Differential Trafficking of Oxidized LDL and Oxidized LDL Immune Complexes in Macrophages: Impact on Oxidative Stress

Background

Oxidized low-density lipoproteins (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contribute to formation of lipid-laden macrophages (foam cells). It has been shown that oxLDL-IC are considerably more efficient than oxLDL in induction of foam cell formation, inflammatory cytokines secretion, and cell survival promotion. Whereas oxLDL is taken up by several scavenger receptors, oxLDL-IC are predominantly internalized through the FCγ receptor I (FCγ RI). This study examined differences in intracellular trafficking of lipid and apolipoprotein moieties of oxLDL and oxLDL-IC and the impact on oxidative stress.

Methodology/Findings

Fluorescently labeled lipid and protein moieties of oxLDL co-localized within endosomal and lysosomal compartments in U937 human monocytic cells. In contrast, the lipid moiety of oxLDL-IC was detected in the endosomal compartment, whereas its apolipoprotein moiety advanced to the lysosomal compartment. Cells treated with oxLDL-IC prior to oxLDL demonstrated co-localization of internalized lipid moieties from both oxLDL and oxLDL-IC in the endosomal compartment. This sequential treatment likely inhibited oxLDL lipid moieties from trafficking to the lysosomal compartment. In RAW 264.7 macrophages, oxLDL-IC but not oxLDL induced GFP-tagged heat shock protein 70 (HSP70) and HSP70B'', which co-localized with the lipid moiety of oxLDL-IC in the endosomal compartment. This suggests that HSP70 family members might prevent the degradation of the internalized lipid moiety of oxLDL-IC by delaying its advancement to the lysosome. The data also showed that mitochondrial membrane potential was decreased and generation of reactive oxygen and nitrogen species was increased in U937 cell treated with oxLDL compared to oxLDL-IC.

Conclusions/Significance

Findings suggest that lipid and apolipoprotein moieties of oxLDL-IC traffic to separate cellular compartments, and that HSP70/70B'' might sequester the lipid moiety of oxLDL-IC in the endosomal compartment. This mechanism could ultimately influence macrophage function and survival. Furthermore, oxLDL-IC might regulate the intracellular trafficking of free oxLDL possibly through the induction of HSP70/70B''.     

Preventive effect of Ligularia fischeri on inhibition of nitric oxide in lipopolysaccharide-stimulated RAW 264.7 macrophages depending on cooking method

Background

Ligularia fischeri (common name Gomchwi) is known for its pharmaceutical properties and used in the treatment of jaundice, scarlet-fever, rheumatoidal arthritis, and hepatic diseases; however, little is known about its anti-inflammatory effect. In this study the influence of blanching and pan-frying on the anti-inflammatory activity of Ligularia fischeri (LF) was evaluated.

Results

Fresh LF and cooked LF showed no significant effect on the viability of macrophages after 24 h incubation. Fresh LF was found to be the most potent inhibitor of nitric oxide (NO) production at 100 μg/ml, while pan-fried LF showed little inhibitory effect on lipoloysaccharide (LPS) stimulated murine machrophage RAW264.7 cells. In contrast with its effect on NO production, pan-fried LF showed significant attenuation of the expression of inducible nitiric oxide synthase (iNOS) compared with fresh LF. In the cooking method of LF, PGE₂ production was not affected in the LPS-induced RAW 264.7 cells. In LPS-induced RAW 264.7 cells, pretreatment by fresh and cooked LF increased COX2 mRNA expression. The 3-O-caffeoylquinic acid content of blanching and pan-frying LF increased by 4.92 and 9.7 fold with blanching and pan-frying respectively in comparison with uncooked LF.

Conclusions

Regardless of the cooking method, Ligularia fischeri exhibited potent inhibition of NO production through expression of iNOS in LPS-induced RAW264.7 cells.     

Intermittent Hypoxia Effect on Osteoclastogenesis Stimulated by Neuroblastoma Cells

Background

Neuroblastoma is the most common extracranial pediatric solid tumor. Intermittent hypoxia, which is characterized by cyclic periods of hypoxia and reoxygenation, has been shown to positively modulate tumor development and thereby induce tumor growth, angiogenic processes, and metastasis. Bone is one of the target organs of metastasis in advanced neuroblastoma Neuroblastoma cells produce osteoclast-activating factors that increase bone resorption by the osteoclasts. The present study focuses on how intermittent hypoxia preconditioned SH-SY5Y neuroblastoma cells modulate osteoclastogenesis in RAW 264.7 cells compared with neuroblastoma cells grown at normoxic conditions.

