Heat-killed Lancefieldella Rimae Induces Bone Resorption by Promoting Osteoclast Differentiation.

INTRODUCTION: Apical periodontitis, mainly caused by bacterial infection in the dental pulp, is often accompanied by abscess, periapical inflammation, and alveolar bone loss. Lancefieldella rimae has been detected in the root canals of patients with apical periodontitis. Here, we investigated whether L. rimae is associated with bone resorption. METHODS: L. rimae was anaerobically cultured and heat-killed (HKLr). A mouse calvarial implantation model was used to determine the bone resorption in vivo. Committed osteoclasts prepared from C57BL/6 wild-type or Toll-like receptor 2 (TLR2)-deficient mice were differentiated into mature osteoclasts in the presence or absence of HKLr. The mRNA expression of tartrate-resistant acid phosphatase (TRAP), ATPase H+ transporting V0 subunit D2, cathepsin K, interleukin-6, tumor necrosis factor-α, and glyceraldehyde 3-phosphate dehydrogenase was quantified using real-time reverse transcription-polymerase chain reaction. The protein levels of c-Fos and NFATc1 were determined by Western blot analysis. RESULTS: Implantation of HKLr onto the mouse calvaria induced the bone destruction with an increase of TRAP-positive areas. While HKLr enhanced the differentiation of osteoclasts, this effect was not observed in TLR2-deficient osteoclasts. HKLr dose-dependently increased the mRNA expression of genes associated with osteoclast differentiation including TRAP, ATPase H+ transporting V0 subunit D2, and cathepsin K. In addition, HKLr enhanced the expression of c-Fos and NFATc1, which are important transcription factors for osteoclast differentiation. Moreover, HKLr increased the expression of interleukin-6 and tumor necrosis factor-α. CONCLUSION: L. rimae induces bone resorption by enhancing osteoclast differentiation through the TLR2 signaling pathway, implying that L. rimae is a causative agent responsible for the alveolar bone resorption accompanying apical periodontitis.

Muramyl dipeptide alleviates estrogen deficiency-induced osteoporosis through canonical Wnt signaling.

Wnt signaling is a positive regulator of bone formation through the induction of osteoblast differentiation and down-regulation of osteoclast differentiation. We previously reported that muramyl dipeptide (MDP) increases bone volume by increasing osteoblast activity and attenuating osteoclast activity in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoporotic model mice. In this study, we investigated whether MDP could alleviate post-menopausal osteoporosis through Wnt signaling regulation in an ovariectomy (OVX)-induced mouse osteoporosis model. MDP-administered OVX mice exhibited higher bone volume and bone mineral density than mice of the control group. MDP significantly increased P1NP in the serum of OVX mice, implying increased bone formation. The expression of pGSK3ß and ß-catenin in the distal femur of OVX mice was lower than that in the distal femur of sham-operated mice. Yet, the expression of pGSK3ß and ß-catenin was increased in MDP-administered OVX mice compared with OVX mice. In addition, MDP increased the expression and transcriptional activity of ß-catenin in osteoblasts. MDP inhibited the proteasomal degradation of ß-catenin via the down-regulation of its ubiquitination by GSK3ß inactivation. When osteoblasts were pretreated with Wnt signaling inhibitors, DKK1 or IWP-2, the induction of pAKT, pGSK3ß, and ß-catenin was not observed. In addition, nucleotide oligomerization domain-containing protein 2-deficient osteoblasts were not sensitive to MDP. MDP-administered OVX mice exhibited fewer tartrate-resistant acid phosphatase (TRAP)-positive cells than did OVX mice, attributed to a decrease in the RANKL/OPG ratio. In conclusion, MDP alleviates estrogen deficiency-induced osteoporosis through canonical Wnt signaling and could be an effective therapeutic for the treatment of post-menopausal bone loss. © 2023 The Pathological Society of Great Britain and Ireland.

Bacterial Lipoproteins Induce BAFF Production via TLR2/MyD88/JNK Signaling Pathways in Dendritic Cells.

