BACKGROUND AND AIMS Laser capture microdissection (LCM) is a powerful tool for the isolation of specific tissue compartments. We aimed to investigate the mucosal immune response that takes place in different intestinal compartments of IBD patients, dissected by LCM, analyzing cytokines expression profile and endoplasmic reticulum (ER) stress markers. METHODS Frozen sections of gut were obtained from patients with Crohn's disease (CD), ulcerative colitis (UC) and from controls. Using LCM, surface epithelium (SE) and lamina propria (LP) compartments were isolated and total RNA extracted. The relative expression of Th1, Th17 and Treg cytokines was evaluated by quantitative reverse transcriptase real-time PCR (qRT-PCR), in addition to the assessment of mRNA splicing of the transcription factor X-box binding protein-1 (XBP1). Human neutrophil elastase (HNE) and the transcription factor forkhead box P3 (Foxp3) were also analyzed by immunohistochemistry. RESULTS The increased expression of IL-17 was observed in both intestinal compartments of IBD patients when compared to controls. IFN- γ, TNF-α , IL-10, HNE and Foxp3 were overexpressed in the LP compartment of both IBD patients as compared to controls. An upregulation of IFN-γ and an infiltration of HNE+ cells was found in the SE of patients with UC. Splicing of XBP1 mRNA was recognized in both intestinal compartments of IBD patients when compared to controls. CONCLUSIONS In IBD patients, both intestinal compartments are involved in Th17 response, whereas, LP compartment plays a prominent role in Th1 and Treg immune responses. Nevertheless, high level of IFN- γ was found in the SE of UC patients, suggesting that this compartment is involved in the Th1 immune response. Our data also suggested that ER stress signalling is active in both LP and SE compartment of IBD patients, thus advocating that ER stress and immunity are intertwined.

中文翻译：

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IBD：肠小室在粘膜免疫反应中的作用。

背景与目的激光捕获显微切割术（LCM）是用于隔离特定组织隔室的强大工具。我们旨在研究通过LCM解剖的IBD患者不同肠腔中发生的粘膜免疫反应，分析细胞因子表达谱和内质网（ER）应激标志物。方法从克罗恩病（CD），溃疡性结肠炎（UC）患者和对照组中获得肠道的冷冻切片。使用LCM，分离表面上皮（SE）和固有层（LP）隔室并提取总RNA。除了评估转录因子X-box结合蛋白-1（XBP1）的mRNA剪接，还通过定量逆转录酶实时PCR（qRT-PCR）评估了Th1，Th17和Treg细胞因子的相对表达。人体中性粒细胞弹性蛋白酶（HNE）和转录因子叉头盒P3（Foxp3）也进行了免疫组织化学分析。结果与对照组相比，在IBD患者的两个肠腔均观察到IL-17表达增加。与对照组相比，在两个IBD患者的LP区室中IFN-γ，TNF-α，IL-10，HNE和Foxp3均过表达。在UC患者的SE中发现IFN-γ的上调和HNE +细胞的浸润。与对照组相比，IBD患者的两个肠腔都可以识别XBP1 mRNA的剪接。结论在IBD患者中，两个肠区都参与Th17应答，而LP区在Th1和Treg免疫应答中起重要作用。不过，在UC患者的SE中发现了高水平的IFN-γ，提示该区室参与了Th1免疫反应。我们的数据还表明，ER应激信号传导在IBD患者的LP和SE隔室中均活跃，因此主张ER应激和免疫力是相互交织的。