Structural and antigenic characterization of a novel genotype of Mfa1 fimbriae in Porphyromonas gingivalis.

Background: Mfa1 fimbriae of the periodontal pathogen Porphyromonas gingivalis are responsible for biofilm formation and comprise five proteins: Mfa1-5. Two major genotypes, mfa170 and mfa153, encode major fimbrillin. The mfa170 genotype is further divided into the mfa170A and mfa170B subtypes. The properties of the novel mfa170B remain unclear. Methods: Fimbriae were purified from P. gingivalis strains JI-1 (mfa170A), 1439 (mfa170B), and Ando (mfa153), and their components and their structures were analyzed. Protein expression and variability in the antigenic specificity of fimbrillins were compared using Coomassie staining and western blotting using polyclonal antibodies against Mfa170A, Mfa170B, and Mfa153 proteins. Cell surface expression levels of fimbriae were analyzed by filtration enzyme-linked immunosorbent assays. Results: The composition and structures of the purified Mfa1 fimbriae of 1439 was similar to that of JI-1. However, each Mfa1 protein of differential subtype/genotype was specifically detected by western blotting. Mfa170B fimbriae were expressed in several strains such as 1439, JKG9, B42, 1436, and Kyudai-3. Differential protein expression and antigenic heterogeneities were detected in Mfa2-5 between strains. Conclusion: Mfa1 fimbriae from the mfa170A and mfa170B genotypes indicated an antigenic difference suggesting the mfa170B, is to be utilized for the novel classification of P. gingivalis.

Porphyromonas gingivalis Fimbriae Induce Osteoclastogenesis via Toll-like Receptors in RAW264 Cells.

The effect of Mfa1 fimbriae of Porphyromonas gingivalis on the progression of bone resorption remains unclear, especially compared with another fimbriae, FimA. We investigated the effect of Mfa1 on osteoclastogenesis together with FimA. We also investigated the role of Toll-like receptors (TLRs) in Mfa1 recognition during osteoclast differentiation. Receptor activator of nuclear factor κß ligand (RANKL)-prestimulated RAW264 cells were used to examine the effects of purified Mfa1 fimbriae. The number of osteoclasts was examined by tartrate-resistant acid phosphate (TRAP) staining, osteoclast activation was investigated by bone resorption assays, and gene expression of differentiation markers was examined by quantitative real-time PCR. Transfection of Tlr2 and Tlr4 siRNAs into RAW264 cells was also employed and their role in Mfa1 recognition was investigated. Mfa1 effectively induced the formation of TRAP-positive multinucleated cells and activated osteoclasts. Mfa1 also increased gene expression of Acp5, Mmp9, and Ctsk in RANKL-prestimulated RAW264 cells compared with the control. The osteoclastogenesis induced by Mfa1 was significantly decreased in cells transfected with Tlr2 or Tlr4 siRNAs compared with control siRNA. Our results revealed the role of Mfa1 fimbriae in osteoclastogenesis that may contribute to the partial elucidation of the mechanisms of periodontal disease progression and the development of new therapeutic strategies.

Effects of cleaning sports mouthguards with ethylene-vinyl acetate on oral bacteria.

Background: Sports mouthguards, worn in the oral cavity to prevent sports injuries, are constantly exposed to various microorganisms that cause oral infections. Hence, the optimal cleaning methods for sports mouthguards have been thoroughly examined. In this study, we evaluated the efficiency of cleaning effects with a mouthguard cleaner (MC) on microbial biofilm formation in sports mouthguards in vitro and in vivo. Methods: We evaluated the cleaning effects of the discs produced by ethylene-vinyl acetate (EVA) on bacterial biofilms formed by the commensal bacterium Streptococcus oralis, the cariogenic bacterium Streptococcus mutans, and the opportunistic pathogen Staphylococcus aureus in vitro. EVA discs with biofilm were subjected to sterile distilled water (CTRL) and ultrasonic washing (UW), followed by treatment with MC and sodium hypochlorite (NaClO) as positive controls. Thereafter, the viable bacterial cell counts were determined. The bacteria adhering to the sheets before and after the treatment were observed under an electron microscope. The degree of cleanliness and measurement of viable microbial cell counts for total bacteria, Streptococci and Candida, opportunistic fungi, were evaluated on the used experimental sports mouthguards with and without UW and MC treatment in vivo. Results: The number of bacterial cells significantly decreased against all the tested biofilm bacteria upon treatment with MC, compared with CTRL and UW. Electron microscopy analysis revealed the biofilm formation by all bacteria on the EVA discs before cleaning. We observed fewer bacteria on the EVA discs treated with MC than those treated with CTRL and UW. Furthermore, the degree of cleanliness of the used experimental sports mouthguards cleaned using MC was significantly higher than that of the CTRL-treated mouthguards. Moreover, the viable microbial cell counts on the used experimental sports mouthguard were considerably lower than those on the CTRL ones. Conclusion: The cleaning effect of MC against oral bacteria was more effective than that of UW. MC treatment might have a potential future application as a cleaning method for sports mouthguards to protect athletes from oral infection.

