Cholesterol 25-hydroxylase prevents type 2 diabetes mellitus induced cardiomyopathy by alleviating cardiac lipotoxicity.

OBJECTIVES: Diabetic cardiomyopathy (DCM) is the leading cause of mortality in type 2 diabetes mellitus (T2DM) patients, with its underlying mechanisms still elusive. This study aims to investigate the role of cholesterol-25-monooxygenase (CH25H) in T2DM induced cardiomyopathy. METHODS: High fat diet combined with streptozotocin (HFD/STZ) were used to establish a T2DM model. CH25H and its product 25-hydroxycholesterol (25HC) were detected in the hearts of T2DM model. Gain- or loss-of-function of CH25H were performed by receiving AAV9-cTNT-CH25H or CH25H knockout (CH25H-/-) mice with HFD/STZ treatment. Cardiac function was evaluated using echocardiography, and cardiac tissues were collected for immunoblot analysis, histological assessment and quantitative polymerase chain reaction (qPCR). Mitochondrial morphology and function were evaluated using transmission electron microscopy (TEM) and Seahorse XF Cell Mito Stress Test Kit. RNA-sequence analysis was performed to determine the molecular changes associated with CH25H deletion. RESULTS: CH25H and 25HC were significantly decreased in the hearts of T2DM mice. CH25H-/- mice treated with HFD/STZ exhibited impaired mitochondrial function and structure, increased lipid accumulation, and aggregated cardiac dysfunction. Conversely, T2DM mice receiving AAV9-CH25H displayed cardioprotective effects. Mechanistically, RNA sequencing and qPCR analysis revealed that CH25H deficiency decreased peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and its target gene expression. Additionally, administration of ZLN005, a potent PGC-1α activator, partially protected against high glucose and palmitic acid induced mitochondria dysfunction and lipid accumulation in vitro. CONCLUSION: Our study provides compelling evidence supporting the protective role of CH25H in T2DM-induced cardiomyopathy. Furthermore, the regulation of PGC-1α may be intricately involved in this cardioprotective process.

25-Hydroxycholesterol regulates lysosome AMP kinase activation and metabolic reprogramming to educate immunosuppressive macrophages.

Macrophages are critical to turn noninflamed "cold tumors" into inflamed "hot tumors". Emerging evidence indicates abnormal cholesterol metabolites in the tumor microenvironment (TME) with unclear function. Here, we uncovered the inducible expression of cholesterol-25-hydroxylase (Ch25h) by interleukin-4 (IL-4) and interleukin-13 (IL-13) via the transcription factor STAT6, causing 25-hydroxycholesterol (25HC) accumulation. scRNA-seq analysis confirmed that CH25Hhi subsets were enriched in immunosuppressive macrophage subsets and correlated to lower survival rates in pan-cancers. Targeting CH25H abrogated macrophage immunosuppressive function to enhance infiltrating T cell numbers and activation, which synergized with anti-PD-1 to improve anti-tumor efficacy. Mechanically, lysosome-accumulated 25HC competed with cholesterol for GPR155 binding to inhibit the kinase mTORC1, leading to AMPKα activation and metabolic reprogramming. AMPKα also phosphorylated STAT6 Ser564 to enhance STAT6 activation and ARG1 production. Together, we propose CH25H as an immunometabolic checkpoint, which manipulates macrophage fate to reshape CD8+ T cell surveillance and anti-tumor response.

Hypoxia-induced ALKBH5 aggravates synovial aggression and inflammation in rheumatoid arthritis by regulating the m6A modification of CH25H.