Methods

We inhibited HIF-1α and HIF-2α in neuroblastoma SH-SY5Y cells by siRNA/shRNA approaches. Protein expression of HIF-1α, HIF-2α and MAPKs were investigated by western blotting. Expression of osteoclastogenic factors were determined by real-time RT-PCR. The influence of intermittent hypoxia and HIF-1α siRNA on migration of neuroblastoma cells and in vitro differentiation of RAW 264.7 cells were assessed. Intratibial injection was performed with SH-SY5Y stable luciferase-expressing cells and in vivo bioluminescence imaging was used in the analysis of tumor growth in bone.

Results

Upregulation of mRNAs of osteoclastogenic factors VEGF and RANKL was observed in intermittent hypoxia-exposed neuroblastoma cells. Conditioned medium from the intermittent hypoxia-exposed neuroblastoma cells was found to enhance osteoclastogenesis, up-regulate the mRNAs of osteoclast marker genes including TRAP, CaSR and cathepsin K and induce the activation of ERK, JNK, and p38 in RAW 264.7 cells. Intermittent hypoxia-exposed neuroblastoma cells showed an increased migratory pattern compared with the parental cells. A significant increase of tumor volume was found in animals that received the intermittent hypoxia-exposed cells intratibially compared with parental cells.

Conclusions

Intermittent hypoxic exposure enhanced capabilities of neuroblastoma cells in induction of osteoclast differentiation in RAW 264.7 cells. Increased migration and intratibial tumor growth was observed in intermittent hypoxia-exposed neuroblastoma cells compared with parental cells.     

Inositol Hexakisphosphate Inhibits Osteoclastogenesis on RAW 264.7 Cells and Human Primary Osteoclasts

Background

Inoxitol hexakisphosphate (IP6) has been found to have an important role in biomineralization and a direct effect inhibiting mineralization of osteoblasts in vitro without impairing extracellular matrix production and expression of alkaline phosphatase. IP6 has been proposed to exhibit similar effects to those of bisphosphonates on bone resorption, however, its direct effect on osteoclasts (OCL) is presently unknown.

Methodology/Principal Findings

The aim of the present study was to investigate the effect of IP6 on the RAW 264.7 monocyte/macrophage mouse cell line and on human primary osteoclasts. On one hand, we show that IP6 decreases the osteoclastogenesis in RAW 264.7 cells induced by RANKL, without affecting cell proliferation or cell viability. The number of TRAP positive cells and mRNA levels of osteoclast markers such as TRAP, calcitonin receptor, cathepsin K and MMP-9 was decreased by IP6 on RANKL-treated cells. On the contrary, when giving IP6 to mature osteoclasts after RANKL treatment, a significant increase of bone resorption activity and TRAP mRNA levels was found. On the other hand, we show that 1 µM of IP6 inhibits osteoclastogenesis of human peripheral blood mononuclear cells (PBMNC) and their resorption activity both, when given to undifferentiated and to mature osteoclasts.

Conclusions/Significance

Our results demonstrate that IP6 inhibits osteoclastogenesis on human PBMNC and on the RAW264.7 cell line. Thus, IP6 may represent a novel type of selective inhibitor of osteoclasts and prove useful for the treatment of osteoporosis.     

New model of macrophage acquisition of the lymphatic endothelial phenotype

Background

Macrophage-derived lymphatic endothelial cell progenitors (M-LECPs) contribute to new lymphatic vessel formation, but the mechanisms regulating their differentiation, recruitment, and function are poorly understood. Detailed characterization of M-LECPs is limited by low frequency in vivo and lack of model systems allowing in-depth molecular analyses in vitro. Our goal was to establish a cell culture model to characterize inflammation-induced macrophage-to-LECP differentiation under controlled conditions.

