Adherence and Cytokine Induction in Caco-2 Cells by Bacterial Populations from a Three-Stage Continuous-Culture Model of the Large Intestine

Adherence of bacteria to epithelial cells is an important step in colonization and immune modulation in the large bowel. The aims of this study were to use a three-stage continuous-culture system (CCS) to investigate how environmental factors affect bacterial attachment to Caco-2 cells and modulation of cytokine expression by gut microorganisms, including a probiotic Bifidobacterium longum strain, DD2004. The CCS simulated environmental conditions in the proximal large intestine (vessel 1 [V1]) and distal colon (V2 and V3) at two different system retention times (R) within the range of normal colonic transits (20 and 60 h). The model was inoculated with human fecal material, and fluorescence in situ hybridization (FISH) was used to characterize microbial populations and to assess bacterial attachment to Caco-2 cells. Real-time quantitative PCR (qPCR) was employed to measure cytokine gene expression following challenge with bacteria from different components of the CCS in the presence and absence of B. longum. At an R of 60 h, bacterial adherence increased from V1 to V3, but this trend was reversed at an R of 20 h. Atopobia were the predominant adherent organisms detected at both system retention times in each culture vessel. Modulation of transforming growth factor beta 1 (TGF-beta 1), interleukin 6 (IL-6), and IL-18 gene expression by CCS bacteria was marked at an R of 60 h, while at an R of 20 h, IL-4, IL-10, TGF-beta 2, IL-1 alpha, and tumor necrosis factor alpha (TNF-alpha) were significantly affected. The addition of B. longum affected cytokine expression significantly at both retention times. This study demonstrates that environmental determinants regulate the adherence properties of intestinal bacteria and their abilities to regulate cytokine synthesis.