Human osteoclastogenesis in Epstein-Barr virus-induced erosive arthritis in humanized NOD/Shi-scid/IL-2Rγnull mice.

Many human viruses, including Epstein-Barr virus (EBV), do not infect mice, which is challenging for biomedical research. We have previously reported that EBV infection induces erosive arthritis, which histologically resembles rheumatoid arthritis, in humanized NOD/Shi-scid/IL-2Rγnull (hu-NOG) mice; however, the underlying mechanisms are not known. Osteoclast-like multinucleated cells were observed during bone erosion in this mouse model, and therefore, we aimed to determine whether the human or mouse immune system activated bone erosion and analyzed the characteristics and origin of the multinucleated cells in hu-NOG mice. Sections of the mice knee joint tissues were immunostained with anti-human antibodies against certain osteoclast markers, including cathepsin K and matrix metalloproteinase-9 (MMP-9). Multinucleated cells observed during bone erosion stained positively for human cathepsin K and MMP-9. These results indicate that human osteoclasts primarily induce erosive arthritis during EBV infections. Human osteoclast development from hematopoietic stem cells transplanted in hu-NOG mice remains unclear. To confirm their differentiation potential into human osteoclasts, we cultured bone marrow cells of EBV-infected hu-NOG mice and analyzed their characteristics. Multinucleated cells cultured from the bone marrow cells stained positive for human cathepsin K and human MMP-9, indicating that bone marrow cells of hu-NOG mice could differentiate from human osteoclast progenitor cells into human osteoclasts. These results indicate that the human immune response to EBV infection may induce human osteoclast activation and cause erosive arthritis in this mouse model. Moreover, this study is the first, to our knowledge, to demonstrate human osteoclastogenesis in humanized mice. We consider that this model is useful for studying associations of EBV infections with rheumatoid arthritis and human bone metabolism.

Inhibition of Ctsk modulates periodontitis with arthritis via downregulation of TLR9 and autophagy.

OBJECTIVES: The mechanisms underlying the effects of Toll-like receptor 9 (TLR9) and autophagy on rheumatoid arthritis (RA)-aggravated periodontitis are unclear. We aimed to explore a novel target, cathepsin K (Ctsk)-mediated TLR9-related autophagy, during the progress of periodontitis with RA. MATERIALS AND METHODS: DBA/J1 mouse model of periodontitis with RA was created by local colonization of Porphyromonas gingivalis (Pg) and injection of collagen. The expression of Ctsk was inhibited by adeno-associated virus (AAV). Micro-CT, immunohistochemistry (IHC), Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of TLR9-related autophagy in periodontitis with RA. Small interfering RNA (siRNA) and CpG oligodeoxynucleotides (CpG ODN) were applied in macrophages. Western blot, immunofluorescence (IF) and qRT-PCR were used to verify the in vivo results. RESULTS: RA can promote periodontitis bone destruction in the lesion area, while inhibiting Ctsk could effectively alleviate this effect. The infiltration of macrophages, TLR9, autophagy proteins (TFEB and LC3) and inflammatory cytokines increased in the periodontitis-with-RA group and was reduced by the inhibition of Ctsk in the periodontal region. Macrophage stimulation confirmed the in vivo results. With the activation of TLR9 by CpG ODN, inhibition of Ctsk could suppress both TLR9 downstream signalling proteins and autophagy-related proteins. CONCLUSIONS: This study advanced a novel role for Ctsk in TLR9 and autophagy to explain the interaction between periodontitis and RA.

Meningeal Foam Cells and Ependymal Cells in Axolotl Spinal Cord Regeneration.

