FTO regulates osteoclast development by modulating the proliferation and apoptosis of osteoclast precursors in inflammatory conditions.

Periodontitis is an oral inflammatory disease that causes alveolar bone destruction by activating osteoclast. FTO, a crucial demethylase of N6-methyladenosine(m6A), exerts essential function in maintaining bone homeostasis. However, the effects of FTO on periodontitis-related bone destruction remain unknown. To investigate its role in inflammatory osteoclastogenesis, we overexpressed FTO in osteoclast precursor cells; RNA-seq revealed that differentially expressed genes were mainly enriched in cell cycle, DNA replication, DNA damage response and apoptosis in FTO overexpression cells during RANKL and LPS-stimulated osteoclast differentiation. FTO overexpression upregulated the expression of S phase-related proteins (Cyclin A2, CDK2), and decreased the expression of DNA damage related proteins in osteoclast precursor cells. FTO promoted cell proliferation demonstrated by EdU and CCK8 assay, and reduced apoptotic rate and the expression of apoptosis-related proteins in osteoclast precursor cell. Conversely, FTO inhibitor FB23-2 produced the reverse effect. Mechanistically, FTO overexpression promoted the stability of CyclinA2 and CDK2 mRNA. These results were consistent in m6A binding protein YTHDF2 knockdown cells. Moreover, FB23-2 suppressed osteoclast-related gene expression, osteoclast formation and bone resorption ability. Treatment of FB23-2 reduced the alveolar bone loss in mice of experimental periodontitis. Collectively, our findings revealed that FTO enhanced the mRNA stability and expression of Cyclin A2, CDK2 in a YTHDF2-dependent manner in osteoclast precursor cells, promoted cell proliferation and inhibited cell apoptosis. FB23-2 reduced the formation of osteoclasts, resulted in alleviating the bone destruction in periodontitis mice. These findings indicated that FTO might be the potential target of the treatment of bone loss in periodontitis.

METTL3 promotes osteoblast ribosome biogenesis and alleviates periodontitis.

BACKGROUND: Periodontitis is a highly prevalent oral disease characterized by bacterium-induced periodontal inflammation and alveolar bone destruction. Osteoblast function is impaired in periodontitis with a global proteome change. METTL3 is the pivotal methyltransferase of N6-methyladenosine (m6A) that is recently proved to exert a crucial role in osteoblast differentiation. This study aims to investigate the role of METTL3 in osteoblast ribosome biogenesis in periodontitis progression. RESULTS: METTL3 was knocked down in osteoblasts, and the downregulated genes were enriched in ribosome and translation. METTL3 knockdown inhibited ribosome biogenesis and oxidative phosphorylation in LPS-stimulated osteoblasts, whereas METTL3 overexpression facilitated ribosomal and mitochondrial function. Mechanistically, METTL3 mediated osteoblast biological behaviors by activating Wnt/ß-catenin/c-Myc signaling. METTL3 depletion enhanced the mRNA expression and stability of Dkk3 and Sostdc1 via YTHDF2. In periodontitis mice, METTL3 inhibitor SAH promoted alveolar bone loss and local inflammatory status, which were partially rescued by Wnt/ß-catenin pathway activator CHIR-99021 HCl. CONCLUSIONS: METTL3 promoted ribosome biogenesis and oxidative phosphorylation by activating Wnt/ß-catenin/c-Myc signaling in LPS-treated osteoblasts and alleviated the inflammatory alveolar bone destruction in periodontitis mice.

Mettl3/Ythdf2 regulate macrophage inflammation and ROS generation by controlling Pyk2 mRNA stability.

