25-Hydroxycholesterol Inhibits Kaposi's Sarcoma Herpesvirus and Epstein-Barr Virus Infections and Activates Inflammatory Cytokine Responses.

Oncogenic gammaherpesviruses express viral products during latent and lytic infection that block the innate immune response. Previously, we found that Kaposi's sarcoma herpesvirus (KSHV/human herpesvirus-8) viral microRNAs (miRNAs) downregulate cholesterol biogenesis, and we hypothesized that this prevents the production of 25-hydroxycholesterol (25HC), a cholesterol derivative. 25HC blocks KSHV de novo infection of primary endothelial cells at a postentry step and decreases viral gene expression of LANA (latency-associated nuclear antigen) and RTA. Herein we expanded on this observation by determining transcriptomic changes associated with 25HC treatment of primary endothelial cells using RNA sequencing (RNA-Seq). We found that 25HC treatment inhibited KSHV gene expression and induced interferon-stimulated genes (ISGs) and several inflammatory cytokines (interleukin 8 [IL-8], IL-1α). Some 25HC-induced genes were partially responsible for the broadly antiviral effect of 25HC against several viruses. Additionally, we found that 25HC inhibited infection of primary B cells by a related oncogenic virus, Epstein-Barr virus (EBV/human herpesvirus-4) by suppressing key viral genes such as LMP-1 and inducing apoptosis. RNA-Seq analysis revealed that IL-1 and IL-8 pathways were induced by 25HC in both primary endothelial cells and B cells. We also found that the gene encoding cholesterol 25-hydroxylase (CH25H), which converts cholesterol to 25HC, can be induced by type I interferon (IFN) in human B cell-enriched peripheral blood mononuclear cells (PBMCs). We propose a model wherein viral miRNAs target the cholesterol pathway to prevent 25HC production and subsequent induction of antiviral ISGs. Together, these results answer some important questions about a widely acting antiviral (25HC), with implications for multiple viral and bacterial infections. IMPORTANCE A cholesterol derivative, 25-hydroxycholesterol (25HC), has been demonstrated to inhibit infections from widely different bacteria and viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, its mechanism of activity is still not fully understood. In this work, we look at gene expression changes in the host and virus after 25HC treatment to find clues about its antiviral activity. We likewise demonstrate that 25HC is also antiviral against EBV, a common cancer-causing virus. We compared our results with previous data from antiviral screening assays and found the same pathways resulting in antiviral activity. Together, these results bring us closer to understanding how a modified form of cholesterol works against several viruses.

The cholesterol metabolite 25-hydroxycholesterol restrains the transcriptional regulator SREBP2 and limits intestinal IgA plasma cell differentiation.

Diets high in cholesterol alter intestinal immunity. Here, we examined how the cholesterol metabolite 25-hydroxycholesterol (25-HC) impacts the intestinal B cell response. Mice lacking cholesterol 25-hydroxylase (CH25H), the enzyme generating 25-HC, had higher frequencies of immunoglobulin A (IgA)-secreting antigen-specific B cells upon immunization or infection. 25-HC did not affect class-switch recombination but rather restrained plasma cell (PC) differentiation. 25-HC was produced by follicular dendritic cells and increased in response to dietary cholesterol. Mechanistically, 25-HC restricted activation of the sterol-sensing transcription factor SREBP2, thereby regulating B cell cholesterol biosynthesis. Ectopic expression of SREBP2 in germinal center B cells induced rapid PC differentiation, whereas SREBP2 deficiency reduced PC output in vitro and in vivo. High-cholesterol diet impaired, whereas Ch25h deficiency enhanced, the IgA response against Salmonella and the resulting protection from systemic bacterial dissemination. Thus, a 25-HC-SREBP2 axis shapes the humoral response at the intestinal barrier, providing insight into the effect of high dietary cholesterol in intestinal immunity.

Antiviral Actions of 25-Hydroxycholesterol in Fish Vary With the Virus-Host Combination.

