Figure 3.

Untreated and heat-treated bifidobacteria strains differently interact with epithelial and immune cells in vitro. (A) Engulfment by airway epithelial cells and (B) transfer between airway epithelial cells and dendritic cells of untreated (UN, blue bars) and heat-treated (HT, orange bars) bifidobacteria. Epithelial cells (TC-1 cells) were incubated overnight with SYTO™ 9-stained bacteria cells. After washing of bacteria, epithelial cells were co-cultured with PKH26-stained JAWS II cells for 4 h. Fluorescence of double stained dendritic cells was analyzed by FACS and expressed as mean fluorescence units (MFI). The level of (C) MCP-1 and (D) IL-6 cytokine production from TC-1 cells was measured by ELISA in culture supernatants after 24-h stimulation with bifidobacteria (UN or HT) or PBS. The level of (E) IL-10 and (F) IL-12p70 cytokine production was determined in supernatants of BMDCs after 20-h stimulation with UN or HT bifidobacteria or PBS. Activation of (G) TLR2 and (H) NOD2 receptors was determined using human embryonic kidney cells (HEK293) stably transfected with human TLR2 or NOD2 expressing vectors and expressed as production of IL-8 cytokine after 20-h stimulation by UN or HT bifidobacteria strains. TLR2 ligand Pam3CSK4 (1 µg/ml) and NOD2 ligand muramyl dipeptide (MDP; 100 ng/ml) were used as positive controls. Cells treated with PBS were used as negative control. Data are collected from three independent experiments. Data are shown as mean ± SD and analyzed with unpaired Student’s t-test between untreated and heat-treated bacteria. *p < 0.05, **p < 0.01 statistically significant difference. Comparison between bifidobacterial strains was calculated by one-way ANOVA Dunett’s multiple comparison test. ^#p < 0.05.