CD206+ M2-like macrophages protect against intervertebral disc degeneration partially by targeting R-spondin-2.

OBJECTIVE: This study aimed to explore the specific function of M2 macrophages in intervertebral disc degeneration (IDD). METHODS: Intervertebral disc (IVD) samples from normal (n = 4) and IDD (n = 6) patients were collected, and the expression of M2-polarized macrophage marker, CD206, was investigated using immunohistochemical staining. Nucleus pulposus cells (NPCs) in a TNF-α environment were obtained, and a mouse caudal IVD puncture model was established. Mice with Rheb deletions, specifically in the myeloid lineage, were generated and subjected to surgery-induced IDD. IDD-induced damage and cell apoptosis were measured using histological scoring, X-ray imaging, immunohistochemical staining, and TdT-mediated dUTP nick end labeling (TUNEL) assay. Finally, mice and NPCs were treated with R-spondin-2 (Rspo2) or anti-Rspo2 to investigate the role of Rspo2 in IDD. RESULTS: Accumulation of CD206 in human and mouse IDD tissues was detected. Rheb deletion in the myeloid lineage (RheBcKO) increased the number of CD206+ M2-like macrophages (mean difference 18.6% [15.7-21.6%], P < 0.001), decreased cell apoptosis (mean difference -15.6% [-8.9 to 22.2%], P = 0.001) and attenuated the IDD process in the mouse IDD model. NPCs treated with Rspo2 displayed increased extracellular matrix catabolism and apoptosis; co-culture with a conditioned medium derived from RheBcKO mice inhibited these changes. Anti-Rspo2 treatment in the mouse caudal IVD puncture model exerted protective effects against IDD. CONCLUSIONS: Promoting CD206+ M2-like macrophages could reduce Rspo2 secretion, thereby alleviating experimental IDD. Rheb deletion may help M2-polarized macrophages accumulate and attenuate experimental IDD partially by inhibiting Rspo2 production. Hence, M2-polarized macrophages and Rspo2 may serve as therapeutic targets for IDD.

Effect of hepatic NPC1L1 on cholesterol gallstone disease and its mechanism.

Cholesterol gallstone disease (CGD) is associated with bile cholesterol supersaturation. The Niemann-Pick C1-like 1 (NPC1L1), the inhibitory target of ezetimibe (EZE), is a critical sterol transporter of cholesterol absorption. Intestinal NPC1L1 facilitates the absorption of cholesterol, whereas hepatic NPC1L1 promotes cholesterol uptake by hepatocytes and reduces bile cholesterol supersaturation. The potential of hepatic NPC1L1 to prevent CGD has yet to be established due to its absence in the mice model. In this study, we generated mice expressing hepatic NPC1L1 using adeno-associated virus (AAV) gene delivery. The biliary cholesterol saturations and gallstone formations were explored under chow diet and lithogenic diet (LD) with or without EZE treatment. The long-term (8-week) LD-fed AAV-mNPC1L1 mice exhibited no significant differences in biliary cholesterol saturation and gallstone formation compared to WT mice. EZE effectively prevented CGD in both WT and AAV-mNPC1L1 mice. Mechanistically, prolonged LD feeding induced the degradation of hepatic NPC1L1, whereas short-term (2-week) LD feeding preserved the expression of hepatic NPC1L1. In conclusion, our findings suggest that hepatic NPC1L1 is unable to prevent CGD, whereas EZE functions as an efficient bile cholesterol desaturator during CGD development.

FGF15 promotes hepatic NPC1L1 degradation in lithogenic diet-fed mice.