Porphyromonas gingivalis Components/Secretions Synergistically Enhance Pneumonia Caused by Streptococcus pneumoniae in Mice.

Streptococcus pneumoniae is an important causative organism of respiratory tract infections. Although periodontal bacteria have been shown to influence respiratory infections such as aspiration pneumonia, the synergistic effect of S. pneumoniae and Porphyromonas gingivalis, a periodontopathic bacterium, on pneumococcal infections is unclear. To investigate whether P. gingivalis accelerates pneumococcal infections, we tested the effects of inoculating P. gingivalis culture supernatant (PgSup) into S. pneumoniae-infected mice. Mice were intratracheally injected with S. pneumoniae and PgSup to induce pneumonia, and lung histopathological sections and the absolute number and frequency of neutrophils and macrophages in the lung were analyzed. Proinflammatory cytokine/chemokine expression was examined by qPCR and ELISA. Inflammatory cell infiltration was observed in S. pneumoniae-infected mice and S. pnemoniae and PgSup mixed-infected mice, and mixed-infected mice showed more pronounced inflammation in lung. The ratios of monocytes/macrophages and neutrophils were not significantly different between the lungs of S. pneumoniae-infected mice and those of mixed-infected mice. PgSup synergistically increased TNF-α expression/production and IL-17 production compared with S. pneumoniae infection alone. We demonstrated that PgSup enhanced inflammation in pneumonia caused by S. pneumoniae, suggesting that virulence factors produced by P. gingivalis are involved in the exacerbation of respiratory tract infections such as aspiration pneumonia.

Porphyromonas gingivalis Mfa1 Induces Chemokine and Cell Adhesion Molecules in Mouse Gingival Fibroblasts via Toll-Like Receptors.

Porphyromonas gingivalis Mfa1 fimbriae are thought to act as adhesion factors and to direct periodontal tissue destruction but their immunomodulatory actions are poorly understood. Here, we investigated the effect of Mfa1 stimulation on the immune and metabolic mechanisms of gingival fibroblasts from periodontal connective tissue. We also determined the role of Toll-like receptor (TLR) 2 and TLR4 in Mfa1 recognition. Mfa1 increased the expression of genes encoding chemokine (C-X-C motif) ligand (CXCL) 1, CXCL3, intercellular adhesion molecule (ICAM) 1 and Selectin endothelium (E) in gingival fibroblasts, but did not have a significant effect on genes that regulate metabolism. Mfa1-stimulated up-regulation of genes was significantly suppressed in Tlr4 siRNA-transfected cells compared with that in control siRNA-transfected cells, which indicates that recognition by TLR4 is essential for immunomodulation by Mfa1. Additionally, suppression of Tlr2 expression partially attenuated the stimulatory effect of Mfa1. Overall, these results help explain the involvement of P. gingivalis Mfa1 fimbriae in the progression of periodontal disease.

Sendai virus V protein decreases nitric oxide production by inhibiting RIG-I signaling in infected RAW264.7 macrophages.

