Deoxynivalenol modulated mucin expression and proinflammatory cytokine production, affecting susceptibility to enteroinvasive Escherichia coli infection in intestinal epithelial cells

Abstract

Deoxynivalenol (DON) is a common mycotoxin in crops that could induce intestinal inflammation, affecting the susceptibility of intestinal epithelial cells (IECs) to pathogen infection. This study aimed to investigate DON's effects on mucin and cytokine production as part of the local immune system and how it affected intestinal susceptibility to pathogen infection. Caco-2 cells were exposed to DON followed by acute enteroinvasive Escherichia coli (EIEC) infection. An increase in EIEC attachment to DON-exposed cells was observed, probably in part, mediated by secretory MUC5AC mucins and membrane-bound MUC4 and MUC17 mucins. Additionally, DON with EIEC posttreatment led to significant changes in the gene expression of several proinflammatory cytokines (IL1α, IL1β, IL6, IL8, TNFα, and MCP-1), which may be in part, mediated by NK-κB and/or MAPK signaling pathways. These data suggested DON may exert immunomodulatory effects on IECs, altering the IEC susceptibility to bacterial infection. PRACTICAL APPLICATION: The results suggested that DON might modulate immune responses by affecting mucus and cytokine production, which may affect the susceptibility of intestinal epithelial cells to pathogen infection.

Keywords: bacterial infection; deoxynivalenol; enteroinvasive Escherichia coli; inflammatory responses; mucins.

FIGURE 1

(a, b) Effects of DON without or with EIEC postinfection on cell viability. (a) Preliminary screening of DON concentrations for subsequent experiments. Cell viability data of caco‐2 cells treated with different concentrations of DON (0, 2, 4, 8, and 16 µM) for 24 h. (b) Cell viability data of Caco‐2 cells treated with DON (8 and 16 µM) for 24 h without or with EIEC bacteria posttreatment at a multiplicity of infection (MOI) of 250:1 to the cells for 1 h. Control received appropriate carriers. Results were shown as mean ± SEM, which are from four independent experiments performed in six replicates. *^, **^, ***^, ***p < 0.05, 0.01, 0.001, and 0.0001 compared with PBS control. One‐way ANOVA post‐Dunnet's test. (c, d) Effects of 24 h of DON incubation on Caco‐2 cells without or with EIEC postinfection (1 h) on bacterial adhesion and invasion. The percentage of (c) adhering and (d) invaded bacteria were calculated as described in Materials and Methods. Results were shown as mean of ±SEM, which are from four separate experiments performed in duplicates. *^, **p < 0.05 and 0.01 compared with PBS control. One‐way ANOVA post‐Dunn's test.

FIGURE 2

(a, b) Effects of 24 h DON incubation without or with EIEC postinfection (1 h) on mucus production visualized by Alcian blue/PAS staining. (a) Alcian blue staining of Caco‐2 cells treated with DON for 24 h without or with EIEC postinfection (1 h). Blue areas indicate acidic mucin deposition (20× objective lens). Scale bar = 200 µm. (b) PAS staining of Caco‐2 cells treated with DON for 24 h without or with EIEC postinfection (1 h). Fuchsia areas indicate neutral mucin deposition (20× objective lens). Scale bar = 200 µm. (c) Effects of 24 h DON incubation without or with EIEC postinfection (1 h) on mucin‐like glycoprotein production as measured by the enzyme‐linked lectin assay (ELLA). Results were shown as mean of ±SEM from six independent experiments. *p < 0.05 compared with PBS control. One‐way ANOVA post‐Dunn's test.

FIGURE 3

(a–f) Effects of 24 h DON incubation without or with EIEC postinfection (1 h) on mucin (MUC) gene expression. (a, b) Secretory MUC5AC and MUC5B, and (c–f) membrane‐bound MUC1, MUC3, MUC4, and MUC17 mRNA expression was measured by qPCR, with GAPDH as the internal control. Results were shown as mean of ±SEM from five independent experiments. *^, **^, *** p < 0.05, 0.01, and 0.001 compared with PBS control. One‐way ANOVA post‐Dunnet's test.

FIGURE 4

(a–e) Effects of 24 h DON incubation without or with EIEC postinfection (1 h) on cytokine and chemokine gene expression. IL1B, IL6, IL8, TNFA, and CCL2 mRNA expression were measured by qPCR, with GAPDH as the internal control. Results were shown as mean of ±SEM from five independent experiments. *^, ***^, ****p < 0.05, 0.001, and 0.0001 compared with PBS control. One‐way ANOVA post‐Dunn's test.

FIGURE 5

(a–c) Effects of 24 h DON incubation without or with EIEC postinfection (1 h) on NF‐κB and MAPK signaling pathways. (a) RELA mRNA expression was measured by qPCR, with GAPDH as the internal control. Results were shown as mean of ±SEM from six independent experiments. (b) NF‐κB p65 protein expression was measured by Western blotting, with GAPDH as the internal control. Representative photos of western blotting of NF‐κB p65 and GAPDH. Quantification of Western blot compared with PBS control from three independent experiments. Inset shows group means. (c) Protein samples were also analyzed by Western blot with phospho‐p38, JNK, and ERK antibodies. The total MAPK levels were used as an internal control. Representative photos of western blotting of MAPKs and GAPDH. Results were shown as mean of ±SEM from four independent experiments. *^, ***^, ****p < 0.05, 0.001, and 0.0001 compared with PBS control. One‐way ANOVA post‐Dunn's test.