Bermudez-Brito Miriam, Muñoz-Quezada Sergio, Gómez-Llorente Carolina, Matencio Esther, Romero Fernando, Gil Angel
Institute of Nutrition and Food Technology "José Mataix", Department of Biochemistry and Molecular Biology II, University of Granada, Biomedical Research Center, Avenida del Conocimiento s/n, 18100 Armilla, Granada, Spain.
Hero Global Technology Center, Hero Spain, S.A., 30820, Alcantarilla, Murcia, Spain.
BMC Microbiol. 2015 Apr 1;15(1):79. doi: 10.1186/s12866-015-0408-6.
The action of probiotics has been studied in vitro in cells isolated from both mice and humans, particularly enterocytes (IECs), dendritic cells (DCs) and co-cultures of peripheral DCs and IECs. Peripheral DCs and murine DCs differ from human gut DCs, and to date there are no data on the action of any probiotic on co-cultured human IECs and human intestinal DCs. To address this issue, a novel transwell model was used. Human IECs (Caco-2 cells) grown in the upper chamber of transwell filters were co-cultured with intestinal-like human DCs grown in the basolateral compartment of the transwells. The system was apically exposed for 4 h to live probiotic L. paracasei CNCM I-4034 obtained from the faeces of breastfed infants or to its cell-free culture supernatant (CFS) and challenged with Salmonella typhi. The secretion of pro- and anti-inflammatory cytokines in the basolateral compartment was determined by immunoassay, and the DC expression pattern of 20 TLR signaling pathway genes was analysed by PCR array.
The presence of the live probiotic alone significantly increased IL-1β, IL-6, IL-8, TGF-β2, RANTES and IP-10 levels and decreased IL-12p40, IL-10, TGF- β1 and MIP-1α levels. This release was correlated with a significant increase in the expression of almost all TLR signaling genes. By contrast, incubation of the co-culture with CFS increased IL-1β, IL-6, TGF-β2 and IP-10 production only when Salmonella was present. This induction was correlated with an overall decrease in the expression of all TLR genes except TLR9, which was strongly up-regulated.
The data presented here clearly indicate that L. paracasei CNCM I-4034 significantly increases the release of pro-inflammatory cytokines, enhances TLR signaling pathway activation and stimulates rather than suppresses the innate immune system. Furthermore, our findings provide evidence that the effects of probiotics in the presence of IECs and DCs differ from the effects of probiotics on cultures of each cell type alone, as reported by us earlier. Thus, co-culture systems such as the one described here are needed to characterise the effects of probiotics in vitro, highlighting the potential utility of such co-cultures as a model system.
益生菌的作用已在从小鼠和人类分离的细胞中进行了体外研究，特别是肠上皮细胞（IECs）、树突状细胞（DCs）以及外周DCs和IECs的共培养物。外周DCs和小鼠DCs与人类肠道DCs不同，迄今为止，尚无关于任何益生菌对共培养的人类IECs和人类肠道DCs作用的数据。为解决这一问题，使用了一种新型的Transwell模型。在Transwell滤器上室生长的人类IECs（Caco-2细胞）与在Transwells基底外侧隔室生长的类肠道人类DCs共培养。该系统顶端暴露于从母乳喂养婴儿粪便中获得的活益生菌副干酪乳杆菌CNCM I-4034或其无细胞培养上清液（CFS）4小时，然后用伤寒沙门氏菌进行攻击。通过免疫测定法测定基底外侧隔室中促炎和抗炎细胞因子的分泌，并通过PCR阵列分析20种TLR信号通路基因的DC表达模式。
单独存在活益生菌时显著增加了IL-1β、IL-6、IL-8、TGF-β2、RANTES和IP-10水平，并降低了IL-12p40、IL-10、TGF-β1和MIP-1α水平。这种释放与几乎所有TLR信号基因表达的显著增加相关。相比之下，仅在存在沙门氏菌时，用CFS孵育共培养物才会增加IL-1β、IL-6、TGF-β2和IP-10的产生。这种诱导与除TLR9外所有TLR基因表达的总体下降相关，而TLR9则强烈上调。
此处呈现的数据清楚地表明，副干酪乳杆菌CNCM I-4034显著增加促炎细胞因子的释放，增强TLR信号通路激活，并刺激而非抑制先天免疫系统。此外，我们的研究结果提供了证据，表明益生菌在存在IECs和DCs时的作用不同于我们之前报道的益生菌对每种细胞类型单独培养的作用。因此，需要像这里描述的共培养系统来体外表征益生菌的作用，突出这种共培养作为模型系统的潜在用途。
