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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 CD73CD104 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 CD73CD104 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 CD73CD104 population. We performed fluorescent-activated cell sorting to purify CD73CD104 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 CD73CD104 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 CD73CD104 cells. The CD73CD104 population had enhanced organoid formation compared with the CD73CD104 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 CD73CD104 cells. Lastly, omeprazole treatment prevented the effects of IL4 and IL13 on the CD73CD104 population.

CONCLUSIONS

This study addressed the role of CD73CD104 cells in epithelial renewal and homeostasis in the context of EoE. The depletion of the CD73CD104 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.

背景与目的

尽管基底细胞增生是嗜酸性食管炎（EoE）的组织学标志，但对于 EoE 炎症环境中上皮细胞更新和分化的能力知之甚少。在鼠食管上皮中，存在自我更新和缓慢增殖的基底样干细胞，其特征是同时表达 CD73（5'-核苷酸酶外切酶）和 CD104（整合素β4）。在这里，我们研究了人食管上皮基底细胞群中的 CD73CD104 细胞，并阐明了这些细胞在 EoE 上皮中的生物学意义。

方法

我们对来自 EoE 和非 EoE 患者的食管活检样本进行流式细胞术，以确定上皮中 CD73CD104 细胞的数量。通过模拟 EoE 环境，我们用白细胞介素（IL）4 和 IL13 刺激原代患者来源和永生化细胞系衍生的食管类器官，并通过流式细胞术、免疫组织化学和定量逆转录聚合酶链反应进行分析。我们在 IL13 刺激的情况下对原代类器官进行单细胞 RNA 测序，并评估 CD73CD104 群体。我们进行荧光激活细胞分选以纯化 CD73CD104 和 CD73 CD104 群体，并将这些群体接种到类器官培养物中，以评估类器官形成率和类器官大小。我们使用 RNA 干扰敲低食管类器官中的 CD73，以评估类器官形成率和大小。我们通过 RNA 干扰、STAT6 抑制剂 AS1517499 和质子泵抑制剂奥美拉唑评估 STAT6 信号转导抑制的作用。

结果

EoE 患者表现出上皮 CD73CD104 细胞含量减少。IL4 和 IL13 刺激导致 3 维类器官中该群体耗竭，单细胞 RNA 测序证实了基底细胞增生的重制，这表明 CD73CD104 细胞耗竭。与 CD73CD104 群体相比，CD73CD104 群体具有增强的类器官形成能力。同样，敲低 CD73 导致类器官形成率降低。STAT6 的遗传和药物抑制可防止辅助性 T 细胞 2 细胞因子诱导的 CD73CD104 细胞耗竭。最后，奥美拉唑治疗可预防 IL4 和 IL13 对 CD73CD104 群体的影响。