Effects of a HP0859 (rfaD) knockout mutation on lipopolysaccharide structure of Helicobacter pylori 26695 and the bacterial adhesion on AGS cells

Cholesteryl ester transfer protein (CETP) and apolipoprotein E (apoE) are secreted by macrophages. Apolipoprotein A–I (apoA–I) is a potent inducer of apoE secretion from lipid-loaded macrophages, but its effect on CETP is not known. We aimed to identify the signaling pathways involved in apoA–I and HDL-mediated regulation of CETP and apoE secretion from lipid-loaded macrophages. THP-1 macrophages were loaded with lipids by incubation with human copper-oxidized LDL. The cells were subsequently exposed to human purified apoA–I or HDL₃ with/without inhibitors of NF-κB (TPCK) or PKA (H89). CETP and apoE in the cultured cells and media were quantified by real-time PCR and Western blot. Results showed that in lipid-loaded macrophages: (i) CETP and apoE gene expression and secretion were increased in the presence of apoA–I, and further increased by inhibition of NF-kB with TPCK; (ii) CETP and apoE gene expression and secretion were reduced by the inhibition of PKA with H89; (iii) PKA-gamma subunit was activated by oxidized LDL and moreover by apoA–I. We also showed that: (i) siRNA-mediated CETP gene silencing diminished apoE secretion from both non-loaded and lipid-loaded macrophages; (ii) addition of apoA–I partially restored apoE secretion from lipid-loaded macrophages with the silenced CETP gene. In conclusion, our data suggest a new mechanism by which apoA–I stimulates CETP secretion, in addition to apoE, from lipid loaded macrophages, a process involving NF-κB inhibition and/or PKA pathway activation.     

Berry anthocyanins suppress the expression and secretion of proinflammatory mediators in macrophages by inhibiting nuclear translocation of NF-κB independent of NRF2-mediated mechanism

The objectives of this study were to compare the anti-inflammatory effects of anthocyanins from blueberry (BBA), blackberry (BKA), and blackcurrant (BCA) and to determine the relationship between their antioxidant capacity and anti-inflammatory effect in macrophages. Major anthocyanins in BBA, BKA and BCA were malvidin-3-glucoside (16%), cyanidin-3-glucoside (98%) and delphinidin-3-rutinoside (44%), respectively. BKA showed higher total antioxidant capacity than BBA and BCA. RAW 264.7 macrophages were incubated with 0–20 μg/ml of BBA, BKA and BCA, and subsequently activated by lipopolysaccharide (LPS) to measure proinflammatory cytokine production. Interleukin 1β (IL-1β) messenger RNA (mRNA) levels were significantly decreased by all berry anthocyanins at 10 μg/ml or higher. Tumor necrosis factor α (TNFα) mRNA levels and secretion were also significantly decreased in LPS-treated macrophages. The levels of the repression were comparable for all berry anthocyanins. LPS-induced nuclear factor κB (NF-κB) p65 translocation to the nucleus was markedly attenuated by all of the berry anthocyanins. In bone marrow-derived macrophages (BMMs) from nuclear factor E2-related factor 2 wild-type (Nrf2^+/+) mice, BBA, BKA and BCA significantly decreased cellular reactive oxygen species (ROS) levels with a concomitant decrease in IL-1β mRNA levels upon LPS stimulation. However, in the BMM from Nrf2^−/− mice, the anthocyanin fractions were able to significantly decrease IL-1β mRNA despite the fact that ROS levels were not significantly affected. In conclusion, BBA, BKA and BCA exert their anti-inflammatory effects in macrophages, at least in part, by inhibiting nuclear translocation of NF-κB independent of the NRF2-mediated pathways.     

Nasunin inhibits the lipopolysaccharide-induced pro-inflammatory mediator production in RAW264 mouse macrophages by suppressing ROS-mediated activation of PI3 K/Akt/NF-κB and p38 signaling pathways

Nasunin is a major anthocyanin in eggplant peel. The purpose of this study was to examine the anti-inflammatory effects of nasunin in lipopolysaccharide (LPS)-stimulated RAW264 macrophages and to identify the molecular mechanisms underlying these effects. We found that nasunin reduced the LPS-induced secretion of tumor necrosis factor-α, interleukin-6 and nitric oxide, and expression of inducible nitric oxide synthase in a dose-dependent manner. Nasunin diminished LPS-induced nuclear factor-κB (NF-κB) activation by suppressing the degradation of inhibitor of κB-α and nuclear translocation of p65 subunit of NF-κB. Nasunin also attenuated the phosphorylation of Akt and p38, signaling molecules involved in pro-inflammatory mediator production. Moreover, nasunin inhibited the intracellular accumulation of ROS, leading to the suppression of NF-κB activation, Akt and p38 phosphorylation, and subsequent pro-inflammatory mediator production. These findings suggest that nasunin exerts an anti-inflammatory effect and this effect is mediated, at least in part, by its antioxidant activity.     

