Escherichia coli-derived outer-membrane vesicles induce immune activation and progression of cirrhosis in mice and humans.

BACKGROUND AND AIMS: Decompensated cirrhosis with fibrosis progression causes portal hypertension followed by an oedematous intestinal tract. These conditions weaken the barrier function against bacteria in the intestinal tract, a condition called leaky gut, resulting in invasion by bacteria and bacterial components. Here, we investigated the role of outer-membrane vesicles (OMVs) of Escherichia coli, which is the representative pathogenic gut-derived bacteria in patients with cirrhosis in the pathogenesis of cirrhosis. METHODS: We investigated the involvement of OMVs in humans using human serum and ascites samples and also investigated the involvement of OMVs from E. coli in mice using mouse liver-derived cells and a mouse cirrhosis model. RESULTS: In vitro, OMVs induced inflammatory responses to macrophages and neutrophils, including the upregulation of C-type lectin domain family 4 member E (Clec4e), and induced the suppression of albumin production in hepatocytes but had a relatively little direct effect on hepatic stellate cells. In a mouse cirrhosis model, administration of OMVs led to increased liver inflammation, especially affecting the activation of macrophages, worsening fibrosis and decreasing albumin production. Albumin administration weakened these inflammatory changes. In addition, multiple antibodies against bacterial components were increased with a progressing Child-Pugh grade, and OMVs were detected in ascites of patients with decompensated cirrhosis. CONCLUSIONS: In conclusion, OMVs induce inflammation, fibrosis and suppression of albumin production, affecting the pathogenesis of cirrhosis. We believe that our study paves the way for the future prevention and treatment of cirrhosis.

Triterpenoid saponin from Panax ginseng increases the sensitivity of methicillin-resistant Staphylococcus aureus to ß-lactam and aminoglycoside antibiotics.

The triterpenoid saponins, ginsenosides, are the major bioactive compound of red ginseng and can exert various physiological activities. In the present study, we examined whether red ginseng extract (RGE) exerts antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). RGE had no bactericidal activity, at least in the range of dissolvable concentration. However, RGE reduced 0.03-0.25-fold the minimum inhibitory concentration (MIC) values of ß-lactam antibiotics (oxacillin, ampicillin, carbenicillin, and cefazolin) and aminoglycoside antibiotics (kanamycin and gentamicin) against the two laboratory strains of MRSA. Moreover, the fractional inhibitory concentration index indicated the synergistic activity of RGE with each of the antibiotics. RGE also increased the kanamycin sensitivity of 15 MRSA strains isolated from human volunteers and increased the ampicillin sensitivity of five MRSA strains isolated from dairy cows diagnosed with bovine mastitis. In contrast, RGE did not alter the MIC values of fosfomycin, tetracycline, and erythromycin, suggesting that RGE acts selectively. In contrast, Triton X-100, which was reported to reduce the MIC value of ß-lactam antibiotics to MRSA by increasing membrane permeability, reduced the MIC values of fosfomycin and tetracycline. These results indicate that RGE increases the bactericidal effect of antibiotics via a mechanism different from that used by Triton X-100. We found that ginsenoside Rg3 (Rg3), a component of RGE, was an essential compound that exhibits synergy activity with antibiotics. Furthermore, the non-natural compound K, which contains a common protopanaxadiol aglycon moiety with Rg3, also showed synergistic activity with antibiotics. Thus, Rg3 and compound K are potentially new antibiotic adjuvants against MRSA.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant organism that is prevalent worldwide. Therefore, the research and development of new agents against MRSA are required. We first found that ginsenoside Rg3 (Rg3) in red ginseng, made from the roots of Panax ginseng C. A. Meyer, increased the sensitivity of ß-lactam antibiotics and aminoglycoside antibiotics to MRSA. Furthermore, we identified that compound K, an unnatural ginsenoside analog, also increased the sensitivity of antibiotics to MRSA, similar to Rg3. By contrast, neither Rg3 nor compound K increased the sensitivity of fosfomycin, tetracycline, and erythromycin to MRSA, suggesting that these act selectively. In the present study, the natural compound Rg3 and its structural isomer, compound K, are potentially new antibiotic adjuvants against MRSA. Currently, multiple antibiotics are used to treat MRSA, but the use of these adjuvants is expected to enable the treatment of MRSA with a single antibiotic and low concentrations of antibiotics.

Escherichia coli-Derived Outer Membrane Vesicles Relay Inflammatory Responses to Macrophage-Derived Exosomes.

Extracellular vesicles are considered to be an inflammatory factor in several acute and chronic inflammatory diseases. The present study shows that exosomes from macrophages (Mφ) infected with live Escherichia coli induced secretion of proinflammatory factors by uninfected Mφ. Inflammatory responses induced by exosomes derived from Mφ infected with heat-inactivated E. coli or lipopolysaccharide were significantly weaker than those elicited by outer membrane vesicles (OMVs) released from live E. coli. Proteome analysis of exosomes from Mφ infected with live or heat-inactivated E. coli revealed that E. coli proteins OmpA, GroL1, DegP, CirA, and FepA are candidate triggers of exosome-mediated inflammatory responses. OMVs from a cirA-deleted strain suppressed exosome-mediated inflammatory responses by uninfected Mφ. The C terminus of the CirA protein (residues 158 to 633), which was relayed from E. coli-derived OMV to Mφ-derived exosomes, promoted exosome-mediated inflammatory responses by uninfected Mφ. These results suggest an alternative mechanism by which extracellular vesicles from E. coli OMV-elicited Mφ transmit proinflammatory responses to uninfected Mφ. IMPORTANCE Recently, extracellular membrane vesicles (EVs) were regarded as drivers that carry cargo such as proteins, lipids, metabolites, RNA, and DNA for intracellular signaling transduction. Mammalian cells release various types of EVs, including microvesicles shed from the plasma membrane, exosomes from endosomes, apoptotic bodies, and others. EVs have been reported to mediate inflammatory signals between mammalian cells. In addition, bacteria are also known to release EVs to carry various bacterial factors. In this study, we show that bacterial EVs lead host mammalian cells to release stimulatory EVs that enhance inflammatory responses. Our results provide a novel example that bacterial EVs transduce biological signals to mammalian EVs.

Suppression of the doxorubicin response by hypoxia-inducible factor-1α is strictly dependent on oxygen concentrations under hypoxic conditions.

Hypoxia-inducible factor-1α (HIF-1α) and p53 are involved in anticancer drug resistance under hypoxic conditions. Here, we found that the cytotoxicity of anticancer drugs (doxorubicin, gemcitabine, and cisplatin) was lower at 1% O2 than at 5% O2. We examined the effects of these drugs on HIF-1α and p53 expression under different hypoxic oxygen concentrations. At 5% O2, the drugs decreased HIF-1α expression and increased p53 levels. At 1% O2, the drugs increased HIF-1α expression but did not alter p53 levels. When the HIF-1α protein was stabilized by DMOG under normoxic conditions, doxorubicin did not increase the level of p53 expression. These results show that the maintenance of HIF-1α expression blocked doxorubicin-dependent increases in p53 expression. We hypothesized the mechanism of HIF-1α protein translation might be different between at 5% and at 1% O2, because many reports indicate that the same mechanism of HIF-1α protein stabilization occurs under hypoxic conditions, such as 5% and 1% O2. The level of phosphorylated-4E-BP1, which causes translation of HIF-1α, was higher at 1% O2 than at 5% O2. Our results suggest that the sensitivity of tumor cells to anticancer drugs is dependent oxygen concentrations under hypoxic conditions, and involves 4E-BP1-dependent stabilization of the HIF-1α protein.