Jihyun Yang

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.

Impaired osteoclastogenesis by staphylococcal lipoteichoic acid through Toll-like receptor 2 with partial involvement of MyD88

Degenerative bone disease, marked by excessive loss of calcified matrix, is often associated with bacterial infections. Osteoclasts, which mediate the bone‐resorptive process, are derived mainly from myeloid precursor cells of the monocyte/macrophage lineage, from which cells with phagocytic and inflammatory capacities may alternatively arise. Here, we investigated the effect of LTA, a major cell‐wall virulence factor of Gram‐positive bacteria, on osteoclast differentiation. Osteoclast precursors were prepared from C57BL/6 mouse BM using M‐CSF and RANKL. When osteoclastogenesis was induced in the presence of staphylococcal LTA, LTA dose‐dependently inhibited the differentiation of osteoclast precursors to mature osteoclasts. A corresponding inhibition of bone‐resorptive function was observed in the reduced resorption area on calcium phosphate‐coated culture plates. In contrast, the phagocytic and inflammatory potential of the osteoclast precursors increased in the presence of LTA. TLR2, known to recognize LTA, might be essential for the LTA inhibition of osteoclastogenesis, as the inhibition did not occur in the precursors from TLR2‐deficient mice. Importantly, MyD88‐dependent and MyD88‐independent pathways would participate in the inhibition, as determined using MyD88‐deficient cells. Moreover, LTA inhibited phosphorylation of ERK and JNK in osteoclast precursors stimulated with M‐CSF and RANKL, concomitantly with a decreased DNA‐binding activity of AP‐1. These results suggest that staphylococcal LTA inhibits osteoclast differentiation primarily through TLR2 but also in part through MyD88 signaling, which in turn, inhibits activation of ERK, JNK, and AP‐1.

Anti-bacterial and anti-toxic immunity induced by a killed whole-cell-cholera toxin B subunit cholera vaccine is essential for protection against lethal bacterial infection in mouse pulmonary cholera model.

The lack of appropriate animal model for studying protective immunity has limited vaccine development against cholera. Here, we demonstrate a pulmonary cholera model conferred by intranasal administration of mice with live Vibrio cholerae. The bacterial components, but not cholera toxin, caused lethal and acute pneumonia by inducing massive inflammation. Intranasal immunization with Dukoral, comprising killed whole bacteria and recombinant cholera toxin B subunit (rCTB), developed both mucosal and systemic antibody responses with protection against the lethal challenge. Either rCTB-free Dukoral or rCTB alone partially protected the mice against the challenge. However, reconstitution of rCTB-free Dukoral with rCTB restored full protection. Parenteral immunization with Dukoral evoked strong systemic immunity without induction of mucosal immunity or protection from the challenge. These results suggest that both anti-bacterial and anti-toxic immunity are required for protection against V. cholerae–induced pneumonia, and this animal model is useful for pre-clinical evaluation of candidate cholera vaccines.

The 4-1BB ligand and 4-1BB expressed on osteoclast precursors enhance RANKL-induced osteoclastogenesis via bi-directional signaling.

The 4–1BB is a costimulatory molecule similar to the receptor activator of NF‐κB ligand (RANKL), both of which are key factors for the differentiation of osteoclasts and are expressed mainly by activated T cells. The 4–1BB shares common signaling pathways with RANK, suggesting a potential role in osteoclastogenesis. In this study, the role of 4–1BB and 4–1BB ligand (4–1BBL) in osteoclastogenesis was investigated using 4–1BB–/– and 4–1BB+/+ mice. Osteoclast precursors normally express 4–1BB and 4–1BBL after exposure to RANKL, which was confirmed by semi‐quantitative RT‐PCR and flow cytometry. The 4–1BB–/– mice had a slightly increased bone mass accompanied by a reduced osteoclastogenic ability of 4–1BB–/– bone marrow‐derived macrophages (BMM) ex vivo. In addition, 4–1BB–/– BMM demonstrated hypophosphorylation of JNK and p38 and decreased induction of c‐Fos in response to RANKL stimulation. Retroviral transduction of wild‐type as well as partial‐length 4–1BB, which lacks TNF receptor‐associated factor 2‐binding sites for signaling, restored the osteoclastogenic ability of 4–1BB–/– BMM. Furthermore, both recombinant 4–1BB and 4–1BBL enhanced RANKL‐induced osteoclastogenesis by 4–1BB+/+ BMM and the induction of c‐Fos and NFATc1.Together, these results indicate that 4–1BBL and 4–1BB expressed on osteoclast precursors enhance RANKL‐induced osteoclastogenesis via bi–directional signaling, findings that may delineate the complex nature of the 4–1BBL and 4–1BB interaction.

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.

Bacillus anthracis lethal toxin attenuates lipoteichoic acid-induced maturation and activation of dendritic cells through a unique mechanism

Lethal toxin (LT), produced by the gram-positive bacterium Bacillus anthracis, was identified as a major etiologic agent causing anthrax due to its strong immunotoxicity. Gram-positive bacteria express lipoteichoic acid (LTA), which is considered as a counterpart to lipopolysaccharide (LPS) of gram-negative bacteria, but differs from LPS in the structure and function. Since dendritic cells (DCs) are essential for the appropriate initiation of immune response, we investigated the effect of LT on LTA-induced DC maturation using immature DCs prepared by differentiation of C57BL/6 mouse bone marrow cells. When immature DCs were matured with LTA in the presence of LT, the expression of representative markers for DC maturation such as CD80, CD83, and CD86 together with MHC class I and II molecules was inhibited. LT ameliorated the attenuation of endocytic capacity during DC maturation by LTA while such effect was not observed in LPS-matured DCs. Furthermore, exposure to LT resulted in a decrease in the expression of pro-inflammatory cytokines including IL-6, TNF-alpha, and IL-12p40 in LTA-stimulated DCs as in LPS-stimulated DCs. Interestingly, LT showed a minimal change in LTA-induced IL-1beta expression while LT highly enhanced the LPS-induced IL-1beta expression. Those inhibitory effects might be associated with LT interference of LTA-signaling pathways mediated through mitogen-activated protein kinases (MAPKs) since LT suppressed phosphorylation of MAPK, which was induced by LTA. Meanwhile, no change was observed in the expression of putative anthrax toxin receptors, TEM8 and CMG2, or Toll-like receptor 2. These results suggest that LT suppresses the maturation and activation of DCs stimulated with LTA, similar to the suppression in the LPS-stimulated DCs, but via a distinct mechanism.

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 β-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, β-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.

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.

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‐1β, 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.