Long-term glucocorticoid treatment impairs the survival and bone formation of osteogenic cells, leading to bone mass loss. The Wnt inhibitor Dickkopf-1 (DKK1) acts as a potent bone-remodeling factor that mediates several types of skeletal disorders. Whereas excess glucocorticoid is known to disturb Wnt signaling in osteogenic cells, modulation of the skeletally deleterious effects of DKK1 to alleviate glucocorticoid induction of bone loss has not been tested. In this study, knockdown of DKK1 expression by end-capped phosphorothioate DKK1 antisense oligonucleotide (DKK1-AS) abrogated dexamethasone suppression of alkaline phosphatase activity and osteocalcin expression in MC3T3-E1 preosteoblasts. Exogenous DKK1-AS treatment alleviated dexamethasone suppression of mineral density, trabecular bone volume, osteoblast surface, and bone formation rate in bone tissue and ex vivo osteogenesis of primary bone-marrow mesenchymal cells. The DKK1-AS inhibited adipocyte volume in the marrow cavity of steroid-treated bone tissue. Immunohistochemical observation revealed that DKK1-AS abrogated dexamethasone-induced DKK1 expression and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling of osteoblasts adjacent to trabecular bone. Knocking down DKK1 abrogated dexamethasone-modulated expression of nuclear beta-catenin and phosphorylated Ser(473)-Akt and survival of osteoblasts and adipocytic differentiation of mesenchymal progenitor cell cultures. Taken together, knocking down DKK1 alleviated the deleterious effect of glucocorticoid on bone microstructure. The DKK1-AS treatment appeared to protect bone tissue by modulating beta-catenin and Akt-mediated survival as well as the osteogenic and adipogenic activities of glucocorticoid-stressed osteoprogenitor cells. Interference with the osteogenesis-inhibitory action of DKK1 has therapeutic potential for preventing glucocorticoid induction of osteopenia.

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CpG-oligodeoxynucleotides (CpG-ODNs) are potent immune stimuli currently under investigation as antimicrobial agents for different species. Toll-like receptor (TLR) 9 and TLR21 are the cellular receptors of CpG-ODN in mammals and chickens, respectively. The avian genomes lack TLR9, whereas mammalian genomes lack TLR21. Although fish contain both of these genes, the biological functions of fish TLR9 and TLR21 have not been investigated previously. In this study, we comparatively investigated zebrafish TLR9 (zebTLR9) and TLR21 (zebTLR21). The two TLRs have similar expression profiles in zebrafish. They are expressed during early development stages and are preferentially expressed in innate immune function-related organs in adult fish. Results from cell-based activation assays indicate that these two zebrafish TLRs are functional, responding to CpG-ODN but not to other TLR ligands. zebTLR9 broadly recognized CpG-ODN with different CpG motifs, but CpG-ODN with GACGTT or AACGTT had better activity to this TLR. In contrast, zebTLR21 responded preferentially to CpG-ODN with GTCGTT motifs. The distinctive ligand recognition profiles of these two TLRs were determined by their ectodomains. Activation of these two TLRs by CpG-ODN occurred inside the cells and was modulated by UNC93B1. The biological functions of these two TLRs were further investigated. The CpGODNs that activate both zebTLR9 and zebTLR21 were more potent than others that activate only zebTLR9 in the activation of cytokine productions and were more bactericidal in zebrafish. These results suggest that zebTLR9 and zebTLR21 cooperatively mediate the antimicrobial activities of CpG-ODN. Overall, this study provides a molecular basis for the activities of CpG-ODN in fish.pattern recognition receptor | innate immunity | adjuvant B acterial and viral CpG-deoxynucleotides containing DNA (CpG-DNA) represent a type of pathogen-associated molecular pattern (PAMP) that activates immune cells and triggers host responses to microbial infections (1-3). Synthetic phosphorothioate-modified CpG-oligodeoxynucleotides (CpG-ODNs) mimic the functions of CpG-DNA and have been investigated as immune modulators for their adjuvant and antimicrobial activities in different species (4-7). In general, a CpG-ODN contains one or more copies of CpG-deoxynucleotides containing hexamer motifs (CpG motifs). A CpG-ODN's immunostimulatory activities are dependent on its length, the number of CpG motifs, and the position, spacing, and surrounding bases of these CpG motifs.A CpG-ODN can have varying immunostimulatory activity in different species. This species-specific property is determined by the nucleotide context of the CpG motifs within the CpG-ODN. For example, CpG-ODNs containing a purine-purine-CGpyrimidine-pyrimidine motif, such as a GACGTT motif, are more potent in activating murine cells compared with those containing a GTCGTT motif. In contrast, the GTCGTT motif containing CpG-ODN generates stronger immune responses in humans and various domestic animals (8, 9).Toll-like receptors (...

Psoriasis is a chronic inflammatory skin disorder that affects ~2%–3% of the worldwide population. Inappropriate and excessive activation of endosomal Toll-like receptors 7, 8, and 9 (TLRs 7–9) at the psoriatic site has been shown to play a pathogenic role in the onset of psoriasis. Macrophage is a major inflammatory cell type that can be differentiated into phenotypes M1 and M2. M1 macrophages produce proinflammatory cytokines, and M2 macrophages produce anti-inflammatory cytokines. The balance between these two types of macrophages determines the progression of various inflammatory diseases; however, whether macrophage polarization plays a role in psoriatic inflammation activated by endosomal TLRs has not been investigated. In this study, we investigated the function and mechanism of macrophages related to the pathogenic role of TLRs 7–9 in the progression of psoriasis. Analysis of clinical data in database revealed significantly increased expression of macrophage markers and inflammatory cytokines in psoriatic tissues over those in normal tissues. In animal studies, depletion of macrophages in mice ameliorated imiquimod, a TLR 7 agonist-induced psoriatic response. Imiquimod induced expression of genes and cytokines that are signature of M1 macrophage in the psoriatic lesions. In addition, treatment with this TLR 7 agonist shifted macrophages in the psoriatic lesions to a higher M1/M2 ratio. Both of the exogenous and endogenous TLR 7–9 ligands activated M1 macrophage polarization. M1 macrophages expressed higher levels of proinflammatory cytokines and TLRs 7–9 than M2 macrophages. These results suggest that by rendering macrophages into a more inflammatory status and capable of response to their ligands in the psoriatic sites, TLR 7–9 activation drives them to participate in endosomal TLR-activated psoriatic inflammation, resulting in an amplified inflammatory response. Our results also suggest that blocking M1 macrophage polarization could be a strategy which enables inhibition of psoriatic inflammation activated by these TLRs.