The impact of gut bacteria producing long chain homologs of vitamin K2 on colorectal carcinogenesis.

Colorectal cancer (CRC) is one of the foremost causes of cancer-related deaths. Lately, a close connection between the course of CRC and the intestinal microbiota has been revealed. Vitamin K2 (VK2) is a bacterially derived compound that plays a crucial role in the human body. Its significant anti-cancer properties may result, inter alia, from a quinone ring possessing a specific chemical structure found in many chemotherapeutics. VK2 can be supplied to our body exogenously, i.e., through dietary supplements or fermented food (e.g., yellow cheese, fermented soybeans -Natto), and endogenously, i.e., through the production of bacteria that constantly colonize the human microbiome of the large intestine.This paper focuses on endogenous K2 synthesized by the most active members of the human gut microbiome. This analysis tested 86 intestinally derived bacterial strains, among which the largest VK2 producers (Lactobacillus, Bifidobacterium, Bacillus) were selected. Moreover, based on the chosen VK2-MK4 homolog, the potential of VK2 penetration into Caco-2 cells in an aqueous environment without the coexistence of fats, pancreatic enzymes, or bile salts has been displayed. The influence of three VK2 homologs: VK2-MK4, VK2-MK7 and VK2-MK9 on apoptosis and necrosis of Caco-2 cells was tested proving the lack of their harmful effects on the tested cells. Moreover, the unique role of long-chain homologs (VK2-MK9 and VK2-MK7) in inhibiting the secretion of pro-inflammatory cytokines such as IL-8 (for Caco-2 tissue) and IL-6 and TNFα (for RAW 264.7) has been documented.

Lipid raft-dependent endocytosis negatively regulates responsiveness of J774 macrophage-like cells to LPS by down regulating the cell surface expression of LPS receptors.

Acting through CD14 and TLR4/MD-2, lipopolysaccharide (LPS) triggers strong pro-inflammatory activation of macrophages, which, if not appropriately controlled, may lead to lethal septic shock. Therefore, numerous mechanisms of negative regulation of responses to LPS exist, but whether they include down-regulation of LPS receptors is not clear. We have found that in J774 cells, the clathrin-dependent endocytic pathway enables activation of TRIF-dependent TLR4 signaling within endosomes, but is not associated with the down-regulation of TLR4 or CD14 surface expression. In contrast, lipid raft-dependent endocytosis negatively regulates the basal cell surface expression of LPS receptors and, consequently, responsiveness to LPS. Together with observations that treatments, known to selectively disrupt lipid rafts, do not inhibit LPS-stimulated cytokine production, our results suggest that lipid rafts may serve as sites in which LPS receptors are sorted for endocytosis, rather than being platforms for the assembly of TLR4-centered signaling complexes, as suggested previously.

The impact of lactoferrin with different levels of metal saturation on the intestinal epithelial barrier function and mucosal inflammation.

Translocation of bacteria, primarily Gram-negative pathogenic flora, from the intestinal lumen into the circulatory system leads to sepsis. In newborns, and especially very low birth weight infants, sepsis is a major cause of morbidity and mortality. The results of recently conducted clinical trials suggest that lactoferrin, an iron-binding protein that is abundant in mammalian colostrum and milk, may be an effective agent in preventing sepsis in newborns. However, despite numerous basic studies on lactoferrin, very little is known about how metal saturation of this protein affects a host's health. Therefore, the main objective of this study was to elucidate how iron-depleted, iron-saturated, and manganese-saturated forms of lactoferrin regulate intestinal barrier function via interactions with epithelial cells and macrophages. For these studies, a human intestinal epithelial cell line, Caco-2, was used. In this model, none of the tested lactoferrin forms induced higher levels of apoptosis or necrosis. There was also no change in the production of tight junction proteins regardless of lactoferrin metal saturation status. None of the tested forms induced a pro-inflammatory response in Caco-2 cells or in macrophages either. However, the various lactoferrin forms did effectively inhibit the pro-inflammatory response in macrophages that were activated with lipopolysaccharide with the most potent effect observed for apolactoferrin. Lactoferrin that was not bound to its cognate receptor was able to bind and neutralize lipopolysaccharide. Lactoferrin was also able to neutralize microbial-derived antigens, thereby potentially reducing their pro-inflammatory effect. Therefore, we hypothesize that lactoferrin supplementation is a relevant strategy for preventing sepsis.

The class A scavenger receptor SR-A/CD204 and the class B scavenger receptor CD36 regulate immune functions of macrophages differently.

SR-A/CD204 and CD36 are major receptors responsible for oxidized lipoproteins uptake by macrophages in atherosclerotic plaques. Both receptors also share the role as receptors for different pathogens, but studies on their signaling have been hampered by the lack of selective ligands. We report that, upon specific ligation by Ab, SR-A does not induce cytokine production, but mediates inhibition of LPS-stimulated production of IL-6 and IL-12/23p40, enhancement of IL-10 release, and has no effect on TNF-α and RANTES production in murine macrophages. In contrast, anti-CD36 Ab alone stimulated production of all these cytokines, with IL-10 production being exceptionally high. Effects of anti-CD36 Ab, except of IL-10 production, were mediated by CD14 and TLR2, whereas those of SR-A ligation by heterotrimeric Gi/o proteins and by phosphatidylinositol 3-kinases. Surprisingly, we found that LPS uptake by macrophages was mediated in part by CD36 cooperating with CD14, whereas SR-A was not involved in this process. Finely, during in vitro Ag presentation to naïve CD4(+) lymphocytes, pre-incubation of macrophages with anti-CD36 Ab enhanced IFN-γ production in the co-culture, but exerted the opposite effect under conditions enabling IL-10 accumulation. In contrast, anti-SR-A Ab was ineffective alone, but reversed the Th1-polarizing effect of LPS.

Immunoregulatory potential of exopolysaccharide from Lactobacillus rhamnosus KL37: effects on the production of inflammatory mediators by mouse macrophages.

The ability to produce exopolysaccharides (EPS) is widespread among lactobacilli including Lactobacillus rhamnosus, the commonly used probiotic bacteria. Exopolysaccharides are a major component of the bacterial biofilm with a well-documented impact on adherence of bacteria to host cells. However, their immunoregulatory properties are unknown. The aim of this study was to examine the immunostimulatory potential of EPS derived from L. rhamnosus KL37. We investigated the effect of EPS on the production of inflammatory mediators by mouse peritoneal macrophages and compared it with the effect of Lipopolysaccharide (LPS). Exopolysaccharides, at concentrations higher than those of LPS, stimulated production of both pro-inflammatory (TNF-α, IL-6, IL-12) and anti-inflammatory (IL-10) cytokines. Interestingly, analysis of the balance of TNF-α/IL-10 production showed a potential pro-inflammatory effect of EPS. Furthermore, our data demonstrate that exposure of macrophages to LPS induced a state of hyporesponsiveness, as indicated by reduced production of TNF-α after restimulation with either LPS or EPS ('cross-tolerance'). By contrast, EPS could make cells tolerant only to subsequent stimulation by the same stimulus. We also examined the relationship between TNF-α production and activation of mitogen-activated protein kinases (MAPKs) by EPS and LPS. Pretreatment of macrophages with specific inhibitors of p38 and ERK MAPKs reduced TNF-α production induced by both stimuli to the same extent. In conclusion, these data demonstrate that EPS can effectively stimulate production of inflammatory mediators by macrophages in vitro. However, to predict whether EPS could be clinically useful as an immunomodulatory agent, further in vivo studies with highly purified EPS are necessary.