Disruption of membrane cholesterol stimulates MyD88-dependent NF-kappaB activation in immature B cells

Agents that extract or sequester membrane cholesterol stimulate IkappaB degradation and lead to NF-kappaB activation in a subset of B cells. Although the extraction of cholesterol by methyl-beta-cyclodextrin is the most potent stimulus of NF-kappaB, other agents that sequester cholesterol have similar effects. B cells and B cell lines with an immature phenotype are significantly more sensitive to the effects of cholesterol perturbation than their mature B cell counterparts. NF-kappaB activation does not involve signaling from the B cell receptor complex. Instead, the disruption of membrane cholesterol activates NF-kappaB through a MyD88-dependent pathway involving the pattern recognition receptor, Toll-like receptor 4. We suggest that lipid raft microdomains may serve not only to orchestrate receptor signaling, but to sequester signaling components one from one another, which serves to prevent receptor-mediated signaling from occurring. A role for this process during B cell development is suggested.     

Endogenous macrophage migration inhibitory factor modulates glucocorticoid sensitivity in macrophages via effects on MAP kinase phosphatase-1 and p38 MAP kinase

The pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is induced by glucocorticoids (GCs), but it was not previously known if MIF regulates cellular sensitivity to GC. Here we show in GC and LPS-treated peritoneal macrophages derived from MIF-/- and wt mice that the absence of endogenous MIF is associated with increased sensitivity to GC of TNF release. This is associated with increased expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), concomitant decreased phosphorylation of p38 MAPK, but no effect of MIF on nuclear factor kappaB (NF-kappaB). These results demonstrate that MIF regulates GC sensitivity by phosphorylation of p38, and provides a cellular mechanism for this observation, indicating that MKP-1 is a central target of this regulation.     

Lipopolysaccharide inhibits GPR120 expression in macrophages via Toll‐like receptor 4 and p38 MAPK activation

Free fatty acid receptor G protein‐coupled receptor 120 (GPR120) is highly expressed in macrophages and was reported to inhibit lipopolysaccharide (LPS)‐stimulated cytokine expression. Under inflammation, macrophages exhibit striking functional changes, but changes in GPR120 expression and signaling are not known. In this study, the effects of LPS treatment on macrophage GPR120 expression and activation were investigated. The results showed that LPS inhibited GPR120 expression in mouse macrophage cell line Ana‐1 cells. Moreover, LPS treatment inhibited GPR120 expression in mouse alveolar macrophages both in vitro and in vivo. The inhibitory effect of LPS on GPR120 expression was blocked by Toll‐like receptor 4 (TLR4) inhibitor TAK242 and p38 mitogen‐activated protein kinase inhibitor LY222820, but not by ERK1/2 inhibitor U0126 and c‐Jun N‐terminal kinase inhibitor SP600125. LPS‐induced inhibition of GPR120 expression was not attenuated by GPR120 agonists TUG891 and GW9508. TUG891 inhibited the phagocytosis of alveolar macrophages, and LPS treatment counteracted the effects of TUG891 on phagocytosis. These results indicate that pretreatment with LPS inhibits GPR120 expression and activation in macrophages. It is suggested that LPS‐induced inhibition of GPR120 expression is a reaction enhancing the LPS‐induced pro‐inflammatory response of macrophages.     

Angiotensin II stimulates intercellular adhesion molecule-1 via an AT1 receptor/nuclear factor-kappaB pathway in brain microvascular endothelial cells

