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.

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.

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.

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.

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.

A Toll-like receptor 2 ligand, Pam3CSK4, augments interferon-γ-induced nitric oxide production via a physical association between MyD88 and interferon-γ receptor in vascular endothelial cells.

The effect of Pam3CSK4, a Toll-like receptor 2 (TLR2) ligand, on interferon-γ (IFN-γ) -induced nitric oxide (NO) production in mouse vascular endothelial END-D cells was studied. Pre-treatment or post-treatment with Pam3CSK4 augmented IFN-γ-induced NO production via enhanced expression of an inducible NO synthase (iNOS) protein and mRNA. Pam3CSK4 augmented phosphorylation of Janus kinase 1 and 2, followed by enhanced phosphorylation of signal transducer and activator of transcription 1 (STAT1) at tyrosine 701. Subsequently, the enhanced STAT1 activation augmented IFN-γ-induced IFN-regulatory factor 1 expression leading to the iNOS expression. Pam3CSK4 also induced the activation of p38 and subsequent phosphorylation of STAT1 at serine 727. A pharmacological p38 inhibitor abolished the augmentation of IFN-γ-induced NO production by Pam3CSK4. Surprisingly, Pam3CSK4 enhanced a physical association of MyD88 and IFN-γ receptor. Together, these findings suggest that Pam3CSK4 up-regulates IFN-γ signalling in vascular endothelial cells via the physical association between MyD88 and IFN-γ receptor α, and p38-dependent serine 727 STAT1 phosphorylation.

Lipopolysaccharide prevents valproic acid-induced apoptosis via activation of nuclear factor-κB and inhibition of p53 activation.

The effect of lipopolysaccharide (LPS) on valproic acid (VPA)-induced cell death was examined by using mouse RAW 264.7 macrophage cells. LPS inhibited the activation of caspase 3 and poly (ADP-ribose) polymerase and prevented VPA-induced apoptosis. LPS inhibited VPA-induced p53 activation and pifithrin-α as a p53 inhibitor as well as LPS prevented VPA-induced apoptosis. LPS abolished the increase of Bax/Bcl-2 ratio, which is a critical indicator of p53-mediated mitochondrial damage, in response to VPA. The nuclear factor (NF)-κB inhibitors, Bay 11-7082 and parthenolide, abolished the preventive action of LPS on VPA-induced apoptosis. A series of toll-like receptor ligands, Pam3CSK4, poly I:C, and CpG DNA as well as LPS prevented VPA-induced apoptosis. Taken together, LPS was suggested to prevent VPA-induced apoptosis via activation of anti-apoptotic NF-κB and inhibition of pro-apoptotic p53 activation. The detailed inhibitory mechanism of VPA-induced apoptosis by LPS is discussed.

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.

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.

An ADP ribosylation factor-GTPase activating protein negatively regulates the production of proinflammatory mediators in response to lipopolysaccharide.

An ADP ribosylation factor-GTPase activating protein (ASAP1) is highly expressed in a variety of tumor cells and is involved in the cell motility, invasion, and metastasis. In order to elucidate the involvement of ASAP1 in lipopolysaccharide (LPS)-mediated inflammatory response, the effect of ASAP1 silencing on LPS-induced proinflammatory mediators production was examined by using RAW 264.7 macrophage-like cells. ASAP1 was constitutively expressed in the cells and the expression was augmented by LPS stimulation. Silencing of ASAP1 with small interfering RNA enhanced the production of tumor necrosis factor-α, interleukin 6, interferon-ß, and nitric oxide in response to LPS. ASAP1 silencing augmented the activation of nuclear factor (NF)-κB and several mitogen-activated protein kinases (MAPKs). On the other hand, ASAP1 silencing did not affect the expression of IRAK4, TRAF6, and Akt as the upstream molecules of NF-κB signaling. A series of toll-like receptor ligands as well as LPS augmented the ASAP1 expression. Taken together, ASAP1 was suggested to negatively regulate LPS-induced proinflammatory mediators production through down-regulating LPS signaling. The feedback function of ASAP1 in LPS-mediated inflammatory response is discussed.

Thalidomide inhibits interferon-γ-mediated nitric oxide production in mouse vascular endothelial cells.

Thalidomide is known as an anti-angiogenic, anti-tumor, and anti-proliferative agent, widely used in the treatment of some immunological disorders and cancers. The effect of thalidomide on interferon (IFN)-γ induced nitric oxide (NO) production in mouse vascular endothelial cells was examined in order to elucidate the anti-angiogenic or anti-inflammatory action. Thalidomide inhibited IFN-γ-induced NO production in mouse END-D cells via reduced expression of an inducible type of NO synthase (iNOS) protein and mRNA. Since thalidomide did not alter the cell surface expression of IFN-γ receptor, the NO inhibition was suggested to be due to the impairment of IFN-γ-induced intracellular event by thalidomide. Thalidomide inhibited the phosphorylation of IRF1, which was required for the iNOS expression. Moreover, it inhibited the phosphorylation of STAT1, an upstream molecule of IRF1, in IFN-γ signaling. Thalidomide did not inhibit the JAK activation in response to IFN-γ. A phosphatase inhibitor, sodium orthovanadate, abolished the inhibitory action of thalidomide. Therefore, thalidomide was suggested to inhibit IFN-γ-induced NO production via impaired STAT1 phosphorylation.

