RNA sequencing screening and gene function analysis uncover G protein-coupled receptor 183 as a key mediator for methionine to stimulate milk synthesis in mouse mammary epithelial cells.

Methionine (Met) can activate the mechanistic target of rapamycin (mTOR) to promote milk synthesis in mammary epithelial cells. However, it is largely unknown which G protein-coupled receptor can mediate the stimulation of Met on mTOR activation. In this study, we employed transcriptome sequencing to analyse which G protein-coupled receptors were associated with the role of Met and further used gene function study approaches to explore the role of G protein-coupled receptor 183 (GPR183) in Met stimulation on mTOR activation in HC11 cells. We identified nine G protein-coupled receptors including GPR183 whose expression levels were upregulated by Met treatment through RNA sequencing and subsequent quantitative real-time PCR analysis. Using GPR183 knockdown and overexpression technology, we demonstrate that GPR183 is a positive regulator of milk protein and fat synthesis and proliferation of HC11 cells. Met affected GPR183 expression in a dose-dependent manner, and GPR183 mediated the stimulation of Met (0·6 mM) on milk protein and fat synthesis, cell proliferation and mTOR phosphorylation and mRNA expression. The inhibition of phosphoinositide 3-kinase blocked the phosphorylation of mTOR and AKT stimulated by GPR183 activation. In summary, through RNA sequencing and gene function study, we uncover that GPR183 is a key mediator for Met to activate the phosphoinositide 3-kinase-mTOR signalling and milk synthesis in mouse mammary epithelial cells.

First report on chemometrics-driven multilayered lead prioritization in addressing oxysterol-mediated overexpression of G protein-coupled receptor 183.

Contemporary research has convincingly demonstrated that upregulation of G protein-coupled receptor 183 (GPR183), orchestrated by its endogenous agonist, 7α,25-dihydroxyxcholesterol (7α,25-OHC), leads to the development of cancer, diabetes, multiple sclerosis, infectious, and inflammatory diseases. A recent study unveiled the cryo-EM structure of 7α,25-OHC bound GPR183 complex, presenting an untapped opportunity for computational exploration of potential GPR183 inhibitors, which served as our inspiration for the current work. A predictive and validated two-dimensional QSAR model using genetic algorithm (GA) and multiple linear regression (MLR) on experimental GPR183 inhibition data was developed. QSAR study highlighted that structural features like dissimilar electronegative atoms, quaternary carbon atoms, and CH2RX fragment (X: heteroatoms) influence positively, while the existence of oxygen atoms with a topological separation of 3, negatively affects GPR183 inhibitory activity. Post assessment of true external set prediction capability, the MLR model was deployed to screen 12,449 DrugBank compounds, followed by a screening pipeline involving molecular docking, druglikeness, ADMET, protein-ligand stability assessment using deep learning algorithm, molecular dynamics, and molecular mechanics. The current findings strongly evidenced DB05790 as a potential lead for prospective interference of oxysterol-mediated GPR183 overexpression, warranting further in vitro and in vivo validation.

Single-cell transcriptomics reveals tumor-infiltrating B cell function after neoadjuvant pembrolizumab and chemotherapy in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most pervasive lung cancer subtype. Recent studies have shown that immune checkpoint inhibitors achieved favorable clinical benefits in resectable NSCLC; however, the associated mechanism remains unclear. The role of T cells in antitumor immunity has received considerable attention, while the antitumor effects of tumor-infiltrating B cells (TIBs) in NSCLC remain poorly understood. Here, we conducted a single-cell RNA-seq analysis of immune cells isolated from 12 patients with IIIA NSCLC to investigate B cell subtypes and their functions following neoadjuvant chemoimmunotherapy. We confirmed the simultaneous existence of the four B cell subtypes. Among them, memory B cells were found to be associated with a positive therapeutic effect to neoadjuvant chemoimmunotherapy. Furthermore, we found that G Protein-Coupled Receptor 183 (GPR183) was most prevalent in memory B cells and associated with a positive therapeutic response. Multiplex immunofluorescence and flow cytometry experiments in an additional cohort of 22 treatment-naïve and 30 IIIA/IIIB NSCLC patients treated with neoadjuvant chemoimmunotherapy verified these findings. Overall, our analysis revealed the functions of TIBs and their potential effect on clinical treatment in NSCLC.

Skin γδ T cell inflammatory responses are hardwired in the thymus by oxysterol sensing via GPR183 and calibrated by dietary cholesterol.