Microvascular changes in the brain are significant causes of cerebral edema and ischemia injury. A number of studies suggest that angiotensin (Ang) II may be involved in the initiation and regulation of processes occurring in brain ischemia. We recently reported that Ang II injures brain microvascular endothelial cells (BMEC) partially via stimulating intercellular adhesion molecule-1 (ICAM-1) expression. However, the signaling cascade leading to Ang II-induced ICAM-1 expression in BMEC was unclear. The present study tested the hypothesis that Ang II induces ICAM-1 expression via an AT1 receptor/nuclear factor-kappaB (NF-kappaB) pathway in BMEC. Ang II directly stimulated the expression of ICAM-1 mRNA and protein in primary cultured BMEC. Ang II treatment also resulted in the degradation of IkappaBalpha and increase of NF-kappaB p65 subunit in the nucleus as well as the DNA binding activity of nuclear NF-kappaB. These effects were abolished by pretreatment with the selective AT1 receptor antagonists, losartan and compound EXP-2528, or losartan plus the AT2 receptor antagonist PD123319, but not by PD123319 alone. Moreover, there were no significant differences between the losartan and losartan plus PD123319 groups. These findings indicate that Ang II-induced ICAM-1 upregulation in brain microvascular endothelial cells may be mediated via an AT1 receptor/NF-kappaB pathway.     

Apoptosis induced by NO via phosphorylation of p38 MAPK that stimulates NF-kappaB, p53 and caspase-3 activation in rabbit articular chondrocytes

Nitric oxide (NO), reported as an important inducer of apoptosis, plays a considerable role in the pathogenetic mechanisms of articular diseases. This research aimed at investigating the role of p38 MAPK signal transduction pathway on apoptosis induced by NO in rabbit articular chondrocytes. In the present study, NO was produced by a novel NO donor NOC-18. Rabbit articular chondrocytes were cultured as monolayer, and the first passage cells were used for the experiments. We detected apoptosis induced by NO using Annexin V-FITC/PI flow cytometry and TUNEL assay. Measurement of caspase-3 has reflected its activity level. Western blotting was performed to show the protein expressions of p38, NF-kappaB, p53 and caspase-3. Furthermore, we examined the inhibitory effects in the NO pathway with p38-specific inhibitor SB203580. Treatment with NOC-18 caused accelerated apoptosis in a concentration dependent manner. This acceleration was able to be reduced when added to SB203580. Besides, the inhibitor could significantly decrease NO-induced p38, NF-kappaB, p53 and caspase-3 protein expressions, as well as caspase-3 intracellular activity (P<0.05). These results suggest that p38 MAPK signal transduction pathway is critical to NO-induced chondrocyte apoptosis, and p38 plays a role by way of stimulating NF-kappaB, p53 and caspase-3 activation.     

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress the TNF-alpha gene expression by reducing phosphorylation of ERK1/2 and p38 MAPK in macrophages

We previously reported that excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress gene expression and production of tumour necrosis factor-alpha in murine macrophages stimulated with lipopolysaccharide. The present study investigated the suppressive mechanisms of tumour necrosis factor-alpha mRNA by excretory/secretory products in lipopolysaccharide-stimulated murine macrophages. Electrophoretic mobility shift assay and supershift assay revealed that neither nuclear translocation of nuclear factor-kappa B nor conformation of the p50/p65 nuclear factor-kappa B subunits was affected by the treatment of excretory/secretory products in lipopolysaccharide-stimulated macrophages. Inhibition of extracellular signal-regulated protein kinase 1/2 with PD98059 or p38 mitogen-activated protein kinase with SB203580 partially reduced tumour necrosis factor-alpha mRNA expression, and a combination of the two inhibitors additionally suppressed the level of tumour necrosis factor-alpha mRNA, revealing that both pathways are crucial for full induction of the gene. Northern blot analysis showed that excretory/secretory products additionally suppressed tumour necrosis factor-alpha mRNA expression in cells treated with PD98059 or SB208530 and, in turn, we found that excretory/secretory products reduced phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated macrophages by Western blot analysis. This is the first report demonstrating that excretory/secretory products from parasites suppress tumour necrosis factor-alpha mRNA expression by reducing phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase without any effect on nuclear factor-kappa B activity in macrophages stimulated with lipopolysaccharide. We hypothesise that excretory/secretory products may enable this parasite to survive within the host.     