Anti-oxidant and anti-inflammatory mechanisms of amlodipine action to improve endothelial cell dysfunction induced by irreversibly glycated LDL

Amlodipine, alone or in combination with other drugs, was successfully used to treat hypertension. Our aim was to evaluate the potential of amlodipine (Am) to restore endothelial dysfunction induced by irreversibly glycated low density lipoproteins (AGE-LDL), an in vitro model mimicking the diabetic condition. Human endothelial cells (HEC) from EA.hy926 line were incubated with AGE-LDL in the presence/absence of Am and the oxidative and inflammatory status of the cells was evaluated along with the p38 MAPK and NF-κB signalling pathways. The cellular NADPH activity, 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine levels in the culture medium and the adhesion of human monocytes to HEC were measured by chemiluminescence, UHPLC, Western Blot and spectrofluorimetric techniques. The gene expression of NADPH subunits (p22^phox, NOX4), eNOS and inflammatory molecules (MCP-1, VCAM-1) were determined by Real Time PCR, while the protein expression of p22^phox, MCP-1, iNOS, phospho-p38 MAPK and phospho-p65 NF-κB subunit were measured by Western Blot. Results showed that in HEC incubated with AGE-LDL, Am led to: (i) decrease of the oxidative stress: by reducing p22^phox, NOX4, iNOS expression, NADPH oxidase activity, 4-HNE and 3-nitrotyrosine levels; (ii) decrease of the inflammatory stress: by the reduction of MCP-1 and VCAM-1 expression, as well as of the number of monocytes adhered to HEC; (iii) inhibition of ROS-sensitive signalling pathways: by decreasing phosphorylation of p38 MAPK and p65 NF-κB subunits. In conclusion, the reported data demonstrate that amlodipine may improve endothelial dysfunction in diabetes through anti-oxidant and anti-inflammatory mechanisms.     

RNA-binding protein AUF1 regulates lipopolysaccharide-induced IL10 expression by activating IkappaB kinase complex in monocytes

Exposure of monocytes and macrophages to endotoxin/lipopolysaccharide (LPS) from Gram-negative bacteria activates the NF-κB signaling pathway. At early times, this leads to their production of proinflammatory cytokines, but subsequently, they produce anti-inflammatory interleukin-10 (IL-10) to quell the immune response. LPS-mediated induction of IL10 gene expression requires the p40 isoform of the RNA-binding protein AUF1. As LPS exerts modest effects upon IL10 mRNA stability, we hypothesized that AUF1 controls the expression of signaling proteins. Indeed, knockdown of AUF1 impairs LPS-mediated p38 mitogen-activated protein kinase (MAPK) and NF-κB signaling, and the expression of an RNA interference-refractory p40(AUF1) cDNA restores both signaling pathways. To define the molecular mechanisms by which p40(AUF1) controls IL10 expression, we focused on the NF-κB pathway in search of AUF1-regulated targets. Here, we show that p40(AUF1) serves to maintain proper levels of the kinase TAK1 (transforming growth factor-β-activated kinase), which phosphorylates the IKKβ subunit within the IκB kinase complex to activate NF-κB-regulated genes. However, p40(AUF1) does not control the TAK1 mRNA levels but instead promotes the translation of the mRNA. Thus, p40(AUF1) regulates a critical node within the NF-κB signaling pathway to permit IL10 induction for the anti-inflammatory arm of an innate immune response.     