Sendai virus V protein is a known antagonist of RIG-I-like receptors (RLRs) RIG-I and MDA5, which activate transcription factors IRF3, leading to activation of ISGF3 and NF-κB. These transcription factors are known activators of inducible NO synthase (iNOS) and increase the production of nitric oxide (NO). By inhibiting ISGF3 and NF-κB, the V protein acts as an indirect negative regulator of iNOS and NO. Here we report that the V gene knockout Sendai virus [SeV V(-)] markedly enhanced iNOS expression and subsequent NO production in infected macrophages compared to wild-type SeV. The knockout of RIG-I in cells inhibited SeV V(-)-induced iNOS expression and subsequent NO production. To understand the underlying mechanism of the V protein-mediated negative regulation of iNOS activation, we transfected HEK293T cells with RIG-I and the RIG-I regulatory protein TRIM25. Our results demonstrated that the V protein inhibited iNOS activation via the RIG-I/TRIM25 pathway. Moreover, the V protein inhibited TRIM25-mediated K63-linked ubiquitination of RIG-I, as well as its CARD-dependent interaction with mitochondrial antiviral signaling (MAVS) molecules. These results suggest that the V protein downregulates iNOS activation and inhibits NO production by preventing the RIG-I-MAVS interaction, possibly through its effect on the ubiquitination status of RIG-I.

NF­κB inhibitor DHMEQ inhibits titanium dioxide nanoparticle­induced interleukin­1ß production: Inhibition of the PM2.5­induced inflammation model.

PM2.5 is a particle with a diameter <2.5 µm that is often involved in air pollution. Nanoparticles <100 nm are thought to invade the trachea and lungs to cause inflammation, possibly through the activation of macrophages. On the other hand, titanium dioxide (TiO2) particles can be used in models of nano­micro­sized particles, as one can prepare the particles with such sizes. TiO2 particles are classified into Rutile, Anatase, and Brookite types by their crystal structure. Among them, Anatase­type TiO2 particles with a primary diameter of 50 nm (A50) were reported to induce interleukin (IL)­1ß production and secretion effectively in phorbol 12­myristate 13­acetate­treated human monocytic leukemia THP­1 cells (THP­1 macrophages). We previously designed and synthesized dehydroxymethyl­epoxyqinomicin (DHMEQ) as an inhibitor of NF­κB. The present study investigated whether the NF­κB inhibitor DHMEQ inhibits TiO2 nanoparticle­induced IL­1ß production in THP­1 macrophages, and determined the mechanism. As a result, DHMEQ inhibited A50­induced IL­1ß secretion in ELISA assays at nontoxic concentrations. It decreased the expression of IL­1ß mRNA, which was dependent on NF­κB. Although NLR family pyrin domain containing 3 (NLRP3)­inflammasome­caspase­1 activation is required for the maturation of IL­1ß, and DHMEQ reduced the NLRP3 mRNA expression and caspase­1 activity; a caspase­1 inhibitor did not influence the A50­induced IL­1ß production. Therefore, it is likely that inhibition of pro­IL­1ß expression by DHMEQ may be sufficient to inhibit mature IL­1ß production. Thus, DHMEQ may be useful for the amelioration of inflammation in the trachea and lungs caused by inhalation of PM2.5.

Sendai virus C protein limits NO production in infected RAW264.7 macrophages.

To suppress virus multiplication, infected macrophages produce NO. However, it remains unclear how infecting viruses then overcome NO challenge. In the present study, we report the effects of accessory protein C from Sendai virus (SeV), a prototypical paramyxovirus, on NO output. We found that in RAW264.7 murine macrophages, a mutant SeV without C protein (4C(-)) significantly enhanced inducible NO synthase (iNOS) expression and subsequent NO production compared to wild type SeV (wtSeV). SeV 4C(-) infection caused marked production of IFN-ß, which is involved in induction of iNOS expression via the JAK-STAT pathway. Addition of anti-IFN-ß Ab, however, resulted in only marginal suppression of NO production. In contrast, NF-κB, a primarily important factor for transcription of the iNOS gene, was also activated by 4C(-) infection but not wtSeV infection. Induction of NO production and iNOS expression by 4C(-) was significantly suppressed in cells constitutively expressing influenza virus NS1 protein that can sequester double-stranded (ds)RNA, which triggers activation of signaling pathways leading to activation of NF-κB and IRF3. Therefore, C protein appears to suppress NF-κB activation to inhibit iNOS expression and subsequent NO production, possibly by limiting dsRNA generation in the context of viral infection.