As one of the most prevalent modifications on RNA, N6-methyladenosine (m6A) has been recently found implicated in various pathological processes. Emerging studies have demonstrated the role of m6A and its writer Mettl3 in fine-tuning the immune response, which now becomes a research hotspot owing to its potential therapeutic value. However, the results are inconsistent and even contradictory, suggesting that there might be multiple Mettl3 target genes involved in different pathways. To delve deeper into the function of Mettl3 in the cellular inflammatory response, we first conducted bioinformatics analysis using RNA-seq in Mettl3 ablation macrophages, and found that Mettl3 might attenuate LPS-induced proinflammatory pathways and reactive oxygen species (ROS) generation process. Mettl3 knockdown significantly increased the LPS-induced IL-6, TNF-α, NOXs (Nox1, Nox2, Ncf1, and Ncf2) expression, ROS generation, and the phosphorylation of MAPKs and AKT signaling. Combining the results of RNA-seq and m6A mapping, we found that Pyk2 might be the target gene of Mettl3 affecting the inflammatory response. Mettl3 and Ythdf2 depletion increased the expression and mRNA stability of Pyk2, and RIP-PCR showed that Ythdf2 directly targeting Pyk2 was Mettl3 dependent. Moreover, the upregulated expression of TNF-α, IL-6, NOXs, ROS generation, and the phosphorylation of MAPKs and AKT signaling were downregulated by Pyk2 inhibitor in Mettl3 knockdown cells. All of these results suggest that Mettl3 regulates the mRNA stability and expression of Pyk2 in a Ythdf2-dependent way, which consequently triggers the activation of MAPKs and AKT signaling and upregulation of NOXs, thus promoting the generation of proinflammatory cytokines and ROS.

NAT10 regulates the LPS-induced inflammatory response via the NOX2-ROS-NF-κB pathway in macrophages.

Periodontitis is a chronic osteolytic inflammatory disease resulting from complex dynamic interactions among bacterial pathogens and the host immune response. Macrophages play a vital role in the pathogenesis of periodontitis by triggering periodontal inflammation and inducing periodontium destruction. N-Acetyltransferase 10 (NAT10) is an acetyltransferase that has been shown to catalyse N4-acetylcytidine (ac4C) mRNA modification and is related to cellular pathophysiological processes, including the inflammatory immune response. Nevertheless, whether NAT10 regulates the inflammatory response of macrophages in periodontitis remains unclear. In this study, the expression of NAT10 in macrophages was found to decrease during LPS-induced inflammation. NAT10 knockdown significantly reduced the generation of inflammatory factors, while NAT10 overexpression had the opposite effect. RNA sequencing revealed that the differentially expressed genes were enriched in the NF-κB signalling pathway and oxidative stress. Both the NF-κB inhibitor Bay11-7082 and the ROS scavenger N-acetyl-L-cysteine (NAC) could reverse the upregulation of inflammatory factors. NAC inhibited the phosphorylation of NF-κB, but Bay11-7082 had no effect on the production of ROS in NAT10-overexpressing cells, suggesting that NAT10 activated the LPS-induced NF-κB signalling pathway by regulating ROS generation. Furthermore, the expression and stability of Nox2 was promoted after NAT10 overexpression, indicating that Nox2 may be a potential target of NAT10. In vivo, the NAT10 inhibitor Remodelin reduced macrophage infiltration and bone resorption in ligature-induced periodontitis mice. In summary, these results showed that NAT10 accelerated LPS-induced inflammation via the NOX2-ROS-NF-κB pathway in macrophages and that its inhibitor Remodelin might be of potential therapeutic significance in periodontitis treatment.

METTL3 Regulates Osteoclast Biological Behaviors via iNOS/NO-Mediated Mitochondrial Dysfunction in Inflammatory Conditions.

Excessive differentiation of osteoclasts contributes to the disruption of bone homeostasis in inflammatory bone diseases. Methyltransferase-like 3 (METTL3), the core methyltransferase that installs an N6-methyladenosine (m6A) modification on RNA, has been reported to participate in bone pathophysiology. However, whether METTL3-mediated m6A affects osteoclast differentiation in inflammatory conditions remains unelucidated. In this study, we observed that the total m6A content and METTL3 expression decreased during LPS-induced osteoclastogenesis. After knocking down METTL3, we found reduced levels of the number of osteoclasts, osteoclast-related gene expression and bone resorption area. A METTL3 deficiency increased osteoclast apoptosis and pro-apoptotic protein expression. RNA sequencing analysis showed that differentially expressed genes in METTL3-deficient cells were mainly associated with the mitochondrial function. The expression of the mitochondrial function-related genes, ATP production and mitochondrial membrane potential decreased after METTL3 knockdown. Moreover, the most obviously upregulated gene in RNA-Seq was Nos2, which encoded the iNOS protein to induce nitric oxide (NO) synthesis. METTL3 knockdown increased the levels of Nos2 mRNA, iNOS protein and NO content. NOS inhibitor L-NAME rescued the inhibited mitochondrial function and osteoclast formation while suppressing osteoclast apoptosis in METTL3-silenced cells. Mechanistically, a METTL3 deficiency promoted the stability and expression of Nos2 mRNA, and similar results were observed after m6A-binding protein YTHDF1 knockdown. Further in vivo evidence revealed that METTL3 knockdown attenuated the inflammatory osteolysis of the murine calvaria and suppressed osteoclast formation. In conclusion, these data suggested that METTL3 knockdown exacerbated iNOS/NO-mediated mitochondrial dysfunction by promoting a Nos2 mRNA stability in a YTHDF1-dependent manner and further inhibited osteoclast differentiation and increased osteoclast apoptosis in inflammatory conditions.

