Lactobacillus acidophilus induces a slow but more sustained chemokine and cytokine response in naïve foetal enterocytes compared to commensal Escherichia coli

Abstract

Background: The first exposure to microorganisms at mucosal surfaces is critical for immune maturation and gut health. Facultative anaerobic bacteria are the first to colonise the infant gut, and the impact of these bacteria on intestinal epithelial cells (IEC) may be determinant for how the immune system subsequently tolerates gut bacteria.

Results: To mirror the influence of the very first bacterial stimuli on infant IEC, we isolated IEC from mouse foetuses at gestational day 19 and from germfree neonates. IEC were stimulated with gut-derived bacteria, Gram-negative Escherichia coli Nissle and Gram-positive Lactobacillus acidophilus NCFM, and expression of genes important for immune regulation was measured together with cytokine production. E. coli Nissle and L. acidophilus NCFM strongly induced chemokines and cytokines, but with different kinetics, and only E. coli Nissle induced down-regulation of Toll-like receptor 4 and up-regulation of Toll-like receptor 2. The sensitivity to stimulation was similar before and after birth in germ-free IEC, although Toll-like receptor 2 expression was higher before birth than immediately after.

Conclusions: In conclusion, IEC isolated before gut colonisation occurs at birth, are highly responsive to stimulation with gut commensals, with L. acidophilus NCFM inducing a slower, but more sustained response than E. coli Nissle. E. coli may induce intestinal tolerance through very rapid up-regulation of chemokine and cytokine genes and down-regulation of Toll-like receptor 4, while regulating also responsiveness to Gram-positive bacteria.

Figure 1

L. acidophilus and E. coli strongly induce chemokine expression in foetal primary epithelial cells in vitro. Epithelial cells from foetuses at Day-1 were stimulated for 2 h and 4 h with LPS (10 μg/ml), L. acidophilus NCFM (10, 30 and 100 μg/ml) or E. coli Nissle (1, 10, 30 μg/ml). Gene expression was measured by RT-PCR. Symbols indicate mean fold increase and SD of 4 independent experiments with cells pooled from 6-10 pups. ***p < 0.001, **p < 0.01, *p < 0.05 compared to "unstimulated cells". Data were normalised to Actb and then to the average of "unstimulated cells 2 h" from the 4 experiments, which was defined to 1.

Figure 2

L. acidophilus and E. coli up-regulate expression of pro-inflammatory and regulatory cytokines in foetal epithelial cells in vitro. Epithelial cells from foetuses at Day-1 were stimulated for 2 h and 4 h with LPS (10 μg/ml), L. acidophilus NCFM (10, 30 and 100 μg/ml) or E. coli Nissle (1, 10, 30 μg/ml). Gene expression was measured by RT-PCR. Symbols indicate mean fold increase and SD of 4 independent experiments with cells pooled from 6-10 pups. ***p < 0.001, **p < 0.01, *p < 0.05 compared to "unstimulated cells". Data were normalised to Actb and then to the average of "unstimulated cells 2 h" from the 4 experiments, which was defined to 1.

Figure 3

Foetal epithelial cells produce cytokines upon in vitro stimulation with G⁺ and G^- commensals with different kinetics. Cytokine and chemokine production in epithelial cells from foetuses at Day-1 after 2 h, 4 h and 18 h stimulation with LPS (10 μg/ml), L. acidophilus NCFM (10, 30 and 100 μg/ml) or E. coli Nissle (1, 10, 30 μg/ml) measured by ELISA. Symbols indicate mean and SD of 4 independent experiments with cells pooled from 6-10 pups. ***p < 0.001, **p < 0.01, *p < 0.05 compared to "unstimulated cells" represented by dotted lines.

Figure 4

E. coli is more potent than L. acidophilus in up-regulating Tlr2, Nfkb1, and Nfkb2 expression and in down-regulating expression of Tlr4 and Clec7a in foetal epithelial cells. Epithelial cells from foetuses at Day-1 were stimulated for 2 h and 4 h with LPS (10 μg/ml), L. acidophilus NCFM (10, 30 and 100 μg/ml) or E. coli Nissle (1, 10, 30 μg/ml). Gene expression was measured by RT-PCR. Symbols indicate mean and SD of 4 independent experiments with cells pooled from 6-10 pups. ***p < 0.001, **p < 0.01, *p < 0.05 compared to "unstimulated cells". Data were normalised to Actb and then to the average of "unstimulated cells 2 h" from the 4 experiments, which was defined to 1.

Figure 5

Age dependent gut maturation does not influence early responses of epithelial cells towards G+ and G- commensals. Epithelial cells from foetuses at Day-1 and neonates at post natal day (PND) 1 and PND6 from germfree mothers stimulated for 2 h with L. acidophilus NCFM or E. coli Nissle. Bars indicate mean and SD of 4 independent experiments with cells pooled from 6-10 pups (Day-1) or 2-3 pups (PND1 and PND6). ***p < 0.001, **p < 0.01, *p < 0.05. Data were normalised to Actb and then to the average of "Day -1 unstimulated" from the 4 experiments, which was defined to 1.