Previous studies have shown that epigenetic factors are involved in the occurrence and development of rheumatoid arthritis (RA). However, the role of N6-methyladenosine (m6A) methylation in RA has not been determined. The aim of this study was to investigate the role and regulatory mechanisms of hypoxia-induced expression of the m6A demethylase alkB homolog 5 (ALKBH5) in RA fibroblast-like synoviocytes (FLSs). Synovial tissues were collected from RA and osteoarthritis (OA) patients, and RA FLSs were obtained. ALKBH5 expression in RA FLSs and collagen-induced arthritis (CIA) model rats was determined using quantitative reverse transcription-PCR (qRT-PCR), western blotting and immunohistochemistry (IHC). Using ALKBH5 overexpression and knockdown, we determined the role of ALKBH5 in RA FLS aggression and inflammation. The role of ALKBH5 in RA FLS regulation was explored using m6A-methylated RNA sequencing and methylated RNA immunoprecipitation coupled with quantitative real-time PCR. The expression of ALKBH5 was increased in RA synovial tissues, CIA model rats and RA FLSs, and a hypoxic environment increased the expression of ALKBH5 in FLSs. Increased expression of ALKBH5 promoted the proliferation and migration of RA-FLSs and inflammation. Conversely, decreased ALKBH5 expression inhibited the migration of RA-FLSs and inflammation. Mechanistically, hypoxia-induced ALKBH5 expression promoted FLS aggression and inflammation by regulating CH25H mRNA stability. Our study elucidated the functional roles of ALKBH5 and mRNA m6A methylation in RA and revealed that the HIF1α/2α-ALKBH5-CH25H pathway may be key for FLS aggression and inflammation. This study provides a novel approach for the treatment of RA by targeting the HIF1α/2α-ALKBH5-CH25H pathway.

25-hydroxycholesterol aggravates oxygen-glucose deprivation/reoxygenation-induced pyroptosis through promoting activation of NLRP3 inflammasome in H9C2 cardiomyocytes

25-hydroxycholesterol (25-HC) plays a role in the regulation of cell survival and immunity. However, the effect of 25-HC on myocardial ischemia/reperfusion (MI/R) injury remains unknown. Our present study aimed to investigate whether 25-HC aggravated MI/R injury through NLRP3 inflammasome-mediated pyroptosis. The overlapping differentially expressed genes (DEGs) in MI/R were identified from the GSE775, GSE45818, GSE58486, and GSE46395 datasets in Gene Expression Omnibus (GEO) database. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the database of Annotation, Visualization and Integration Discovery (DAVID). The protein-protein interaction (PPI) network of the overlapping DEGs was established using the Search Tool for the Retrieval of Interacting Genes (STRING) database. These bioinformatics analyses indicated that cholesterol 25-hydroxylase (CH25H) was one of the crucial genes in MI/R injury. The oxygen-glucose deprivation/reoxygenation (OGD/R) cell model was established to simulate MI/R injury. Western blot and RT-qPCR analysis demonstrated that CH25H was significantly upregulated in OGD/R-stimulated H9C2 cardiomyocytes. Moreover, knockdown of CH25H inhibited the OGD/R-induced pyroptosis and nod-like receptor protein 3 (NLRP3) inflammasome activation, as demonstrated by cell counting kit-8 (CCK8), lactate dehydrogenase (LDH), RT-qPCR, and western blotting assays. Conversely, 25-HC, which is synthesized by CH25H, promoted activation of NLRP3 inflammasome in OGD/R-stimulated H9C2 cardiomyocytes. In addition, the NLRP3 inhibitor BAY11-7082 attenuated 25-HC-induced H9C2 cell injury and pyroptosis under OGD/R condition. In conclusion, 25-HC could aggravate OGD/R-induced pyroptosis through promoting activation of NLRP3 inflammasome in H9C2 cells.

Quantitative Determination of Cholesterol Hydroxylase Specificities by GC-MS/MS in Living Mammalian Cells.

Cholesterol is oxygenated by a variety of cholesterol hydroxylases; oxysterols play diverse important roles in physiological and pathophysiological conditions by regulating several transcription factors and cell-surface receptors. Each oxysterol has distinct and overlapping functions. The expression of cholesterol hydroxylases is highly regulated, but their physiological and pathophysiological roles are not fully understood. Although the activity of cholesterol hydroxylases has been characterized biochemically using radiolabeled cholesterol as the substrate, their specificities remain to be comprehensively determined quantitatively. To better understand their roles, a highly sensitive method to measure the amount of various oxysterols synthesized by cholesterol hydroxylases in living mammalian cells is required. Our method described here, with gas chromatography coupled with tandem mass spectrometry (GC-MS/MS), can quantitatively determine a series of oxysterols endogenously synthesized by forced expression of one of the four major cholesterol hydroxylases-CH25H, CYP7A1, CYP27A1, and CYP46A1-or induction of CH25H expression by a physiological stimulus. This protocol can also simultaneously measure the amount of intermediate sterols, which serve as markers for cellular cholesterol synthesis activity. Key features • Allows measuring the amount of a variety of oxysterols synthesized endogenously by cholesterol hydroxylases using GC-MS/MS. • Comprehensive and quantitative analysis of cholesterol hydroxylase specificities in living mammalian cells. • Simultaneous quantification of intermediate sterols to assess cholesterol synthesis activity.