Arousal as a universal embedding for spatiotemporal brain dynamics.

Neural activity in awake organisms shows widespread and spatiotemporally diverse correlations with behavioral and physiological measurements. We propose that this covariation reflects in part the dynamics of a unified, arousal-related process that regulates brain-wide physiology on the timescale of seconds. Taken together with theoretical foundations in dynamical systems, this interpretation leads us to a surprising prediction: that a single, scalar measurement of arousal (e.g., pupil diameter) should suffice to reconstruct the continuous evolution of multimodal, spatiotemporal measurements of large-scale brain physiology. To test this hypothesis, we perform multimodal, cortex-wide optical imaging and behavioral monitoring in awake mice. We demonstrate that spatiotemporal measurements of neuronal calcium, metabolism, and blood-oxygen can be accurately and parsimoniously modeled from a low-dimensional state-space reconstructed from the time history of pupil diameter. Extending this framework to behavioral and electrophysiological measurements from the Allen Brain Observatory, we demonstrate the ability to integrate diverse experimental data into a unified generative model via mappings from an intrinsic arousal manifold. Our results support the hypothesis that spontaneous, spatially structured fluctuations in brain-wide physiology-widely interpreted to reflect regionally-specific neural communication-are in large part reflections of an arousal-related process. This enriched view of arousal dynamics has broad implications for interpreting observations of brain, body, and behavior as measured across modalities, contexts, and scales.

Expression and prognostic relevance of CRP, PCT, and ll-15 in patients with postoperative infection due to spinal injury.

C-reactive protein (CRP), procalcitonin (PCT), interleukin-15 (ll-15) expression and prognostic relevance were analyzed in patients with postoperative infection due to spinal injury. For this purpose, a total of 169 cases of spinal injury patients who underwent surgical treatment from July 2021 to July 2022 were selected, and the patients were divided into the uninfected group (148 cases), infected group (21 cases) according to the presence or absence of infection after surgery. Looking at the site of infection in both groups, the levels of CRP, PCT, and ll-15 in the two groups were detected using an enzyme-linked immunosorbent assay, and the expression of the three in postoperative infection of spinal injury and the correlation with prognosis were analyzed. Results showed that compared with the uninfected group, the infected group had higher levels of CRP, PCT, and ll-15, which were different (P < 0.05). There were no significant differences in the CRP levels between the superficial infection of the incision and the deep infection of the incision as well as other systemic infection populations at 1 d after surgery (P >0.05). CRP levels were higher in the group with deep infection of the incision as well as other systemic infections compared to the group with superficial infection at 3D and 7d after surgery (P < 0.05). There were no significant differences in the level of PCT between patients with superficial infection of the incision and those with deep infection of the incision as well as other systemic infections at 1 d after surgery (P > 0.05). The level of PCT was higher in the population with deep infection of incision as well as other systemic infections compared to the population with superficial infection at 3D and 7d after the operation (P<0.05). There were no statistically significant differences in the levels of ll-15 between patients with superficial infection of the incision and those with deep infection of the incision as well as other systemic infections at 1 D postoperatively (P>0.05). At 3D and 7d postoperatively, compared to the population with superficial incisions, the population with deep incisions as well as other systemic infections had higher levels of ll-15 with statistical significance (P < 0.05). CRP and PCT showed a positive correlation (r=7.192, P=0.001). CRP, ll-15 showed a positive correlation (r = 5.231, P = 0.001). PCT, ll-15 showed a positive correlation (r=9.029, P=0.001). CRP, PCT, ll-15 levels are closely related to postoperative infection in spinal injury. CRP, PCT, ll-15 showed high expression in postoperative infection of spinal injury and compared with the infection of the superficial part of the incision, the infection of the deep part of the incision, other systems have higher levels of CRP, PCT, ll-15. Moreover, CRP, PCT, and ll-15 were significantly associated with prognosis.