TY - JOUR
T1 - CD73+ Epithelial Progenitor Cells That Contribute to Homeostasis and Renewal Are Depleted in Eosinophilic Esophagitis
AU - Hara, Takeo
AU - Kasagi, Yuta
AU - Wang, Joshua
AU - Sasaki, Masaru
AU - Aaron, Bailey
AU - Karami, Adam
AU - Shimonosono, Masataka
AU - Shimonosono, Rieko
AU - Maekawa, Hisatsugu
AU - Dolinsky, Lauren
AU - Wilkins, Benjamin
AU - Klein, Jeremy
AU - Wei, Jane
AU - Nunes, Kathryn
AU - Lynch, Kristle
AU - Spergel, Jonathan M.
AU - Hamilton, Kathryn E.
AU - Ruffner, Melanie A.
AU - Karakasheva, Tatiana A.
AU - Whelan, Kelly A.
AU - Nakagawa, Hiroshi
AU - Muir, Amanda B.
N1 - Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.
PY - 2022/1
Y1 - 2022/1
N2 - Background & Aims: Although basal cell hyperplasia is a histologic hallmark of eosinophilic esophagitis (EoE), little is known about the capabilities of epithelial renewal and differentiation in the EoE inflammatory milieu. In murine esophageal epithelium, there are self-renewing and slowly proliferating basal stem-like cells characterized by concurrent expression of CD73 (5'-nucleotidase ecto) and CD104 (integrin β4). Here, we investigated CD73+CD104+ cells within the basal population of human esophageal epithelium and clarified the biological significance of these cells in the EoE epithelium. Methods: We performed flow cytometry on esophageal biopsy samples from EoE and non-EoE patients to determine the quantity of CD73+CD104+ cells in the epithelium. Simulating the EoE milieu we stimulated primary patient-derived and immortalized cell line–derived esophageal organoids with interleukin (IL)4 and IL13 and analyzed by flow cytometry, immunohistochemistry, and quantitative reverse-transcription polymerase chain reaction. We performed single-cell RNA sequencing on primary organoids in the setting of IL13 stimulation and evaluated the CD73+CD104+ population. We performed fluorescent-activated cell sorting to purify CD73+CD104+ and CD73- CD104+ populations and seeded these groups in organoid culture to evaluate the organoid formation rate and organoid size. We used RNA interference to knock down CD73 in esophageal organoids to evaluate organoid formation rates and size. We evaluated the effects of signal transducer and activator of transcription 6 (STAT6) signaling inhibition by RNA interference, a STAT6 inhibitor, AS1517499, as well as the proton pump inhibitor omeprazole. Results: EoE patients showed decreased epithelial CD73+CD104+ cell content. IL4 and IL13 stimulation depleted this population in 3-dimensional organoids with a recapitulation of basal cell hyperplasia as corroborated by single-cell RNA sequencing of the organoids, which suggests depletion of CD73+CD104+ cells. The CD73+CD104+ population had enhanced organoid formation compared with the CD73-CD104+ population. Similarly, knock-down of CD73 resulted in decreased organoid formation rate. Genetic and pharmacologic inhibition of STAT6 prevented T helper 2 cytokine-induced depletion of CD73+CD104+ cells. Lastly, omeprazole treatment prevented the effects of IL4 and IL13 on the CD73+CD104+ population. Conclusions: This study addressed the role of CD73+CD104+ cells in epithelial renewal and homeostasis in the context of EoE. The depletion of the CD73+CD104+ self-renewal population by helper T cell 2 cytokines in EoE milieu may be perpetuating epithelial injury. Future therapies targeting epithelial restitution in EoE could decrease the need for immune modulation and steroid therapy.
AB - Background & Aims: Although basal cell hyperplasia is a histologic hallmark of eosinophilic esophagitis (EoE), little is known about the capabilities of epithelial renewal and differentiation in the EoE inflammatory milieu. In murine esophageal epithelium, there are self-renewing and slowly proliferating basal stem-like cells characterized by concurrent expression of CD73 (5'-nucleotidase ecto) and CD104 (integrin β4). Here, we investigated CD73+CD104+ cells within the basal population of human esophageal epithelium and clarified the biological significance of these cells in the EoE epithelium. Methods: We performed flow cytometry on esophageal biopsy samples from EoE and non-EoE patients to determine the quantity of CD73+CD104+ cells in the epithelium. Simulating the EoE milieu we stimulated primary patient-derived and immortalized cell line–derived esophageal organoids with interleukin (IL)4 and IL13 and analyzed by flow cytometry, immunohistochemistry, and quantitative reverse-transcription polymerase chain reaction. We performed single-cell RNA sequencing on primary organoids in the setting of IL13 stimulation and evaluated the CD73+CD104+ population. We performed fluorescent-activated cell sorting to purify CD73+CD104+ and CD73- CD104+ populations and seeded these groups in organoid culture to evaluate the organoid formation rate and organoid size. We used RNA interference to knock down CD73 in esophageal organoids to evaluate organoid formation rates and size. We evaluated the effects of signal transducer and activator of transcription 6 (STAT6) signaling inhibition by RNA interference, a STAT6 inhibitor, AS1517499, as well as the proton pump inhibitor omeprazole. Results: EoE patients showed decreased epithelial CD73+CD104+ cell content. IL4 and IL13 stimulation depleted this population in 3-dimensional organoids with a recapitulation of basal cell hyperplasia as corroborated by single-cell RNA sequencing of the organoids, which suggests depletion of CD73+CD104+ cells. The CD73+CD104+ population had enhanced organoid formation compared with the CD73-CD104+ population. Similarly, knock-down of CD73 resulted in decreased organoid formation rate. Genetic and pharmacologic inhibition of STAT6 prevented T helper 2 cytokine-induced depletion of CD73+CD104+ cells. Lastly, omeprazole treatment prevented the effects of IL4 and IL13 on the CD73+CD104+ population. Conclusions: This study addressed the role of CD73+CD104+ cells in epithelial renewal and homeostasis in the context of EoE. The depletion of the CD73+CD104+ self-renewal population by helper T cell 2 cytokines in EoE milieu may be perpetuating epithelial injury. Future therapies targeting epithelial restitution in EoE could decrease the need for immune modulation and steroid therapy.
KW - 5'-Nucleotidase/therapeutic use
KW - Animals
KW - Cytokines
KW - Eosinophilic Esophagitis/drug therapy
KW - Homeostasis
KW - Humans
KW - Hyperplasia/pathology
KW - Interleukin-13/pharmacology
KW - Interleukin-4/therapeutic use
KW - Mice
KW - Omeprazole/pharmacology
KW - Stem Cells/metabolism
UR - http://www.scopus.com/inward/record.url?scp=85126906074&partnerID=8YFLogxK
UR - https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=purepublist2023&SrcAuth=WosAPI&KeyUT=WOS:000796482500003&DestLinkType=FullRecord&DestApp=WOS
U2 - 10.1016/j.jcmgh.2022.01.018
DO - 10.1016/j.jcmgh.2022.01.018
M3 - Article
C2 - 35108658
SN - 2352-345X
VL - 13
SP - 1449
EP - 1467
JO - CMGH
JF - CMGH
IS - 5
ER -