University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Magee-Womens Research Institute and Foundation, Pittsburgh, PA, USA.
University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
J Immunol Methods. 2018 Dec;463:39-46. doi: 10.1016/j.jim.2018.08.009. Epub 2018 Sep 12.
There are limited data on the immunological responsiveness of healthy intestinal tissue when it is cultured and stimulated ex vivo. Such an ex vivo model has the potential to be a valuable tool in understanding disease pathogenesis and as a preclinical tool for the assessment of candidate therapeutic agents used to treat inflammatory bowel disease (IBD).
We undertook a comprehensive study to evaluate ex vivo immunological responses of intestinal tissue and isolated mucosal mononuclear cells (MMC) to a broad range of stimuli.
Colorectal biopsies (explants) were obtained from healthy participants by flexible sigmoidoscopy and were placed either directly into culture or digested to isolate MMC prior to placement in culture. Explants or MMC were treated with polyinosinic:polycytidylic acid (Poly IC), phytohemagglutinin (PHA), lipopolysacccharides from E Coli (LPS), anti-CD3/CD28 antibodies, or IL-1β/TNF-α for 24 h. Supernatants were assayed for 40 inflammatory biomarkers using multiplexed enzyme-linked immunosorbent assay (ELISA). The isolated MMCs were further characterized using twelve color flow cytometry.
Explants have greater weight adjusted constitutive expression of inflammatory biomarkers than MMCs. Biomarker responses varied as a function of immunogen and use of intact tissue or isolated cells. PHA applied to intact explants was the most effective agent in inducing biomarker changes. Stimulation induced activated and memory cellular phenotypes in both explants and MMCs.
The breadth and magnitude of responses from intact and enzymatically digested intestinal tissue explants stimulated with exogenous immunogens are complex and vary by tissue form and treatment. Overall, PHA stimulation of intact explants produced the most robust responses in normal human colorectal tissue. This system could potentially serve as a preliminary model of the disease state, suitable for small scale screening of new therapeutic agents prior to using IBD patient derived tissue.
关于培养和体外刺激健康肠组织的免疫反应性的数据有限。这种体外模型有可能成为理解疾病发病机制的有用工具，并成为评估用于治疗炎症性肠病 (IBD) 的候选治疗剂的临床前工具。
我们进行了一项全面的研究，以评估肠组织和分离的粘膜单核细胞 (MMC) 对广泛刺激物的体外免疫反应。
通过柔性乙状结肠镜从健康参与者中获得结肠活检（外植体），并将其直接放入培养物中，或在放入培养物之前消化以分离 MMC。将外植体或 MMC 用聚肌苷酸：聚胞苷酸 (Poly IC)、植物血凝素 (PHA)、大肠杆菌脂多糖 (LPS)、抗 CD3/CD28 抗体或 IL-1β/TNF-α 处理 24 小时。使用多重酶联免疫吸附试验 (ELISA) 测定上清液中 40 种炎症生物标志物。使用十二色流式细胞术进一步表征分离的 MMC。
与 MMC 相比，经重量调整的外植体固有表达的炎症生物标志物更多。生物标志物的反应随免疫原和使用完整组织或分离细胞而变化。应用于完整外植体的 PHA 是诱导生物标志物变化最有效的药物。刺激诱导了外植体和 MMC 中激活和记忆细胞表型。
用外源性免疫原刺激完整和酶消化的肠组织外植体产生的反应的广度和幅度是复杂的，并且取决于组织形式和处理方式。总体而言，PHA 刺激完整外植体在正常人类结直肠组织中产生最强烈的反应。该系统有可能作为疾病状态的初步模型，适用于在使用 IBD 患者来源组织之前对新型治疗剂进行小规模筛选。
