Lactobacillus rhamnosus GG increases cyclooxygenase-2 expression and prostaglandin E2 secretion in colonic myofibroblasts via a MyD88-dependent mechanism during homeostasis

Abstract

Prostaglandin E2 (PGE₂ ) plays a critical role in intestinal mucosal tolerance and barrier integrity. Cyclooxygenase-2 (COX-2)-dependent PGE₂ production involves mobilisation of arachidonic acid. Lactobacillus rhamnosus GG (LbGG) is one of the most widely used probiotics reported to colonise the colonic mucosa. LbGG contributes to the protection of the small intestine against radiation injury through the repositioning of mucosal COX-2 expressing cells. However, it is unknown if LbGG modulates PGE₂ production in the colonic mucosa under homeostasis and the major cellular elements involved in these processes. Colonic epithelial and CD90⁺ mesenchymal stromal cells, also known as (myo) fibroblasts (CMFs), are abundant innate immune cells in normal colonic mucosa able to produce PGE₂ . Herein, we tested the hypothesis that under colonic mucosal homeostasis, LbGG modulates the eicosanoid pathway resulting in increased PGE₂ production in both epithelial and stromal cells. Among the five tested human colonic epithelial cell lines, only exposure of Caco-2 to LbGG for 24 hr led to the mobilisation of arachidonic acid with concomitant increase in the components within the leukotriene and COX-2-dependent PGE₂ pathways. By contrast, CMFs isolated from the normal human colonic mucosa responded to LbGG with increased expression of COX-2 and PGE₂ in the prostaglandin pathway, but not 5-LO in the leukotriene pathway. Oral gavage of C57BL/6 mice for 5 days with LbGG (5 × 10⁸ Colony-Forming Unit (CFU)/dose) increased COX-2 expression in the colonic mucosa. The majority of cells upregulating COX-2 protein expression were located in the colonic lamina propria and colocalised with α-SMA⁺ cells corresponding to the CMF phenotype. This process was myeloid differentiation factor-88-dependent, because silencing of myeloid differentiation factor-88 expression in CMFs abrogated LbGG-induced upregulation of COX-2 in culture and in vivo. Taken together, our data suggest that LbGG increases release of COX-2-mediated PGE₂ , contributing to the maintenance of mucosal homeostasis in the colon and CMFs are among the major contributors to this process.

Keywords: COX-2; lactic acid bacteria; mechanism of action; metabolic processes; microbial cell interaction.

Figure 1. Lactobacillus rhamnosus

GG (LbGG) induces an increase in cPLA2, release of AA, an increase in COX-2 expression, and increased PGE₂ production in Caco-2 cells. Caco-2 cells were exposed to LbGG (10 bacteria: 1 epithelial cell) for 24 h and harvested for Western blot and ELISA analysis. (A) Western blot using anti-cPLA2 revealed a significant increase in cPLA2 protein expression in Caco-2 cells following exposure to LbGG. Protein density was normalized to the housekeeping protein β-actin (relative density) and adjusted density was calculated for each sample by dividing the relative density of the experimental condition by those obtained for a control sample. (B) Arachidonic acid (AA) levels were determined by gas chromatography in Caco-2 cell. Exposure to LbGG significantly enhanced the release of AA. Western blot analysis demonstrated an increase in (C) COX-2 and (D) 5-LO protein expression following LbGG stimulation. (E) PGE₂ concentrations (ELISA analysis) were measured in the conditioned media of Caco-2 cells after exposure to different strains of lactobacilli. The results are shown as means ± SEM. Student’s t test was used to calculate the significance of the obtained results when comparing two groups (panel A, B, C, and D). One-way ANOVA test was used to analyze significance of the obtained results when comparing more than two groups (panel E). n=3, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure 2

Only Caco-2 among the five tested colonic epithelial cell lines (LS174T, SW480, HCT116, Caco-2, and HT-29) responded to LbGG with an increase in COX-2 expression and PGE₂ production. The epithelial cell lines were co-cultured with LbGG (10 bacteria: 1 epithelial cell) for 24 h, and harvested for western blot analysis and ELISA. (A) Representative western blot and summary of relative density data are shown and demonstrate that LbGG induces COX-2 protein expression in Caco-2 cells but not other cell lines. (B) Caco-2, but not other tested cell lines respond to LbGG stimulation with a significant increase in PGE₂ production. The results are shown as means ± SEM. The summary results shown on panel (A) and (B) are expressed as means ± SEM. One-way ANOVA was used to calculate the significance of the obtained results, three experimental repeat per tested cell line were run in duplicate (n=6), ****p<0.0001.

