Indoleamine 2,3-dioxygenase-1 involves in CD8+T cell exhaustion in glioblastoma via regulating tryptophan levels.

Indoleamine 2,3-dioxygenase-1 (IDO-1) is an enzyme that catalyzes the metabolism of tryptophan (Trp). It is expressed in limited amounts in normal tissues but significantly upregulated during inflammation and infection. Various inflammatory factors, especially IFN-γ, can induce the expression of IDO-1. While extensive research has been conducted on the role of IDO-1 in tumors, its specific role in complex central nervous system tumors such as glioblastoma (GBM) remains unclear. This study aims to explore the role of IDO-1 in the development of GBM and analyze its association with tryptophan levels and CD8+T cell exhaustion in the tumor region. To achieve this, we constructed an orthotopic mouse glioblastoma tumor model to investigate the specific mechanisms between IDO-1, GBM, and CD8+T cell exhaustion. Our results showed that IDO-1 can promote CD8+T cell exhaustion by reducing tryptophan levels. When IDO-1 was knocked down in glioblastoma cells, other cells within the tumor microenvironment upregulated IDO-1 expression to compensate for the loss and enhance immunosuppressive effects. Therefore, the data suggest that the GBM microenvironment controls tryptophan levels by regulating IDO-1 expression, which plays a critical role in immune suppression. These findings support the use of immune therapy in combination with IDO-1 inhibitors or tryptophan supplementation as a potential treatment strategy.

Exogenous Angiotensin-(1-7) Provides Protection Against Inflammatory Bone Resorption and Osteoclastogenesis by Inhibition of TNF-α Expression in Macrophages.

Renin-angiotensin-aldosterone system plays a crucial role in the regulation of blood pressure and fluid homeostasis. It is reported to be involved in mediating osteoclastogenesis and bone loss in diseases of inflammatory bone resorption such as osteoporosis. Angiotensin-(1-7), a product of Angiotensin I and II (Ang I, II), is cleaved by Angiotensin-converting enzyme 2 and then binds to Mas receptor to counteract inflammatory effects produced by Ang II. However, the mechanism by which Ang-(1-7) reduces bone resorption remains unclear. Therefore, we aim to elucidate the effects of Ang-(1-7) on lipopolysaccharide (LPS)-induced osteoclastogenesis. In vivo, mice were supracalvarial injected with Ang-(1-7) or LPS ± Ang-(1-7) subcutaneously. Bone resorption and osteoclast formation were compared using micro-computed tomography, tartrate-resistant acid phosphatase (TRAP) stain, and real-time PCR. We found that Ang-(1-7) attenuated tumor necrosis factor (TNF)-α, TRAP, and Cathepsin K expression from calvaria and decreased osteoclast number along with bone resorption at the suture mesenchyme. In vitro, RANKL/TNF-α ± Ang-(1-7) was added to cultures of bone marrow-derived macrophages (BMMs) and osteoclast formation was measured via TRAP staining. The effect of Ang-(1-7) on LPS-induced osteoblasts RANKL expression and peritoneal macrophages TNF-α expression was also investigated. The effect of Ang-(1-7) on the MAPK and NF-κB pathway was studied by Western blotting. As a result, Ang-(1-7) reduced LPS-stimulated macrophages TNF-α expression and inhibited the MAPK and NF-κB pathway activation. However, Ang-(1-7) did not affect osteoclastogenesis induced by RANKL/TNF-α nor reduce osteoblasts RANKL expression in vitro. In conclusion, Ang-(1-7) alleviated LPS-induced osteoclastogenesis and bone resorption in vivo via inhibiting TNF-α expression in macrophages.

Effect of age on orthodontic tooth movement in mice.