6-Gingerol inhibits ROS and iNOS through the suppression of PKC-α and NF-κB pathways in lipopolysaccharide-stimulated mouse macrophages

Inflammation is involved in numerous diseases, including chronic inflammatory diseases and the development of cancer. Many plants possess a variety of biological activities, including antifungal, antibacterial and anti-inflammatory activities. However, our understanding of the anti-inflammatory effects of 6-gingerol is very limited. We used lipopolysaccharide (LPS)-stimulated macrophages as a model of inflammation to investigate the anti-inflammatory effects of 6-gingerol, which contains phenolic structure. We found that 6-gingerol exhibited an anti-inflammatory effect. 6-Gingerol could decrease inducible nitric oxide synthase and TNF-α expression through suppression of I-κBα phosphorylation, NF-κB nuclear activation and PKC-α translocation, which in turn inhibits Ca²⁺ mobilization and disruption of mitochondrial membrane potential in LPS-stimulated macrophages. Here, we demonstrate that 6-gingerol acts as an anti-inflammatory agent by blocking NF-κB and PKC signaling, and may be developed as a useful agent for the chemoprevention of cancer or inflammatory diseases.     

Vitamin E ameliorates ox-LDL-induced foam cells formation through modulating the activities of oxidative stress-induced NF-κB pathway

The role of antioxidant supplementation with vitamin E in the prevention of atherosclerosis has been a topic of considerable recent interest. The relevance of vitamin E for macrophage-derived foam cell formation, a hallmark of atherosclerosis, however, has not been unequivocally resolved. Here, we investigated the effect of oxidized LDL (ox-LDL) and vitamin E on lipid accumulation and total cholesterol content in U937 macrophages, reactive oxygen species generation and expression of nuclear factor-κB (NF-κB) signaling pathway. The results showed that the mRNA expression and protein levels of P-selectin were evident in U937 macrophages treated with ox-LDL and vitamin E, which indicating that expression of P-selectin is important in macrophage-derived foam cell formation. Moreover, P-selectin changes in ox-LDL-induced foam cell formation can be mediated by vitamin E through activities of nuclear NF-κB activated by serine phosphorylation of NF-κB inhibitor α, suggesting that activation of NF-κB pathway by macrophages may occur. Taken together, these data suggested that vitamin E can prevent ox-LDL-induced foam cell macrophages formation through modulating the activities of oxidative stress-induced NF-κB pathway.     

ABCA1 Protein Enhances Toll-like Receptor 4 (TLR4)-stimulated Interleukin-10 (IL-10) Secretion through Protein Kinase A (PKA) Activation

Nonresolving inflammatory response from macrophages is a major characteristic of atherosclerosis. Macrophage ABCA1 has been previously shown to suppress the secretion of proinflammatory cytokine. In the present study, we demonstrate that ABCA1 also promotes the secretion of IL-10, an anti-inflammatory cytokine critical for inflammation resolution. ABCA1^+/+ bone marrow-derived macrophages secrete more IL-10 but less proinflammatory cytokines than ABCA1^−/− bone marrow-derived macrophages, similar to alternatively activated (M2) macrophages. We present evidence that ABCA1 activates PKA and that this elevated PKA activity contributes to M2-like inflammatory response from ABCA1^+/+ bone marrow-derived macrophages. Furthermore, cholesterol lowering by statins, methyl-β-cyclodextrin, or filipin also activates PKA and, consequently, transforms macrophages toward M2-like phenotype. Conversely, cholesterol enrichment suppresses PKA activity and promotes M1-like inflammatory response. As the primary function of ABCA1 is cholesterol removal, our results suggest that ABCA1 activates PKA by regulating cholesterol. Indeed, forced cholesterol enrichment in ABCA1-expressing macrophages suppresses PKA activation and elicits M1-like response. Collectively, these findings reveal a novel protective process by ABCA1-activated PKA in macrophages. They also suggest cholesterol lowering in extra-hepatic tissues by statins as an anti-inflammation strategy.     

Hesperetin suppresses RANKL-induced osteoclastogenesis and ameliorates lipopolysaccharide-induced bone loss

Destructive bone diseases caused by osteolysis are increasing in incidence. They are characterized by an excessive imbalance of osteoclast formation and activation. During osteolysis, the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways are triggered by receptor activator of NF-κB ligand (RANKL), inflammatory factors, and oxidative stress. Previous studies have indicated that the common flavanone glycoside compound hesperetin exhibits anti-inflammatory and antioxidant activity by inhibition of NF-κB and MAPK signaling pathways. However, the direct relationship between hesperetin and osteolysis remain unclear. In the present study, we investigated the effects of hesperetin on lipopolysaccharide (LPS)-induced osteoporosis and elucidated the related mechanisms. Hesperetin effectively suppressed RANKL-induced osteoclastogenesis, osteoclastic bone resorption, and F-actin ring formation in a dose-dependent manner. It also significantly suppressed the expression of osteoclast-specific markers including tartrate-resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, c-Fos, and nuclear factor of activated T-cells cytoplasmic 1. Furthermore, it inhibited osteoclastogenesis by inhibiting activation of NF-κB and MAPK signaling, scavenging reactive oxygen species, and activating the nuclear factor E2 p45-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling pathway. Consistent with in vitro results, hesperetin effectively ameliorated LPS-induced bone loss, reduced osteoclast numbers, and decreased the RANKL/OPG ratio in vivo. As such, our results suggest that hesperetin may be a great candidate for developing a novel drug for destructive bone diseases such as periodontal disease, tumor bone metastasis, rheumatoid arthritis, and osteoporosis.     