A previously unreported population of foam cells (foamy macrophages) accumulates in the invasive fibrotic meninges during gap regeneration of transected adult Axolotl spinal cord (salamander Ambystoma mexicanum) and may act beneficially. Multinucleated giant cells (MNGCs) also occurred in the fibrotic meninges. Actin-label localization and transmission electron microscopy showed characteristic foam cell and MNGC podosome and ruffled border-containing sealing ring structures involved in substratum attachment, with characteristic intermediate filament accumulations surrounding nuclei. These cells co-localized with regenerating cord ependymal cell (ependymoglial) outgrowth. Phase contrast-bright droplets labeled with Oil Red O, DiI, and DyRect polar lipid live cell label showed accumulated foamy macrophages to be heavily lipid-laden, while reactive ependymoglia contained smaller lipid droplets. Both cell types contained both neutral and polar lipids in lipid droplets. Foamy macrophages and ependymoglia expressed the lipid scavenger receptor CD36 (fatty acid translocase) and the co-transporter toll-like receptor-4 (TLR4). Competitive inhibitor treatment using the modified fatty acid Sulfo-N-succinimidyl Oleate verified the role of the lipid scavenger receptor CD36 in lipid uptake studies in vitro. Fluoromyelin staining showed both cell types took up myelin fragments in situ during the regeneration process. Foam cells took up DiI-Ox-LDL and DiI-myelin fragments in vitro while ependymoglia took up only DiI-myelin in vitro. Both cell types expressed the cysteine proteinase cathepsin K, with foam cells sequestering cathepsin K within the sealing ring adjacent to the culture substratum. The two cell types act as sinks for Ox-LDL and myelin fragments within the lesion site, with foamy macrophages showing more Ox-LDL uptake activity. Cathepsin K activity and cellular localization suggested that foamy macrophages digest ECM within reactive meninges, while ependymal cells act from within the spinal cord tissue during outgrowth into the lesion site, acting in complementary fashion. Small MNGCs also expressed lipid transporters and showed cathepsin K activity. Comparison of 3H-glucosamine uptake in ependymal cells and foam cells showed that only ependymal cells produce glycosaminoglycan and proteoglycan-containing ECM, while the cathepsin studies showed both cell types remove ECM. Interaction of foam cells and ependymoglia in vitro supported the dispersion of ependymal outgrowth associated with tissue reconstruction in Axolotl spinal cord regeneration.

Characterization and initial functional analysis of cathepsin K in turbot (Scophthalmus maximus L.).

Cathepsins are the best-known group of proteases in lysosomes, playing a significant role in immune responses. Cathepsin K (CTSK) is abundantly and selectively expressed in osteoclasts, dendritic cells and monocyte-derived macrophages, where it is involved in ECM degradation and bone remodeling. A growing body of evidences have indicated the vital roles of cathepsin K in innate immune responses. Here, one CTSK gene was captured in turbot (SmCTSK) with a 993 bp open reading frame (ORF). The genomic structure analysis showed that SmCTSK had 7 exons similar to other vertebrate species. The syntenic analysis revealed that CTSK had the same neighboring genes across all the selected species, which suggested the synteny encompassing CTSK region was conserved during vertebrate evolution. Subsequently, SmCTSK was widely expressed in all the examined tissues, with the highest expression level in spleen and the lowest expression level in liver. In addition, SmCTSK was significantly down-regulated in intestine following Gram-negative bacteria Vibrio anguillarum immersion challenge, but up-regulated in three tissues (gill, skin and intestine) following Gram-positive bacteria Streptococcus iniae immersion challenge. Finally, the rSmCTSK showed strong binding ability to all the examined microbial ligands. Taken together, our results suggested SmCTSK played vital roles in fish innate immune responses against infection. However, the knowledge of SmCTSK is still limited in teleost species, further studies should be carried out to better characterize its comprehensive roles in teleost mucosal immunity.

Mouse models of psoriasis and their relevance.

Psoriasis is an inflammatory skin disorder that includes dynamic interactions between the immune system and skin and is clinically characterized by keratinocyte proliferation and distinct inflammatory cell infiltrates. Cross-talk between keratinocytes and immunocytes is essential for the development of psoriasis given that it mediates the production of cytokines, chemokines and growth factors. To resolve the pathogenesis of psoriasis, numerous experimental animal models have been generated. In this review, we discuss recent findings from mouse models, their relevancy to psoriasis and use, including the discovery of new therapies.

Inhibition of lipopolysaccharide-induced osteoclast formation and bone resorption in vitro and in vivo by cysteine proteinase inhibitors.

Inflammation-induced bone destruction is a major treatment target in many inflammatory skeletal diseases. The aim of this study was to investigate if the cysteine proteinase inhibitors cystatin C, fungal cysteine proteinase inhibitor (E-64), and N-benzyloxycarbonyl-arginyl-leucyl-valyl-glycyl-diazomethane acetate (Z-RLVG-CHN2) can inhibit LPS-induced osteoclast formation. Mouse bone marrow macrophages (BMMs) were isolated and primed with receptor activator of NF-κB ligand (RANKL) for 24 h, followed by stimulation with LPS, with and without inhibitors. Adult mice were injected locally with LPS and then treated with E-64 and osteoclast formation assessed by the number of cathepsin K+ multinucleated cells. Cystatin C inhibited LPS-induced osteoclast formation time and concentration dependently (IC50 = 0.3 µM). The effect was associated with decreased mRNA and protein expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K and of the osteoclastogenic transcription factors c-Fos and NFATc1. LPS-induced osteoclast formation on bone slices was also inhibited by cystatin C, resulting in decreased pit formation and release of bone matrix proteins. Similar data were obtained with E-64 and Z-RLVG-CHN2 Cystatin C was internalized in BMMs stimulated by LPS but not in unstimulated BMMs. Osteoclast formation induced by LPS was dependent on TNF-α, and the 3 inhibitors abolished LPS-induced TNF superfamily 2 (gene encoding TNF-α; Tnfsf2) mRNA expression without affecting Il1b, Il6, or oncostatin M (Osm) expression. Formation of osteoclasts in the skull bones after local LPS stimulation was inhibited by E-64. It is concluded that cysteine proteinase inhibitors effectively inhibit LPS-induced osteoclast formation in vivo and in vitro by inhibition of TNF-α expression. The targeting of cysteine proteinases might represent a novel treatment modality for prevention of inflammatory bone loss.