B-cell activating factor (BAFF) plays a crucial role in survival, differentiation, and antibody secretion of B cells. Microbial products with B-cell mitogenic properties can indirectly promote expansion and activation of B cells by stimulating accessory cells, such as dendritic cells (DCs), to induce BAFF. Although bacterial lipoproteins are potent B-cell mitogen like lipopolysaccharides (LPSs), it is uncertain whether they can stimulate DCs to induce BAFF expression. Here, we evaluated the effect of bacterial lipoproteins on BAFF expression in mouse bone marrow-derived DCs. Lipoprotein-deficient Staphylococcus aureus mutant induced relatively low expression level of membrane-bound BAFF (mBAFF) and the mRNA compared with its wild-type strain, implying that bacterial lipoproteins can positively regulate BAFF induction. The synthetic lipopeptides Pam2CSK4 and Pam3CSK4, which mimic bacterial lipoproteins, dose-dependently induced BAFF expression, and their BAFF-inducing capacities were comparable to those of LPS in DCs. Induction of BAFF by the lipopeptide was higher than the induction by other microbe-associated molecular patterns, including peptidoglycan, flagellin, zymosan, lipoteichoic acid, and poly(I:C). Pam3CSK4 induced both mBAFF and soluble BAFF expression in a dose- and time-dependent manner. BAFF expression by Pam3CSK4 was completely absent in DCs from TLR2- or MyD88-deficient mice. Among various MAP kinase inhibitors, only JNK inhibitors blocked Pam3CSK4-induced BAFF mRNA expression, while inhibitors blocking ERK or p38 kinase had no such effect. Furthermore, Pam3CSK4 increased the DNA-binding activities of NF-κB and Sp1, but not that of C/EBP. Pam3CSK4-induced BAFF promoter activity via TLR2/1 was blocked by NF-κB or Sp1 inhibitor. Collectively, these results suggest that bacterial lipoproteins induce expression of BAFF through TLR2/MyD88/JNK signaling pathways leading to NF-κB and Sp1 activation in DCs, and BAFF derived from bacterial lipoprotein-stimulated DCs induces B-cell proliferation.

Modulation of macrophage subtypes by IRF5 determines osteoclastogenic potential.

Bone-resorbing osteoclasts are differentiated from macrophages (MΦ) by M-CSF and RANKL. MΦ can be mainly classified into M1 and M2 MΦ, which are proinflammatory and anti-inflammatory, respectively, but little is known about their osteoclastogenic potential. Here, we investigated the osteoclastogenic potential of MΦ subtypes. When the two MΦ subtypes were differentiated into osteoclasts using M-CSF and RANKL, M2 MΦ more potently differentiated into osteoclasts than M1 MΦ. M2 MΦ generated with IL-4 or IL-10 also showed enhanced osteoclast differentiation compared with M1 MΦ induced by IFN-γ and lipopolysaccharide. In addition, robust bone-resorptive capacity and giant actin rings, which are features of mature osteoclasts, were observed in M2, but not M1 MΦ, under the osteoclast differentiation condition. Osteoclast differentiation was significantly increased in CD206+ M2 MΦ but not in CD86+ M1 MΦ. Compared with M1 MΦ, c-Fms and RANK were highly expressed in M2 MΦ. Enhanced osteoclastogenesis of M2 MΦ was mediated through sustained ERK activation, followed by efficient c-Fos and NFATc1 induction. Notably, the osteoclastogenic potential of M1 MΦ converted into M2 MΦ by exposure to M-CSF was higher than that of M2 MΦ converted into M1 MΦ by exposure to GM-CSF. Silencing IRF5, which is responsible for M1 MΦ polarization, increased osteoclast differentiation by enhancing c-Fms expression and activation of ERK, c-Fos, CREB, and NFATc1, which was inhibited by overexpression of IRF5. Collectively, M2 MΦ are suggested to be more efficient osteoclast precursors than M1 MΦ because of the attenuated expression of IRF5.

Cyclic Dinucleotides Inhibit Osteoclast Differentiation Through STING-Mediated Interferon-ß Signaling.