Anti-osteoclastic effect of caffeic acid phenethyl ester in murine macrophages depends upon the suppression of superoxide anion production through the prevention of an active-Nox1 complex formation

This study investigated the anti-osteoclastic effect of caffeic acid phenethyl ester (CAPE) through suppression of Nox1-mediated superoxide anions production. The multi-nucleated cells were counted and followed by measuring their tartrate-resistant acid phosphatase (TRAP) activity. The superoxide anion production was determined by using fluorescent probe dihydroethidium (DHE). After one day of exposure to the receptor activator of nuclear factor-κB ligand (RANKL), the expression of the proteins involved in superoxide anion production was determined by western blotting. A potent anti-osteoclastic effect of CAPE was observed; the superoxide anion level reached a maximum value after one day of incubation. CAPE attenuated the expression of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1) and Rac1, and mitigated the RANKL-induced translocation of p47^phox to the cell membrane. In addition, CAPE suppressed the expression of nuclear factor-kappa B (NF-κB p65), its translocation to the nucleus, and the activation of NF-κB inhibitor (IκBα) and its kinase (IKKβ). Furthermore, CAPE diminished the expression and activation of the c-jun N-terminal kinase (JNK) and the expression of protein-1 activators (AP-1) such as c-Fos and c-Jun. The expression of Nox1 was suppressed by CAPE through the down-regulation of IKKβ/IκBα/NF-κB and JNK/AP-1 signal pathway. This study provides evidence that the anti-osteoclastic effect of CAPE depends upon the attenuated superoxide anion production, which is closely related with interruption of an active Nox1 complex formation due to the attenuated catalytic subunit Nox1 expression resulting from suppression of the IKKβ/IκBα/NF-κB and JNK/AP-1 signaling pathway and the down-regulation of the p47^phox subunit translocation to the cell membrane.     

Real-time monitoring of inflammation status in 3T3-L1 adipocytes possessing a secretory Gaussia luciferase gene under the control of nuclear factor-kappa B response element

We have established 3T3-L1 cells possessing a secretory Gaussia luciferase (GLuc) gene under the control of nuclear factor-kappa B (NF-κB) response element. The 3T3-L1 cells named 3T3-L1-NF-κB-RE-GLuc could differentiate into adipocyte as comparably as parental 3T3-L1 cells. Inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β induced GLuc secretion of 3T3-L1-NF-κB-RE-GLuc adipocytes in a concentration- and time-dependent manner. GLuc secretion of 3T3-L1-NF-κB-RE-GLuc adipocytes was also induced when cultured with RAW264.7 macrophages and was dramatically enhanced by lipopolysaccharide (LPS)-activated macrophages. An NF-κB activation inhibitor BAY-11-7085 and an antioxidant N-acetyl cysteine significantly suppressed GLuc secretion induced by macrophages. Finally, we found that rosemary-derived carnosic acid strongly suppressed GLuc secretion induced by macrophages and on the contrary up-regulated adiponectin secretion. Collectively, by using 3T3-L1-NF-κB-RE-GLuc adipocytes, inflammation status can be monitored in real time and inflammation-attenuating compounds can be screened more conveniently.     

