Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
Cell Biology Program, Research Institute, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada.
Microbiology (Reading). 2010 Nov;156(Pt 11):3288-3297. doi: 10.1099/mic.0.040139-0. Epub 2010 Jul 23.
The intestinal epithelium forms a protective barrier against luminal contents and the external environment, mediated via intercellular tight junctions (TJs). The TJ can be disrupted via cell signalling induced by either enteric pathogens or pro-inflammatory cytokines, thereby contributing to various intestinal disorders ranging from acute infectious diarrhoea to chronic inflammatory bowel diseases. Probiotics, such as Lactobacillus rhamnosus GG (LGG), are reported to confer beneficial effects on epithelial cells, including antagonizing infections and reducing overt pro-inflammatory responses, but the underlying mechanisms of these observed effects require further characterization. We hypothesized that probiotics preserve barrier function by interfering with pro-inflammatory cytokine signalling. Caco-2bbe cells were seeded into Transwells to attain polarized monolayers with intercellular TJs. Monolayers were inoculated apically with the probiotic LGG 3 h prior to the addition of IFN-γ (100 ng ml(-1)) to the basolateral medium overnight. The monolayers were then placed in fresh basal medium±TNF-α (10 ng ml(-1)) and transepithelial electrical resistance (TER) measurements were taken over the time-course of TNF-α stimulation. To complement the TER findings, cells were processed for zona occludens-1 (ZO-1) immunofluorescence staining. As a measure of TNF-α downstream signalling, cells were immunofluorescently stained for NF-κB p65 subunit and CXCL-8 mRNA was quantified by qRT-PCR. Basal cell culture medium was collected after overnight TNF-α stimulation to measure secreted chemokines, including CXCL-8 (interleukin-8) and CCL-11 (eotaxin). Following LGG inoculation, IFN-γ priming and 24 h TNF-α stimulation, epithelial cells maintained TER and ZO-1 distribution. LGG diminished the nuclear translocation of p65, demonstrated by both immunofluorescence and CXCL-8 mRNA expression. CXCL-8 and CCL-11 protein levels were decreased in LGG-inoculated, cytokine-challenged cells. These findings indicate that LGG alleviates the effects of pro-inflammatory cytokines on epithelial barrier integrity and inflammation, mediated, at least in part, through inhibition of NF-κB signalling.
肠上皮形成了一道针对腔内容物和外部环境的保护屏障，这一屏障通过细胞间紧密连接（TJ）实现。TJ 可通过肠病原体或促炎细胞因子诱导的细胞信号转导而被破坏，从而导致从急性感染性腹泻到慢性炎症性肠病等各种肠道疾病。已报道益生菌，如鼠李糖乳杆菌 GG（LGG），对上皮细胞具有有益作用，包括拮抗感染和减少明显的促炎反应，但这些观察到的作用的潜在机制需要进一步表征。我们假设益生菌通过干扰促炎细胞因子信号转导来维持屏障功能。将 Caco-2bbe 细胞接种到 Transwell 中，在细胞间 TJ 处形成极化单层。用 IFN-γ（100ng/ml）在基底外侧培养基中孵育过夜之前，将益生菌 LGG 3 h 接种到顶端。然后将单层置于新鲜基底培养基±TNF-α（10ng/ml）中，并在 TNF-α 刺激过程中测量跨上皮电阻（TER）。为了补充 TER 发现，对细胞进行紧密连接蛋白-1（ZO-1）免疫荧光染色。作为 TNF-α 下游信号的测量，通过免疫荧光染色对 NF-κB p65 亚基进行染色，并通过 qRT-PCR 定量 CXCL-8 mRNA。在 TNF-α 刺激过夜后收集基础细胞培养基，以测量分泌趋化因子，包括 CXCL-8（白细胞介素-8）和 CCL-11（嗜酸粒细胞趋化因子）。在用 LGG 接种、IFN-γ 预刺激和 24 h TNF-α 刺激后，上皮细胞维持 TER 和 ZO-1 分布。LGG 减少了 p65 的核易位，这一点通过免疫荧光和 CXCL-8 mRNA 表达都得到了证明。在 LGG 接种、细胞因子挑战的细胞中，CXCL-8 和 CCL-11 蛋白水平降低。这些发现表明，LGG 减轻了促炎细胞因子对上皮屏障完整性和炎症的影响，至少部分通过抑制 NF-κB 信号转导介导。