Cyclic dinucleotides (CDNs), such as cyclic diadenylate monophosphate and cyclic diguanylate monophosphate, are commensal bacteria-derived second messengers in the gut that modulate bacterial survival, colonization, and biofilm formation. Recently, CDNs have been discovered to have an immunomodulatory activity by inducing the expression of type I interferon (IFN) through STING signaling pathway in macrophages. Because CDNs are possibly absorbed and delivered into the bone marrow, where bone-resorbing osteoclasts are derived from monocyte/macrophage lineages, CDNs could affect bone metabolism by regulating osteoclast differentiation. In this study, we investigated the effect of CDNs on the differentiation and function of osteoclasts and osteoblasts. When bone marrow-derived macrophages (BMMs) were differentiated into osteoclasts with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) in the presence of CDNs, the differentiation was inhibited by CDNs in a dose-dependent manner. In contrast, CDNs did not influence the differentiation of committed osteoclasts or osteoblast precursors. STING signaling pathway appeared to be critical for CDNs-mediated inhibition of osteoclast differentiation since CDNs induced the phosphorylation of TBK1 and IRF3, a representative feature of STING activation, and osteoclast differentiation was restored in STING knockdown BMMs with siRNA. Moreover, CDNs increased the mRNA expression of STING-meditated IFN-ß, which is a negative regulator of osteoclastogenesis. In addition, CDNs also induced the phosphorylation of STAT1, which mediates IFN-α/ß receptor (IFNAR) signal transduction. The inhibitory effects of CDNs on osteoclast differentiation were not observed in the presence of antibody blocking IFNAR or in macrophages derived from IFNAR1-/- mice. Experiments using a mouse calvarial implantation model showed that RANKL-induced bone resorption was inhibited by CDNs. Taken together, these results suggest that CDNs inhibit osteoclast differentiation and bone resorption through induction of IFN-ß via the STING signaling pathway. © 2019 American Society for Bone and Mineral Research.

Streptococcus gordonii induces bone resorption by increasing osteoclast differentiation and reducing osteoblast differentiation.

Streptococcus gordonii is commonly found in the periapical endodontic lesions of patients with apical periodontitis, a condition characterized by inflammation and periapical bone loss. Since bone metabolism is controlled by osteoclastic bone resorption and osteoblastic bone formation, we investigated the effects of S. gordonii on the differentiation and function of osteoclasts and osteoblasts. For the determination of bone resorption activity in vivo, collagen sheets soaked with heat-killed S. gordonii were implanted on mouse calvaria, and the calvarial bones were scanned by micro-computed tomography. Mouse bone marrow-derived macrophages (BMMs) were stimulated with M-CSF and RANKL for 2 days and then differentiated into osteoclasts in the presence or absence of heat-killed S. gordonii. Tartrate-resistant acid phosphatase staining was performed to determine osteoclast differentiation. Primary osteoblast precursors were differentiated into osteoblasts with ascorbic acid and ß-glycerophosphate in the presence or absence of heat-killed S. gordonii. Alkaline phosphatase staining and alizarin red S staining were conducted to determine osteoblast differentiation. Western blotting was performed to examine the expression of transcription factors including c-Fos, NFATc1, and Runx2. Heat-killed S. gordonii induced bone destruction in a mouse calvarial implantation model. The differentiation of RANKL-primed BMMs into osteoclasts was enhanced in the presence of heat-killed S. gordonii. Heat-killed S. gordonii increased the expression of c-Fos and NFATc1, which are essential transcription factors for osteoclast differentiation. On the other hand, heat-killed S. gordonii inhibited osteoblast differentiation and reduced the expression of Runx2, an essential transcription factor for osteoblast differentiation. S. gordonii exerts bone resorptive activity by increasing osteoclast differentiation and reducing osteoblast differentiation, which may be involved in periapical bone resorption.

A 15-amino acid C-terminal peptide of beta-defensin-3 inhibits bone resorption by inhibiting the osteoclast differentiation and disrupting podosome belt formation.

Human beta-defensin-3 (HBD3), which is secreted from cells in the skin, salivary gland, and bone marrow, exhibits antimicrobial and immunomodulatory activities. Its C-terminal end contains a 15-amino acid polypeptide (HBD3-C15) that is known to effectively elicit antimicrobial activity. Recently, certain antimicrobial peptides are known to inhibit osteoclast differentiation and, thus, we investigated whether HBD3-C15 hinders osteoclast differentiation and bone destruction to assess its potential use as an anti-bone resorption agent. HBD3-C15 inhibited the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation and formation of resorption pits. In addition, HBD3-C15 disrupted the formation of RANKL-induced podosome belt which is a feature typically found in mature osteoclasts with bone-resorbing capacity. HBD3-C15 downregulated cortactin, cofilin, and vinculin, which are involved in the podosome belt formation. Furthermore, bone loss induced by RANKL was significantly reduced in a mouse calvarial implantation model that was treated with HBD3-C15. Similar inhibitory effects were observed on the osteoclast differentiation and podosome belt formation induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide (AaLPS). Concordantly, HBD3-C15 attenuated the resorption in the calvarial bone of AaLPS-implanted mouse. Collectively, these results suggest that HBD3-C15 has an anti-bone resorption effect in developing osteoclasts and that this occurs via its disruption of podosome belt formation. HBD3-C15 could be a potential therapeutic agent for the inhibition of bone destruction. KEY MESSAGES: HBD3-C15 inhibits osteoclast differentiation and bone resorption capacity. HBD3-C15 disrupts the podosome belt formation in osteoclasts. HBD3-C15 alleviates the bone loss by RANKL or A. actinomycetemcomitans LPS in vivo.

