Clostridium difficile toxin B intoxicated mouse colonic epithelial CT26 cells stimulate the activation of dendritic cells

Abstract

Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis mainly through two exotoxins TcdA and TcdB that target intestinal epithelial cells. Dendritic cells (DCs) play an important role in regulating intestinal inflammatory responses. In the current study, we explored the interaction of TcdB-intoxicated epithelial cells with mouse bone marrow-derived DCs. TcdB induced cell death and heat shock protein translocation in mouse intestinal epithelial CT26 cells. The intoxicated epithelial cells promoted the phagocytosis and the TNF-α secretion by DCs. Incubation with TcdB-intoxicated CT26 cells stimulated DC maturation. Moreover, TcdB-treated CT26 cells induced DC immigration when they were injected into mice subcutaneously. Taken together, these data demonstrate that TcdB-intoxicated intestinal epithelial cells are able to stimulate DC activation in vitro and attract DCs in vivo, indicating that epithelial cells may be able to regulate DC activation under the exposure of TcdB during C. difficile infection.

Keywords: Clostridium difficile; TcdB; dendritic cells; epithelial cells; inflammation.

Graphical Abstract Figure.

Intestinal epithelial cells, after their exposure to Clostridium difficile toxin B, activate dendritic cells and regulate their function.

Figure 1.

Cytopathic and cytotoxic effects of TcdB on CT26 cells. (a) SEM shows the rapid CT26 cell rounding triggered by TcdB exposure. CT26 cells were untreated (left panel) or exposed to 10 ng mL⁻¹ of TcdB (right panel) for 4 h before harvesting for imaging. Scale bar: 20 μm. (b) CT26 cells are highly sensitive to TcdB in a MTT viability assay. CT26 cells were exposed to the indicated concentrations of TcdB for 72 h, and cell viability was measured by the MTT assay. These data represent the mean of three independent determinations ±SEM.

Figure 2.

Cell surface translocation of CRT in TcdB-treated CT26 cells. (a) Immunofluorescence detection of CRT on the cell surface (in non-permeabilized live cells; NP) or within the cell (with fixation and permeabilization; P) was performed using confocal microscopy (100×). The nuclei were visualized with Hoechst33342 (blue). (b and c) Flow cytometry analysis of PI-negative cells for surface expression of CRT. Quantitative analysis of three independent results from flow cytometry is shown in c (Data were analyzed by unpaired two-tailed t-test; ** represents P < 0.01, *** represents P < 0.001; error bars, SEM).

Figure 3.

Upregulation and secretion of HSP in CT26 cells induced by TcdB intoxication. CT26 cells were incubated with 500 ng mL⁻¹ of TcdB for the indicated times. (a) Cells were lysed and the expression of HSP70 was measured using Western blotting. Quantitative data (lower panel) from three independent experiments were analyzed by one-way ANOVA (** represents P < 0.01). Error bars show the standard error of mean (SEM). (b) The supernatant was collected and concentrated for measuring the secretion of HSP70 or HSP90 by intoxicated CT26 cells by Western blotting. Quantitative data (lower panel) from three independent were analyzed by one-way ANOVA (* represents P < 0.05; ** represents P < 0.01). Error bars, SEM. F/T means repeatedly frozen-thawed cell lysate.

Figure 4.

Phagocytosis of intoxicated CT26 cells and TNF-α production by DCs. (a) DC phagocytosis of intoxicated CT26 cells. After co-culturing TcdB-intoxicated and CFSE-labeled CT26 cells with CD11c-positive mouse BMDCs, the cell mixture was harvested and analyzed by FACS. (b) The production of TNF-α by DCs was detected by flow cytometry. CT26 cells alone or TcdB-intoxicated epithelial cells with or without anti-TcdB polysera (polyAb) treatment were used to stimulated DCs. Brefeldin A was added in the last 12 h of co-culture, and the cells were harvested to measure intracellular TNF-α expression. These data show the gated CD11c-positive cells under FACS analysis.

Figure 5.

Maturation of DCs incubated with TcdB-intoxicated CT26 cells. DCs were incubated with CT26 cells alone (medium), TcdB-treated CT26 cells, or LPS. The cell mixtures were harvested and the cell surface expression of CD40, CD80 or CD86 for the CD11c-positive gated population was analyzed by FACS.

Figure 6.

DC immigration induced by TcdB-exposed CT26 cells in vivo. Mouse tissues were collected 12, 24 or 48 h after injection of TcdB-treated CT26 cells. Tissue DCs were detected by immunohistochemistry performed on sections from paraffin-embedded tissue blocks using CD11c antibodies and DAB visualization; the nuclei were stained with hematoxylin. (a) Quantitative analysis of the number of DCs from five equivalent fields in each tissue section. These data represent the mean of five-field determinations ± SEM and were analyzed by unpaired two-tailed t-test. * represents P < 0.05 for TcdB group (CT26 + TcdB) vs antitoxin pretreatment group (CT26 + TcdB + polysera). (b) The images from representative fields, for different groups at 24 h, were taken with a Nikon digital camera attached to a microscope (200×) and arrows point CD11c-positive staining.