Cysteine cathepsin activity regulation by glycosaminoglycans.

Cysteine cathepsins are a group of enzymes normally found in the endolysosomes where they are primarily involved in intracellular protein turnover but also have a critical role in MHC II-mediated antigen processing and presentation. However, in a number of pathologies cysteine cathepsins were found to be heavily upregulated and secreted into extracellular milieu, where they were found to degrade a number of extracellular proteins. A major role in modulating cathepsin activities play glycosaminoglycans, which were found not only to facilitate their autocatalytic activation including at neutral pH, but also to critically modulate their activities such as in the case of the collagenolytic activity of cathepsin K. The interaction between cathepsins and glycosaminoglycans will be discussed in more detail.

Cathepsin K is involved in development of psoriasis-like skin lesions through TLR-dependent Th17 activation.

Cathepsins (CTSs) are lysosomal cysteine proteases that play an important role in the turnover of intracellular proteins and extracellular proteins, such as the degradation of extracellular matrices and the processing of antigenic proteins. A CTS inhibitor, NC-2300, not only suppresses bone erosion by inhibition of cathepsin K (CTSK), but also ameliorates paw swelling at inflamed joints in adjuvant-induced arthritis in rats. It has been demonstrated that the amelioration of joint inflammation by NC-2300 is mediated by the downregulation of cytokine expression in dendritic cells, which are essential for Th17 activation. In this work, we studied the role for CTSs in the pathogenesis of psoriasis-like lesion in K5.Stat3C mice, a mouse model of psoriasis, in which Th17 contributes to lesion development similar to psoriasis. Psoriatic lesions expressed increased levels of Ctsk and Ctss mRNA compared with uninvolved skin and normal control skin. Similarly, the epidermis and dermis in K5.Stat3C mice demonstrated increased CTSK activities, which were sensitive to NC-2300. Topical treatment with NC-2300 significantly ameliorated 12-O-tetradecanoylphorbol-13-acetate-induced psoriasis-like lesions in K5.Stat3C mice, and downregulated the expression of IL-12, IL-23, and Th17 cytokines. In vitro experiments revealed that TLR7 activation of bone marrow-derived myeloid dendritic cells led to increase in IL-23 at mRNA and protein levels, which were downregulated by NC-2300. These results suggest that CTSK plays a role in development of psoriatic lesions through TLR7-dependent Th17 polarization.

Cathepsin K deficiency aggravates lung injury in hyperoxia-exposed newborn mice.

Cathepsin K (CatK) is a potent collagenase and elastase and may be involved in the development of neonatal bronchopulmonary dysplasia. The authors evaluated the effects of CatK deletion on neonatal lung development and response to prolonged hyperoxic challenge. CatK deficiency resulted in thinner alveolar walls than wild-type littermates on postnatal day (PN) 7. However, no morphological difference could be detected between CatK-deficient and control groups on PN 14. Exposure to 90% oxygen for 7 days after birth caused intensive CatK expression in the bronchial epithelium and alveolar macrophages of wild-type mice. Hyperoxia caused fatal respiratory distress in both groups of mice. However, whereas ∼20% of wild-type mice survived for 2 weeks in hyperoxia, all CatK-deficient mice died within the first 9 postnatal days. Hyperoxia-exposed lungs of CatK-deficient mice contained high number of macrophages and multinucleated giant cells and had increased content of reduced glutathione, indicating intensified pulmonary oxidative stress. These results suggest that CatK is involved in pulmonary development and it may be an important host-defence protease in the oxygen-stressed newborn lung.

S100A8 enhances osteoclastic bone resorption in vitro through activation of Toll-like receptor 4: implications for bone destruction in murine antigen-induced arthritis.