Expression Pattern of Cholesterol 25-Hydroxylase and Serum Level of 25-Hydroxycholesterol and Relevant Inflammatory Cytokines in Patients with Varying Disease Severity of COVID-19.

Cholesterol 25-hydroxylase (CH25H) and its product 25-hydroxycholesterol (25HC) showed antiviral effects against various viruses in vitro. CH25H expression is regulated in mice by pro-inflammatory cytokine interferons (IFNs) in mice but data on its possible correlation with IFNs in humans are still unclear. We examined gene expression of CH25H, IFN-α, and IFN-ß and serum levels of 25HC in Iranian patients with mild and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Fifty intensive care unit (ICU) patients and outpatients with SARS-CoV-2 and 25 healthy controls were studied. Gene expression of CH25H and relevant inflammatory cytokines was quantified in peripheral blood mononuclear cells by real-time polymerase chain reaction. The expression of CH25H and serum levels of 25HC were significantly higher in ICU patients with SARS-CoV-2. Notably, IFN-α levels increased in healthy controls. However, compared to healthy controls, IFN-ß was considerably higher in outpatients. Finally, statistical analysis shows that no correlation was found between CH25H and IFN-α expression; nevertheless, a lower correlation was found with IFN-ß. The data revealed that CH25H and 25HC levels increase after SARS-CoV-2 infection. In other words, decreased levels of those factors in severe patients compared with mild patients may indicate the importance of their function in controlling the progression of the disease.

The role of cholesterol 25-hydroxylase in viral infections: Mechanisms and implications.

Viral infections pose significant threats to human health, causing various diseases with varying severity. The intricate interactions between viruses and host cells determine the outcome of infection, including viral replication, immune responses, and disease progression. Cholesterol 25-hydroxylase (CH25H) is an enzyme that catalyzes the conversion of cholesterol to 25-hydroxycholesterol (25HC), a potent antiviral molecule. In recent years, increasing evidence has highlighted the critical involvement of CH25H in modulating immune responses and influencing viral infections. Notably, the review discusses the implications of CH25H in viral pathogenesis and the development of therapeutic strategies. It examines the interplay between CH25H and viral immune evasion mechanisms, highlighting the potential of viral antagonism of CH25H to enhance viral replication and pathogenesis. Furthermore, it explores the therapeutic potential of targeting CH25H or modulating its downstream signaling pathways as a strategy to control viral infections and enhance antiviral immune responses. This comprehensive review demonstrates the crucial role of CH25H in viral infections, shedding light on its mechanisms of action in viral entry, replication, and immune modulation. Understanding the complex interplay between CH25H and viral infections may pave the way for novel therapeutic approaches and the development of antiviral strategies aimed at exploiting the antiviral properties of CH25H and enhancing host immune responses against viral pathogens. In the current review, we tried to provide an overview of the antiviral activity and importance of CH25H in viral pathogenesis.

Inhibition of Marek's Disease Virus Replication and Spread by 25-hydroxycholesterol and 27-hydroxycholesterol In Vitro.