Muramyl Dipeptide, a Shared Structural Motif of Peptidoglycans, Is a Novel Inducer of Bone Formation through Induction of Runx2.

Peptidoglycan fragments released from gut microbiota can be delivered to the bone marrow and affect bone metabolism. We investigated the regulation of bone metabolism by muramyl dipeptide (MDP), which is a shared structural unit of peptidoglycans. Increased bone and mineral density by enhanced bone formation were observed in mice administered with MDP. Remarkably, pretreatment or posttreatment with MDP alleviated bone loss in RANKL-induced osteoporosis mouse models. MDP directly augmented osteoblast differentiation and bone-forming gene expression by Runx2 activation. Despite no direct effect, MDP indirectly attenuated osteoclast differentiation through downregulation of the RANKL/osteoprotegerin (OPG) ratio. MDP increased the expression of the MDP receptor, Nod2, and MDP-induced bone formation and osteoblast activation did not occur during Nod2 deficiency. Other Nod2 ligands also increased bone formation through the induction of Runx2, as MDP did. In conclusion, we suggest that MDP is a novel inducer of bone formation that could potentially be a new therapeutic molecule to protect against osteoporosis. © 2017 American Society for Bone and Mineral Research.

Lipoteichoic Acid of Enterococcus faecalis Inhibits the Differentiation of Macrophages into Osteoclasts.

INTRODUCTION: Enterococcus faecalis is associated with persistent endodontic infection and refractory apical periodontitis. Recently, we have shown that heat-killed E. faecalis attenuates osteoclast differentiation. Because lipoteichoic acid (LTA) is a major virulence factor of gram-positive bacteria, we investigated the effect of LTA from E. faecalis (EfLTA) on osteoclast differentiation. METHODS: EfLTA was purified through organic solvent extraction, hydrophobic interaction column chromatography, and ion exchange column chromatography. Bone marrow cells from C57BL/6 or Toll-like receptor 2-deficient mice were incubated with macrophage colony-stimulating factor (M-CSF) for 5 days to generate macrophages (bone marrow-derived macrophages [BMMs]). The cells were differentiated into osteoclasts with M-CSF and receptor activator of NF-κB ligand (RANKL) in the presence or absence of EfLTA. The degree of osteoclast differentiation was determined by tartrate-resistant acid phosphatase staining. The expression of NFATc1 and c-Fos transcription factors was determined by Western blotting. A phagocytosis assay was performed by measuring the uptake of carboxyfluorescein diacetate succinimidyl ester-stained E. faecalis. An enzyme-linked immunosorbent assay was used to determine the amount of cytokines and chemokines. RESULTS: When BMMs were treated with EfLTA, osteoclast differentiation was attenuated. EfLTA inhibited the RANKL-induced expression of NFATc1 and c-Fos. EfLTA inhibition of osteoclast differentiation was not observed in TLR2-deficient BMMs. In addition, EfLTA sustained the phagocytic capacity of BMMs even after the differentiation into osteoclasts, whereas it induced the expression of inflammatory cytokines and chemokines. CONCLUSIONS: EfLTA inhibits the differentiation of macrophages into osteoclasts and thereby maintains the phagocytic and inflammatory capacities of macrophages, potentially contributing to refractory apical periodontitis.

Systemic administration of RANKL overcomes the bottleneck of oral vaccine delivery through microfold cells in ileum.