Methodology/Principal Findings

Time-course analysis of diaphragms from lipopolysaccharide (LPS)-treated mice revealed rapid mobilization of bone marrow-derived and peritoneal macrophages to the proximity of lymphatic vessels followed by widespread (∼50%) incorporation of M-LECPs into the inflamed lymphatic vasculature. A differentiation shift toward the lymphatic phenotype was found in three LPS-induced subsets of activated macrophages that were positive for VEGFR-3 and many other lymphatic-specific markers. VEGFR-3 was strongly elevated in the early stage of macrophage transition to LECPs but undetectable in M-LECPs prior to vascular integration. Similar transient pattern of VEGFR-3 expression was found in RAW264.7 macrophages activated by LPS in vitro. Activated RAW264.7 cells co-expressed VEGF-C that induced an autocrine signaling loop as indicated by VEGFR-3 phosphorylation inhibited by a soluble receptor. LPS-activated RAW264.7 macrophages also showed a 68% overlap with endogenous CD11b⁺/VEGFR-3⁺ LECPs in the expression of lymphatic-specific genes. Moreover, when injected into LPS- but not saline-treated mice, GFP-tagged RAW264.7 cells massively infiltrated the inflamed diaphragm followed by integration into 18% of lymphatic vessels.

Conclusions/Significance

We present a new model for macrophage-LECP differentiation based on LPS activation of cultured RAW264.7 cells. This system designated here as the “RAW model” mimics fundamental features of endogenous M-LECPs. Unlike native LECPs, this model is unrestricted by cell numbers, heterogeneity of population, and ability to change genetic composition for experimental purposes. As such, this model can provide a valuable tool for understanding the LECP and lymphatic biology.     

The Role of Siglec-1 and SR-BI Interaction in the Phagocytosis of Oxidized Low Density Lipoprotein by Macrophages

Background

Macrophages play a proatherosclerotic role in atherosclerosis via oxLDL uptake. As an adhesion molecular of I-type lectins, Siglec-1 is highly expressed on circulating monocytes and plaque macrophages of atherosclerotic patients, but the exact role of Siglec-1 has not been elucidated.

Methods

In this study, oxLDL was used to stimulate Siglec-1 and some oxLDL receptors (SR-BI, CD64, CD32B, LOX-1 and TLR-4) expression on bone marrow-derived macrophages, whereas small interfering RNA was used to down-regulate Siglec-1. Meanwhile, an ELISA-based assay for Siglec-1-oxLDL interaction was performed, and co-immunoprecipitation (co-IP) and laser scanning confocal microscopy (LSCM) were used to determine the role of Siglec-1 in oxLDL uptake by macrophages.

Results

We found that oxLDL could up-regulate the expression of various potential oxLDL receptors, including Siglec-1, in a dose-dependent manner. Moreover, down-regulation of Siglec-1 could attenuate oxLDL uptake by Oil red O staining. LSCM revealed that Siglec-1 and CD64/SR-BI may colocalize on oxLDL-stimulated macrophage surface, whereas co-IP showed that Siglec-1 and SR-BI can be immunoprecipitated by each other. However, no direct interaction between Siglec-1 and oxLDL was found in the in vitro protein interaction system.

Conclusions

Thus, Siglec-1 can interact with SR-BI in the phagocytosis of oxLDL by macrophages, rather than act as an independent receptor for oxLDL.     

Resveratrol Inhibits LPS-Induced MAPKs Activation via Activation of the Phosphatidylinositol 3-Kinase Pathway in Murine RAW 264.7 Macrophage Cells

Background

Resveratrol is a natural polyphenolic compound that has cardioprotective, anticancer and anti-inflammatory properties. We investigated the capacity of resveratrol to protect RAW 264.7 cells from inflammatory insults and explored mechanisms underlying inhibitory effects of resveratrol on RAW 264.7 cells.

Methodology/Principal Findings

Murine RAW 264.7 cells were treated with resveratrol (1, 5, and 10 µM) and/or LPS (5 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by ELISA, RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of Akt, cyclic AMP-responsive element-binding protein (CREB), mitogen-activated protein kinases (MAPKs) cascades, AMP-activated protein kinase (AMPK) and expression of SIRT1(Silent information regulator T1) were measured by western blot. Wortmannin (1 µM), a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, was used to determine if PI3-K/Akt signaling pathway might be involved in resveratrol’s action on RAW 264.7 cells. Resveratrol significantly attenuated the LPS-induced expression of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW 264.7 cells. Resveratrol increased Akt phosphorylation in a time-dependent manner. Wortmannin, a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, blocked the effects of resveratrol on LPS-induced RAW 264.7 cells activation. In addition, PI3-K inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of cyclic AMP-responsive element-binding protein (CREB) and mitogen-activated protein kinases (MAPKs) cascades. Meanwhile, PI3-K is essential for resveratrol-mediated phosphorylation of AMPK and expression of SIRT1.