Dietary components and metabolites have a profound impact on immunity and inflammation. Here, we investigated how sensing of cholesterol metabolite oxysterols by γδ T cells impacts their tissue residency and function. We show that dermal IL-17-producing γδ T (Tγδ17) cells essential for skin-barrier homeostasis require oxysterols sensing through G protein receptor 183 (GPR183) for their development and inflammatory responses. Single-cell transcriptomics and murine reporter strains revealed that GPR183 on developing γδ thymocytes is needed for their maturation by sensing medullary thymic epithelial-cell-derived oxysterols. In the skin, basal keratinocytes expressing the oxysterol enzyme cholesterol 25-hydroxylase (CH25H) maintain dermal Tγδ17 cells. Diet-driven increases in oxysterols exacerbate Tγδ17-cell-mediated psoriatic inflammation, dependent on GPR183 on γδ T cells. Hence, cholesterol-derived oxysterols control spatially distinct but biologically linked processes of thymic education and peripheral function of dermal T cells, implicating diet as a focal parameter of dermal Tγδ17 cells.

Identification of a potential structure-based GPCR drug for interstitial cystitis/bladder pain syndrome: in silico protein structure analysis and molecular docking.

INTRODUCTION AND HYPOTHESIS: There is currently no effective treatment for interstitial cystitis / bladder pain syndrome (IC/BPS) and thus seriously reduces the quality of life of patients. The purpose of this study is to analyze the structure and function of G protein coupled receptors related to IC/BPS by integrating bioinformatics and provide basis for the development of new drugs for IC/BPS. METHODS: We used ProtParam and DNAMAN to analyze the physical and chemical properties of GPR18 and GPR183 proteins. The secondary and tertiary structure, conservative domain, phosphorylation site of both proteins were predicted by ProtScale, PredictProtein, SWISS-MODEL and GPS5.0 respectively. Multiple sequence alignment of the proteins were carried out by DNAMAN and the phylogenetic tree was constructed by MEGA. Further, the molecular docking verification of cannabidiol and both proteins were carried out by using AutoDock Vin. RESULTS: GPR18 and GPR183 proteins were composed of 331 and 361 amino acids respectively. α-helix is the highest in the secondary structure of the two proteins. Both proteins contain seven transmembrane domains specific to G protein coupled receptors. And homology analysis showed that the two proteins had high homology. In terms of molecular docking, cannabidiol, a non psychoactive component extracted from the cannabis, can form effective molecular binding with GPR18 and GPR183 proteins. CONCLUSIONS: We identified the structures of GPR18 and GPR183 proteins and their highly homologous evolutionary properties. Furthermore, both proteins can form effective binding with cannabidiol which provides new insights for the development of IC/BPS drugs by targeting G protein coupled receptors.

Evaluation of endogenous and therapeutic 25-hydroxycholesterols in murine models of pulmonary SARS-CoV-2 infection.

Oxysterols (i.e., oxidized cholesterol species) have complex roles in biology. 25-hydroxycholesterol (25HC), a product of activity of cholesterol-25-hydroxylase (CH25H) upon cholesterol, has recently been shown to be broadly antiviral, suggesting therapeutic potential against SARS-CoV-2. However, 25HC can also amplify inflammation and tissue injury and be converted by CYP7B1 to 7α,25HC, a lipid with chemoattractant activity via the G protein-coupled receptor, EBI2/GPR183. Here, using in vitro studies and two different murine models of SARS-CoV-2 infection, we investigate the effects of these two oxysterols on SARS-CoV-2 pneumonia. We show that while 25HC and enantiomeric-25HC are antiviral in vitro against human endemic coronavirus-229E, they did not inhibit SARS-CoV-2; nor did supplemental 25HC reduce pulmonary SARS-CoV-2 titers in the K18-human ACE2 mouse model in vivo. 25HC treatment also did not alter immune cell influx into the airway, airspace cytokines, lung pathology, weight loss, symptoms, or survival but was associated with increased airspace albumin, an indicator of microvascular injury, and increased plasma pro-inflammatory cytokines. Conversely, mice treated with the EBI2/GPR183 inhibitor NIBR189 displayed a modest increase in lung viral load only at late time points, but no change in weight loss. Consistent with these findings, although Ch25h was upregulated in the lungs of SARS-CoV-2-infected WT mice, lung viral titers and weight loss in Ch25h-/- and Gpr183-/- mice infected with the beta variant were similar to control animals. Taken together, endogenous 25-hydroxycholesterols do not significantly regulate early SARS-CoV-2 replication or pathogenesis and supplemental 25HC may have pro-injury rather than therapeutic effects in SARS-CoV-2 pneumonia.

