Extracellular MUC3 mucin secretion follows adherence of Lactobacillus strains to intestinal epithelial cells in vitro

Abstract

Background: Mucins are large complex glycoproteins that protect intestinal mucosal surfaces by limiting access of environmental matter to their epithelial cells. Several mucin genes have been described, including MUC3 that is a membrane associated mucin of the small intestine. Increased MUC3 mRNA transcription is induced by incubation of intestinal epithelial cells with a Lactobacillus strain known to be adherent to them.

Aims: To determine whether increased epithelial cell MUC3 mucin expression in response to Lactobacillus strains results in increased extracellular secretion of MUC3 mucins and the importance of epithelial cell adherence in modulation of MUC3 mucin expression.

Methods: HT29 cells grown to enhance expression of MUC3 mucins were incubated with selected Lactobacillus strains. Spent cell culture medium was collected for detection of secreted MUC3 mucins using dot blot immunoassay with a generated MUC3 antibody. Post-incubation HT29 cell RNA was collected for analysis of MUC3 expression by northern blot analysis using a MUC3 cDNA probe. In vitro binding studies using Lactobacillus strains incubated alone or coincubated with enteropathogenic Escherichia coli strain E2348/69 were used for adherence and inhibition of adherence studies, respectively.

Results: Lactobacillus strains with minimal ability to adhere to HT29 cells failed to induce upregulation of mucin gene expression. There was a direct correlation between upregulation of MUC3 mucin mRNA expression and extracellular secretion of MUC3 mucin. The same Lactobacillus strains that increased extracellular secretion of MUC3 mucin led to reduced adherence of enteropathogen E coli E2348/69 during coincubation experiments.

Conclusion: Probiotic microbes induce MUC3 mucin transcription and translation with extracellular secretion of the MUC3 mucins. Epithelial cell adherence enhances the effects of probiotics on eukaryotic mucin expression.

Figure 1

(A) Lactobacillus adherence to HT29 cells. Lactobacillus strains 10⁶ colony forming unit (CFU)/well were incubated for four hours with HT29 cells grown in glucose free galactose containing cell culture medium to enhance MUC3 expression. Non-adherent bacteria were washed off (×4) and adherent bacteria were quantified by CFU determination on MRS agar plates. Lp299v, Lactobacillus plantarum strain 299v; LrGG, L rhamnosus strain GG; LaDDS, L acidophilus strain DDS-1. (B) Similar experiments were performed using the parent Lp299v strain and its adhesin negative genetic mutant Lp adh− strain. In both, five experiments were run in triplicate and results are expressed as mean (SEM).

Figure 2

Mucin mRNA expression induced by Lactobacillus strains. Lactobacilli (4.5×10¹⁰ colony forming units) were added to flasks in an equivalent amount as cell surface contact area of 12 well plates. After one hour of incubation, northern blots were hybridised using random-primed P labelled cDNA probe to the MUC3 tandem repeat. Mucin mRNA levels were quantified by area integration of Phosphor screen autoradiography. Results were normalised to 28s RNA levels on agarose gels used for northern blots. Results are expressed as means (SEM) of five experiments for HT29 cells were grown in a glucose free galactose containing cell culture medium to enhance MUC3 expression. As shown in (A), MUC3 expression for cells incubated with Lactobacillus plantarum strain 299v (Lp299v) and L rhamnosus strain GG (LrGG) were increased compared with controls without bacteria added and HT29 cells with L acidophilus strain DDS-1 (LaDDS) added (*p<0.05). (B) MUC3 mRNA expression was increased for Lp299v compared with the ligand negative Lp adh− strain (*p<0.05).

Figure 3

Immunoassay of HT29 spent cell culture medium. Immune serum generated against a 9 mer peptide (TSRRTTRIT) of deduced MUC3 sequence was incubated with MUC2 (A) and MUC3 mucins (B) isolated from HT29 cell culture medium. Mucin protein (20 μg) was blotted onto nitrocellulose. A 1:100 dilution of mouse antisera was added. After washings, bound antibody was detected by a horseradish peroxidase colorisation reaction. A black dot in each frame is pencil marking used to indicate where medium was blotted initially. High reactivity was observed for spent culture medium of cells grown in galactose containing medium to enhance MUC3 expression. In contrast, there was less reactivity to material from spent culture medium of HT29 cells grown in a glucose containing medium that is associated with high MUC2 mucin mRNA expression and low MUC3 mucin mRNA expression.

Figure 4

Indirect immunofluorescence of human duodenum. The photomicrograph using mouse antihuman MUC3 mucin antibody as the primary antibody is shown. The cross section view of a villus from a biopsy taken from the second part of the duodenum shows fluorescence of the epithelial cell cytoplasm with increased fluorescence at the surface of the epithelial cells (arrows). Controls using preimmune serum, FITC conjugate alone, and phosphate buffered saline were negative (not shown). Magnification 40×.

Figure 5

Immunoassay for MUC3 mucins in spent cell culture medium from Lactobacillus strains. HT29 cells were grown in glucose free galactose containing medium to enhance MUC3 expression and incubated with equivalent colony forming units of different Lactobacillus strains. Cell culture medium (10 μg protein) was blotted onto nitrocellulose. A 1:100 dilution of mouse anti-MUC3 antisera was added. After washings, bound antibody was detected using a peroxidase colorisation reaction. A black dot in each frame is pencil marking used to indicate where medium was blotted initially. Greater immunoreactive MUC3 mucin was detected in cell culture medium incubated with Lactobacillus plantarum strain 299v (Lp299v) and L rhamnosus strain GG (LrGG) than with L acidophilus strain DDS-1 (LaDDS) and control cells without a probiotic added (none).

Figure 6

Comparative inhibition of enteropathogenic Escherichia coli (EPEC) adherence to MUC3 expressing HT29 cells. Each well received 10⁹ colony forming units (CFU)/well Lactobacillus that were incubated at 37°C with the HT29 cells for an hour prior to the addition of 10⁶ CFU/well of EPEC E2348/69. After a three hour incubation, EPEC adherent to cells were quantified by determining CFU on MacConkey agar. Results are expressed as means (SEM) of at least five independent experiments performed in triplicate. As shown in (A), both Lactobacillus plantarum strain 299v (Lp299v) and L rhamnosus strain GG (LrGG) decreased adherence of EPEC compared with controls of EPEC alone but L acidophilus strain DDS-1 (LaDDS) did not (*p<0.05). (B) The ligand negative strain Lp adh− showed less adherence than the parent ligand positive strain Lp299v (*p<0.05).