Conclusion and Implications

This investigation demonstrates that PI3-K/Akt activation is an important signaling in resveratrol-mediated activation of AMPK phosphorylation and SIRT1 expression, and inhibition of phosphorylation of CREB and MAPKs activation, proinflammatory mediators and cytokines production in response to LPS in RAW 264.7 cells.     

Inhibition of anthrax lethal toxin-induced cytolysis of RAW264.7 cells by celastrol

Background

Bacillus anthracis is the bacterium responsible for causing anthrax. The ability of B. anthracis to cause disease is dependent on a secreted virulence factor, lethal toxin, that promotes survival of the bacteria in the host by impairing the immune response. A well-studied effect of lethal toxin is the killing of macrophages, although the molecular mechanisms involved have not been fully characterized.

Methodology/Principal Findings

Here, we demonstrate that celastrol, a quinone methide triterpene derived from a plant extract used in herbal medicine, inhibits lethal toxin-induced death of RAW264.7 murine macrophages. Celastrol did not prevent cleavage of mitogen activated protein kinase kinase 1, a cytosolic target of the toxin, indicating that it did not inhibit the uptake or catalytic activity of lethal toxin. Surprisingly, celastrol conferred almost complete protection when it was added up to 1.5 h after intoxication, indicating that it could rescue cells in the late stages of intoxication. Since the activity of the proteasome has been implicated in intoxication using other pharmacological agents, we tested whether celastrol blocked proteasome activity. We found that celastrol inhibited the proteasome-dependent degradation of proteins in RAW264.7 cells, but only slightly inhibited proteasome-mediated cleavage of fluorogenic substrates in vitro. Furthermore, celastrol blocked stimulation of IL-18 processing, indicating that celastrol acted upstream of inflammasome activation.

Conclusions/Significance

This work identifies celastrol as an inhibitor of lethal toxin-mediated macrophage lysis and suggests an inhibitory mechanism involving inhibition of the proteasome pathway.     

A Novel Role for Adipose Ephrin-B1 in Inflammatory Response

Aims

Ephrin-B1 (EfnB1) was selected among genes of unknown function in adipocytes or adipose tissue and subjected to thorough analysis to understand its role in the development of obesity.

Methods and Results

EfnB1 mRNA and protein levels were significantly decreased in adipose tissues of obese mice and such reduction was mainly observed in mature adipocytes. Exposure of 3T3-L1 adipocytes to tumor necrosis factor-α (TNF-α) and their culture with RAW264.7 cells reduced EFNB1 levels. Knockdown of adipose EFNB1 increased monocyte chemoattractant protein-1 (Mcp-1) mRNA level and augmented the TNF-α-mediated THP-1 monocyte adhesion to adipocytes. Adenovirus-mediated adipose EFNB1-overexpression significantly reduced the increase in Mcp-1 mRNA level induced by coculture of 3T3-L1 adipocytes with RAW264.7 cells. Monocyte adherent assay showed that adipose EfnB1-overexpression significantly decreased the increase of monocyte adhesion by coculture with RAW264.7 cells. TNF-α-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was reduced by EFNB1-overexpression.

Conclusions

EFNB1 contributes to the suppression of adipose inflammatory response. In obesity, reduction of adipose EFNB1 may accelerate the vicious cycle involved in adipose tissue inflammation.     

Mycobacterial Nucleoside Diphosphate Kinase Blocks Phagosome Maturation in Murine Raw 264.7 Macrophages

Background

Microorganisms capable of surviving within macrophages are rare, but represent very successful pathogens. One of them is Mycobacterium tuberculosis (Mtb) whose resistance to early mechanisms of macrophage killing and failure of its phagosomes to fuse with lysosomes causes tuberculosis (TB) disease in humans. Thus, defining the mechanisms of phagosome maturation arrest and identifying mycobacterial factors responsible for it are key to rational design of novel drugs for the treatment of TB. Previous studies have shown that Mtb and the related vaccine strain, M. bovis bacille Calmette-Guérin (BCG), disrupt the normal function of host Rab5 and Rab7, two small GTPases that are instrumental in the control of phagosome fusion with early endosomes and late endosomes/lysosomes respectively.