OBJECTIVE: Rheumatoid arthritis, which is associated with elevated levels of S100A8 and S100A9, is characterized by severe bone erosions caused by enhanced osteoclast formation and activity. The aim of the present study was to investigate the role of S100A8 and S100A9 in osteoclastic bone destruction in murine antigen-induced arthritis (AIA). METHODS: Bone destruction was analyzed in the arthritic knee joints of S100A9-deficient mice in which S100A8 protein expression was also lacking, and in wild-type (WT) controls. Osteoclast precursors from S100A9-deficient and WT mice were differentiated into osteoclasts in vitro. Additionally, precursors were stimulated with S100A8, S100A9, or S100A8/A9 during osteoclastogenesis. Receptor involvement was investigated using an anti-receptor for advanced glycation end products (anti-RAGE)-blocking antibody, soluble RAGE, or Toll-like receptor 4 (TLR-4)-deficient osteoclast precursors. The formation of osteoclasts and actin rings, the regulation of osteoclast markers, and bone resorption were analyzed. RESULTS: Bone erosions and cathepsin K staining were significantly suppressed in S100A9-deficient mice after AIA induction. However, osteoclast precursors from S100A9-deficient mice developed normally into functional osteoclasts, which excludes a role for intrinsic S100A8/A9. In contrast to the results observed with S100A9 and S100A8/A9, the addition of S100A8 during osteoclastogenesis resulted in stimulation of osteoclast formation in conjunction with enhanced actin ring formation and increased bone resorption. Analysis of the putative receptor for S100A8 in osteoclastogenesis revealed that osteoclast differentiation and function could not be inhibited by blocking RAGE, whereas the increase in osteoclast numbers and enhanced bone resorption were completely abrogated using TLR-4-deficient osteoclast precursors. CONCLUSION: These results demonstrate that S100A8 stimulated osteoclast formation and activity and suggest that both S100A8 and TLR-4 are important factors in mediating osteoclastic bone destruction in experimental arthritis.

Immune response and expression analysis of cathepsin K in goldfish during Aeromonas hydrophila infection.

The innate immunity and expression profiles of cathepsins D were determined in the goldfish (Carassius auratus) tissues after challenge with a fish pathogen Aeromonas hydrophila. The innate immunity of reactive oxygen species (ROS) and reactive nitrogen species (RNS) were determined by peripheral blood leucocytes. Blood and tissue samples of the muscle, gills, liver, kidney, heart, spleen, and intestine were sampled at 1, 3, 6 and 12 h post-infection for cathepsin D expression by semi-quantitative RT-PCR. The ROS and RNS production did not significantly increase at 1 h post-challenged goldfish. However, the ROS and RNS production was significantly increased after 3 h post-challenged fish compared to the control. The cathepsin D expression was found very low in muscle and kidney of the control fish, other tissues was not found the expression. A similar pattern was found in goldfish at 1 h post-challenge with A. hydrophila. However, at 3 h post-challenge goldfish, the cathepsin D expression was high only in the heart. At 6 h post-challenge goldfish, the cathepsin D expression was seen high all the tissues, except in the spleen. However, the expression was decreased at 12 h post-infection samples. This result was suggested that the goldfish infected with A. hydrophila decreased the innate immunity level in peripheral blood and expressed the cathepsin D in tissues.

The interaction of monocytes with rheumatoid synovial cells is a key step in LIGHT-mediated inflammatory bone destruction.

Formation of osteoclasts and consequent joint destruction are hallmarks of rheumatoid arthritis (RA). Here we show that LIGHT, a member of the tumour necrosis factor (TNF) superfamily, induced the differentiation into tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) of CD14(+) monocytes cocultured with nurse-like cells isolated from RA synovium, but not of freshly isolated CD14(+) monocytes. Receptor activator of nuclear factor-kappaB ligand (RANKL) enhanced this LIGHT-induced generation of TRAP-positive MNCs. The MNCs showed the phenotypical and functional characteristics of osteoclasts; they showed the expression of osteoclast markers such as cathepsin K, actin-ring formation, and the ability to resorb bone. Moreover, the MNCs expressed both matrix metalloproteinase 9 (MMP-9) and MMP-12, but the latter was not expressed in osteoclasts induced from CD14(+) monocytes by RANKL. Immunohistochemical analysis showed that the MMP-12-producing MNCs were present in the erosive areas of joints in RA, but not in the affected joints of osteoarthritic patients. These findings suggested that LIGHT might be involved in the progression of inflammatory bone destruction in RA, and that osteoclast progenitors might become competent for LIGHT-mediated osteoclastogenesis via interactions with synoviocyte-like nurse-like cells.