Changes in the astronaut serum proteome during prolonged spaceflight.

The molecular mechanisms associated with spaceflight-induced biological adaptations that may affect many healthy tissue functions remain poorly understood. In this study, we analyzed temporal changes in the serum proteome of six astronauts during prolonged spaceflight missions using quantitative comprehensive proteome analysis performed with the data-independent acquisition method of mass spectrometry (DIA-MS). All six astronauts participated in a spaceflight mission for approximately 6 months and showed a decreasing trend in T-scores at almost all sites where dual-energy X-ray absorptiometry scans were performed. DIA-MS successfully identified 624 nonredundant proteins in sera and further quantitative analysis for each sampling point provided information on serum protein profiles closely related to several time points before (pre-), during (in-), and after (post-) spaceflight. Changes in serum protein levels between spaceflight and on the ground suggest that abnormalities in bone metabolism are induced in astronauts during spaceflight. Furthermore, changes in the proteomic profile occurring during spaceflight suggest that serum levels of bone metabolism-related proteins, namely ALPL, COL1A1, SPP1, and POSTN, could serve as highly responsive indicators of bone metabolism status in spaceflight missions. This study will allow us to accelerate research to improve our understanding of the molecular mechanisms of biological adaptations associated with prolonged spaceflight.

Dipotassium glycyrrhizate and hinokitiol enhance macrophage efferocytosis by regulating recognition, uptake, and metabolism of apoptotic cells in vitro.

BACKGROUND AND OBJECTIVE: Efferocytosis is a process whereby macrophages remove apoptotic cells, such as neutrophils, that have accumulated in tissues, which is required for resolution of inflammation. Efferocytosis is impaired in individuals with increasing age and in those with various systemic diseases. Recently, efferocytosis has been reported to be related to the pathogenesis and progression of periodontitis, and enhancement of efferocytosis, especially in the subjects with impaired efferocytosis, was suggested to lead to periodontitis prevention and care. Various anti-inflammatory ingredients are used in oral care products, but their effect on efferocytosis is unclear. Here, we aimed to identify ingredients contained in oral care products that are effective for efferocytosis regulation. METHODS: The ability of dead cells to induce inflammation in human gingival fibroblast (HGF) cells were evaluated by measuring IL-6 secretion. Six ingredients in oral care products used as anti-inflammatory agents were evaluated for their effect on efferocytosis using flow cytometry. The expression of various efferocytosis-related molecules, such as MERTK and LRP1 involved in recognition, and LXRα and ABCA1 that function in metabolism, were measured in RAW264.7 cells with or without ingredient treatment. Rac1 activity, which is related to the uptake of dead cells, was measured using the G-LISA kit. RESULTS: Dead cells elicited IL-6 secretion in HGF cells. Among the six ingredients, GK2 and hinokitiol enhanced efferocytosis activity. GK2 and hinokitiol significantly increased the expression of MERTK and LRP1, and also enhanced LXRα and ABCA1 expression after efferocytosis. Furthermore, they increased Rac1 activity in the presence of dead cells. CONCLUSION: Among the six ingredients tested, GK2 and hinokitiol promoted efferocytosis by regulating apoptotic cell recognition, uptake, and metabolism-related molecules. Efferocytosis upregulation may be one of the mechanisms of GK2 and hinokitiol in the treatment of inflammatory diseases, such as periodontitis.

A cross-sectional study of relationships between periodontal disease and general health: The Hitachi Oral Healthcare Survey.