Background/purpose: The number of middle-aged and elderly orthodontic patients is increasing due to changes in age composition. It is important to investigate the detailed mechanisms of bone remodeling in orthodontic tooth movement (OTM) in the elderly. However, there are few reports on the mechanism of tooth movement in the elderly. The purpose of the present study was to analyze OTM and osteoclastogenesis in aged mice and to elucidate the mechanism. Materials and methods: It has been reported that tumor necrosis factor (TNF)-α plays an important role in osteoclast formation and OTM. First, 8-week-old and 78-week-old male C57BL/6J mice were subcutaneously injected with TNF-α into the calvaiae, and micro-CT, tartrate-resistant acid phosphatase (TRAP) staining, and real-time PCR were performed to evaluate osteoclast formation and bone resorption. Furthermore, osteoclastogenesis by TNF-α and receptor activator of nuclear factor-kappa B ligand (RANKL) using bone marrow cells was evaluated in vitro. Finally, a nickel-titanium closed-coil spring was attached, mesial movement of the maxillary left first molar was performed, and tooth movement distance and osteoclast formation were evaluated. Results: Compared to 8-week-old mice, 78-week-old mice had decreased TNF-α-induced bone resorption, osteoclastogenesis, and TRAP and cathepsin K expression in the calvariae. In vitro osteoclast formation also decreased in 78-week-old mice. Furthermore, tooth movement distance and osteoclastogenesis were reduced. Conclusion: OTM decreased in aged mice, which was shown to be caused by a decrease in osteoclastogenesis. Therefore, it was suggested that it is necessary to keep in mind that tooth movement may be suppressed when treating elderly patients.

Serum ITIH5 as a novel diagnostic biomarker in cholangiocarcinoma.

Cholangiocarcinoma often remains undetected until advanced stages due to the lack of reliable diagnostic markers. Our goal was to identify a unique secretory protein for cholangiocarcinoma diagnosis and differentiation from other malignancies, benign hepatobiliary diseases, and chronic liver conditions. We conducted bulk RNA-seq analysis to identify genes specifically upregulated in cholangiocarcinoma but not in most other cancers, benign hepatobiliary diseases, and chronic liver diseases focusing on exocrine protein-encoding genes. Single-cell RNA sequencing examined subcellular distribution. Immunohistochemistry and enzyme-linked immunosorbent assays assessed tissue and serum expression. Diagnostic performance was evaluated via receiver-operating characteristic (ROC) analysis. Inter-alpha-trypsin inhibitor heavy chain family member five (ITIH5), a gene encoding an extracellular protein, is notably upregulated in cholangiocarcinoma. This elevation is not observed in most other cancer types, benign hepatobiliary diseases, or chronic liver disorders. It is specifically expressed by malignant cholangiocytes. ITIH5 expression in cholangiocarcinoma tissues exceeded that in nontumorous bile duct, hepatocellular carcinoma, and nontumorous hepatic tissues. Serum ITIH5 levels were elevated in cholangiocarcinoma compared with controls (hepatocellular carcinoma, benign diseases, chronic hepatitis B, and healthy individuals). ITIH5 yielded areas under the ROC curve (AUCs) from 0.839 to 0.851 distinguishing cholangiocarcinoma from controls. Combining ITIH5 with carbohydrate antigen 19-9 (CA19-9) enhanced CA19-9's diagnostic effectiveness. In conclusion, serum ITIH5 may serve as a novel noninvasive cholangiocarcinoma diagnostic marker.

(D-Ala2)GIP Inhibits Inflammatory Bone Resorption by Suppressing TNF-α and RANKL Expression and Directly Impeding Osteoclast Formation.

Glucose-insulinotropic polypeptide (GIP) is an incretin hormone that induces insulin secretion and decreases blood glucose levels. In addition, it has been reported to suppress osteoclast formation. Native GIP is rapidly degraded by dipeptidyl peptidase-4 (DPP-4). (D-Ala2)GIP is a newly developed GIP analog that demonstrates enhanced resistance to DPP-4. This study aimed to evaluate the influence of (D-Ala2)GIP on osteoclast formation and bone resorption during lipopolysaccharide (LPS)-induced inflammation in vivo and in vitro. In vivo, mice received supracalvarial injections of LPS with or without (D-Ala2)GIP for 5 days. Osteoclast formation and bone resorption were evaluated, and TNF-α and RANKL expression were measured. In vitro, the influence of (D-Ala2)GIP on RANKL- and TNF-α-induced osteoclastogenesis, LPS-triggered TNF-α expression in macrophages, and RANKL expression in osteoblasts were examined. Compared to the LPS-only group, calvariae co-administered LPS and (D-Ala2)GIP led to less osteoclast formation, lower bone resorption, and decreased TNF-α and RANKL expression. (D-Ala2)GIP inhibited osteoclastogenesis induced by RANKL and TNF-α and downregulated TNF-α expression in macrophages and RANKL expression in osteoblasts in vitro. Furthermore, (D-Ala2)GIP suppressed the MAPK signaling pathway. The results suggest that (D-Ala2)GIP dampened LPS-triggered osteoclast formation and bone resorption in vivo by reducing TNF-α and RANKL expression and directly inhibiting osteoclastogenesis.