Cholesterol is essential for building and maintaining cell membranes and is critical for several steps in the replication cycle of viruses, especially for enveloped viruses. In mammalian cells virus infections lead to the accumulation of the oxysterol 25-hydroxycholesterol (25HC), an antiviral factor, which is produced from cholesterol by the cholesterol 25 hydroxylase (CH25H). Antiviral responses based on CH25H are not well studied in fish. Therefore, in the present study putative genes encoding for CH25H were identified and amplified in common carp and rainbow trout cells and an HPLC-MS method was applied for determination of oxysterol concentrations in these cells under virus infection. Our results give some evidence that the activation of CH25H could be a part of the antiviral response against a broad spectrum of viruses infecting fish, in both common carp and rainbow trout cells in vitro. Quantification of oxysterols showed that fibroblastic cells are capable of producing 25HC and its metabolite 7α,25diHC. The oxysterol 25HC showed an antiviral activity by blocking the entry of cyprinid herpesvirus 3 (CyHV-3) into KFC cells, but not spring viremia of carp virus (SVCV) or common carp paramyxovirus (Para) in the same cells, or viral haemorrhagic septicaemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) into RTG-2 cells. Despite the fact that the CH25H based antiviral response coincides with type I IFN responses, the stimulation of salmonid cells with recombinant type I IFN proteins from rainbow trout could not induce ch25h_b gene expression. This provided further evidence, that the CH25H-response is not type I IFN dependent. Interestingly, the susceptibility of CyHV-3 to 25HC is counteracted by a downregulation of the expression of the ch25h_b gene in carp fibroblasts during CyHV-3 infection. This shows a unique interplay between oxysterol based immune responses and immunomodulatory abilities of certain viruses.

Hydroxycholesterol binds and enhances the anti-viral activities of zebrafish monomeric c-reactive protein isoforms.

C-reactive proteins (CRPs) are among the faster acute-phase inflammation-responses proteins encoded by one gene (hcrp) in humans and seven genes (crp1-7) in zebrafish (Danio rerio) with importance in bacterial and viral infections. In this study, we described novel preferential bindings of 25-hydroxycholesterol (25HOCh) to CRP1-7 compared with other lipids and explored the antiviral effects of both 25HOCh and CRP1-7 against spring viremia carp virus (SVCV) infection in zebrafish. Both in silico and in vitro results confirmed the antiviral effect of 25HOCh and CRP1-7 interactions, thereby showing that the crosstalk between them differed among the zebrafish isoforms. The presence of oxidized cholesterols in human atherosclerotic plaques amplifies the importance that similar interactions may occur for vascular and/or neurodegenerative diseases during viral infections. In this context, the zebrafish model offers a genetic tool to further investigate these interactions.

Regulating Innate and Adaptive Immunity for Controlling SIV Infection by 25-Hydroxycholesterol.

Persistent inflammation and extensive immune activation have been associated with HIV-1/SIV pathogenesis. Previously, we reported that cholesterol-25-hydroxylase (CH25H) and its metabolite 25-hydroxycholesterol (25-HC) had a broad antiviral activity in inhibiting Zika, Ebola, and HIV-1 infection. However, the underlying immunological mechanism of CH25H and 25-HC in inhibiting viral infection remains poorly understood. We report here that 25-HC effectively regulates immune responses for controlling viral infection. CH25H expression was interferon-dependent and induced by SIV infection in monkey-derived macrophages and PBMC cells, and 25-HC inhibited SIV infection both in permissive cell lines and primary monkey lymphocytes. 25-HC also strongly inhibited bacterial lipopolysaccharide (LPS)-stimulated inflammation and restricted mitogen-stimulated proliferation in primary monkey lymphocytes. Strikingly, 25-HC promoted SIV-specific IFN-γ-producing cellular responses, but selectively suppressed proinflammatory CD4+ T lymphocytes secreting IL-2 and TNF-α cytokines in vaccinated mice. In addition, 25-HC had no significant immunosuppressive effects on cytotoxic CD8+ T lymphocytes or antibody-producing B lymphocytes. Collectively, 25-HC modulated both innate and adaptive immune responses toward inhibiting HIV/SIV infection. This study provides insights into improving vaccination and immunotherapy regimes against HIV-1 infection.

25-Hydroxycholesterol and 27-hydroxycholesterol inhibit human rotavirus infection by sequestering viral particles into late endosomes.