Sendai Virus V Protein Inhibits the Secretion of Interleukin-1ß by Preventing NLRP3 Inflammasome Assembly.

Inflammasomes play a key role in host innate immune responses to viral infection by caspase-1 (Casp-1) activation to facilitate interleukin-1ß (IL-1ß) secretion, which contributes to the host antiviral defense. The NLRP3 inflammasome consists of the cytoplasmic sensor molecule NLRP3, adaptor protein ASC, and effector protein pro-caspase-1 (pro-Casp-1). NLRP3 and ASC promote pro-Casp-1 cleavage, leading to IL-1ß maturation and secretion. However, as a countermeasure, viral pathogens have evolved virulence factors to antagonize inflammasome pathways. Here we report that V gene knockout Sendai virus [SeV V(-)] induced markedly greater amounts of IL-1ß than wild-type SeV in infected THP1 macrophages. Deficiency of NLRP3 in cells inhibited SeV V(-)-induced IL-1ß secretion, indicating an essential role for NLRP3 in SeV V(-)-induced IL-1ß activation. Moreover, SeV V protein inhibited the assembly of NLRP3 inflammasomes, including NLRP3-dependent ASC oligomerization, NLRP3-ASC association, NLRP3 self-oligomerization, and intermolecular interactions between NLRP3 molecules. Furthermore, a high correlation between the NLRP3-binding capacity of V protein and the ability to block inflammasome complex assembly was observed. Therefore, SeV V protein likely inhibits NLRP3 self-oligomerization by interacting with NLRP3 and inhibiting subsequent recruitment of ASC to block NLRP3-dependent ASC oligomerization, in turn blocking full activation of the NLRP3 inflammasome and thus blocking IL-1ß secretion. Notably, the inhibitory action of SeV V protein on NLRP3 inflammasome activation is shared by other paramyxovirus V proteins, such as Nipah virus and human parainfluenza virus type 2. We thus reveal a mechanism by which paramyxovirus inhibits inflammatory responses by inhibiting NLRP3 inflammasome complex assembly and IL-1ß activation.IMPORTANCE The present study demonstrates that the V protein of SeV, Nipah virus, and human parainfluenza virus type 2 interacts with NLRP3 to inhibit NLRP3 inflammasome activation, potentially suggesting a novel strategy by which viruses evade the host innate immune response. As all members of the Paramyxovirinae subfamily carry similar V genes, this new finding may also lead to identification of novel therapeutic targets for paramyxovirus infection and related diseases.

A Wnt Pathway Activator Induces Apoptosis and Cell Death in Mouse Monocytic Leukemia Cells.

A Wnt agonist, 2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl) pyrimidine, is a cell-permeable pyrimidine compound that has been shown to mimic the effect of Wnt. In this study, leukemic mouse cell lines, RAW 264.7 and J774.1, were incubated with the Wnt agonist. The Wnt agonist showed cell death in the concentration of 1-10 µM. The Wnt agonist did not show inhibition of GSK-3ß activity but induced ß-catenin accumulation in the nucleus. The Wnt agonist showed caspase-independent cell death, but no further involvement in cell death ER stress signaling. Here we discuss the possible mechanism of Wnt agonist-induced apoptotic cell death in RAW 264.7 cells.

GSK2656157, a PERK inhibitor, reduced LPS-induced IL-1ß production through inhibiting Caspase 1 activation in macrophage-like J774.1 cells.