Methodology/Principal Findings

Here we show that recombinant Mtb nucleoside diphosphate kinase (Ndk) exhibits GTPase activating protein (GAP) activity towards Rab5 and Rab7. Then, using a model of latex bead phagosomes, we demonstrated that Ndk inhibits phagosome maturation and fusion with lysosomes in murine RAW 264.7 macrophages. Maturation arrest of phagosomes containing Ndk-beads was associated with the inactivation of both Rab5 and Rab7 as evidenced by the lack of recruitment of their respective effectors EEA1 (early endosome antigen 1) and RILP (Rab7-interacting lysosomal protein). Consistent with these findings, macrophage infection with an Ndk knocked-down BCG strain resulted in increased fusion of its phagosome with lysosomes along with decreased survival of the mutant.

Conclusion

Our findings provide evidence in support of the hypothesis that mycobacterial Ndk is a putative virulence factor that inhibits phagosome maturation and promotes survival of mycobacteria within the macrophage.     

Bisgma Stimulates Prostaglandin E2 Production in Macrophages via Cyclooxygenase-2, Cytosolic Phospholipase A2, and Mitogen-Activated Protein Kinases Family

Background

Bisphenol A-glycidyl-methacrylate (BisGMA) employs as a monomer in dental resins. The leakage of BisGMA from composite resins into the peripheral environment can result in inflammation via macrophage activation. Prostaglandin E2 (PGE2) is a key regulator of immunopathology in inflammatory reactions. Little is known about the mechanisms of BisGMA-induced PGE2 expression in macrophage. The aim of this study was to evaluate the signal transduction pathways of BisGMA-induced PGE2 production in murine RAW264.7 macrophages.

Methodology/Principal Findings

Herein, we demonstrate that BisGMA can exhibit cytotoxicity to RAW264.7 macrophages in a dose- and time-dependent manner (p<0.05). In addition, PGE2 production, COX-2 expression, and cPLA2 phosphorylation were induced by BisGMA on RAW264.7 macrophages in a dose- and time-dependent manner (p<0.05). Moreover, BisGMA could induce the phosphorylation of ERK1/2 pathway (MEK1/2, ERK1/2, and Elk), p38 pathway (MEK3/6, p38, and MAPKAPK2), and JNK pathway (MEK4, JNK, and c-Jun) in a dose- and time-dependent manner (p<0.05). Pretreatment with AACOCF3, U0126, SB203580, and SP600125 significantly diminished the phosphorylation of cPLA2, ERK1/2, p38, and JNK stimulated by BisGMA, respectively (p<0.05). BisGMA-induced cytotoxicity, cPLA2 phosphorylation, PGE2 generation, and caspases activation were reduced by AACOCF3, U0126, SB203580, and SP600125, respectively (p<0.05).

Conclusions

These results suggest that BisGMA induced-PGE2 production may be via COX-2 expression, cPLA2 phosphorylation, and the phosphorylation of MAPK family. Cytotoxicity mediated by BisGMA may be due to caspases activation through the phosphorylation of cPLA2 and MAPKs family.     

Synthetic Toll like receptor-4 (TLR-4) agonist peptides as a novel class of adjuvants

Background

Adjuvants serve as catalysts of the innate immune response by initiating a localized site of inflammation that is mitigated by the interactions between antigens and toll like receptor (TLR) proteins. Currently, the majority of vaccines are formulated with aluminum based adjuvants, which are associated with various side effects. In an effort to develop a new class of adjuvants, agonists of TLR proteins, such as bacterial products, would be natural candidates. Lipopolysaccharide (LPS), a major structural component of gram negative bacteria cell walls, induces the systemic inflammation observed in septic shock by interacting with TLR-4. The use of synthetic peptides of LPS or TLR-4 agonists, which mimic the interaction between TLR-4 and LPS, can potentially regulate cellular signal transduction pathways such that a localized inflammatory response is achieved similar to that generated by adjuvants.