A novel innate immune strategy, involving specific cholesterol oxidation products as effectors, has begun to reveal connections between cholesterol metabolism and immune response against viral infections. Indeed, 25-hydroxycholesterol (25HC) and 27-hydroxycholesterol (27HC), physiologically produced by enzymatic oxidation of cholesterol, act as inhibitors of a wide spectrum of enveloped and non-enveloped human viruses. However, the mechanisms underlying their protective effects against non-enveloped viruses are almost completely unexplored. To get insight into this field, we investigated the antiviral activity of 25HC and 27HC against a non-enveloped virus causing acute gastroenteritis in children, the human rotavirus (HRV). We found that 25HC and 27HC block the infectivity of several HRV strains at 50% inhibitory concentrations in the low micromolar range in the absence of cell toxicity. Both molecules affect the final step of virus penetration into cells by preventing the association of two cellular proteins: the oxysterol binding protein (OSBP) and the vesicle-associated membrane protein-associated protein-A (VAP-A). By altering the activity of these cellular mediators, 25HC and 27HC disturb the recycling of cholesterol between the endoplasmic reticulum and the late endosomes which are exploited by HRV to penetrate into the cell. The substantial accumulation of cholesterol in the late endosomal compartment results in sequestering viral particles inside these vesicles thereby preventing cytoplasmic virus replication. These findings suggest that cholesterol oxidation products of enzymatic origin might be primary effectors of host restriction strategies to counteract HRV infection and point to redox active lipids involvement in viral infections as a research area of focus to better focus in order to identify novel antiviral agents targets.

Interferon-independent antiviral activity of 25-hydroxycholesterol in a teleost fish.

Oxysterols are a family of cholesterol oxygenated derivatives with diverse roles in many biological activities and have recently been linked with the induction of a cellular antiviral state. The antiviral effects of 25-hydroxycholesterol (25HC) extend to several mammalian enveloped and non-enveloped viruses. It has been reported that the expression of the gene encoding cholesterol 25-hydroxylase (CH25H) is induced by interferons (IFNs). In this work, five ch25h genes were identified in the zebrafish (Danio rerio) genome. The ch25h genes showed different tissue expression patterns and differed in their expression after immune stimulation with lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (PolyI:C) and Spring Viremia Carp Virus (SVCV). Only one of the 5 genes, ch25hb, was overexpressed after the administration of the treatments. Synteny and phylogenetic analyses revealed that ch25hb is the putative homolog of mammalian Ch25h in zebrafish, while the remaining zebrafish ch25h genes are products of duplications within the teleost lineage. Interestingly, its modulation was not mediated by type I IFNs, contrasting previous reports on mammalian orthologs. Nevertheless, in vivo overexpression of ch25hb in zebrafish larvae significantly reduced mortality after SVCV challenge. Viral replication was also negatively affected by 25HC administration to the zebrafish cell line ZF4. In conclusion, the interferon-independent antiviral role of 25HC was extended to a non-mammalian species for the first time, and dual activity that both protects the cells and interacts with the virus cannot be discarded.

Immune oxysterols: Role in mycobacterial infection and inflammation.

Infection remains an important cause of morbidity and mortality. Natural defenses to infection are mediated by intrinsic/innate and adaptive immune responses. While our understanding is considerable it is incomplete and emerging areas of research such as those related to the immune-metabolic axis are only beginning to be appreciated. There is increasing evidence showing a connection between immune signalling and the regulation of sterol and fatty acid metabolism. In particular, metabolic intermediates of cholesterol biosynthesis and its oxidized metabolites (oxysterols) have been shown to regulate adaptive immunity and inflammation and for innate immune signalling to regulate the dynamics of cholesterol synthesis and homeostasis. The side-chain oxidized oxysterols, 25-hydroxycholesterol (25HC) and vitamin D metabolites (vitamin D3 and vitamin D2), are now known to impart physiologically profound effects on immune responses. Macrophages play a frontline role in this process connecting immunity, infection and lipid biology, and collaterally are a central target for infection by a wide range of pathogens including viruses and bacteria, especially intracellular bacteria such as mycobacteria. Clinical manifestations of disease severity in the infected host are likely to pay tribute to perturbations of the metabolic-immune phenomena found in lymphocytes and myeloid cells. Historically and consistent with this notion, vitamin D based oxysterols have had a long association with promoting clinical improvements to patients infected with Mycobacterium tuberculosis. Hence understanding the role of early metabolic mediators of inflammatory responses to infection in particular oxysterols, will aid in the development of urgently needed host directed therapeutic and diagnostic design innovation to combat adverse infection outcomes and antibiotic resistance.