Marek's disease virus (MDV) causes a deadly lymphoproliferative disease in chickens, resulting in huge economic losses in the poultry industry. It has been suggested that MDV suppresses the induction of type I interferons and thus escapes immune control. Cholesterol 25-hydroxylase (CH25H), a gene that encodes an enzyme that catalyses cholesterol to 25-hydroxycholesterol (25-HC), is an interferon-stimulating gene (ISG) known to exert antiviral activities. Other oxysterols, such as 27-hydroxycholesterols (27-HC), have also been shown to exert antiviral activities, and 27-HC is synthesised by the catalysis of cholesterol via the cytochrome P450 enzyme oxidase sterol 27-hydroxylase A1 (CYP27A1). At 24 h post infection (hpi), MDV stimulated a type I interferon (IFN-α) response, which was significantly reduced at 48 and 72 hpi, as detected using the luciferase assay for chicken type I IFNs. Then, using RT-PCR, we demonstrated that chicken type I IFN (IFN-α) upregulates chicken CH25H and CYP27A1 genes in chicken embryo fibroblast (CEF) cells. In parallel, our results demonstrate a moderate and transient upregulation of CH25H at 48 hpi and CYP27A1 at 72hpi in MDV-infected CEF cells. A significant reduction in MDV titer and plaque sizes was observed in CEFs treated with 25-HC or 27-HC in vitro, as demonstrated using a standard plaque assay for MDV. Taken together, our results suggest that 25-HC and 27-HC may be useful antiviral agents to control MDV replication and spread.

Impact of interferon-γ on the target cell tropism of nanoparticles.

Interferon-γ (IFN-γ) is well known to reduce the infectivity of viral pathogens by altering their tissue tropism. This effect is induced by upregulation of cholesterol 25-hydroxylase (CH25H). Given the similarity of viral pathogens and ligand-functionalized nanoparticles in the underlying strategy of receptor-mediated cell recognition, it appears conceivable that IFN-γ exceeds similar effects on nanoparticles. Concretely, IFN-γ-induced activation of CH25H could decrease nanoparticle avidity for target cells via depletion of clathrin-coated pits. We hypothesized that this effect would cause deterioration of target-cell specific accumulation of nanoparticles. To prove our hypothesis, we investigated the cell tropism of angiotensin II functionalized nanoparticles (NPLys-Ang II) in a co-culture system of angiotensin II subtype 1 receptor (AT1R) positive rat mesangial target cells (rMCs) and AT1R-negative HeLa off-target cells. In the presence of IFN-γ we observed an up to 5-fold loss of target cell preference for NPLys-Ang II. Thus, our in vitro results suggest a strong influence of IFN-γ on nanoparticle distribution, which is relevant in the context of nanotherapeutic approaches to cancer treatment, as IFN-γ is strongly expressed in tumors. For the target cell tropism of viruses, our results provide a conclusive hypothesis for the underlying mechanism behind non-directed viral distribution in the presence of IFN-γ.

The combination of machine learning and untargeted metabolomics identifies the lipid metabolism -related gene CH25H as a potential biomarker in asthma.

BACKGROUND: Lipids, significant signaling molecules, regulate a multitude of cellular responses and biological pathways in asthma which are closely associated with disease onset and progression. However, the characteristic lipid genes and metabolites in asthma remain to be explored. It is also necessary to further investigate the role of lipid molecules in asthma based on high-throughput data. OBJECTIVE: To explore the biomarkers and molecular mechanisms associated with lipid metabolism in asthma. METHODS: In this study, we selected three mouse-derived datasets and one human dataset (GSE41665, GSE41667, GSE3184 and GSE67472) from the GEO database. Five machine learning algorithms, LASSO, SVM-RFE, Boruta, XGBoost and RF, were used to identify core gene. Additionally, we used non-negative matrix breakdown (NMF) clustering to identify two lipid molecular subgroups and constructed a lipid metabolism score by principal component analysis (PCA) to differentiate the subtypes. Finally, Western blot confirmed the altered expression levels of core genes in OVA (ovalbumin) and HDM+LPS (house dust mite+lipopolysaccharide) stimulated and challenged BALB/c mice, respectively. Results of non-targeted metabolomics revealed multiple differentially expressed metabolites in the plasma of OVA-induced asthmatic mice. RESULTS: Cholesterol 25-hydroxylase (CH25H) was finally localized as a core lipid metabolism gene in asthma and was verified to be highly expressed in two mouse models of asthma. Five-gene lipid metabolism constructed from CYP2E1, CH25H, PTGES, ALOX15 and ME1 was able to distinguish the subtypes effectively. The results of non-targeted metabolomics showed that most of the aberrantly expressed metabolites in the plasma of asthmatic mice were lipids, such as LPC 16:0, LPC 18:1 and LPA 18:1. CONCLUSION: Our findings imply that the lipid-related gene CH25H may be a useful biomarker in the diagnosis of asthma.