MicroRNA-181a regulates the activation of the NLRP3 inflammatory pathway by targeting MEK1 in THP-1 macrophages stimulated by ox-LDL

Atherosclerosis (AS) is a chronic inflammatory disease that is characterized by the deposition of lipids in the vascular wall and the formation of foam cells. Macrophages play a critical role in the development of this chronic inflammation. An increasing amount of research shows that microRNAs affect many steps of inflammation. The goal of our study was to investigate the regulatory effect of miR-181a on the NLRP3 inflammasome pathway and explore its possible mechanism. Compared with the control group, the expression of miR-181a was downregulated in the carotid tissue of AS group mice, while the expression of MEK1 and NLRP3-related proteins was upregulated significantly. In vitro, when THP-1 macrophages were stimulated with oxidized low-density lipoprotein (ox-LDL), the expression of miR-181a was decreased, the MEK/ERK/NF-κB inflammatory pathways were activated and the expression of NLRP3 inflammasome-related proteins was upregulated. Exogenous overexpression of miR-181a downregulated the activation of the MEK/ERK/NF-κB pathway and decreased the expression of NLRP3 inflammasome-related proteins (such as NLRP3, caspase-1, interleukin-18 [IL-18], IL-1β, etc). Exogenous miR-181a knockdown showed the opposite results to those of overexpression group. A luciferase reporter assay proved that miR-181a inhibited the expression of MEK1 by binding to its 3′-untranslated region. When we knocked down miR-181a and then treated cells with U0126 before ox-LDL stimulation, we found that U0126 reversed the increased activation of the MEK/ERK/NF-κB pathway and upregulation of NLRP3 inflammasome-related proteins (NLRP3, caspase-1, IL-18, IL-1β) that resulted from miR-181a knockdown. Our study suggests that miR-181a regulates the activation of the NLRP3 inflammatory pathway by altering the activity of the MEK/ERK/NF-κB pathway via targeting of MEK1.     

Pellino proteins are more than scaffold proteins in TLR/IL-1R signalling: a role as novel RING E3-ubiquitin-ligases

Members of the Toll-like receptor (TLR) and IL-1 receptor (IL-1R) family initiate signalling pathways that shape innate immunity. Pellino proteins have recently been implicated as evolutionary conserved scaffold proteins in TLR/IL-1R signalling leading to nuclear factor-kappaB and mitogen activated protein kinase-dependent gene expression. We found that Pellino proteins contain a new RING-like motif. Because RING motifs are a feature of a subclass of E3-ubiquitin-ligases that target specific proteins for ubiquitination, we suggest that Pellino proteins are involved in TLR/IL-1R signalling not only as scaffold proteins but also as RING E3-ubiquitin-ligases. In support of this hypothesis we show that Pellino proteins induce IRAK-1 polyubiquitination in a RING-dependent manner. We further propose a model in which Pellino-mediated IRAK-1 polyubiquitination regulates TLR/IL-1R signalling.     

Hydrogen inhalation reduced epithelial apoptosis in ventilator-induced lung injury via a mechanism involving nuclear factor-kappa B activation

We recently demonstrated the inhalation of hydrogen gas, a novel medical therapeutic gas, ameliorates ventilator-induced lung injury (VILI); however, the molecular mechanisms by which hydrogen ameliorates VILI remain unclear. Therefore, we investigated whether inhaled hydrogen gas modulates the nuclear factor-kappa B (NFκB) signaling pathway. VILI was generated in male C57BL6 mice by performing a tracheostomy and placing the mice on a mechanical ventilator (tidal volume of 30 ml/kg or 10 ml/kg without positive end-expiratory pressure). The ventilator delivered either 2% nitrogen or 2% hydrogen in balanced air. NFκB activation, as indicated by NFκB DNA binding, was detected by electrophoretic mobility shift assays and enzyme-linked immunosorbent assay. Hydrogen gas inhalation increased NFκB DNA binding after 1 h of ventilation and decreased NFκB DNA binding after 2 h of ventilation, as compared with controls. The early activation of NFκB during hydrogen treatment was correlated with elevated levels of the antiapoptotic protein Bcl-2 and decreased levels of Bax. Hydrogen inhalation increased oxygen tension, decreased lung edema, and decreased the expression of proinflammatory mediators. Chemical inhibition of early NFκB activation using SN50 reversed these protective effects. NFκB activation and an associated increase in the expression of Bcl-2 may contribute, in part, to the cytoprotective effects of hydrogen against apoptotic and inflammatory signaling pathway activation during VILI.     