BACKGROUND: This cross-sectional study performed to clarify the relationship between periodontal disease and non-communicable diseases (NCDs), such as obesity, diabetes mellitus, impaired glucose tolerance (IGT), chronic obstructive pulmonary disease (COPD), and atherosclerotic cardiovascular disease (ASCVD) by introducing dental examinations into the annual health examinations conducted by Japanese companies, and to highlights the importance of a medical system that connects dental and medical professionals. METHODS: A total of 1.022 Hitachi Ltd. employees were enrolled in this cross-sectional study. We examined correlations and odds ratios (ORs) between the dental and overall health of employees using stratification and multiple logistic regression analyses based on the periodontal health indicators, general health indicators, and occlusal force. RESULTS: The adjusted OR of PPD for obesity (OR, 1.42; 95% confidence interval [CI], 1.09-1.84; p = 0.009), IGT (OR, 1.48; 95% CI, 1.00-2.20; p = 0.049), and COPD (OR, 1.38; 95% CI, 1.02-1.88; p = 0.038) significantly differed. The adjusted OR of body mass index (OR, 1.28; 95% CI 1.15-1.42; p < 0.001), haemoglobin A1C (HbA1c) (OR, 4.34; 95% CI, 1.89-9.98; p < 0.001), fasting blood glucose (FBG) levels (OR, 1.08; 95% CI 1.04-1.11; p < 0.001), postbronchodilator forced expiratory volume in one second/forced vital capacity ratio (%FEV1) (OR, 0.95; 95% CI 0.91-1.00; p = 0.031) and smoking (OR, 2.32; 95% CI 1.62-3.33; p < 0.001) for severe periodontal disease also significantly differed. Occlusal force was significantly reduced in employees aged 50-59 years compared to those aged 40-49 years. Both PPD, HbA1c, FBG levels were significantly associated with occlusal force among employees with moderate/severe periodontitis. PPD was significantly associated with occlusal force among employees with and moderate COPD, and ASCVD. %FEV1 was significantly associated with occlusal force among employees with IGT. CONCLUSIONS: This cross-sectional study revealed mutual relationships among periodontal disease, NCDs, and occlusal force on Japanese corporate workers. We demonstrated that a comprehensive, regional healthcare system centred on annual integrated dental and physical health examinations in the workplace will benefit employees and positively impact corporate health insurance.

OC-STAMP promotes osteoclast fusion for pathogenic bone resorption in periodontitis via up-regulation of permissive fusogen CD9.

Cell fusion-mediated formation of multinuclear osteoclasts (OCs) plays a key role in bone resorption. It is reported that 2 unique OC-specific fusogens [ i.e., OC-stimulatory transmembrane protein (OC-STAMP) and dendritic cell-specific transmembrane protein (DC-STAMP)], and permissive fusogen CD9, are involved in OC fusion. In contrast to DC-STAMP-knockout (KO) mice, which show the osteopetrotic phenotype, OC-STAMP-KO mice show no difference in systemic bone mineral density. Nonetheless, according to the ligature-induced periodontitis model, significantly lower level of bone resorption was found in OC-STAMP-KO mice compared to WT mice. Anti-OC-STAMP-neutralizing mAb down-modulated in vitro: 1) the emergence of large multinuclear tartrate-resistant acid phosphatase-positive cells, 2) pit formation, and 3) mRNA and protein expression of CD9, but not DC-STAMP, in receptor activator of NF-κB ligand (RANKL)-stimulated OC precursor cells (OCps). While anti-DC-STAMP-mAb also down-regulated RANKL-induced osteoclastogenesis in vitro, it had no effect on CD9 expression. In our mouse model, systemic administration of anti-OC-STAMP-mAb suppressed the expression of CD9 mRNA, but not DC-STAMP mRNA, in periodontal tissue, along with diminished alveolar bone loss and reduced emergence of CD9+ OCps and tartrate-resistant acid phosphatase-positive multinuclear OCs. The present study demonstrated that OC-STAMP partners CD9 to promote periodontal bone destruction by up-regulation of fusion during osteoclastogenesis, suggesting that anti-OC-STAMP-mAb may lead to the development of a novel therapeutic regimen for periodontitis.-Ishii, T., Ruiz-Torruella, M., Ikeda, A., Shindo, S., Movila, A., Mawardi, H., Albassam, A., Kayal, R. A., Al-Dharrab, A. A., Egashira, K., Wisitrasameewong, W., Yamamoto, K., Mira, A. I., Sueishi, K., Han, X., Taubman, M. A., Miyamoto, T., Kawai, T. OC-STAMP promotes osteoclast fusion for pathogenic bone resorption in periodontitis via up-regulation of permissive fusogen CD9.

Endogenous acid ceramidase protects epithelial cells from Porphyromonas gingivalis-induced inflammation in vitro.