BACKGROUND: Cholesterol gallstone disease (CGD) is accompanied by biliary cholesterol supersaturation. Hepatic Niemann-Pick C1-like 1 (NPC1L1), which is present in humans but not in wild-type (WT) mice, promotes hepatocyte cholesterol uptake and decreases biliary cholesterol supersaturation. In contrast, intestinal NPC1L1 promotes intestinal cholesterol absorption, increasing biliary cholesterol supersaturation. Ezetimibe (EZE) can inhibit both hepatic and intestinal NPC1L1. However, whether hepatic NPC1L1 can affect CGD progress remains unknown. METHODS: Mice expressing hepatic NPC1L1 (NPC1L1hepatic-OE mice) were generated using Adeno-associated viruses (AAV) gene delivery. The protein level and function of hepatic NPC1L1 were examined under chow diet, high fat-cholesterol diet (HFCD), and lithogenic diet (LD) feeding. Gallstone formation rates were examined with or without EZE treatment. Fibroblast growth factor 15 (FGF15) treatment and inhibition of fibroblast growth factor receptor 4 (FGFR4) were applied to verify the mechanism of hepatic NPC1L1 degradation. RESULTS: The HFCD-fed NPC1L1hepatic-OE mice retained the biliary cholesterol desaturation function of hepatic NPC1L1, whereas EZE treatment decreased biliary cholesterol saturation and did not cause CGD. The ubiquitination and degradation of hepatic NPC1L1 were discovered in LD-fed NPC1L1hepatic-OE mice. Treatment of FGF15 during HFCD feeding and inhibition of FGFR4 during LD feeding could affect the protein level and function of hepatic NPC1L1. CONCLUSIONS: LD induces the ubiquitination and degradation of hepatic NPC1L1 via the FGF15-FGFR4 pathway. EZE may act as an effective preventative agent for CGD.

Tumor-Infiltrating Lymphocyte-Based Risk Score for Predicting Prognosis in Gastric Cancer.

Tumor-infiltrating lymphocytes (TILs) in gastric cancer are closely related to clinical prognosis; however, little is known regarding the immune microenvironment in this disease. Thus, RNA-sequencing data from gastric cancer patients were downloaded from the Gene Expression Omnibus (GEO). The proportion of immune cells was determined based on a deconvolution algorithm (CIBERSORT), and gene expression profiles were analyzed in the context of clinical outcomes to construct an immune risk score. Data were analyzed using least absolute shrinkage and selection operator (LASSO) and multivariable Cox regression, to identify prognostic markers of gastric cancer survival. The model included four immune cell types: neutrophils, plasma cells, activated CD4+ memory T cells, and T follicular helper cells. Patients were classified into two subgroups based on risk score, and a significant difference in overall survival (OS) was seen between the subgroups in both the training and testing cohorts, particularly in patients with tumor stages ≥T3. Multivariable analysis revealed that both T-stage and risk score were independent prognostic factors for gastric cancer survival [hazard ratio (HR) 1.505; 95% confidence interval (CI) 1.043-2.173, HR 1.686; 95% CI 1.367-2.080]. Risk scores and clinical factors were then integrated into a nomogram to build a model with both good discriminatory power and accuracy in predicting clinical outcomes. Further analysis using gene set enrichment analysis (GSEA) identified strong associations of immune risk with TGF-ß and tumor metastasis-related pathways, which could inform research on the molecular mechanisms of gastric cancer. Collectively, the data presented here suggest that an immune risk model can make an important contribution to predictions prognosis in gastric cancer patients.

N6-methyladenosine (m6A) RNA methylation signature as a predictor of stomach adenocarcinoma outcomes and its association with immune checkpoint molecules.

OBJECTIVE: Although N6-methyladenosine (m6A) RNA methylation is the most common mRNA modification process, few studies have examined the role of m6A in stomach adenocarcinomas (STADs). METHODS: In this retrospective study, we analyzed 293 STAD samples from The Cancer Genome Atlas with complete clinicopathological feature profiles. The m6A methylation risk signature was derived from LASSO-Cox regression analyses with 15 m6A regulators. Statistical analysis was performed and figures were prepared using R software (https://www.R-project.org/). RESULTS: The m6A signature was established as follows: risk score = FTO × 0.127 + YTHDF1 × 0.004 + KIAA1429 × 0.044 + YTHDC2 × 0.112 - RBM15 × 0.135 - ALKBH5 × 0.019 - YTHDF2 × 0.028, which was confirmed as an independent prognostic indicator to predict overall survival of patients with STAD. Risk scores and tumor grades were closely associated. Cell cycle, p53 signaling pathways, DNA mismatch repair, and RNA degradation were enriched in the low-risk subgroup. This subgroup showed significantly higher expression of immune checkpoint molecules including PD-1 (programmed death 1), PD-L1 (programmed death-ligand 1), and CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4), suggesting that the signature may be a useful immunotherapy predictor. CONCLUSIONS: We established an m6A methylation signature as an independent prognostic tool to predict overall survival, which may also be useful as an immunotherapy predictor.