Generating Bone Marrow Chimeric Mouse Using GPR120 Deficient Mouse for the Study of DHA Inhibitory Effect on Osteoclast Formation and Bone Resorption.

Docosahexaenoic acid (DHA) is an omega-3 fatty acid that exerts physiological effects via G protein-coupled receptor 120 (GPR120). In our previous studies, we figured out the inhibitory effects of DHA on TNF-α (Tumor necrosis factor-α)-induced osteoclastogenesis via GPR120 in vivo. Moreover, DHA directly suppressed RANKL expression in osteoblasts via GPR120 in vitro. In this study, we generated bone marrow chimeric mice using GPR120 deficient mice (GPR120-KO) to study the inhibitory effects of DHA on bone resorption and osteoclast formation. Bone marrow cells of wild-type (WT) or GPR120-KO mice were transplanted into irradiated recipient mice, which were WT or GPR120 deficient mice. The resulting chimeric mice contained stromal cells from the recipient and bone marrow cells, including osteoclast precursors, from the donor. These chimeric mice were used to perform a series of histological and microfocus computed tomography (micro-CT) analyses after TNF-α injection for induction of osteoclast formation with or without DHA. Osteoclast number and bone resorption were found to be significantly increased in chimeric mice, which did not express GPR120 in stromal cells, compared to chimeric mice, which expressed GPR120 in stromal cells. DHA was also found to suppress specific signaling pathways. We summarized that DHA suppressed TNF-α-induced stromal-dependent osteoclast formation and bone resorption via GPR120.

Azilsartan inhibits inflammation-triggered bone resorption and osteoclastogenesis in vivo via suppression of TNF-α expression in macrophages.

Introduction: Hypertension is a major risk factor for cardiovascular disease (CVD) and is associated with increased bone loss due to excessive activity of the local renin-angiotensin system (RAS). Angiotensinogen/Angiotensin (ANG) II/Angiotensin II type 1 receptor (AT1R) axis is considered as the core axis regulating RAS activity. Azilsartan is an FDA-approved selective AT1R antagonist that is used to treat hypertension. This study aimed to determine whether azilsartan affects formation of osteoclast, resorption of bone, and the expression of cytokines linked with osteoclastogenesis during lipopolysaccharide (LPS)-triggered inflammation in vivo. Methods: In vivo, following a 5-day supracalvarial injection of LPS or tumor necrosis factor-alpha (TNF-α) with or without azilsartan, the proportion of bone resorption and the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, which are identified as osteoclasts on mice calvariae were counted. The mRNA expression levels of TRAP, cathepsin K, receptor activator of NF-κB ligand (RANKL), and TNF-α were also evaluated. In vitro, the effect of azilsartan (0, 0.01, 0.1, 1, and 10 µM) on RANKL and TNF-α-triggered osteoclastogenesis were investigated. Also, whether azilsartan restrains LPS-triggered TNF-α mRNA and protein expression in macrophages and RANKL expression in osteoblasts were assessed. Furthermore, western blotting for analysis of mitogen-activated protein kinases (MAPKs) signaling was conducted. Results: Azilsartan-treated calvariae exhibited significantly lower bone resorption and osteoclastogenesis than those treated with LPS alone. In vivo, LPS with azilsartan administration resulted in lower levels of receptor activator of RANKL and TNF-α mRNA expression than LPS administration alone. Nevertheless, azilsartan did not show inhibitory effect on RANKL- and TNF-α-triggered osteoclastogenesis in vitro. Compared to macrophages treated with LPS, TNF-α mRNA and protein levels were lower in macrophages treated by LPS with azilsartan. In contrast, RANKL mRNA and protein expression levels in osteoblasts were the same in cells co-treated with azilsartan and LPS and those exposed to LPS only. Furthermore, azilsartan suppressed LPS-triggered MAPKs signaling pathway in macrophages. After 5-day supracalvarial injection, there is no difference between TNF-α injection group and TNF-α with azilsartan injection group. Conclusion: These findings imply that azilsartan prevents LPS-triggered TNF-α production in macrophages, which in turn prevents LPS-Triggered osteoclast formation and bone resorption in vivo.