LAPTM5 Protein Is a Positive Regulator of Proinflammatory Signaling Pathways in Macrophages

LAPTM5 (lysosomal-associated protein transmembrane 5) is a protein that is preferentially expressed in immune cells, and it interacts with the Nedd4 family of ubiquitin ligases. Recent studies in T and B cells identified LAPTM5 as a negative regulator of T and B cell receptor levels at the plasma membrane. Here we investigated the function of LAPTM5 in macrophages. We demonstrate that expression of LAPTM5 is required for the secretion of proinflammatory cytokines in response to Toll-like receptor ligands. We also show that RAW264.7 cells knocked down for LAPTM5 or macrophages from LAPTM5(-/-) mice exhibit reduced activation of NF-κB and MAPK signaling pathways mediated by the TNF receptor, as well as multiple pattern recognition receptors in various cellular compartments. TNF stimulation of LAPTM5-deficient macrophages leads to reduced ubiquitination of RIP1 (receptor-interacting protein 1), suggesting a role for LAPTM5 at the receptor-proximate level. Interestingly, we find that macrophages from LAPTM5(-/-) mice display up-regulated levels of A20, a ubiquitin-editing enzyme responsible for deubiquitination of RIP1 and subsequent termination of NF-κB activation. Our studies thus indicate that, in contrast to its negative role in T and B cell activation, LAPTM5 acts as a positive modulator of inflammatory signaling pathways and hence cytokine secretion in macrophages. They also highlight a role for the endosomal/lysosomal system in regulating signaling via cytokine and pattern recognition receptors.     

Enalapril inhibits nuclear factor-κB signaling in intestinal epithelial cells and peritoneal macrophages and attenuates experimental colitis in mice

Aims

Enalapril, an angiotensin-converting enzyme (ACE) inhibitor, has pleiotropic effects such as anti-inflammatory effects. This study investigated the effect of enalapril on the nuclear factor-kappa B (NF-κB) pathway and on experimental colitis.

Main methods

The human intestinal epithelial cell (IEC) line COLO 205 and peritoneal macrophages from C57BL/6 wild-type mice and IL-10-deficient (IL-10^−/−) mice were prepared and subsequently stimulated with lipopolysaccharide (LPS) alone or LPS plus enalapril. The effect of enalapril on NF-κB signaling was examined by western blotting to detect IκBα phosphorylation/degradation; an electrophoretic mobility shift assay (EMSA) to assess the DNA binding activity of NF-κB; and ELISAs to qualify IL-8, TNF-α, IL-6, and IL-12 production. In in vivo studies, dextran sulfate sodium (DSS)-induced acute colitis in wild-type mice and chronic colitis in IL-10^−/− mice were treated with or without enalapril. Colitis was quantified by histologic scoring, and the phosphorylation of IκBα in the colonic mucosa was assessed using immunohistochemistry.

Key findings

Enalapril significantly inhibited LPS-induced IκBα phosphorylation/degradation, NF-κB binding activity, and pro-inflammatory cytokine production in both IEC and peritoneal macrophages. The administration of enalapril significantly reduced the severity of colitis, as assessed based on histology in both murine colitis models. Furthermore, in colon tissue, the up-regulation of IκBα phosphorylation with colitis induction was attenuated in enalapril-treated mice.

Significance

Enalapril may block the NF-κB signaling pathway, inhibit the activation of IECs and macrophages, and attenuate experimental murine colitis by down-regulating IκBα phosphorylation. These findings suggest that enalapril is a potential therapeutic agent for inflammatory bowel disease.     

Visfatin contributes to the differentiation of monocytes into macrophages through the differential regulation of inflammatory cytokines in THP-1 cells

Visfatin is a novel multifunctional adipocytokine with inflammatory properties. Although a link between visfatin and atherosclerosis has recently been suggested, its actions in the development of atherosclerosis remain unknown. Therefore, we investigated a potential role and underlying mechanism(s) of visfatin in monocytes/macrophages differentiation, a critical early step in atherogenesis, using phorbol-12-myristate-13-acetate (PMA)-stimulated THP-1 cell models. The co-incubation of PMA with visfatin-induced CD36 expression with a concomitant increase in the phagocytosis of latex beads compared with PMA alone treatment. Moreover, visfatin markedly increased interleukin (IL)-1β secretion by enhancing IL-1β mRNA stability in a short-term incubation. Visfatin also significantly elevated the secretion of IL-6 as well as IL-1β in a longer incubation period, which was partially suppressed by nuclear factor-κB (NF-κB) inhibitor, BAY11-7082, and c-Jun-N-terminal kinase (JNK) inhibitor, SP600125. Furthermore, silencing IL-1β successfully blocked IL-6 secretion, CD36 expression, and NF-κB activation in response to visfatin. Collectively, these results suggest that visfatin enhances the IL-1β-dependent induction of IL-6 and CD36 via distinct signaling pathways mediated by JNK and NF-κB, respectively, and consequently, leading to the acceleration of monocytes/macrophages differentiation.