A successful delivery of antigen through oral route requires to overcome several barriers, such as enzymatic barrier of gastrointestinal tract and epithelial barrier that constitutes of microfold cells (M cells) for antigen uptake. Although each barrier represents a critical step in determining the final efficiency of antigen delivery, the transcytosis of antigen by M cells in the follicle-associated epithelium (FAE) to Peyer's patches appears to be a major bottleneck. Considering the systemic administration of receptor activator of nuclear factor (NF)-ĸB ligand (RANKL) induces differentiation of receptor activator of nuclear factor (NF)-ĸB (RANK)-expressing enterocytes into M cells, here, we illustrated a promising approach of antigen delivery using full length transmembrane RANKL (mRANKL). The results showed that the intraperitoneal injection of mRANKL increased the population of dendritic cells and macrophages in mesenteric lymph nodes and spleen. Subsequently, systemic administration of mRANKL resulted in significantly higher number of functional GP2(+) M cells leading higher transcytosis of fluorescent beads through them. To corroborate the effect of mRANKL in antigen delivery through M cells, we orally delivered microparticulate antigen to mice treated with mRANKL. Oral immunization induced strong protective IgA and systemic IgG antibody responses against orally delivered antigen in mRANKL-treated mice. The higher antibody responses are attributed to the higher transcytosis of antigens through M cells. Ultimately, the higher memory B cells and effector memory CD4 T cells after oral immunization in RANKL-treated mice confirmed potency of RANKL-mediated antigen delivery. To the best of our knowledge, this is the first study to demonstrate significant induction of mucosal and humoral immune responses to M cell targeted oral vaccines after the systemic administration of RANKL.

Serum amyloid A inhibits osteoclast differentiation to maintain macrophage function.

Serum amyloid A is an acute phase protein that is elevated under inflammatory conditions. Additionally, the serum levels of serum amyloid A are associated with the progression of inflammatory arthritis; thus, serum amyloid A might be involved in the regulation of osteoclast differentiation. In the present study, we examined the effects of serum amyloid A on osteoclast differentiation and function. When bone marrow-derived macrophages, as osteoclast precursors, were stimulated with serum amyloid A in the presence of M-CSF and receptor activator of nuclear factor-κB ligand, osteoclast differentiation and its bone-resorption activity were substantially inhibited. TLR2 was important in the inhibitory effect of serum amyloid A on osteoclast differentiation, because serum amyloid A stimulated TLR2. The inhibitory effect was absent in bone marrow-derived macrophages obtained from TLR2-deficient mice. Furthermore, serum amyloid A inhibited the expression of c-Fos and nuclear factor of activated T cells c1, which are crucial transcription factors for osteoclast differentiation, but prevented downregulation of IFN regulatory factor-8, a negative regulator of osteoclast differentiation. In contrast, serum amyloid A sustained the endocytic capacity of bone marrow-derived macrophages and their ability to induce the proinflammatory cytokines, IL-6, IL-1ß, and TNF-α. Taken together, these results suggest that serum amyloid A, when increased by inflammatory conditions, inhibits differentiation of macrophages to osteoclasts, likely to maintain macrophage function for host defense.

Enterococcus faecalis Inhibits Osteoblast Differentiation and Induces Chemokine Expression.

INTRODUCTION: Enterococcus faecalis is commonly found in root canals of patients with refractory apical periodontitis, often accompanying inflammation and malfunctioning bone regeneration. In this study, we investigated the effect of E. faecalis on osteoblast differentiation and the ability to induce chemokine expression to recruit inflammatory cells. METHODS: Osteoblast precursors from mouse calvaria were differentiated into osteoblasts with ascorbic acid and ß-glycerophosphate in the absence or presence of heat-killed E. faecalis (HKEF). Alizarin red S staining was performed to determine the degree of mineralization. Reporter gene and reverse-transcription polymerase chain reaction assays were performed to examine the activity of the Runx2 transcription factor and the expression of osteogenic marker genes, respectively. Secretion of the chemokines keratinocyte-derived chemokine and monocyte chemotactic protein-1 was measured by the enzyme-linked immunosorbent assay, and their functions were analyzed by measuring the migration of peripheral blood mononuclear cells using a transwell system. RESULTS: HKEF inhibited osteoblast mineralization and Runx2 transcriptional activity, which are typical features of osteoblast differentiation. HKEF also decreased the expression of Runx2, osterix, ß-catenin, osteocalcin, and type I collagen. Interestingly, however, the expression of keratinocyte-derived chemokine and monocyte chemotactic protein-1 was increased by HKEF, and the culture supernatant of HKEF-stimulated osteoblasts increased the transmigration of peripheral blood mononuclear cells. CONCLUSIONS: HKEF inhibits osteoblast differentiation and induces chemokine expression, which might be involved in refractory apical periodontitis and bone loss.

