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. 2006 Jun;118(2):202-15.
doi: 10.1111/j.1365-2567.2006.02358.x.

Functional Modulation of Human Intestinal Epithelial Cell Responses by Bifidobacterium Infantis and Lactobacillus Salivarius

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Free PMC article

Functional Modulation of Human Intestinal Epithelial Cell Responses by Bifidobacterium Infantis and Lactobacillus Salivarius

Ann M O'Hara et al. Immunology. .
Free PMC article

Abstract

Intestinal epithelial cells (IECs) and dendritic cells (DCs) play a pivotal role in antigen sampling and the maintenance of gut homeostasis. However, the interaction of commensal bacteria with the intestinal surface remains incompletely understood. Here we investigated immune cell responses to commensal and pathogenic bacteria. HT-29 human IECs were incubated with Bifidobacterium infantis 35624, Lactobacillus salivarius UCC118 or Salmonella typhimurium UK1 for varying times, or were pretreated with a probiotic for 2 hr prior to stimulation with S. typhimurium or flagellin. Gene arrays were used to examine inflammatory gene expression. Nuclear factor (NF)-kappaB activation, interleukin (IL)-8 secretion, pathogen adherence to IECs, and mucin-3 (MUC3) and E-cadherin gene expression were assayed by TransAM assay, enzyme-linked immunosorbent assay (ELISA), fluorescence, and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. IL-10 and tumour necrosis factor (TNF)-alpha secretion by bacteria-treated peripheral blood-derived DCs were measured using ELISA. S. typhimurium increased expression of 36 of the 847 immune-related genes assayed, including NF-kappaB and IL-8. The commensal bacteria did not alter expression levels of any of the 847 genes. However, B. infantis and L. salivarius attenuated both IL-8 secretion at baseline and S. typhimurium-induced pro-inflammatory responses. B. infantis also limited flagellin-induced IL-8 protein secretion. The commensal bacteria did not increase MUC3or E-cadherin expression, or interfere with pathogen binding to HT-29 cells, but they did stimulate IL-10 and TNF-alpha secretion by DCs. The data demonstrate that, although the intestinal epithelium is immunologically quiescent when it encounters B. infantis or L. salivarius, these commensal bacteria exert immunomodulatory effects on intestinal immune cells that mediate host responses to flagellin and enteric pathogens.