Ceramidases are a group of enzymes that degrade pro-inflammatory ceramide by cleaving a fatty acid to form anti-inflammatory sphingosine lipid. Thus far, acid, neutral and alkaline ceramidase isozymes have been described. However, the expression patterns of ceramidase isoforms as well as their role in periodontal disease pathogenesis remain unknown. In this study, expression patterns of ceramidase isoforms were quantified by real-time PCR and immunohistochemistry in gingival samples of patients with periodontitis and healthy subjects, as well as in EpiGingivalTM-3D culture and OBA-9 gingival epithelial cells both of which were stimulated with or without the presence of live Porphyromonas gingivalis (ATCC 33277 strain). A significantly lower level of acid ceramidase expression was detected in gingival tissues from periodontal patients compared to those from healthy subjects. In addition, acid-ceramidase expression in EpiGingival™ 3D culture and OBA-9 cells was suppressed by stimulation with P. gingivalis in vitro. No significant fluctuation was detected for neutral or alkaline ceramidases in either gingival samples or cell cultures. Next, to elucidate the role of acid ceramidase in P. gingivalis-induced inflammation in vitro, OBA-9 cells were transduced with adenoviral vector expressing the human acid ceramidase (Ad-ASAH1) gene or control adenoviral vector (Ad-control). In response to stimulation with P. gingivalis, ASAH1-over-expressing OBA-9 cells showed significantly lower mRNA expressions of caspase-3 as well as the percentage of Annexin V-positive cells, when compared with OBA-9 cells transduced with Ad-control vector. Furthermore, in response to stimulation with P. gingivalis, ASAH1-over-expressing OBA-9 cells produced less TNF-α, IL-6, and IL1ß pro-inflammatory cytokines than observed in OBA-9 cells transduced with Ad-control vector. Collectively, our data show the novel discovery of anti-inflammatory and anti-apoptotic effects of acid ceramidase in host cells exposed to periodontal bacteria, and the attenuation of the expression of host-protective acid ceramidase in periodontal lesions.

Phosphoglycerol dihydroceramide, a distinctive ceramide produced by Porphyromonas gingivalis, promotes RANKL-induced osteoclastogenesis by acting on non-muscle myosin II-A (Myh9), an osteoclast cell fusion regulatory factor.

Among several virulence factors produced by the periodontal pathogen Porphyromonas gingivalis (Pg), a recently identified novel class of dihydroceramide lipids that contains a long acyl-chain has the potential to play a pathogenic role in periodontitis because of its higher level of tissue penetration compared to other lipid classes produced by Pg. However, the possible impact of Pg ceramides on osteoclastogenesis is largely unknown. In the present study, we report that the phosphoglycerol dihydroceramide (PGDHC) isolated from Pg enhanced osteoclastogenesis in vitro and in vivo. Using RAW264.7 cells, in vitro assays indicated that PGDHC can promote RANKL-induced osteoclastogenesis by generating remarkably larger TRAP+ multinuclear osteoclasts compared to Pg LPS in a TLR2/4-independent manner. According to fluorescent confocal microscopy, co-localization of non-muscle myosin II-A (Myh9) and PGDHC was observed in the cytoplasm of osteoclasts, indicating the membrane-permeability of PGDHC. Loss- and gain-of-function assays using RNAi-based Myh9 gene silencing, as well as overexpression of the Myh9 gene, in RAW264.7 cells showed that interaction of PGDHC with Myh9 enhances RANKL-induced osteoclastogenesis. It was also demonstrated that PGDHC can upregulate the expression of dendritic cell-specific transmembrane protein (DC-STAMP), an important osteoclast fusogen, through signaling that involves Rac1, suggesting that interaction of PGDHC with Myh9 can elicit the cell signal that promotes osteoclast cell fusion. Taken together, our data indicated that PGDHC is a Pg-derived, cell-permeable ceramide that possesses a unique property of promoting osteoclastogenesis via interaction with Myh9 which, in turn, activates a Rac1/DC-STAMP pathway for upregulation of osteoclast cell fusion.

Possible pathogenic engagement of soluble Semaphorin 4D produced by γδT cells in medication-related osteonecrosis of the jaw (MRONJ).

Prior consensus held that medication-related osteonecrosis of the jaw (MRONJ) lesion was composed of necrotic bone; however, more recent studies have identified inflammatory infiltrates in the lesion. Herein, we report that remarkably elevated infiltrating γδT cells (90% of lymphocytes) express Semaphorin 4D (Sema4D) in human patient with MRONJ lesion, whereas γδT cells only account for 2-5% of lymphocytes in blood. Importantly, Sema4D is implicated in the pathogenesis of T cell-mediated inflammatory diseases, such as rheumatoid arthritis and multiple sclerosis. Indeed, in a mouse model of MRONJ, an elevated number of γδT, but not αßT, cells infiltrating in the MRONJ-like lesion was observed. Both elevated soluble Sema4D (sSema4D) production accompanied by pro-inflammatory cytokines, including TNF-α IFN-γ and IL-1ß, and Sema4D-expressing γδT cells were detected in mouse MRONJ-like lesion. Activated γδT cells produced sSema4D in vitro, which could promote TNF-α production from macrophages. Meanwhile, γδT cell-KO mice were resistant to the induction of MRONJ and, hence, showed no elevation of local productions of Sema4D and TNF-α. Finally, systemic administration of anti-Sema4D neutralizing mAb suppressed the onset of MRONJ in wild-type mice in conjunction with diminished level of TNF-α. These results suggested a critical pathogenic engagement of Sema4D produced by γδT cells in the development of MRONJ.