Lipopolysaccharide of Aggregatibacter actinomycetemcomitans induces the expression of chemokines MCP-1, MIP-1α, and IP-10 via similar but distinct signaling pathways in murine macrophages.

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium frequently isolated from lesions of patients with localized aggressive periodontitis. Lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria, stimulates innate immune cells via Toll-like receptor 4 (TLR4) to initiate inflammatory responses. In this study, we purified LPS from A. actinomycetemcomitans (AaLPS) and investigated its ability to induce the expression of chemokines, which play an important role in recruitment of leukocytes to the infection site. AaLPS induced the expression of chemokines, MCP-1, MIP-1α, and IP-10 in murine macrophages, leading to the infiltration of peripheral blood mononuclear cells in a transwell system. Although TLR4 was essential for the induction of all these chemokines by AaLPS, MCP-1 and MIP-1α expressions were MyD88-dependent, but IP-10 expression was MyD88-independent, as determined using macrophages from mice deficient in TLR4 or MyD88. Furthermore, the activation of ERK and JNK were necessary for the expression of MCP-1 and MIP-1α, whereas p38 MAP kinase and JNK activations were required for IP-10 expression. In addition, IFN-ß/STAT1 signaling was exclusively involved in IP-10 expression but not in MCP-1 or MIP-1α expression. AaLPS also activated the transcription factors, NF-κB, AP-1, NF-IL6, and ISRE, all of which are involved in chemokine gene expression. These results suggest that AaLPS induces the expression of chemokines MCP-1, MIP-1α, and IP-10 through TLR4 in murine macrophages. Further, the induction of MCP-1 and MIP-1α requires MyD88, ERK, and JNK, whereas the induction of IP-10 requires JNK, p38 MAP kinase, and IFN-ß/STAT1.

Staphylococcus aureus induces IL-8 expression through its lipoproteins in the human intestinal epithelial cell, Caco-2.

Staphylococcus aureus can cause the intestinal inflammatory diseases. However, little is known about the molecular mechanism of S. aureus infection in the intestine. In the present study, we investigated whether S. aureus could stimulate human intestinal epithelial cells triggering inflammation. When the human intestinal epithelial cell-line, Caco-2, and the primary colon cells were stimulated with ethanol-inactivated S. aureus, IL-8 expression was induced in a dose-dependent manner. The inactivated S. aureus preferentially stimulated Toll-like receptor (TLR) 2 rather than TLR4. Lipoproteins, lipoteichoic acid (LTA), and peptidoglycan (PGN) are considered as potential TLR2 ligands of S. aureus. Interestingly, S aureus lipoproteins and Pam2CSK4 mimicking Gram-positive bacterial lipoproteins, but not LTA and PGN of S. aureus, significantly induced IL-8 expression in Caco-2 cells. Furthermore, lipoprotein-deficient S. aureus mutant strain failed to induce IL-8 production. Collectively, these results suggest that S. aureus stimulates the human intestinal epithelial cells to induce the chemokine IL-8 production through its lipoproteins, potentially contributing the development of intestinal inflammation.

Enterococcus faecalis attenuates the differentiation of macrophages into osteoclasts.

INTRODUCTION: Enterococcus faecalis is closely associated with refractory apical periodontitis, manifesting periapical lesions and alveolar bone loss. Macrophages playing an important role in the induction of inflammation can differentiate into bone-resorbing osteoclasts. In the present study, we investigated the effect of E. faecalis on the differentiation and function of macrophages as osteoclast precursors. METHODS: Bone marrow-derived macrophages (BMMs) were differentiated into osteoclasts with macrophage colony-stimulating factor and receptor activator of nuclear factor kappa B ligand in the presence or absence of heat-killed E. faecalis (HKEF). Tartrate-resistant acid phosphatase-positive multinucleated giant cells were analyzed to determine osteoclast differentiation. Western blotting was performed to examine the expression of c-Fos and NFATc1 transcription factors. Phagocytic capacity was analyzed by measuring uptake of carboxyfluorescein succinimidyl ester-labeled E. faecalis. Secretion of tumor necrosis factor-α, interleukin-6, keratinocyte-derived chemokine, and monocyte chemotactic protein-1 was determined by enzyme-linked immunosorbent assay. RESULTS: Differentiation of BMMs into osteoclasts was attenuated in the presence of HKEF, and expression of c-Fos and NFATc1 was inhibited. HKEF exposure also prevented a reduction in the phagocytic capacity of BMMs after differentiation into osteoclasts. Concomitantly, HKEF induced the expression of chemokines monocyte chemotactic protein-1 and keratinocyte-derived chemokine and proinflammatory cytokines tumor necrosis factor-α and interleukin-6. CONCLUSIONS: E. faecalis attenuated macrophages from differentiating into osteoclasts, allowing them to keep their ability to phagocytose and kill pathogens and to induce proinflammatory cytokine and chemokine secretion.

