Indole modulates lipoteichoic acid-induced inflammatory response in duck intestinal epithelial cells

Abstract

The microbial metabolite indole not only affects gut microbiome function but also regulates intestinal homeostasis and the immune response. Indole is primarily located in the intestinal tract; however, its effects on duck intestinal epithelial cells (IECs) under lipoteichoic acid (LTA) stimulation remain unknown. In this study, primary duck IECs were isolated, and their identity was verified using immunofluorescence staining and specific gene expression profiling. The proliferative activity of IECs was detected using the CCK-8 method. The viability of IECs under varying LTA concentrations was detected using the MTT assay. The mRNA levels of TNF-α and IL-1β under varying LTA concentrations and of TNF-α, IL-6, TGF-β1, claudin-1, and ZO-1 under varying indole concentrations were detected using quantitative real-time polymerase chain reaction (qRT-PCR). The expression levels of NF-κB-Rel and NF-κB-p65 in IECs subjected to a combination of LTA (40 μg/mL, 24 h) and indole (50 mg/L, 24 h) were detected using qRT-PCR and western blotting, respectively. We observed a reduction in IEC viability with increasing LTA concentrations. In vitro, LTA (40 μg/mL, 24 h) treatment significantly induced the mRNA expression of TNF-α and IL-1β in IECs (p < 0.001). However, indole (50 mg/L, 24 h) treatment significantly reduced their expression (p < 0.01), whereas it significantly increased mRNA levels of TGF-β1, claudin-1, and ZO-1 (p < 0.01) under LTA (40 μg/mL, 24 h) treatment. The mRNA expression of NF-κB-Rel significantly decreased in the LTA + indole group (p < 0.001) compared to that in the LTA group. Grayscale value analyses revealed that the LTA + indole group had lower phosphorylated-NF-κB-p65/NF-κB-p65 ratios for the total and cytoplasmic protein fractions of IECs than those of the LTA group (p < 0.001). In conclusion, indole reduces the inflammatory response in LTA-treated duck IECs by regulating the NF-κB signaling pathway, which may also maintain the function of the intestinal epithelial barrier by enhancing the mRNA expression of genes encoding tight junction proteins (claudin-1 and ZO-1) in ducks.