Cholesterol metabolism and immunity.

A study in mice implicates a cholesterol derivative in damping down the inflammatory response mediated by interleukin-1ß and explaining, at least in part, the immunosuppressive effect of type I interferon, which is used in the treatment of multiple sclerosis.

25-Hydroxycholesterols in innate and adaptive immunity.

Cholesterol and components of the cholesterol biosynthetic pathway have fundamental roles in all mammalian cells. Hydroxylated forms of cholesterol are now emerging as important regulators of immune function. This involves effects on the cholesterol biosynthetic pathway and cell membrane properties, which can have antiviral and anti-inflammatory influences. In addition, a dihydroxylated form of cholesterol functions as an immune cell guidance cue by engaging the G protein-coupled receptor EBI2, and it is required for mounting adaptive immune responses. In this Review, we summarize the current understanding of the closely related oxysterols 25-hydroxycholesterol and 7α,25-dihydroxycholesterol, and the growing evidence that they have wide-ranging influences on innate and adaptive immunity.

Transcriptional regulation and functional characterization of the oxysterol/EBI2 system in primary human macrophages.

Oxysterols such as 7 alpha, 25-dihydroxycholesterol (7α,25-OHC) are natural ligands for the Epstein-Barr virus (EBV)-induced gene 2 (EBI2, aka GPR183), a G protein-coupled receptor (GPCR) highly expressed in immune cells and required for adaptive immune responses. Activation of EBI2 by specific oxysterols leads to chemotaxis of B cells in lymphoid tissues. While the ligand gradient necessary for this critical process of the adaptive immune response is established by a stromal cells subset here we investigate the involvement of the oxysterol/EBI2 system in the innate immune response. First, we show that primary human macrophages express EBI2 and the enzymes needed for ligand production such as cholesterol 25-hydroxylase (CH25H), sterol 27-hydroxylase (CYP27A1), and oxysterol 7α-hydroxylase (CYP7B1). Furthermore, challenge of monocyte-derived macrophages with lipopolysaccharides (LPS) triggers a strong up-regulation of CH25H and CYP7B1 in comparison to a transient increase in EBI2 expression. Stimulation of EBI2 expressed on macrophages leads to calcium mobilization and to directed cell migration. Supernatants of LPS-stimulated macrophages are able to stimulate EBI2 signaling indicating that an induction of CH25H, CYP27A1, and CYP7B1 results in an enhanced production and release of oxysterols into the cellular environment. This is a study characterizing the oxysterol/EBI2 pathway in primary monocyte-derived macrophages. Given the crucial functional role of macrophages in the innate immune response these results encourage further exploration of a possible link to systemic autoimmunity.

On the formation and possible biological role of 25-hydroxycholesterol.

The oxysterol 25-hydroxycholesterol is a widely used compound displaying an array of pharmacological actions in in vitro systems and cell based experimental systems. In spite of the frequent use of this compound over the last few decades and a large number of studies in vitro and in vivo, its mechanism of formation in vivo is still not well understood. Cholesterol autoxidation does not seem to be an important contributor to in vivo formation of 25-hydroxycholesterol. A number of different cytochrome P450 enzymes such as CYP27A1 and CYP3A4 have been reported to catalyze the conversion of cholesterol to 25-hydroxycholesterol in vitro, but the importance of these reactions in vivo remains unclear. The dioxygenase enzyme cholesterol 25-hydroxylase has been shown to generate 25-hydroxycholesterol, but in cholesterol 25-hydroxylase knockout mice there are still significant levels of 25-hydroxycholesterol in several tissues. This suggests that cholesterol 25-hydroxylase is not the sole producer of 25-hydroxycholesterol. The relative importance of different mechanisms of formation of 25-hydroxycholesterol in vivo have still to be elucidated. The maintenance of cholesterol homeostasis is of great importance to supply tissues with the appropriate amount of cholesterol and prevent accumulation that may affect health. Numerous articles mention 25-hydroxycholesterol as an important regulator of cholesterol metabolism. However, mice with a disruption of the cholesterol 25-hydroxylase gene regulate cholesterol metabolism normally and patients with highly elevated levels of 25-hydroxycholesterol also display normal cholesterol and bile acid levels. These reports challenge the hypothesis that 25-hydroxycholesterol is an important regulator of cholesterol metabolism. Recent reports suggest that 25-hydroxycholesterol and one of its metabolites may have functions in regulation of humoral immunity. Thus, 25-hydroxycholesterol may be more important as a regulator of immunity than as a regulator of cholesterol metabolism.