Chicken CH25H inhibits ALV-J replication by promoting cellular autophagy.

Autophagy plays an important role in host antiviral defense. The avian leukosis virus subgroup J (ALV-J) has been shown to inhibit autophagy while promoting viral replication. The underlying autophagic mechanisms, however, are unknown. Cholesterol 25-hydroxylase (CH25H) is a conserved interferon-stimulated gene, which converts cholesterol to a soluble antiviral factor, 25-hydroxycholesterol (25HC). In this study, we further investigated the autophagic mechanism of CH25H resistance to ALV-J in chicken embryonic fibroblast cell lines (DF1). Our results found that overexpression of CH25H and treatment with 25HC promoted the autophagic markers microtubule-associated protein 1 light chain 3 II (LC3II) and autophagy-related gene 5(ATG5), while decreased autophagy substrate p62/SQSTM1 (p62) expression in ALV-J infection DF-1 cells. Induction of cellular autophagy also reduces the levels of ALV-J gp85 and p27. ALV-J infection, on the other hand, suppresses autophagic marker protein LC3II expression. These findings suggest that CH25H-induced autophagy is a host defense mechanism that aids in ALV-J replication inhibition. In particular, CH25H interacts with CHMP4B and inhibits ALV-J infection in DF-1 cells by promoting autophagy, revealing a novel mechanism by which CH25H inhibits ALV-J infection. Although the underlying mechanisms are not completely understood, CH25H and 25HC are the first to show inhibiting ALV-J infection via autophagy.

Role of Cholesterol 25-Hydroxylase (Ch25h) in Mediating Innate Immune Responses to Streptococcus pneumoniae Infection.

Alveolar macrophages (AM) are long-lived tissue-resident innate immune cells of the airways. AM are key effectors of recognition, initiation, and resolution of the host defense against microbes and play an essential role in mediating host responses to Streptococcus pneumoniae infection. Lipid metabolism in AM can significantly impact cellular function and biology. Dysregulated metabolism contributes to an accumulation of lipids, unfolded protein response induction, and inflammatory cytokine production. Our study was designed to investigate the impact of Ch25h on mediating innate immune responses by macrophages during S. pneumoniae infection. Using wild-type and Ch25-/- mice, we examined the role of cholesterol metabolism on inflammatory cytokine production and bacterial clearance. Our results demonstrate that Ch25h plays an important role in the initiation and intensity of cytokine and chemokine production in the lung during S. pneumoniae infection. In the absence of Ch25h, there was enhanced phagocytosis and bacterial clearance. Taken together, our findings demonstrate the important role of Ch25h in modulating host responsiveness to S. pneumoniae infection.

Hydroxylation site-specific and production-dependent effects of endogenous oxysterols on cholesterol homeostasis: Implications for SREBP-2 and LXR.

The cholesterol metabolites, oxysterols, play central roles in cholesterol feedback control. They modulate the activity of two master transcription factors that control cholesterol homeostatic responses, sterol regulatory element-binding protein-2 (SREBP-2) and liver X receptor (LXR). Although the role of exogenous oxysterols in regulating these transcription factors has been well established, whether endogenously synthesized oxysterols similarly control both SREBP-2 and LXR remains poorly explored. Here, we carefully validate the role of oxysterols enzymatically synthesized within cells in cholesterol homeostatic responses. We first show that SREBP-2 responds more sensitively to exogenous oxysterols than LXR in Chinese hamster ovary cells and rat primary hepatocytes. We then show that 25-hydroxycholesterol (25-HC), 27-hydroxycholesterol, and 24S-hydroxycholesterol endogenously synthesized by CH25H, CYP27A1, and CYP46A1, respectively, suppress SREBP-2 activity at different degrees by stabilizing Insig (insulin-induced gene) proteins, whereas 7α-hydroxycholesterol has little impact on SREBP-2. These results demonstrate the role of site-specific hydroxylation of endogenous oxysterols. In contrast, the expression of CH25H, CYP46A1, CYP27A1, or CYP7A1 fails to induce LXR target gene expression. We also show the 25-HC production-dependent suppression of SREBP-2 using a tetracycline-inducible CH25H expression system. To induce 25-HC production physiologically, murine macrophages are stimulated with a Toll-like receptor 4 ligand, and its effect on SREBP-2 and LXR is examined. The results also suggest that de novo synthesis of 25-HC preferentially regulates SREBP-2 activity. Finally, we quantitatively determine the specificity of the four cholesterol hydroxylases in living cells. Based on our current findings, we conclude that endogenous side-chain oxysterols primarily regulate the activity of SREBP-2, not LXR.

