Bacterial Metabolite Indole Alters Inflammatory Signaling Cascades In Human Lymphatic Endothelial Cells

Pathological conditions, as metabolic syndrome (MetSyn) or obesity, are closely associated with inflammation‐induced alterations in the gut microbiota metabolites. Indole, a bacterial signal produced by Escherichia coli, has anti‐inflammatory effects on intestinal epithelial cells. As a key signaling molecule in the GI tract, indole has been found to have anti‐inflammatory effects on intestinal epithelial cells. Inflammatory conditions have been shown to directly alter the microbiota in the gut and several studies have linked these changes with onset of obesity and MetSyn, suggesting that the relative levels of indole could be changed in the intestine and mesentery under inflammatory conditions. We have previously shown that high fructose diet‐induced MetSyn in rats causes inflammatory conditions in the mesentery and impairs lymphatic contractile function. Hence, in this study we investigated whether the changes in the gut microbiota that occur under obesity, MetSyn or during other inflammatory conditions would influence lymphatic function. We hypothesize that indole reduces inflammation in lymphatic endothelial cells stimulated with dietary endotoxin, such as lipopolysaccharide (LPS). Human dermal lymphatic endothelial cells (HDLECs) were pretreated with a 100μM concentration of indole overnight, followed by LPS treatment for 24 hours. We used HDLECs treated with LPS or indole alone, and cells with no treatment were used as controls. Effects of indole on localization of NF‐κB and β‐catenin were determined by immunofluorescence. We also carried out quantitative RT‐PCR analysis to quantify the relative levels of expression of inflammatory marker genes, such as IL‐1b, c‐fos, c‐jun, MCP1, MIP1 and IL1A. Further, protein cytokine arrays were carried out to determine the relative levels of various inflammation associated cytokines and chemokines. Our immunofluorescence data indicate that indole decreases LPS‐mediated NF‐κB translocation to the nucleus. Further, the expression pattern of β‐catenin an important component of the adherens junction that controls endothelial cell permeability and normally present on the cell membrane was found to be diffused throughout the cytoplasm after LPS treatment. However, indole significantly decreased the LPS effects on β‐catenin and restored its expression on the cell membrane thereby tightening LEC junctions. Our quantitative RT‐PCR and protein cytokine data indicate that indole reduces the expression of many of the key proinflammatory chemokines, cytokines, and interleukins and thereby determines the balance between suppression of inflammation and progression of inflammatory signaling mechanisms. Thus, the data we have presented here provide the first evidence that a key signaling molecule of the gut microbiota, indole, reduces inflammation in the LECs, and has potential therapeutic value in the treatment of lymphatic inflammation associated pathological conditions.