Use of data-independent acquisition mass spectrometry to identify an objective serum indicator of the need for osteoporotic therapeutic intervention.

Osteoporosis is characterized by weakened bone microstructure and loss of bone mass. Current diagnostic criteria for osteoporosis are based on the T-score, which is a measure of bone mineral density. However, osteoporotic fragility fractures can occur regardless of the T-score, underscoring the need for additional criteria for the early detection of patients at fracture risk. To identify indicators of reduced bone strength, we performed serum proteomic analysis using data-independent acquisition mass spectrometry with serum samples from two patient groups, one with osteoporosis but no fractures and the other with osteopenia and fragility fractures. Collective evaluation of the results identified six serum proteins that changed to a similar extent in both patient groups compared with controls. Of these, extracellular matrix protein 1 (ECM1), which contributes to bone formation, showed the most significant increase in serum levels in both patient groups. An ELISA-based assay suggested that ECM1 could serve as a serum indicator of the need for therapeutic intervention; however, further prospective studies with a larger sample size are necessary to confirm these results. The present findings may contribute to the provision of early and appropriate therapeutic strategies for patients at risk of osteoporotic fractures. SIGNIFICANCE: This study aimed to identify objective serum indicators of the need for therapeutic intervention in individuals at risk of osteoporotic fracture. Comprehensive proteome analyses of serum collected from patients with osteoporosis but no fractures, patients with osteopenia and fragility fractures, and controls were performed by data-independent acquisition mass spectrometry. Collective evaluation of the proteome analysis data and ELISA-based assays identified serum ECM1 as a potential objective marker of the risk of fragility fractures in patients with osteoporosis or osteopenia. The findings are an important step toward the development of appropriate bone health management methods to improve well-being and maintain quality of life.

Identification of gravity-responsive proteins in the femur of spaceflight mice using a quantitative proteomic approach.

Although the microgravity (µ-g) environment that astronauts encounter during spaceflight can cause severe acute bone loss, the molecular mechanism of this bone loss remains unclear. To investigate the gravity-response proteins involved in bone metabolism, it is important to comprehensively determine which proteins exhibit differential abundance associated with mechanical stimuli. However, comprehensive proteomic analysis using small bone samples is difficult because protein extraction in mineralized bone tissue is inefficient. Here, we established a high-sensitivity analysis system for mouse bone proteins using data-independent acquisition mass spectrometry. This system successfully detected 40 proteins in the femoral diaphysis showing differential abundance between mice raised in a µ-g environment, where the bone mass was reduced by gravity unloading, and mice raised in an artificial 1-gravity environment on the International Space Station. Additionally, 22 proteins, including noncollagenous bone matrix proteins, showed similar abundance between the two groups in the mandible, where bone mass was unaltered due to mastication stimuli, suggesting that these proteins are responsive to mechanical stimuli. One of these proteins, SPARCL1, is suggested to promote osteoclastogenesis induced by receptor activator of nuclear factor-κB ligand. We expect these findings to lead to new insights into the mechanisms of bone metabolism induced by mechanical stimuli. SIGNIFICANCE: We aimed to investigate the gravity-response proteins involved in bone metabolism. To this end, we established a comprehensive analysis system for mouse bone proteins using data-independent acquisition mass spectrometry, which is particularly useful in comprehensively analyzing the bone proteome using small sample volumes. In addition, a comprehensive proteomic analysis of the femoral diaphysis and mandible, which exhibit different degrees of bone loss in mice raised on the International Space Station, identified proteins that respond to mechanical stimuli. SPARCL1, a mechanical stimulus-responsive protein, was consequently suggested to be involved in osteoclast differentiation associated with bone remodeling. Our findings represent an important step toward elucidating the molecular mechanism of bone metabolism induced by mechanical stimuli.

Effects of microgravity exposure and fructo-oligosaccharide ingestion on the proteome of soleus and extensor digitorum longus muscles in developing mice.

Short-chain fatty acids produced by the gut bacterial fermentation of non-digestible carbohydrates, e.g., fructo-oligosaccharide (FOS), contribute to the maintenance of skeletal muscle mass and oxidative metabolic capacity. We evaluated the effect of FOS ingestion on protein expression of soleus (Sol) and extensor digitorum longus muscles in mice exposed to microgravity (µ-g). Twelve 9-week-old male C57BL/6J mice were raised individually on the International Space Station under µ-g or artificial 1-g and fed a diet with or without FOS (n = 3/group). Regardless of FOS ingestion, the absolute wet weights of both muscles tended to decrease, and the fiber phenotype in Sol muscles shifted toward fast-twitch type following µ-g exposure. However, FOS ingestion tended to mitigate the µ-g-exposure-related decrease in oxidative metabolism and enhance glutathione redox detoxification in Sol muscles. These results indicate that FOS ingestion mildly suppresses metabolic changes and oxidative stress in antigravity Sol muscles during spaceflight.

