(P14-020-24) The Vegetable Kale Prevents Pro-inflammatory Lipopolysaccharide Effects by Blocking Its Binding to TLR4-MD2 and Increasing the Activity of Intestinal Alkaline Phosphatase

Objectives: The cruciferous vegetable kale is a rich source of bioavailable minerals, vitamins, glucosinolates, and sulforaphane. We have previously shown that in C576BL6 mice kale augments the gut barrier integrity and reduces the pro-inflammatory lipopolysaccharide (P-LPS) production in the gut by increasing the Bacteroidetes to Proteobacteria ratio. Since P-LPS is a potent mediator of several diseases, we sought to study the protective impacts of kale against its stimulatory effects. Specifically, the impact of kale bioactive compounds on the interaction of P-LPS with the TLR4-MD2 receptor and the resultant downstream proinflammatory cytokine production. Secondly, we determined the role of kale on the activity of Intestinal Alkaline Phosphatase (IAP) which dephosphorylates the lipid-A moiety of P-LPS thus deactivating it within the gut.

Methods: Using mouse peripheral blood mononuclear cells (PBMCs) and RAW 264.7 cells, we studied the interaction of P-LPS with the TLR4-MD2 complex by flow cytometry. Cells were incubated with or without the ethanolic extract of kale (100ug/ml) for 2 hours before adding 5ug/ml of FITC labeled P-LPS (E. coli O111:B4) and stained with surface CD14 and MD2/TLR4 antibodies. The TNFa, iNOS, and IL-6 expressions were quantified by qPCR. To determine the IAP activity, Caco-2 cells were treated with kale extract for 24 hours before adding 2% Dextran sodium sulfate (DSS) for 6 hours. FITC labeled Dextran flux and TEER measurements were quantified as a measure of gut permeability. The IAP enzyme activity was determined by quantifying nitrophenol produced from the substrate p-nitrophenol phosphate using a colorimetric kit.

Results: The binding of P-LPS with TLR4-MD2 increased (P < 0.05, n = 4) by P-LPS stimulation. Kale impeded the interaction of P-LPS with the TLR4-MD2 receptor and monocyte CD14 of PBMCs and with the TLR4-MD2 receptor of the macrophages. As a result, P-LPS-induced upregulation of  IL-6, iNOS, and TNFa was prevented. In Caco-2 cells, kale increased the activity of IAP bidirectionally; in both apical and basal layers. (P < 0.05, n = 3)

Conclusions: By modulating the formation of the P-LPS-TLR4-MD2 complex, kale prevents the downstream inflammatory response. Furthermore, by increasing the IAP activity, kale reduces the endotoxicity of P-LPS.

Funding Sources: USDA–NIFA grant, Maryland Agricultural Experimental Station (MAES)