Cigarette smoke induces the release of CXCL-8 from human bronchial epithelial cells via TLRs and induction of the inflammasome

COPD is a chronic airway disease associated with inflammation and cigarette smoking. Airway epithelial cells are the first cells exposed to cigarette smoke (CS) and can release CXCL-8 and IL-1β. These cytokines are involved in acute and chronic inflammatory processes in COPD. The aim of this study was to investigate whether toll-like receptors (TLRs) located in/on epithelial cells were involved in cigarette smoke-induced cytokine production. Here we demonstrate that CS induces the release of CXCL-8 and IL-1β from human bronchial epithelial cells (HBE-14o). CS-induced CXCL-8 production was inhibited by an antibody against TLR4 and by inhibitory ODN suggesting the involvement of TLR4 and TLR9. In addition, exposure of HBE-14o cells to TLR4 or TLR9 ligands resulted in the release of CXCL-8 and IL1β. TLR4 and also TLR9 were present on the cell surface and the expression of both receptors decreased after CS exposure. The molecular mechanism of the CS-induced CXCL-8 production by the epithelial cells was further investigated. It was found that P2X7 receptors and reactive oxygen species were involved. Interestingly, the inflammasome activator monosodium urate crystals (MSU) induced the release of CXCL-8 and IL-1β and the caspase-1 inhibitor Z-VADDCB suppressed the CS-induced release of CXCL-8. In addition, CS, CpGODN, lipopolysaccharide and MSU all increased the expression of caspase-1 and IL-1β. In conclusion, our results demonstrate that CS releases CXCL-8 from HBE-14o cells via TLR4 and TLR9 and inflammasome activation. Therefore, inflammasome signaling in airway epithelial cells may play an important role in pathogenesis of diseases like COPD.     

Fatty acids induce leukotriene C4 synthesis in macrophages in a fatty acid binding protein-dependent manner

Obesity results in increased macrophage recruitment to adipose tissue that promotes a chronic low-grade inflammatory state linked to increased fatty acid efflux from adipocytes. Activated macrophages produce a variety of pro-inflammatory lipids such as leukotriene C₄ (LTC₄) and 5-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) suggesting the hypothesis that fatty acids may stimulate eicosanoid synthesis. To assess if eicosanoid production increases with obesity, adipose tissue of leptin deficient ob/ob mice was analyzed. In ob/ob mice, LTC₄ and 12-HETE levels increased in the visceral (but not subcutaneous) adipose depot while the 5-HETE levels decreased and 15-HETE abundance was unchanged. Since macrophages produce the majority of inflammatory molecules in adipose tissue, treatment of RAW264.7 or primary peritoneal macrophages with free fatty acids led to increased secretion of LTC₄ and 5-HETE, but not 12- or 15-HETE. Fatty acid binding proteins (FABPs) facilitate the intracellular trafficking of fatty acids and other hydrophobic ligands and in vitro stabilize the LTC₄ precursor leukotriene A₄ (LTA₄) from non-enzymatic hydrolysis. Consistent with a role for FABPs in LTC₄ synthesis, treatment of macrophages with HTS01037, a specific FABP inhibitor, resulted in a marked decrease in both basal and fatty acid-stimulated LTC₄ secretion but no change in 5-HETE production or 5-lipoxygenase expression. These results indicate that the products of adipocyte lipolysis may stimulate the 5-lipoxygenase pathway leading to FABP-dependent production of LTC₄ and contribute to the insulin resistant state.     