Proteomic analysis revealed different responses to hypergravity of soleus and extensor digitorum longus muscles in mice.

Investigating protein abundance profiles is important to understand the differences in the slow and fast skeletal muscle characteristics. The profiles in soleus (Sol) and extensor digitorum longus (EDL) muscles in mice exposed to 1 g or 3 g for 28 d were compared. The biological implications of the profiles revealed that hypergravity exposure activated a larger number of pathways involved in protein synthesis in Sol. In contrast, the inactivation of signalling pathways involved in oxidative phosphorylation were conspicuous in EDL. These results suggested that the reactivity of molecular pathways in Sol and EDL differed. Additionally, the levels of spermidine synthase and spermidine, an important polyamine for cell growth, increased in both muscles following hypergravity exposure, whereas the level of spermine oxidase (SMOX) increased in EDL alone. The SMOX level was negatively correlated with spermine content, which is involved in muscle atrophy, and was higher in EDL than Sol, even in the 1 g group. These results indicated that the contribution of SMOX to the regulation of spermidine and spermine contents in Sol and EDL differed. However, contrary to expectations, the difference in the SMOX level did not have a significant impact on the growth of these muscles following hypergravity exposure. SIGNIFICANCE: The skeletal muscle-specific protein abundance profiles result in differences in the characteristics of slow and fast skeletal muscles. We investigated differences in the profiles in mouse slow-twitch Sol and fast-twitch EDL muscles following 28-d of 1 g and 3 g exposure by LC-MS/MS analysis and label-free quantitation. A two-step solubilisation of the skeletal muscle proteins increased the coverage of proteins identified by LC-MS/MS analysis. Additionally, this method reduced the complexity of samples more easily than protein or peptide fractionation by SDS-PAGE and offline HPLC while maintaining the high operability of samples and was reproducible. A larger number of hypergravity-responsive proteins as well as a prominent increase in the wet weights was observed in Sol than EDL muscles. The biological implications of the difference in the protein abundance profiles in 1 g and 3 g groups revealed that the reactivity of each molecular pathway in Sol and EDL muscles to hypergravity exposure differed significantly. In addition, we found that the biosynthetic and interconversion pathway of polyamines, essential factors for cell growth and survival in mammals, was responsive to hypergravity exposure; spermidine and spermine contents in Sol and EDL muscles were regulated by different mechanisms even in the 1 g group. However, our results indicated that the difference in the mechanism regulating polyamine contents is unlikely to have a significant effect on the differences in Sol and EDL muscle growth following hypergravity exposure.

Coffee intake mitigated inflammation and obesity-induced insulin resistance in skeletal muscle of high-fat diet-induced obese mice.

Epidemiologic findings offer the promise that coffee or its many constituents may be useful as a dietary intervention in type 2 diabetes (T2D) prevention. We aimed to elucidate the molecular mechanisms involved in the ameliorative effects of caffeinated coffee (CC), decaffeinated coffee (DC) and unroasted caffeinated green coffee (GC) on skeletal muscle gene expression profiles and their relationships in an obesity animal model. Eight-week-old male C57BL6 mice were raised for 9 weeks ad libitum on a normal diet, a high-fat diet, or high-fat diet containing 2 % freeze-dried CC, or DC, or GC. Total RNA and protein were extracted from skeletal muscle and subjected to microarray (Mouse Genome 430 2.0, Affymetrix) and western blotting analyses, respectively. Coffee intake mitigated the insulin resistance by decreasing plasma glucose levels during an insulin tolerance test and by increasing tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), p85/IRS-1 complex and pAkt/PKB (protein kinase B). In addition, coffee intake down-regulated the anti-inflammatory genes activating transcription factor 3, FBJ osteosarcoma oncogene, heat shock protein 1A, heat shock protein 1B and synuclein, gamma and the inflammation-associated insulin signaling genes stearoyl-coenzyme A desaturase 1 and secreted phosphoprotein 1. These results provide scientific insight on the probable positive effects of coffee intake on impaired insulin signaling, inflammation and obesity, thereby providing a new perspective on the prevention of obesity and T2D.