Methodology/Principal Findings

We report the identification and activity of several peptides isolated using phage display combinatorial peptide technology, which functionally mimicked LPS. The activity of the LPS-TLR-4 interaction was assessed by NF-κB nuclear translocation analyses in HEK-BLUE™-4 cells, a cell culture model that expresses only TLR-4, and the murine macrophage cell line, RAW264.7. Furthermore, the LPS peptide mimics were capable of inducing inflammatory cytokine secretion from RAW264.7 cells. Lastly, ELISA analysis of serum from vaccinated BALB/c mice revealed that the LPS peptide mimics act as a functional adjuvant.

Conclusions/Significance

Our data demonstrate the identification of synthetic peptides that mimic LPS by interacting with TLR-4. This LPS mimotope-TLR-4 interaction will allow for the development and use of these peptides as a new class of adjuvants, namely TLR-4 agonists.     

AMPK Inhibition Blocks ROS-NFκB Signaling and Attenuates Endotoxemia-Induced Liver Injury

Background

AMP-activated protein kinase (AMPK) is an important enzyme in regulation of cellular energy homeostasis. We have previously shown that AMPK activation by 5-aminoimidazole-4-carboxamide (AICAR) results in suppression of immune responses, indicating the pivotal role of AMPK in immune regulation. However, the cellular mechanism underpinning AMPK inhibition on immune response remains largely to be elucidated. The study aimed to investigate the effects of AMPK inhibition on reactive oxygen species (ROS)-nuclear factor κB (NFκB) signaling and endotoxemia-induced liver injury.

Methodology/Principal Findings

RAW 264.7 cells were pretreated with AMPK activator or inhibitor, followed by LPS challenge. In addition, LPS was injected intraperitoneally into mice to induce systemic inflammation. The parameters of liver injury and immune responses were determined, and survival of mice was monitored respectively. LPS challenge in RAW 264.7 cells resulted in AMPK activation which was then inhibited by compound C treatment. Both AMPK activation by AICAR or inhibition by compound C diminished LPS-induced ROS generation, inhibited phosphorylation of IKK, IκB, and NFκB p65, and consequently, decreased TNF production of RAW 264.7 cells. AICAR or compound C treatment decreased ALT, AST, and TNF levels in serum, reduced CD68 expression and MPO activity in liver tissue of mice with endotoxemia. Moreover, AICAR or compound C treatment improved survival of endotoxemic mice.

Conclusions

AICAR or compound C treatment attenuates LPS-induced ROS-NFκB signaling, immune responses and liver injury. Strategies to activate or inhibit AMPK signaling may provide alternatives to the current clinical approaches to inhibit immune responses of endotoxemia.     

Flotillins Are Involved in the Polarization of Primitive and Mature Hematopoietic Cells

Background

Migration of mature and immature leukocytes in response to chemokines is not only essential during inflammation and host defense, but also during development of the hematopoietic system. Many molecules implicated in migratory polarity show uniform cellular distribution under non-activated conditions, but acquire a polarized localization upon exposure to migratory cues.

Methodology/Principal Findings

Here, we present evidence that raft-associated endocytic proteins (flotillins) are pre-assembled in lymphoid, myeloid and primitive hematopoietic cells and accumulate in the uropod during migration. Furthermore, flotillins display a polarized distribution during immunological synapse formation. Employing the membrane lipid-order sensitive probe Laurdan, we show that flotillin accumulation in the immunological synapse is concomittant with membrane ordering in these regions.

Conclusions

Together with the observation that flotillin polarization does not occur in other polarized cell types such as polarized epithelial cells, our results suggest a specific role for flotillins in hematopoietic cell polarization. Based on our results, we propose that in hematopoietic cells, flotillins provide intrinsic cues that govern segregation of certain microdomain-associated molecules during immune cell polarization.     

Nitrogen-containing bisphosphonates inhibit RANKL- and M-CSF-induced osteoclast formation through the inhibition of ERK1/2 and Akt activation

Background

Bisphosphonates are an important class of antiresorptive drugs used in the treatment of metabolic bone diseases. Recent studies have shown that nitrogen-containing bisphosphonates induced apoptosis in rabbit osteoclasts and prevented prenylated small GTPase. However, whether bisphosphonates inhibit osteoclast formation has not been determined. In the present study, we investigated the inhibitory effect of minodronate and alendronate on the osteoclast formation and clarified the mechanism involved in a mouse macrophage-like cell lines C7 and RAW264.7.