Figures

Figure 1
Figure 1
Effects of bacterial treatment on the HT-29 cell line model. HT-29 cells in antibiotic-supplemented media were untreated, or were exposed to Bifidobacterium infantis, Lactobacillus salivarius, or Salmonella typhimurium at a bacterial to epithelial cell ratio of 10 : 1 for up to 24 hr. (a) Epithelial cell viability was measured at the indicated time-points and results are expressed as per cent viable HT-29 cells detected at each time-point. (b) The pH of the corresponding cell culture supernatants recorded at each indicated time-point is shown. (c) Bacterial survival at various times post treatment was determined by quantifying colony-forming units and results are expressed as per cent surviving bacteria. The graphs are representative of n = 3 independent experiments.
Figure 2
Figure 2
Bifidobacterium infantis or Lactobacillus salivarius do not activate nuclear factor (NF)-κB in epithelial cells. (a) Immunohistochemical staining for NF-κB p65 demonstrated a constitutive expression of NF-κB p65 in the cytoplasm of untreated HeLa cells. Incubation with tumour necrosis factor (TNF)-α (20 ng/ml) for 30 min caused p65 nuclear translocation, whereas B. infantis did not induce translocation of p65. (b) HT-29 cells were untreated, or were treated with 10 : 1 B. infantis, L. salivarius, Salmonella typhimurium, or 5 ng/ml TNF-α for 30 min or 1 hr. The DNA-binding activity of NF-κB p65 in HT-29 nuclear extracts was determined using an enzyme-linked immunosorbent assay (ELISA)-based transcription factor assay. The positive control Jurkat nuclear extract provided with the kit was used to verify assay specificity in competition assays with wild-type or mutated NF-κB oligonucleotides. The data represent mean absorbance readings ± standard errors of five separate experiments. *P < 0·05 relative to untreated HT-29 cells.
Figure 3
Figure 3
Salmonella typhimurium-induced interleukin (IL)-8 expression. Subconfluent HT-29 cells were treated with 10 : 1 Bifidobacterium infantis, Lactobacillus salivarius or S. typhimurium for varying times. Untreated cells served as controls. (a) IL-8 mRNA expression was determined after 2 hr by real-time reverse transcriptase–polymerase chain reaction (RT-PCR). The data are presented as normalized (to GAPDH) IL-8 mRNA expression levels and are mean ± standard error (SE) (n = 3). *P < 0·05 relative to untreated cells. (b) IL-8 protein levels (pg/ml) in cell culture supernatants were measured after 24 hr by enzyme-linked immunosorbent assay (ELISA). The data represent mean ± SE (n = 3). *P < 0·05 relative to untreated cells. (c) Time–course of S. typhimurium-induced IL-8 mRNA expression (normalized to GAPDH), intracellular IL-8 protein depletion, and extracellular IL-8 protein accumulation in HT-29 cells. The data are expressed as percentage of maximum IL-8 mRNA/protein expression and are representative of two independent experiments.
Figure 4
Figure 4
Probiotic pretreatment down-regulates interleukin (IL)-8 secretion at baseline. Confluent HT-29 cells were treated with Bifidobacterium infantis or Lactobacillus salivarius at doses of 1 × 105, 1 × 106, or 1 × 107 colony-forming units (CFU)/ml, and IL-8 protein levels were measured after 6 hr. B. infantis and L. salivarius caused a dose-dependent inhibition of baseline IL-8 secretion by confluent HT-29 monolayers (*P < 0·05 compared with untreated monolayers). The data are expressed as pg/ml IL-8 and represent the mean ± standard error (n = 3 independent experiments).
Figure 5
Figure 5
Probiotic pretreatment attenuates Salmonella typhimurium-induced pro-inflammatory responses. Subconfluent HT-29 cells were pretreated with 10 : 1 Bifidobacterium infantis or Lactobacillus salivarius for 2 hr prior to infection with 10 : 1 S. typhimurium. The levels of activated nuclear factor (NF)-κB in nuclear extracts were determined after 1 and 2 hr, and interleukin (IL)-8 protein levels were measured after 24 hr. (a) Pretreatment with B. infantis or L. salivarius reduced NF-κB activation by S. typhimurium after 1 and 2 hr (*P < 0·05 compared with S. typhimurium-infected HT-29 cells). The data are expressed as per cent of S. typhimurium-induced NF-κB activation, and represent the mean ± standard error (SE) (n = 5 independent experiments). (b) Pretreatment with B. infantis or L. salivarius inhibited S. typhimurium-induced IL-8 secretion (*P < 0·05 relative to S. typhimurium-infected HT-29 cells). The data are expressed as pg/ml IL-8 and represent the mean ± SE (n = 6 independent experiments).
Figure 6
Figure 6
Bifidobacterium infantis attenuates flagellin-induced interleukin (IL)-8 secretion. Confluent HT-29 cells were pretreated for 2 hr with B. infantis or Lactobacillus salivarius at doses of 1 × 106 or 1 × 107 colony-forming units (CFU)/ml prior to stimulation with 0·5 µg/ml flagellin for 6 hr. Pretreatment with 1 × 107 CFU/ml B. infantis, but not L. salivarius, significantly inhibited flagellin-induced IL-8 secretion (*P < 0·05 relative to flagellin-treated HT-29 monolayers). The data are expressed as pg/ml IL-8 and represent the mean ± standard error of three independent experiments.
Figure 7
Figure 7
MUC3 mRNA expression is unaltered by probiotic treatment. Confluent HT-29 monolayers were treated with 1 × 107/ml Bifidobacterium infantis or Lactobacillus salivarius for 1 or 2 hr. Real-time reverse transcriptase–polymerase chain reaction (RT-PCR) analysis of MUC3A and MUC3B mRNA expression was performed and the fold difference in gene expression compared with that of untreated samples was calculated using GAPDH as the reference gene. Data are presented as mean ± standard error of three separate experiments.
Figure 8
Figure 8
Commensal bacteria stimulate interleukin (IL)-10 and tumour necrosis factor (TNF)-α secretion by myeloid dendritic cells (DCs). DCs isolated from peripheral blood mononuclear cells (PBMCs) of healthy volunteers (n = 3) were exposed to Bifidobacterium infantis or Lactobacillus salivarius for 48 hr, and IL-10 and TNF-α levels were determined. B. infantis and L. salivarius stimulated IL-10 and TNF-α secretion (*P < 0·05 relative to untreated PBMC-derived DCs). The data are expressed as mean pg/ml ± standard error of three independent experiments.

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