IL-1ß is one of the inflammatory cytokines and is cleaved from pro-IL-1ß proteolytically by activated Caspase 1. For the activation of Caspase 1, inflammasome was formed by two signals, what is called, priming and triggering signals. In this study, it was found that mouse macrophage J774.1 cells, when treated by single large amount of lipopolysaccharide (LPS), produced a significant amount of IL-1ß. On the other hand, IL-1ß production was not detected when treated by a single, small amount of LPS. Then, focusing on endoplasmic reticulum (ER) stress response among stress responses induced by a large amount of LPS, when GSK2656157, a PERK inhibitor, was used for inhibition of ER stress, GSK2656157 reduced IL-1ß production dose-dependently. Next, when Thapsigargin, an ER stress reagent, was added with LPS, IL-1ß production increased more than by LPS alone. Thus, these results suggested that ER stress was involved in LPS-induced IL-1ß production. When the activation of Caspase 1 was examined by fluorescence activated cell sorter analysis, it was found that GSK2656157 inhibited LPS-induced Caspase 1 activation. Further, it was confirmed that GSK2656157 did not affect LPS-induced TNF-α production and activation of NF-κB and specifically inhibited the PERK/eIF-2α pathway. Therefore, it was found that GSK2656157 specifically inhibited ER stress induced by large amount of LPS and reduced LPS-induced IL-1ß production through inhibition of Caspase 1 activation.

Pretreatment of LPS inhibits IFN-ß-induced STAT1 phosphorylation through SOCS3 induced by LPS.

It has been known that LPS activates macrophages and induces IFN-ß production from macrophages. The endogenous IFN-ß produced by LPS stimulates the cells, which plays a role in innate immune. However, it was not elucidated yet if the signaling by exogenous IFN-ß was influenced by LPS stimulation. In this study, it was found pretreatment of LPS interrupted IFN-ß-induced JAK1/STAT1 phosphorylation. LPS pretreatment also reduced IFN-ß-induced ISG54, one of IFN-ß-inducible genes. Pretreatment with LPS for more than 2h shows inhibitory effect on IFN-ß-induced STAT1 phosphorylation but simultaneous treatment or post-treatment of LPS with IFN-ß did not show the inhibitory effect. The study using a neutralizing antibody to IFN-ß indicated that IFN-ß produced by LPS does not take part in the inhibitory effect of LPS. Furthermore, LPS did not affect the expression of IFN αß receptor. A previous report has shown that LPS-induced SOCS3 inhibited IFN-γ-induced STAT1 phosphorylation, likewise, it was also shown in this study that LPS induced SOCS3 expression and its expression inhibited IFN-ß-induced STAT1 phosphorylation which was confirmed by the knockdown study by the siRNA of SOCS3. The real-time PCR and immune-blot studies of SOCS3 indicated that LPS induced SOCS3 is independent of IL-6, IL-10, TNF-α and STAT3, and might depend on p38 activation by LPS. It was suggested that bacterial LPS rather interfere with IFN-ß actions, dependent on the timing of LPS stimulation.

TGF-ß1 inhibits the production of IFN in response to CpG DNA via ubiquitination of TNF receptor-associated factor (TRAF) 6.

The effect of TGF-ß1 on CpG DNA-induced type I IFN production was examined by reconstituting a series of signaling molecules in TLR 3 signaling. TGF-ß1 inhibited CpG DNA-induced IFN-α4 productivity in HeLa cells. Transfection of IFN regulatory factor (IRF)7 but not TNF receptor-associated factor (TRAF)6 and TRAF3 into cells triggered IFN-α4 productivity, and TGF-ß1 inhibited IRF7-mediated type I IFN production in the presence of TRAF6. TGF-ß1 induced ubiquitination of TRAF6, although CpG DNA did not induce it. Moreover, TGF-ß1 accelerated the ubiquitination of TRAF6 in the presence of CpG DNA. TGF-ß1 ubiquitinated TRAF6 at K63 but not K48. TGF-ß1 also induced ubiquitination of IRF7. Further, TGF-ß1 did not impair the interaction of IRF7 and TRAF6. CpG DNA induced the phosphorylation of IRF7 in the presence of TRAF6, whereas TGF-ß1 inhibited the IRF7 phosphorylation. Blocking of TRAF6 ubiquitination abolished the inhibition of CpG DNA-induced type I IFN production by TGF-ß. Taken together, TGF-ß was suggested to inhibit CpG DNA-induced type I IFN production transcriptionally via ubiquitination of TRAF6.

Lipopolysaccharide downregulates the expression of p53 through activation of MDM2 and enhances activation of nuclear factor-kappa B.