Results

It was found that minodronate and alendronate inhibited the osteoclast formation of C7 cells induced by receptor activator of NF-κB ligand and macrophage colony stimulating factor, which are inhibited by the suppression of geranylgeranyl pyrophosphate (GGPP) biosynthesis. It was also found that minodronate and alendronate inhibited the osteoclast formation of RAW264.7 cells induced by receptor activator of NF-κB ligand. Furthermore, minodronate and alendornate decreased phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt; similarly, U0126, a mitogen protein kinase kinase 1/2 (MEK1/2) inhibitor, and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited osteoclast formation.

Conclusions

This indicates that minodronate and alendronate inhibit GGPP biosynthesis in the mevalonate pathway and then signal transduction in the MEK/ERK and PI3K/Akt pathways, thereby inhibiting osteoclast formation. These results suggest a novel effect of bisphosphonates that could be effective in the treatment of bone metabolic diseases, such as osteoporosis.     

Nontypeable Haemophilus Influenzae Infection Upregulates the NLRP3 Inflammasome and Leads to Caspase-1-Dependent Secretion of Interleukin-1β — A Possible Pathway of Exacerbations in COPD

Rationale

Nontypeable Haemophilus influenzae (NTHi) is the most common cause for bacterial exacerbations in chronic obstructive pulmonary disease (COPD). Recent investigations suggest the participation of the inflammasome in the pathomechanism of airway inflammation. The inflammasome is a cytosolic protein complex important for early inflammatory responses, by processing Interleukin-1β (IL-1β) to its active form.

Objectives

Since inflammasome activation has been described for a variety of inflammatory diseases, we investigated whether this pathway plays a role in NTHi infection of the airways.

Methods

A murine macrophage cell line (RAW 264.7), human alveolar macrophages and human lung tissue (HLT) were stimulated with viable or non-viable NTHi and/or nigericin, a potassium ionophore. Secreted cytokines were measured with ELISA and participating proteins detected via Western Blot or immunohistochemistry.

Measurements and Main Results

Western Blot analysis of cells and immunohistochemistry of lung tissue detected the inflammasome key components NLRP3 and caspase-1 after stimulation, leading to a significant induction of IL-1β expression (RAW: control at the lower detection limit vs. NTHi 505±111pg/ml, p<0.01). Inhibition of caspase-1 in human lung tissue led to a significant reduction of IL-1β and IL-18 levels (IL-1β: NTHi 24 h 17423±3198pg/ml vs. NTHi+Z-YVAD-FMK 6961±1751pg/ml, p<0.01).

Conclusion

Our data demonstrate the upregulation of the NRLP3-inflammasome during NTHi-induced inflammation in respiratory cells and tissues. Our findings concerning caspase-1 dependent IL-1β release suggest a role for the inflammasome in respiratory tract infections with NTHi which may be relevant for the pathogenesis of bacterial exacerbations in COPD.     

Propionibacterium acnes CAMP factor and host acid sphingomyelinase contribute to bacterial virulence: potential targets for inflammatory acne treatment

Background

In the progression of acne vulgaris, the disruption of follicular epithelia by an over-growth of Propionibacterium acnes (P. acnes) permits the bacteria to spread and become in contact with various skin and immune cells.

Methodology/Principal Findings

We have demonstrated in the present study that the Christie, Atkins, Munch-Peterson (CAMP) factor of P. acnes is a secretory protein with co-hemolytic activity with sphingomyelinase that can confer cytotoxicity to HaCaT keratinocytes and RAW264.7 macrophages. The CAMP factor from bacteria and acid sphingomyelinase (ASMase) from the host cells were simultaneously present in the culture supernatant only when the cells were co-cultured with P. acnes. Either anti-CAMP factor serum or desipramine, a selective ASMase inhibitor, significantly abrogated the P. acnes-induced cell death of HaCaT and RAW264.7 cells. Intradermal injection of ICR mouse ears with live P. acnes induced considerable ear inflammation, macrophage infiltration, and an increase in cellular soluble ASMase. Suppression of ASMase by systemic treatment with desipramine significantly reduced inflammatory reaction induced by intradermal injection with P. acnes, suggesting the contribution of host ASMase in P. acnes-induced inflammatory reaction in vivo. Vaccination of mice with CAMP factor elicited a protective immunity against P. acnes-induced ear inflammation, indicating the involvement of CAMP factor in P. acnes-induced inflammation. Most notably, suppression of both bacterial CAMP factor and host ASMase using vaccination and specific antibody injection, respectively, cooperatively alleviated P. acnes-induced inflammation.