Curcumol inhibits EMCV replication by activating CH25H and inhibiting the formation of ROs.

BACKGROUND: Zedoary turmeric oil extracted from the roots of curcuma (Curcuma aeruginosa Roxb.) is used for the treatment of myocarditis in China. EMCV infection causes abortion in pregnant sows and myocarditis in piglets. Our previous studies demonstrated that curcumol significantly increased the expression of IFN-ß in EMCV infected HEK-293T cells. The present results showed that curcumol inhibits EMCV replication by interfering the host cell cholesterol homeostasis and reducing ROs production through activation of the JAK/STAT signaling pathway. METHOD: This study was designed to explore whether curcumol can inhibit the replication of encephalomyocarditis viruses (EMCV) in cell culture. The expression level of JAK1, IRF9, STAT2, P-STAT2, CH25H, PI4KA and OSBP in EMCV-infected HEK-293T cells treated with curcumol, ribavirin or hydroxypropyl-ß-CD (HPCD) were determined by Western blotting (WB). The cholesterol level in EMCV infected HEK-293T cells treated with curcumol and HPCD were detected using Amplex™ Red Cholesterol Assay Kit. The antiviral effects of curcumol and HPCD on EMCV were also quantitatively detected by real-time fluorescence quantitative PCR (q-PCR). The amount and morphology of ROs were observed by transmission electron microscopy (TEM). RESULTS: The results demonstrated that curcumol significantly (P < 0.05) increased the expression of JAK1, IRF9, P-STAT2 and CH25H proteins, while that of STAT2, PI4KA and OSBP were remained unchanged. Compared with virus group (0.134 µg.µg-1 proteins), the total cholesterol level was significantly (P < 0.05) reduced by curcumol (0.108 µg.µg-1 proteins) and HPCD (0.089 µg.µg-1 proteins). Compared with virus group (88237 copies), curcumol (41802 copies) and HPCD (53 copies) significantly (P < 0.05) reduced EMCV load. Curcumol significantly reduced the production of ROs in EMCV-infected HEK-293T cells and activated CH25H through the JAK/STAT signaling pathway. CONCLUSION: Curcumol inhibited EMCV replication by affecting the cholesterol homeostasis and the production of ROs in HEK-293T cell.

ATF3 and CH25H regulate effector trogocytosis and anti-tumor activities of endogenous and immunotherapeutic cytotoxic T lymphocytes.

Effector trogocytosis between malignant cells and tumor-specific cytotoxic T lymphocytes (CTLs) contributes to immune evasion through antigen loss on target cells and fratricide of antigen-experienced CTLs by other CTLs. The mechanisms regulating these events in tumors remain poorly understood. Here, we demonstrate that tumor-derived factors (TDFs) stimulated effector trogocytosis and restricted CTLs' tumoricidal activity and viability in vitro. TDFs robustly altered the CTL's lipid profile, including depletion of 25-hydroxycholesterol (25HC). 25HC inhibited trogocytosis and prevented CTL's inactivation and fratricide. Mechanistically, TDFs induced ATF3 transcription factor that suppressed the expression of 25HC-regulating gene-cholesterol 25-hydroxylase (CH25H). Stimulation of trogocytosis in the intratumoral CTL by the ATF3-CH25H axis attenuated anti-tumor immunity, stimulated tumor growth, and impeded the efficacy of chimeric antigen receptor (CAR) T cell adoptive therapy. Through use of armored CAR constructs or pharmacologic agents restoring CH25H expression, we reversed these phenotypes and increased the efficacy of immunotherapies.