The effect of lipopolysaccharide (LPS) on the expression of p53 protein in RAW 264.7 macrophage cells was examined. LPS downregulated the expression of p53 protein 4-24 h after the stimulation. LPS-induced p53 inhibition was restored with pharmacological inhibitors of c-jun N-terminal kinase (JNK) and phosphatidylinositol 3-kinase (PI3K). It was also restored by inhibitors of MDM2 activation and proteasome. LPS-induced p53 inhibition corresponded to activation of MDM2. LPS-induced MDM2 activation was prevented by inhibitors of JNK and PI3K. The expression of p65 NF-κB at a late stage after LPS stimulation was downregulated in the presence of a MDM2 inhibitor. Nutlin-3 as a MDM2 inhibitor reduced LPS-induced production of nitric oxide but not tumor necrosis factor-α. Administration of LPS into mice downregulated the in vivo expression of p53 in the livers. Taken together, LPS was suggested to downregulate the expression of p53 via activation of MDM2 and enhance the activation of NF-κB at a late stage.

Involvement of redox balance in in vitro osteoclast formation of RAW 264.7 macrophage cells in response to LPS.

Here we report that LPS induces osteoclast (OC) formation in murine RAW 264.7 macrophage cells in RPMI-1640 medium but not in α-minimum essential medium (α-MEM) as the original culture medium. LPS-induced OC formation in both media was examined to clarify the differential response. Receptor activator of NF-κB ligand induced OC formation in either α-MEM or RPMI-1640 medium. However, LPS-induced OC formation in RAW 264.7 cells maintained in RPMI-1640 medium, but not α-MEM, which was also supported by mouse bone marrow-derived macrophages, although they were less sensitive to LPS than RAW 264.7 cells. LPS augmented the expression of nuclear factor of activated T-cells (NFATc1) as a key transcription factor of osteoclastogenesis in cells maintained in RPMI-1640 medium, but reduced it in cells maintained in α-MEM. A high concentration of LPS was cytotoxic against cells maintained in α-MEM. Glutathione exclusively present in RPMI-1640 medium prevented LPS-induced cell death in α-MEM and augmented LPS-induced NFATc1 expression, followed by enhanced LPS-induced OC formation. LPS induced higher generation of reactive oxygen species in α-MEM than RPMI-1640 medium. An antioxidant enhanced LPS-induced OC formation, whereas a pro-oxidant reduced it. Taken together, redox balance in the culture condition was suggested to regulate in vitro LPS-induced OC formation.

Sendai virus C protein inhibits lipopolysaccharide-induced nitric oxide production through impairing interferon-ß signaling.

The effect of Sendai virus (SeV) C protein on lipopolysaccharide (LPS)-induced nitric oxide (NO) production was examined using RAW 264.7 macrophage cells. Infection of SeV inhibited LPS-induced NO production via downregulating the expression of an inducible NO synthase protein (iNOS). On the other hand, C gene-knockout 4C(-) SeV inhibited neither NO production nor iNOS expression. Wild type and 4C(-) SeV did not affect LPS-induced production of tumor necrosis factor-α and interleukin-6, and further LPS-induced activation of nuclear factor (NF)-κB and mitogen-activated protein kinases. Although wild type and 4C(-) SeV did not inhibit LPS-induced interferon (IFN)-ß production, wild type SeV but not 4C(-) SeV inhibited the activation of STAT1/2 in the IFN-ß signaling. SeV C protein inhibited LPS-induced iNOS expression and NO production. C protein inhibited the promotor activation of IFN-ß and IFN-sensitive response element (ISRE) in response to LPS whereas the C mutant protein CF170S, which lacks the ability to block the STAT activation, did not inhibit it. Taken together, SeV C protein was suggested to inhibit LPS-induced NO production through impairing IFN-ß signaling.

A novel mechanism for inhibition of lipopolysaccharide-induced proinflammatory cytokine production by valproic acid.