Flavopiridol inhibits lipopolysaccharide-induced TNF-α production through inactivation of nuclear factor-κB and mitogen-activated protein kinases in the MyD88-dependent pathway.

Flavopiridol is a cyclin-dependent kinase inhibitor and inhibits the growth of various cancer cells. The effect of flavopiridol on lipopolysaccharide (LPS)-induced proinflammatory mediator production was examined in RAW 264.7 macrophage-like cells. Flavopiridol significantly reduced the production of tumor necrosis factor-α and, to a lesser extent, nitric oxide in LPS-stimulated cells. Flavopiridol inhibited the activation of nuclear factor-κB and IκB kinase in response to LPS. Flavopiridol also inhibited the activation of a series of mitogen-activated protein kinases, such as p38, stress-activated protein kinase/c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in response to LPS. However, flavopiridol did not alter the expression of tumor necrosis factor receptor-associated factor 6, myeloid differentiation factor 88 (MyD88) or CD14/toll-like receptor (TLR) 4. Flavopiridol inhibited nitric oxide production induced by a MyD88-dependent TLR2 ligand, but not a MyD88-independent TLR3 ligand. Further, flavopiridol did not alter the phosphorylation of interferon regulatory factor 3 in the MyD88-independent pathway. Therefore, it was suggested that flavopiridol exclusively inhibited the activation of nuclear factor-κB and mitogen-activated protein kinases in the MyD88-dependent pathway. Flavopiridol might be useful for the prevention of LPS-induced inflammatory response.

Tumor necrosis factor-a augments lipopolysaccharide-induced suppressor of cytokine signalling 3 (SOCS-3) protein expression by preventing the degradation.

The regulatory role of tumour necrosis factor-a (TNF-a) on the expression of suppressor of cytokine signalling 3 (SOCS-3) in response to lipopolysaccharide (LPS) was examined using peritoneal macrophages from TNF-a-deficient mice. The LPS-induced SOCS-3 expression was markedly augmented in macrophages from wild-type mice whereas such augmentation was not seen in the cells from TNF-a-deficient mice. However, there was no significant difference in the level of SOCS-3 messenger RNA expression between macrophages from wild-type mice and those from TNF-a-deficient mice. The addition of exogenous TNF-a augmented the LPS-induced SOCS-3 expression in macrophages from TNF-a-deficient mice. The pulse chase analysis suggested augmented degradation of LPS-induced SOCS-3 protein in macrophages from TNF-a-deficient mice. Moreover, MG 132, a 26S proteasome inhibitor, sustained the LPS-induced SOCS-3 expression in those cells. The tyrosine phosphorylation of SOCS-3 was definitely induced in LPS-stimulated macrophages from TNF-a-deficient mice but not wild-type mice. A tyrosine phosphatase inhibitor enhanced the tyrosine phosphorylation of SOCS-3 in wild-type mice and accelerated the degradation. Therefore, it was suggested that TNF-a prevented the degradation of SOCS-3 protein via inhibition of the tyrosine phosphorylation in LPS-stimulated macrophages.

Seladin-1 is a novel lipopolysaccharide (LPS)-responsive gene and inhibits the tumour necrosis factor-alpha production and osteoclast formation in response to LPS.

Selective Alzheimer disease indicator-1 (seladin-1) is a broadly expressed oxidoreductase and is related to Alzheimer disease, cholesterol metabolism and carcinogenesis. The effect of lipopolysaccharide (LPS) on the expression of seladin-1 was examined using RAW 264.7 macrophage-like cells and murine peritoneal macrophages. Lipopolysaccharide induced the expression of seladin-1 protein and messenger RNA in those macrophages. The seladin-1 expression was also augmented by a series of Toll-like receptor ligands. The LPS augmented the expression of seladin-1 via reactive oxygen species generation and p38 activation. Seladin-1 inhibited LPS-induced activation of p38 but not nuclear factor-kappaB and inhibited the production of tumour necrosis factor-alpha in response to LPS. Moreover, seladin-1 inhibited LPS-induced osteoclast formation and enhanced LPS-induced alkaline phosphatase activity. Therefore, it was suggested that seladin-1 might be an LPS-responsible gene product and regulate the LPS-induced inflammatory response negatively.

Retinoblastoma protein-interacting zinc finger 1, a tumor suppressor, augments lipopolysaccharide-induced proinflammatory cytokine production via enhancing nuclear factor-kappaB activation.