Rapid GPR183-mediated recruitment of eosinophils to the lung after Mycobacterium tuberculosis infection.

Influx of eosinophils into the lungs is typically associated with type II responses during allergy and fungal and parasitic infections. However, we previously reported that eosinophils accumulate in lung lesions during type I inflammatory responses to Mycobacterium tuberculosis (Mtb) in humans, macaques, and mice, in which they support host resistance. Here we show eosinophils migrate into the lungs of macaques and mice as early as one week after Mtb exposure. In mice this influx is CCR3 independent and instead requires cell-intrinsic expression of the oxysterol receptor GPR183, which is highly expressed on human and macaque eosinophils. Murine eosinophils interact directly with bacilli-laden alveolar macrophages, which upregulate the oxysterol-synthesizing enzyme Ch25h, and eosinophil recruitment is impaired in Ch25h-deficient mice. Our findings show that eosinophils are among the earliest cells from circulation to sense and respond to Mtb infection of alveolar macrophages and reveal a role for GPR183 in the migration of eosinophils into lung tissue.

Dual-Role of Cholesterol-25-Hydroxylase in Regulating Hepatitis B Virus Infection and Replication.

Hepatitis B virus (HBV)-related diseases are among the major diseases that affect millions of people worldwide. These diseases are difficult to eradicate and thus pose a serious global health challenge. There is an urgent need to understand the cross talk mechanism between HBV and the host. Cholesterol-25-hydroxylase (CH25H) and its enzymatic product, 25-hydroxycholesterol (25HC), were previously shown to exhibit effective broad-spectrum antiviral activity. However, the role of CH25H in the regulation of HBV infection and replication remains unclear. The present study reported increased expression of CH25H in HBV-infected patients compared to healthy subjects. Importantly, higher expression of CH25H expression was found to be associated with low HBV replication. Additionally, the present study aimed to identify CH25H mutants, which would lack hydroxylase activity but retain antiviral activity toward HBV infection and replication. Interestingly, it was observed that both CH25H and its mutants interacted with HBx protein and inhibited nuclear translocation of HBx. In particular, CH25H interacted with the C-terminal region of HBx, while transmembrane region 3 of CH25H was found to be critical for CH25H-HBx interaction and inhibition of HBV replication. The study results suggested that 25HC promoted HBV infection but not HBV replication. Thus, the results of the present study suggested the involvement of a dual mechanism in CH25H-mediated regulation of HBV replication. The study clearly demonstrated cross talk between HBV and the host through CH25H-HBx axis. IMPORTANCE The enzymatic product of CH25H, 25-hydroxycholesterol (25HC), has been previously shown to play a critical role in the blockage of the cell-virus fusion in response to viral infection. However, our study indicates a dual role of CH25H in regulating HBV. We find the CH25H-mediated inhibition of HBV replication is independent on its enzyme activity and CH25H binds to HBx and inhibits HBx nucleus translocation. We are interested to find out 25HC promotes HBV infection.

Ch25h and 25-HC prevent liver steatosis through regulation of cholesterol metabolism and inflammation.

Non-alcoholic fatty liver disease (NAFLD) is currently the most prevalent metabolic disorder all over the world, and lipid metabolic disorders and inflammation are closely associated and contribute to the pathogenesis of NAFLD. Cholesterol 25-hydroxylase (Ch25h) and its product, 25-hydroxycholesterol (25-HC), play important roles in cholesterol homeostasis and inflammation, but whether Ch25h and 25-HC are involved in NAFLD remains uncertain. In this study, we use Ch25h knockout mice, hepatic cells and liver biopsies to explore the role of Ch25h and 25-HC in lipid metabolism and accumulation in liver, determine the molecular mechanism of lipid accumulation and inflammation influenced by Ch25h and 25-HC, and assess the regulatory effects of Ch25h and 25-HC on human NAFLD. Our results indicate that mice lacking Ch25h have normal cholesterol homeostasis with normal diet, but under the condition of high fat diet (HFD), the mice show higher total cholesterol and triglyceride in serum, and prone to hepatic steatosis. Ch25h deficiency reduces the cholesterol efflux regulated by liver X receptor α (LXRα), increases the synthesis of cholesterol mediated by sterol-regulatory element binding protein 2 (SREBP-2), and increases the activation of NLRP3 inflammasome, therefore promotes hepatic steatosis. Collectively, our data suggest that Ch25h and 25-HC play important roles in lipid metabolism and inflammation, thereby exerting anti-NAFLD functions.

