The cruciferous vegetable Brassica oleracea L. var. acephala, (Kale) protects against pro-inflammatory- lipopolysaccharide formation, translocation, and endotoxicity.

Abstract

Cruciferous vegetables have been widely studied for cancer prevention and cardiovascular health. Broccoli is a cruciferous vegetable whose phytochemistry and physiological effects have been most extensively studied. Kale is often on the list of ‘most healthy foods,’ but there is a paucity of scientific data on it. Easily cultivated with resistance to extreme weather, kale is rich in phytochemicals and micronutrients. Compared to other Brassica, including broccoli, arugula, and cabbage, kale has the highest density of sulforaphane, lutein, zeaxanthin, β-carotene, quercetin, and minerals (Zn, K, P). The gut microbiota influences host phenotype through direct contact with intestinal cells or indirectly via bacterial metabolites. Lipopolysaccharide (LPS) is one of the most potent activators of innate immune signaling, is a causal or complicating factor in several diseases, and is an important mediator of the microbiome’s influence on host physiology. The structure, chemical properties, and immunogenicity of LPS vary depending on the specific gut bacterial species that produce it. Pro-inflammatory LPS (P-LPS) activates the innate immune system, leading to inflammatory responses. In contrast, immunosuppressive or anti-inflammatory LPS (A-LPS) is not a ligand for Toll-like receptor 4 (TLR4) and therefore does not trigger immune signaling. Host-mediated inflammation, whether from the diet, a pathogen, chemical induction, or deficiency in immunity, favors the growth of aerobic bacterial taxa, e.g., Enterobacteriaceae, the main producers of P-LPS. The P-LPS is a highly immunogenic antigen that further exacerbates inflammation and is detrimental to gut barrier function. The goal of this study was to determine the protective impacts of kale supplementation as a functional food against diet-induced fat accumulation, insulin resistance, and inflammation. In two studies in C57BL6 mice, we found that whole ‘curly green kale’ is protective against systemic low-grade inflammation induced by a high fat (HF) diet and acute inflammation induced chemically by dextran sulfate sodium (DSS). The second goal of the study was to determine mechanisms by which kale protects against the formation, translocation, and functioning of P-LPS. Using C56BL6J mice and in vitro models, we show that kale imparts this protection by changing the gut microbiota composition, reducing the abundance of P-LPS-producing Enterobacteriaceae while increasing the representation of Bacteroidaceae, including species like Bacteroides thetaiotaomicron, which is known to produce immunosuppressive A-LPS. Kale thus modulates the P-LPS to A-LPS ratio. Relative levels of this ratio will exacerbate or inhibit inflammation. In addition, kale promotes the outgrowth of gram-positive taxa, specifically the species Turicibacter sanguinis. In follow-up studies in co-cultures of bacteria and intestinal epithelial cells (Caco-2 cells), we show that T. sanguinis protects gut barrier integrity by attenuating the adhesion and proliferation of E.coli O157:H7 (the source of P-LPS) and enhancing the expression of tight junction proteins. Further experiments in Caco-2 cells treated with P-LPS showed that the extract of kale promotes the expression and activity of intestinal alkaline phosphatase (IAP), an enzyme that deactivates P-LPS by dephosphorylating its lipid A moiety, thus rendering it unable to bind to TLR4 and initiate signaling. Lastly, using flow cytometry, we showed that in mouse primary peripheral blood mononuclear cells (PBMCs) and RAW 264.7 macrophages, kale extract inhibited the ability of P-LPS to bind to the TLR4-MD2 complex and CD14 receptors, thus attenuating the downstream inflammatory responses. In summary, dietary supplementation with kale is protective against inflammation. Particularly inflammation induced by the bacterial metabolite P-LPS. It is protective against (i) P-LPS formation (by modulating the gut microbiota), (ii) P-LPS translocation (by strengthening the gut barrier), and (iii) the functioning of P-LPS (promoting its dephosphorylation and hence detoxification and preventing its binding to TLR4