Dancing with the sterols: Critical roles for ABCG1, ABCA1, miRNAs, and nuclear and cell surface receptors in controlling cellular sterol homeostasis

ATP binding cassette (ABC) transporters represent a large and diverse family of proteins that transport specific substrates across a membrane. The importance of these transporters is illustrated by the finding that inactivating mutations within 17 different family members are known to lead to specific human diseases. Clinical data from humans and/or studies with mice lacking functional transporters indicate that ABCA1, ABCG1, ABCG4, ABCG5 and ABCG8 are involved in cholesterol and/or phospholipid transport. This review discusses the multiple mechanisms that control cellular sterol homeostasis, including the roles of microRNAs, nuclear and cell surface receptors and ABC transporters, with particular emphasis on recent findings that have provided insights into the role(s) of ABCG1. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Immunogenic cell death,DAMPs and anticancer therapeutics: An emerging amalgamation

Immunogenic profile of certain cancer cell death mechanisms has been transmuted by research published over a period of last few years and this change has been so drastic that a new (sub)class of apoptotic cancer cell death, redefined as ‘immunogenic apoptosis’ has started taking shape. In fact, it has been shown that this chemotherapeutic agent-specific immunogenic cancer cell death modality has the capabilities to induce ‘anticancer vaccine effect’, in vivo. These new trends have given an opportunity to combine tumour cell kill and antitumour immunity within a single paradigm, a sort of ‘holy grail’ of anticancer therapeutics. At the molecular level, it has been shown that the immunological silhouette of these cell death pathways is defined by a set of molecules called ‘damage-associated molecular patterns (DAMPs)’. Various intracellular molecules like calreticulin (CRT), heat-shock proteins (HSPs), high-mobility group box-1 (HMGB1) protein, have been shown to be DAMPs exposed/secreted in a stress agent/factor-and cell death-specific manner. These discoveries have motivated further research into discovery of new DAMPs, new pathways for their exposure/secretion, search for new agents capable of inducing immunogenic cell death and urge to solve currently present problems with this paradigm. We anticipate that this emerging amalgamation of DAMPs, immunogenic cell death and anticancer therapeutics may be the key towards squelching cancer-related mortalities, in near future.     

Bioactivity of soy-based fermented foods: A review

For centuries, fermented soy foods have been dietary staples in Asia and, now, in response to consumer demand, they are available throughout the world. Fermentation bestows unique flavors, boosts nutritional values and increases or adds new functional properties. In this review, we describe the functional properties and underlying action mechanisms of soy-based fermented foods such as Natto, fermented soy milk, Tempeh and soy sauce. When possible, the contribution of specific bioactive components is highlighted. While numerous studies with in vitro and animal models have hinted at the functionality of fermented soy foods, ascribing health benefits requires well-designed, often complex human studies with analysis of diet, lifestyle, family and medical history combined with long-term follow-ups for each subject. In addition, the contribution of the microbiome to the bioactivities of fermented soy foods, possibly mediated through direct action or bioactive metabolites, needs to be studied. Potential synergy or other interactions among the microorganisms carrying out the fermentation and the host's microbial community may also contribute to food functionality, but the details still require elucidation. Finally, safety evaluation of fermented soy foods has been limited, but is essential in order to provide guidelines for consumption and confirm lack of toxicity.     

Bio-informatics based analysis of genes implicated in alcohol mediated liver injury

Alcohol induced liver injury has been studied extensively. Using literature search and bioinformatics tools, the present study characterizes the genes involved in alcohol induced liver injury. The cellular and metabolic processes in which genes involved in alcohol induced liver injury are implicated are also discussed. The genes related to alcohol induced liver injury are also involved in affecting certain molecular functions and metabolism of drugs, besides being associated with diseases. In conclusion, the changes in regulation of genes implicated in alcohol induced liver injury apart from causing alcohol mediated hepatic dysfunction may affect other vital processes in the body.