The inhibitory effect of valproic acid (VPA) on lipopolysaccharide (LPS)-induced inflammatory response was studied by using mouse RAW 264.7 macrophage-like cells. VPA pretreatment attenuated LPS-induced phosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt, but not nuclear factor (NF)-κB and mitogen-activated protein kinases. VPA reduced phosphorylation of MDM2, an ubiquitin ligase and then prevented LPS-induced p53 degradation, followed by enhanced p53 expression. Moreover, p53 small interfering RNA (siRNA) abolished the inhibitory action of VPA on LPS-induced NF-κB p65 transcriptional activation and further LPS-induced tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. VPA prevented LPS-induced degradation of phosphatase and tensin homologue deleted on chromosome ten (PTEN) and up-regulated the PTEN expression. Taken together, VPA was suggested to down-regulate LPS-induced NF-κB-dependent transcriptional activity via impaired PI3K/Akt/MDM2 activation and enhanced p53 expression. A detailed mechanism for inhibition of LPS-induced inflammatory response by VPA is discussed.

Lipopolysaccharide impairs insulin sensitivity via activation of phosphoinositide 3-kinase in adipocytes.

The effect of lipopolysaccharide (LPS) on insulin sensitivity in adipocytes were examined by using differentiated 3T3-L1 adipocytes. Insulin-mediated activation of insulin receptor substrate (IRS) 1/2 was inhibited in LPS-pretreated adipocytes and IRS1/2-mediated Akt activation was also attenuated in those cells. LPS inhibited activation of glycogen synthase kinase 3 as a negative regulator of glycogenesis and impaired the glycogen synthesis in response to insulin. LPS-induced activation of phosphoinositide 3-kinase (PI3K) in adipocytes. Involvement of suppressor of cytokine signaling 3 (SOCS3) in LPS-induced IRS1/2 inhibition was excluded. Considering that both insulin and LPS were able to activate the PI3K/Akt signaling pathway, LPS was suggested to impair insulin sensitivity of adipocytes through down-regulating insulin-mediated PI3K/Akt activation.

Mouse pyrin and HIN domain family member 1 (pyhin1) protein positively regulates LPS-induced IFN-ß and NO production in macrophages.

The pyrin and HIN-domain (PYHIN) family member1 (pyhin1) is a member of PYHIN proteins and involved in transcriptional regulation of genes important for cell cycle control, differentiation and apoptosis. The regulatory action of mouse pyhin1 on LPS-induced inflammatory response was examined. LPS augmented the pyhin1 mRNA expression in murine RAW 264.7 macrophage cells and peritoneal macrophages. The augmentation of pyhin1 mRNA expression was abolished by parthenolide, a NF-κB inhibitor. Silencing of pyhin1 with small interfering RNA reduced the production of IFN-ß and NO. However, pyhin1 silencing did not affect the production of TNF-α, IL-6, IL-10 and prostaglandin E2. Reduced IFN-ß production by pyhin1 silencing caused inactivation of STAT1 and reduced expression of IRF1. Pyhin1 silencing inhibited the expression of TRAF6, TBK1 and TRIF, which trigger IFN-ß production in the MyD88-independent pathway. However, pyhin1 silencing did not affect the expression of MyD88, IRAK4 and several mitogen-activated protein kinases in the MyD88-dependent pathway. Taken together, mouse pyhin1 was suggested to be a NF-κB-responsible gene in response to LPS and positively regulate LPS-induced IFN-ß and NO production through up-regulating the MyD88-independent signaling pathway.

Augmentation of LPS-induced vascular endothelial cell growth factor production in macrophages by transforming growth factor-ß1.

The effect of LPS on the production of vascular endothelial growth factor (VEGF) was examined using RAW 264.7 macrophage cells. LPS induced VEGF production in RAW 264.7 cells and mouse peritoneal cells. LPS induced VEGF production via the expression of hypoxia inducible factor-1α and LPS-induced VEGF production was dependent on the activation of p38 MAPK and NF-κB activation· Transforming growth factor (TGF)-ß1 augmented LPS-induced VEGF production, although TGF-ß1 alone did not induce VEGF production. The augmentation of LPS-induced VEGF production by TGF-ß1 was inhibited by a p38 MAPK inhibitor and was correlated with the phosphorylation of Smad3. The enhancing effect of TGF-ß1 on LPS-induced VEGF production was observed in vivo in the skin lesions of mice receiving a subcutaneous injection of LPS. Taken together, it is suggested that LPS induced the VEGF production in macrophages and that it was augmented by TGF-ß1 in vitro and in vivo.