Interleukin-1 Induces the Release of Lubricating Phospholipids from Human Osteoarthritic Fibroblast-like Synoviocytes.

(1) Background: Synovial fluid (SF) from knee joints with osteoarthritis (OA) has increased levels of phospholipids (PL). We have reported earlier that TGF-ß and IGF-1 stimulate fibroblast-like synoviocytes (FLS) to synthesize increased amounts of PLs. The current study examined whether IL-1ß induces the release of PLs in FLS and the underlying mechanism. (2) Methods: Cultured human OA FLS were treated with IL-1ß alone and with pathway inhibitors or with synthetic liver X receptor (LXR) agonists. Cholesterol hydroxylases, ABC transporters, apolipoproteins (APO), LXR, sterol regulatory binding proteins (SREBPs), and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) were analyzed by RT-PCR, Western blot, and ELISA. The release of radiolabeled PLs from FLS was determined, and statistical analysis was performed using R (N = 5-9). (3) Results: Like synthetic LXR agonists, IL-1ß induced a 1.4-fold greater release of PLs from FLS. Simultaneously, IL-1ß upregulated the level of the PL transporter ABCA1 and of cholesterol hydroxylases CH25H and CYP7B1. IL-1ß and T0901317 stimulated the expression of SREBP1c, whereas only T0901317 enhanced SREBP2, HMGCR, APOE, LXRα, and ABCG1 additionally. (4) Conclusions: IL-1ß partially controls PL levels in OA-SF by affecting the release of PLs from FLS. Our data show that IL-1ß upregulates cholesterol hydroxylases and thus the formation of oxysterols, which, as natural agonists of LXR, increase the level of active ABCA1, in turn enhancing the release of PLs.

Hepatic Reduction in Cholesterol 25-Hydroxylase Aggravates Diet-induced Steatosis.

BACKGROUND & AIMS: Cholesterol 25-hydroxylase (Ch25h), converting cholesterol to 25-hydroxycholesterol (25-HC), is critical in modulating cellular lipid metabolism and anti-inflammatory and antiviral activities. However, its role in nonalcoholic fatty liver disease remains unclear. METHODS: Ch25h expression was detected in livers of ob/ob mice and E3 rats fed a high-fat diet (HFD). Gain- or loss-of-function of Ch25h was performed using Ch25h+/+ (wild type [WT]) mice receiving AAV8-Ch25h or Ch25h knockout (Ch25h-/-) mice. WT mice fed an HFD were administered with 25-HC. The Ch25h-LXRα-CYP axis was measured in primary hepatocytes isolated from WT and Ch25h-/- mice. RESULTS: We found that Ch25h level was decreased in livers of ob/ob mice and E3 rats fed an HFD. Ch25h-/- mice fed an HFD showed aggravated fatty liver and decreased level of cytochrome P450 7A1 (CYP7A1), in comparison with their WT littermates. RNA-seq analysis revealed that the differentially expressed genes in livers of HFD-fed Ch25h-/- mice were involved in pathways of positive regulation of lipid metabolic process, steroid metabolic process, cholesterol metabolic process, and bile acid biosynthetic process. As gain-of-function experiments, WT mice receiving AAV8-Ch25h or 25-HC showed alleviated NAFLD, when compared with the control group receiving AAV8-control or vehicle control. Consistently, Ch25h overexpression significantly elevated the levels of primary and secondary bile acids and CYP7A1 but decreased those of small heterodimer partner and FGFR4. CONCLUSIONS: Elevated levels of Ch25h and its enzymatic product 25-HC alleviate HFD-induced hepatic steatosis via regulating enterohepatic circulation of bile acids. The underlying mechanism involves 25-HC activation of CYP7A1 via liver X receptor. These data suggest that targeting Ch25h or 25-HC may have therapeutic advantages against nonalcoholic fatty liver disease.