Enhancement of orthodontic tooth movement and root resorption in ovariectomized mice.

Background/purpose: As the number of patients with osteoporosis requiring orthodontic treatment is increasing with the aging of society, it is necessary to evaluate the relations between bone metabolism in old age and orthodontic tooth movement (OTM). However, the effects of changes in bone metabolism due to osteoporosis on OTM and root resorption are still unclear. Therefore, we investigated the effects of OTM and root resorption in a mouse ovariectomy (OVX)-induced osteoporosis model. Materials and methods: Eight-week-old female wild-type mice underwent OVX or sham surgery (Sham) as controls. One month after treatment, a nickel titanium coil spring was used to apply a mesial force to the maxillary left first molars of OVX or Sham mice for 12 days. The distance between the maxillary first molar and the second molar changed due to OTM and osteoclast formation was evaluated. The odontoclast formation and root resorption along the root surface of the distobuccal root of the first molar was also evaluated by histological analysis and scanning electron microscopy. Results: Distance of tooth movement and osteoclast formation were significantly increased in OVX mice compared to Sham controls. Furthermore, root resorption in the mesial surface of the distal molars induced by orthodontic force was significantly increased in OVX mice. Conclusion: The amount of OTM was significantly increased, and the accompanying root resorption was also increased in OVX mice. Therefore, attention should be paid to the risk of root resorption associated with orthodontic treatment in patients with osteoporosis.

Micro-Osteoperforations Induce TNF-α Expression and Accelerate Orthodontic Tooth Movement via TNF-α-Responsive Stromal Cells.

Micro-osteoperforations (MOPs) have been reported to accelerate orthodontic tooth movement (OTM), and tumor necrosis factor (TNF)-α has been reported to play a crucial role in OTM. In this report, the influence of MOPs during OTM was analyzed. We evaluated the expression of TNF-α with and without MOPs by RT-PCR analysis. A Ni-Ti closed coil spring was fixed between the maxillary left first molar and the incisors as an OTM mouse model to move the first molar in the mesial direction. MOPs were prepared on the lingual side and mesial side of the upper first molars. Furthermore, to investigate the target cell of TNF-α for osteoclast formation during OTM with MOPs in vivo, we created four types of chimeric mice in which bone marrow of wild-type (WT) or TNF receptor 1- and 2-deficient mice (KO) was transplanted into lethally irradiated WT or KO mice. The results showed that MOPs increased TNF-α expression, the distance of tooth movement and osteoclast formation significantly. Furthermore, mice with TNF-α-responsive stromal cells showed a significant increase in tooth movement and number of osteoclasts by MOPs. We conclude that MOPs increase TNF-α expression, and tooth movement is dependent on TNF-α-responsive stromal cells.

Role of the Interaction of Tumor Necrosis Factor-α and Tumor Necrosis Factor Receptors 1 and 2 in Bone-Related Cells.

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine expressed by macrophages, monocytes, and T cells, and its expression is triggered by the immune system in response to pathogens and their products, such as endotoxins. TNF-α plays an important role in host defense by inducing inflammatory reactions such as phagocytes and cytocidal systems activation. TNF-α also plays an important role in bone metabolism and is associated with inflammatory bone diseases. TNF-α binds to two cell surface receptors, the 55kDa TNF receptor-1 (TNFR1) and the 75kDa TNF receptor-2 (TNFR2). Bone is in a constant state of turnover; it is continuously degraded and built via the process of bone remodeling, which results from the regulated balance between bone-resorbing osteoclasts, bone-forming osteoblasts, and the mechanosensory cell type osteocytes. Precise interactions between these cells maintain skeletal homeostasis. Studies have shown that TNF-α affects bone-related cells via TNFRs. Signaling through either receptor results in different outcomes in different cell types as well as in the same cell type. This review summarizes and discusses current research on the TNF-α and TNFR interaction and its role in bone-related cells.

Tumor necrosis factor-α enhances the expression of vascular endothelial growth factor in a mouse orthodontic tooth movement model.