Irradiation by gallium-aluminum-arsenate diode laser enhances the induction of nitric oxide by Porphyromonas gingivalis in RAW 264.7 cells.

BACKGROUND: Low-level laser irradiation promotes cell viability and wound healing in periodontal tissue. However, its effect on periodontal pathogenic bacteria is unknown. The purpose of this study is to investigate the biologic effect of low-level laser irradiation on Porphyromonas gingivalis. METHODS: A murine macrophage cell line (RAW 264.7) was cultured and treated with gallium-aluminum-arsenate (GaAlAs) laser-irradiated P. gingivalis with varying levels of energy fluency. Gene expression of monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), interferon-ß (IFN-ß), and inducible nitric oxide synthase (iNOS) was examined by reverse transcription-polymerase chain reaction. Production of iNOS was determined by Western blot analysis, and nitric oxide (NO) release was assessed using Griess reagent. Flow cytometric analysis was performed to determine the activation of Toll-like receptors (TLRs) in response to P. gingivalis. RESULTS: The laser-irradiated P. gingivalis significantly enhanced messenger RNA and protein levels of iNOS in RAW 264.7. Although the laser irradiation on P. gingivalis did not alter the expression level of MCP-1, IL-6, and IFN-ß, it showed a noticeable effect on NO production in RAW 264.7. Furthermore, the laser-irradiated P. gingivalis accelerated TLR2 activation, but not TLR4 activation. CONCLUSIONS: This study reveals that GaAlAs laser irradiation on P. gingivalis induced iNOS expression at the transcriptional and translation levels and increased NO release in macrophages. Moreover, it is confirmed that this process was mediated specifically by TLR2 activation. These findings suggest that low-level laser irradiation to periodontal pathogenic bacteria could be detrimental to periodontal treatments.

Muramyl dipeptide potentiates staphylococcal lipoteichoic acid induction of cyclooxygenase-2 expression in macrophages.

Gram-positive bacteria contain lipoteichoic acid (LTA) and peptidoglycan (PGN) layers, both of which are considered as major virulence factors associated with inflammation. Cyclooxygenase-2 (COX-2) plays an important role in the inflammation by generating prostaglandins at infections. Since LTA and PGN are thought to cooperate in the establishment of inflammation, we examined the ability of staphylococcal LTA (Sa.LTA) to induce COX-2 expression in the presence of muramyl dipeptide (MDP), which is the minimal structural unit of PGN required for inflammation, in macrophages. While MDP failed to induce COX-2 expression, Sa.LTA alone was sufficient to induce COX-2 production. Treatment with MDP enhanced Sa.LTA-induced COX-2 and prostaglandin E2 production. The cooperative effect between Sa.LTA and MDP was not observed in COX-2 expression by macrophages derived from Toll-like receptor 2 (TLR2)- or nucleotide-binding oligomerization domain 2 (NOD2)-deficient mice. In addition, MDP enhanced Sa.LTA-induced activation of the transcription factors NF-κB and CRE, which are known to modulate COX-2 gene transcription. Conclusively, these results suggest that MDP and Sa.LTA cooperatively induce inflammatory response by overproducing COX-2 through NOD2 and TLR2.

Lipoteichoic acid of Enterococcus faecalis induces the expression of chemokines via TLR2 and PAFR signaling pathways.