Oxysterol gradient generation by lymphoid stromal cells guides activated B cell movement during humoral responses.

7α,25-dihydroxycholesterol (7α,25-OHC) is a ligand for the G protein-coupled receptor EBI2; however, the cellular sources of this oxysterol are undefined. 7α,25-OHC is synthesized from cholesterol by the stepwise actions of two enzymes, CH25H and CYP7B1, and is metabolized to a 3-oxo derivative by HSD3B7. We showed that all three enzymes control EBI2 ligand concentration in lymphoid tissues. Lymphoid stromal cells were the main CH25H- and CYP7B1-expressing cells required for positioning of B cells, and they also mediated 7α,25-OHC inactivation. CH25H and CYP7B1 were abundant at the follicle perimeter, whereas CH25H expression by follicular dendritic cells was repressed. CYP7B1, CH25H, and HSD3B7 deficiencies each resulted in defective T cell-dependent plasma cell responses. These findings establish that CYP7B1 and HSD3B7, as well as CH25H, have essential roles in controlling oxysterol production in lymphoid tissues, and they suggest that differential enzyme expression in stromal cell subsets establishes 7α,25-OHC gradients required for B cell responses.

Coronary atherosclerosis and oxidative stress as reflected by autoantibodies against oxidized low-density lipoprotein and oxysterols.

Clinical studies and animal experiments have demonstrated that oxidized low-density lipoprotein (oxLDL) and oxysterols play important roles in atherogenesis. OxLDL is immunogenic, and autoantibodies (Ab) against oxLDL are detectable in serum. We investigated the relevance of oxysterols and Ab against-oxLDL to coronary artery disease (CAD) in 183 patients undergoing coronary angiography. Patient groups included angiographically normal subjects (< 75% stenosis), others with spasm (> 75% narrowing in response to acetylcholine), and some others with fixed stenosis (> 75%). The group with stenosis was subdivided into patients with stable and unstable angina. Serum concentrations of autoantibodies and 25-, 27-, and 7-beta-hydroxycholesterols were significantly higher in the stenotic group than in the normal group (P < 0.01, P < 0.05, P < 0.05, and P < 0.05, respectively). Antibodies, but not oxysterol concentrations, were significantly greater in subjects with unstable than with stable angina (P < 0.01). We conclude that anti-oxLDL antibody and oxysterol concentrations are associated with coronary artery stenosis, and that oxidative stress may be greatly increased in unstable angina.

Studies on the immunological properties of oxysterols: in vivo actions of 7,25-dihydroxycholesterol upon murine peritoneal cells.

Oxygenated derivatives of cholesterol are potent immunosuppressors. It has been reported previously that 7,25-dihydroxycholesterol (7,25-OHC), synthesized in the URA31, strongly inhibits the early steps of T-cell activation. So far, the mechanisms underlying this type of effect have been mainly investigated in vitro, and the activity of these substances on the immune system has been poorly studied. This study describes that a single i.p. injection of 7,25-OHC induces a strong inflammatory response, consisting of a massive influx of macrophages and neutrophils into the abdominal cavity. Macrophages harvested from 7,25-OHC-treated mice express class II antigen to a lesser extent. Moreover, the 7,25-OHC treatment abolishes the class II induction by bacillus Calmette-Guérin (BCG) or interferon-gamma (IFN-gamma). The inflammatory process triggered by the oxysterol is not the consequence of a non-specific effect due, for instance, to the presence of crystals in the abdominal cavity. Moreover, treatments by inhibitors of the acid arachidonic cascade do not affect the peritoneal exudate cell (PEC) influx induced by these substances. This study could be an important contribution to the mechanism determining the oxysterol-induced immunosuppression.