values less than .05 were considered significant. The Kaplan–Meier method was used to generate survival curves and differences between 2 groups were evaluated by a log-rank test. GraphPad Prism (v8.0; GraphPad Software) was used for these data analyses. Calculation of propensity score and a Cox regression analysis were performed using R v3.5.2.