Enterococcus faecalis is one of the most common opportunistic pathogens responsible for nosocomial infections, and its LTA is known as an important virulence factor causing inflammatory responses. As chemokines play a key role in inflammatory diseases by triggering leukocyte infiltration into the infection site, we purified EfLTA and investigated its effect on the expression of chemokines, IP-10, MIP-1α, and MCP-1, in murine macrophages. EfLTA induced the expression of these chemokines at the mRNA and protein levels. TLR2, CD14, and MyD88 were involved in the EfLTA-induced chemokine expression, as the expression was reduced remarkably in macrophages derived from TLR2-, CD14-, or MyD88-deficient mice. EfLTA induced phosphorylation of MAPKs and enhanced the DNA-binding activity of NF-κB, AP-1, and NF-IL6 transcription factors. The induction of IP-10 required ERK, JNK, p38 MAPK, PKC, PTK, PI3K, and ROS. We noticed that all of these signaling molecules, except p38 MAPK and ROS, were indispensable for the induction of MCP-1 and MIP-1α. Interestingly, the EfLTA-induced chemokine expression was mediated through PAFR/JAK/STAT1 signaling pathways without IFN-ß involvement, which is different from LPS-induced chemokine expression requiring IFN-ß/JAK/STAT1 signaling pathways. Furthermore, the culture supernatant of EfLTA-treated RAW 264.7 cells promoted the platelet aggregation, and exogenous PAF induced the chemokine expression in macrophages derived from WT and TLR2-deficient mice. These results suggest that EfLTA induces the expression of chemokines via signaling pathways requiring TLR2 and PAFR, which is distinct from that of LPS-induced chemokine expression.

Lipoproteins are an important bacterial component responsible for bone destruction through the induction of osteoclast differentiation and activation.

Bacterial infection can cause inflammatory bone diseases accompanied by the bone destruction resulting from excess generation of osteoclasts. Although lipoproteins are one of the major immunostimulating components of bacteria, little is known about their effects on bone metabolism. In this study, we investigated the role of lipoproteins in bacteria-induced bone destruction using Staphylococcus aureus wild type, its lipoprotein-deficient mutant, and synthetic lipopeptides Pam2CSK4 and Pam3CSK4 known to mimic bacterial lipoproteins. Formaldehyde-inactivated S. aureus or the synthetic lipopeptides induced severe bone loss in the femurs of mice after intraperitoneal administration and in a calvarial bone implantation model, whereas the lipoprotein-deficient S. aureus did not show such effects. Mechanism studies further identified three action mechanisms for the lipopeptide-induced osteoclast differentiation and bone resorption via (i) enhancement of osteoclast differentiation through Toll-like receptor 2 and MyD88-dependent signaling pathways; (ii) induction of pro-inflammatory cytokines, TNF-α and IL-6; and (iii) upregulation of RANKL expression with downregulation of osteoprotegerin expression in osteoblasts. Taken together, these results suggest that lipoprotein might be an important bacterial component responsible for bone destruction during bacterial infections through augmentation of osteoclast differentiation and activation.

Functional characterization of a novel FGFR2 mutation, E731K, in craniosynostosis.

Craniosynostosis is a condition in which some or all of the sutures in the skull of an infant close prematurely. Fibroblast growth factor receptor 2 (FGFR2) mutations are a well-known cause of craniosynostosis. Many syndromes that comprise craniosynostosis, such as Apert syndrome, Crouzon syndrome, and Pfeiffer syndrome, have one of the phenotypes that have been reported in FGFR2 mutant patients. FGFRs have been reported in four types (FGFR1-4), and upon binding with FGF ligands, signal transduction occurs inside of cells. Activated FGFR stimulates an osteogenic master transcription factor, Runx2, through the MAP kinase and PKC pathways. We obtained a genetic analysis of six Korean patients who have craniosynostosis as a phenotype. All of the patients had at least one mutation in the FGFR2 gene; five of those mutations have already been reported elsewhere, while one mutation is novel and was hypothesized to lead to Apert syndrome. In this study, we reported and functionally analyzed a novel mutation of the FGFR2 gene found in a craniosynostosis patient, E731K. The mutation is in the 2nd tyrosine kinase domain in the C-terminal cytoplasmic region of the molecule. The mutation caused an enhanced phosphorylation of the FGFR2(E731K) and ERK-MAP kinase, the stimulation of transcriptional activity of Runx2, and consequently, the enhancement of osteogenic marker gene expression. We conclude that the substitution of E731K in FGFR2 is a novel mutation that resulted in a constitutive activation of the receptor and ultimately resulted in premature suture obliteration.