The involvement of retinoblastoma protein-interacting zinc finger 1 (RIZ1), a tumor suppressor, in lipopolysaccharide (LPS)-induced inflammatory responses was investigated by using RAW 264.7 macrophage-like cells. LPS significantly augmented the expression of RIZ1 and the augmentation was mediated by the activation of nuclear factor (NF)-kappaB and Akt. The silencing of RIZ1 with the siRNA led to the inactivation of NF-kappaB in response to LPS. Moreover, the RIZ1 silencing caused the down-regulation of p53 activation and a p53 pharmacological inhibitor attenuated the RIZ1 expression. LPS-induced tumor necrosis factor-alpha and interleukin-6 production was prevented by RIZ1 siRNA or a p53 pharmacological inhibitor. Therefore, RIZ1 was suggested to augment LPS-induced NF-kappaB activation in collaboration with p53 and enhance the production of proinflammatory cytokines in response to LPS.

B1 cells produce nitric oxide in response to a series of toll-like receptor ligands.

The effect of a series of toll-like receptor (TLR) ligands on the production of nitric oxide (NO) in mouse B1 cells was examined by using CD5(+) IgM(+) WEHI 231 cells. The stimulation with a series of TLR ligands, which were Pam3Csk4 for TLR1/2, poly I:C for TLR3, lipopolysaccharide (LPS) for TLR4, imiquimod for TLR7 and CpG DNA for TLR9, resulted in enhanced NO production via augmented expression of an inducible type of NO synthase (iNOS). LPS was most potent for the enhancement of NO production, followed by poly I:C and Pam3Csk4. Imiquimod and CpG DNA led to slight NO production. The LPS-induced NO production was dependent on MyD88-dependent pathway consisting of nuclear factor (NF)-kappaB and a series of mitogen-activated protein kinases (MAPKs). Further, it was also dependent on the MyD88-independent pathway consisting of toll-IL-1R domain-containing adaptor-inducing IFN-beta (TRIF) and interferon regulatory factor (IRF)-3. Physiologic peritoneal B1 cells also produced NO via the iNOS expression in response to LPS. The immunological significance of TLR ligands-induced NO production in B1 cells is discussed.

Retinoblastoma protein-interacting zinc finger 1 (RIZ1) regulates the proliferation of monocytic leukemia cells via activation of p53.

The role of retinoblastoma protein-interacting zinc finger 1 (RIZ1) on the cell growth of mouse and human monocytic leukemia cells was examined. RIZ1 expression was induced in response to tumor necrosis factor (TNF)-α. The expression was dependent on the nuclear factor-κB and AKT signaling. Further, RIZ1 expression led to the augmentation of p53 expression and the silencing of RIZ1 prevented it. On the other hand, a p53 inhibitor enhanced the TNF-α-induced RIZ1 expression. Silencing of RIZ1 augmented the proliferative activity of TNF-α-treated cells. Therefore, it is suggested that RIZ1 negatively regulated the cell proliferation of monocytic leukemia cells via activation of p53.

TLR/MyD88 and liver X receptor alpha signaling pathways reciprocally control Chlamydia pneumoniae-induced acceleration of atherosclerosis.

Experimental and clinical studies link Chlamydia pneumoniae infection to atherogenesis and atherothrombotic events, but the underlying mechanisms are unclear. We tested the hypothesis that C. pneumoniae-induced acceleration of atherosclerosis in apolipoprotein E (ApoE)(-/-) mice is reciprocally modulated by activation of TLR-mediated innate immune and liver X receptor alpha (LXRalpha) signaling pathways. We infected ApoE(-/-) mice and ApoE(-/-) mice that also lacked TLR2, TLR4, MyD88, or LXRalpha intranasally with C. pneumoniae followed by feeding of a high fat diet for 4 mo. Mock-infected littermates served as controls. Atherosclerosis was assessed in aortic sinuses and in en face preparation of whole aorta. The numbers of activated dendritic cells (DCs) within plaques and the serum levels of cholesterol and proinflammatory cytokines were also measured. C. pneumoniae infection markedly accelerated atherosclerosis in ApoE-deficient mice that was associated with increased numbers of activated DCs in aortic sinus plaques and higher circulating levels of MCP-1, IL-12p40, IL-6, and TNF-alpha. In contrast, C. pneumoniae infection had only a minimal effect on atherosclerosis, accumulation of activated DCs in the sinus plaques, or circulating cytokine increases in ApoE(-/-) mice that were also deficient in TLR2, TLR4, or MyD88. However, C. pneumoniae-induced acceleration of atherosclerosis in ApoE(-/-) mice was further enhanced in ApoE(-/-)LXRalpha(-/-) double knockout mice and was accompanied by higher serum levels of IL-6 and TNF-alpha. We conclude that C. pneumoniae infection accelerates atherosclerosis in hypercholesterolemic mice predominantly through a TLR/MyD88-dependent mechanism and that LXRalpha appears to reciprocally modulate and reduce the proatherogenic effects of C. pneumoniae infection.

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.

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.