BACKGROUND/PURPOSE: Tooth movement that is achieved using orthodontic mechanical principles relies on bone resorption which takes place on the compression side via osteoclasts. Tumor necrosis factor-α (TNF-α) has been known to affect osteoclast formation in orthodontic tooth movement (OTM). Vascular endothelial growth factor (VEGF), which is one of the mediators of angiogenesis, also plays an important role in OTM by inducing vascular permeability and chemotaxis of osteoclast precursors. Therefore, the purpose of this research was to evaluate the effect of TNF-α on VEGF expression during OTM. MATERIALS AND METHODS: In order to demonstrate the effect of TNF-α on VEGF expression during OTM, a nickel titanium closed coil spring was fixed to the upper left first molar and the alveolar bone beneath the upper incisors of both wild type (WT) and TNF receptors (TNFRs) deficient mice resulting in a mesial movement of the molar for 12 days. The maxilla was removed for histological analysis and real-time RCR analysis of VEGF expression. RESULTS: Immunohistochemical analysis revealed that there were fewer VEGF-positive cells in the periodontal membrane on the mesial side of the distobuccal root in TNFRs-deficient mice than that in WT mice during the OTM for 12 days. Furthermore, expression of VEGF mRNA is lower level in TNFRs-deficient mice than that in WT mice. CONCLUSION: Our results indicate that TNF-α plays an important role in VEGF expression during tooth movement.

Docosahexaenoic acid inhibits TNF-α-induced osteoclast formation and orthodontic tooth movement through GPR120.

Docosahexaenoic acid (DHA) is an omega-3 fatty acid that has a range of positive impacts on human health, including anti-inflammatory effects and inhibition of osteoclast formation via G-protein-coupled receptor 120 (GPR120). Orthodontic force was reported to induce tumor necrosis factor-α (TNF-α) expression, which activates osteoclast differentiation during orthodontic tooth movement (OTM). The aim of this study was to investigate the influence of DHA on TNF-α-induced osteoclast formation and OTM in vivo. We examined osteoclast formation and bone resorption within the calvaria of both wild-type (WT) and GPR120-deficient (GPR120-KO) mice injected with phosphate-buffered saline (PBS), TNF-α, TNF-α and DHA, or DHA. DHA inhibited TNF-α-induced osteoclast formation and bone resorption in WT mice but had no effect in GPR120-KO mice. OTM experiments were performed in mouse strains with or without regular injection of DHA, and the effects of DHA on osteoclast formation in the alveolar bones during OTM were examined. DHA also suppressed OTM in WT but not GPR120-KO mice. Our data showed that DHA suppresses TNF-α-induced osteoclastogenesis and bone resorption via GPR120. TNF-α has considerable significance in OTM, and therefore, DHA may also inhibit TNF-α-induced osteoclast formation and bone resorption in OTM.

Effect of TNF-α on osteocyte RANKL expression during orthodontic tooth movement.

BACKGROUND/PURPOSE: Orthodontic tooth movement (OTM) is facilitated by two events; bone resorption on the compression side and bone formation on the tension side simultaneously termed bone remodeling. Osteocytes play a critical role in bone remodeling during OTM, as they have been described as the critical source of nuclear factor-κB ligand (RANKL) necessary for bone remodeling during OTM. Tumor necrosis factor (TNF)-α is a cytokine that acts by amplifying RANKL expression in osteocytes. In this study, we evaluated the effects of TNF-α on RANKL expression in osteocyte during OTM. MATERIALS AND METHODS: We assessed whether TNF-α influenced RANKL expression in osteocyte during orthodontic tooth movement by using wild-type (WT) and TNF receptor I and II deficient (TNFRsKO) mice. A Nickel-titanium closed coil spring was attached to the maxillary alveolar bone near the incisors and the upper left first molar, and the first molars were moved mesially in WT and TNFRsKO mice. After OTM, the number of RANKL-positive osteocytes in the alveolar bone was evaluated by immunohistochemistry. RESULTS: The number of RANKL-positive osteocyte in the alveolar bone significantly increased in WT mice than in TNFRsKO mice after OTM. CONCLUSION: The results indicate that TNF-α induces the expression of RANKL in osteocyte during OTM.