Figure 3

LbGG activated the COX-2-dependent PGE₂ pathway in primary human CMFs. Primary CMFs isolated from normal colonic mucosa were exposed to LbGG for 24h at a ratio of 10 bacteria per one CMF. Western blotting (WB) using antibodies specific to (A) cPLA2, (B) COX-2, and (C) 5-Lipoxygenase proteins demonstrated that the total cPLA2 and COX-2 levels are increased in LbGG-treated CMFs cultures compared to the untreated controls. The representative WB and summary of the adjusted density are shown. The summary of adjusted density results for each studied molecule are shown as means ± SEM. Student’s t test was used to calculate the significance of the obtained results n=3, *p<0.05; **p<0.01; ****p<0.0001. (D) PGE₂ concentrations determined by ELISA were increased in the condition media of CMFs after exposure to LbGG. Student’s t test (panel A, B, C) and Wilcoxon signed ranked test (panel D) were used to calculate the significance of the obtained results. Results from three experiments running in duplicate are shown as means ± SEM, n=6, *p<0.05.

Figure 4

Silencing of myD88 gene expression in normal primary human isolates of CMFs inhibits the increase in LbGG-dependent COX-2 production. CMFs were transfected with MyD88 siRNA or control siRNA 10 days prior to exposure to LbGG, COX-2 expression was analyzed 24 h post LbGG exposure using western blot analysis. A representative WB and summary of WB adjusted density analysis for COX-2 expression are shown. One-way ANOVA was used to calculate the significance of the obtained results. Results are shown as means ± SEM, n=3, **p < .01 and ****p< 0.0001.

Figure 5

LbGG activates the arachidonic acid (AA) metabolic pathway in vivo. 5×10⁸ CFU/dose of the LbGG was delivered daily by oral gavage for 5 days to C57BL/6 mice. (A) AA levels in the harvested, homogenized mucosa were determined by ELISA. Exposure to LbGG significantly enhanced the release of AA in the colonic mucosa of LbGG-treated mice. The results are shown as means ± SEM, n=5, ***p< 0.001. (B) Western blot analysis demonstrated an increase in COX-2 expression in the colonic mucosa of LbGG-treated mice. The results are shown as means ± SEM, n=6, *p<0.05. (C) Representative tissue sections of the colonic mucosa from control and LbGG treated mice were immunostained and analyzed by confocal microscopy (see Methods). Expression of COX-2 is increased within the α-SMA⁺ CMFs in the colonic mucosa of mice treated with LbGG. DAPI was used to stain cell nuclei (blue); activated CMFs were detected by anti–α-SMA mAb (green; clone A4);anti-COX-2 mAb (red; clone 33) was also used. A yellow-orange color on merged images indicates co-localization of α-SMA and COX-2 (indicated by arrows). Scale bar represents 20 Tm. Representative cross-sections are shown, n= 5 animals per group. (D) The summary of changes in the Corrected Total Cell Fluorescence (CTCF) fluorescence intensity from in situ COX-2 protein expression in the LbGG-treated and control murine colonic mucosa. Student’s t test was used to calculate the significance of the obtained results. The means ± SEM are shown, n=5 per group **p< 0.01.

Figure 6

MyD88 signaling in α-SMA⁺ CMFs is required for COX-2 production in response to LbGG. 5×10⁸ CFU/dose of the LbGG was delivered daily by oral gavage for 5 days to MF-MyD88 KO or control mice. Control mice negative for Acta2-Cre but positive for MyD88^fl/fl recombination were derived from at least five backcrossing of MF-MyD88 KO with C57BL/6 animals. (A) Immunostaining of colonic mucosal tissue cross-sections was performed, followed by confocal microscopy. DAPI was used to stain cell nuclei (blue); activated CMFs were detected by anti–α-SMA mAb (green; clone A4) and the sections were stained for COX-2 with mAb (red; clone 33). Scale bar represents 20 Tm. (B) Measurement of CTCF fluorescence intensity from confocal microscopy images of COX-2 protein, and (C) western blotting using antibodies specific to COX-2, demonstrated that the total COX-2 levels increased in colonic tissue of LbGG-treated mice compared with the untreated controls. Deletion of MyD88 within CMFs reduced this response. One-way ANOVA was used to calculate the significance of the obtained results. Results are shown as means ± SEM. Four-five animals per group were used in the above experiments, *p < .05.