Conclusions/Significance

These findings envision a novel infectious mechanism by which P. acnes CAMP factor may hijack host ASMase to amplify bacterial virulence to degrade and invade host cells. This work has identified both CAMP factor and ASMase as potential molecular targets for the development of drugs and vaccines against acne vulgaris.     

Immune Suppressive Effect of Cinnamaldehyde Due to Inhibition of Proliferation and Induction of Apoptosis in Immune Cells: Implications in Cancer

Background

Besides its anti-inflammatory effects, cinnamaldehyde has been reported to have anti-carcinogenic activity. Here, we investigated its impact on immune cells.

Methods

Activation of nuclear factor-κB by cinnamaldehyde (0–10 µg/ml) alone or in combination with lipopolysaccharide was assessed in THP1XBlue human monocytic cell line and in human peripheral blood mononuclear cells (PBMCs). Proliferation and secretion of cytokines (IL10 and TNFα) was determined in primary immune cells and the human cell lines (THP1, Jurkat E6-1 and Raji cell lines) stimulated with cinnamaldehyde alone or in conjunction with lipopolysaccharide. Nitric oxide was determined in mouse RAW264.7 cells. Moreover, different treated PBMCs were stained for CD3, CD20 and AnnexinV.

Results

Low concentrations (up to 1 µg/ml) of cinnamaldehyde resulted in a slight increase in nuclar factor-kB activation, whereas higher concentrations led to a dose-dependent decrease of nuclear factor-kB activation (up to 50%) in lipopolysachharide-stimulated THP1 cells and PBMCs. Accordingly, nitric oxide, interleukin 10 secretion as well as cell proliferation were reduced in lipopolysachharide-stimulated RAW264.7 cells, PBMCs and THP1, Raji and Jurkat-E6 immune cells in the presence of cinnamaldehyde in a concentration-dependent manner. Flow cytometric analysis of PBMCs revealed that CD3+ were more affected than CD20+ cells to apopotosis by cinnamaldehyde.

Conclusion

We attribute the anti-inflammatory properties of cinnamaldehyde to its ability to block nuclear factor-κB activation in immune cells. Treatment with cinnamaldehyde led to inhibition of cell viability, proliferation and induced apoptosis in a dose-dependent manner in primary and immortalized immune cells. Therefore, despite its described anti-carcinogenic property, treatment with cinnamaldehyde in cancer patients might be contraindicated due to its ability to inhibit immune cell activation.     

MAP Kinase Phosphatase 2 Regulates Macrophage-Adipocyte Interaction

Objective

Inflammation is critical for the development of obesity-associated metabolic disorders. This study aims to investigate the role of mitogen-activated protein kinase phosphatase 2 (MKP-2) in inflammation during macrophage-adipocyte interaction.

Methods

White adipose tissues (WAT) from mice either on a high-fat diet (HFD) or normal chow (NC) were isolated to examine the expression of MKP-2. Murine macrophage cell line RAW264.7 stably expressing MKP-2 was used to study the regulation of MKP-2 in macrophages in response to saturated free fatty acid (FFA) and its role in macrophage M1/M2 activation. Macrophage-adipocyte co-culture system was employed to investigate the role of MKP-2 in regulating inflammation during adipocyte-macrophage interaction. c-Jun N-terminal kinase (JNK)- and p38-specific inhibitors were used to examine the mechanisms by which MKP-2 regulates macrophage activation and macrophage-adipocytes interaction.

Results

HFD changed the expression of MKP-2 in WAT, and MKP-2 was highly expressed in the stromal vascular cells (SVCs). MKP-2 inhibited the production of proinflammatory cytokines in response to FFA stimulation in macrophages. MKP-2 inhibited macrophage M1 activation through JNK and p38. In addition, overexpression of MKP-2 in macrophages suppressed inflammation during macrophage-adipocyte interaction.

Conclusion

MKP-2 is a negative regulator of macrophage M1 activation through JNK and p38 and inhibits inflammation during macrophage-adipocyte interaction.