Abnormal lower expression of GPR183 in peripheral blood T and B cell subsets of systemic lupus erythematosus patients.

G protein-coupled receptor 183 (GPR183) has been indicated to mediate the migration and localisation of immune cells in T cell-dependent antibody responses. Systemic lupus erythematosus (SLE) is a canonical autoimmune disease involving B cell-mediated tolerance destruction and excessive pathogenic autoantibody production, in which multiple GPCRs play a role. To date, there has been no systematic study regarding the expression of GPR183 in lymphocyte subsets of SLE patients. In this research, firstly, we observed the expression trends of GRP183 in various T and B cell subsets in human tonsil tissues. These lymphocyte subsets include CD4+, CD8+, naïve T, effector T, Tfh, activated Tfh, Th1, Th2, Th17, Treg, CD19+CD27-, CD19+CD27+, naïve B, germinal centre B, memory B, and plasma cells. Further, compared with healthy controls (HCs), GPR183 expression levels in above peripheral blood lymphocyte subsets of patients with SLE were reduced overall. The differential expression of GPR183 expression between inactive and active SLE patients indicates that GPR183 expression may be concerned with the disease activity of SLE. This was further confirmed through the strong negative correlation with SLEDAI score and positive correlation with serum complement protein C3, C4 and C1q levels. Further receiver operating characteristic (ROC) curve analysis revealed that GPR183 expression in circulating CD27-IgD+ B cells may be beneficial in distinguishing between inactive and active SLE patients. In addition, type I interferon stimulation could down-regulate the expression of GPR183 in peripheral blood T and B cell subsets. Aberrant expression of GPR183 may provide some novel insights into disease activity prediction and underlying pathogenesis of SLE.

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.

Structures of oxysterol sensor EBI2/GPR183, a key regulator of the immune response.

Oxysterols induce the migration of B-lymphocytes and dendritic cells to interfollicular regions of lymphoid tissues through binding the EBI2 (GPR183) to stimulate effective adaptive immunity and antibody production during infection. Aberrant EBI2 signaling is implicated in inflammatory bowel disease, sclerosis, and infectious disease. Here, we report the cryo-EM structures of an EBI2-Gi signaling complex with its endogenous agonist 7α,25-OHC and that of an inactive EBI2 bound to the inverse agonist GSK682753A. These structures reveal an agonist binding site for the oxysterol and a potential ligand entrance site exposed to the lipid bilayer. Mutations within the oxysterol binding site and the Gαi interface attenuate G protein signaling and abolish oxysterol-mediated cell migration indicating that G protein signaling directly involves in the oxysterol-EBI2 pathway. Together, these findings provide new insight into how EBI2 is activated by an oxysterol ligand and will facilitate the development of therapeutic approaches that target EBI2-linked diseases.

A Blunted GPR183/Oxysterol Axis During Dysglycemia Results in Delayed Recruitment of Macrophages to the Lung During Mycobacterium tuberculosis Infection.

BACKGROUND: We previously reported that reduced GPR183 expression in blood from tuberculosis (TB) patients with diabetes is associated with more severe TB. METHODS: To further elucidate the role of GPR183 and its oxysterol ligands in the lung, we studied dysglycemic mice infected with Mycobacterium tuberculosis (Mtb). RESULTS: We found upregulation of the oxysterol-producing enzymes CH25H and CYP7B1 and increased concentrations of 25-hydroxycholesterol upon Mtb infection in the lungs of mice. This was associated with increased expression of GPR183 indicative of oxysterol-mediated recruitment of GPR183-expressing immune cells to the lung. CYP7B1 was predominantly expressed by macrophages in TB granulomas. CYP7B1 expression was significantly blunted in lungs from dysglycemic animals, which coincided with delayed macrophage infiltration. GPR183-deficient mice similarly had reduced macrophage recruitment during early infection. CONCLUSIONS: Taken together, we demonstrate a requirement of the GPR183/oxysterol axis for positioning of macrophages to the site of infection and add an explanation to more severe TB in diabetes patients.

GPR183 Is Dispensable for B1 Cell Accumulation and Function, but Affects B2 Cell Abundance, in the Omentum and Peritoneal Cavity.

B1 cells constitute a specialized subset of B cells, best characterized in mice, which is abundant in body cavities, including the peritoneal cavity. Through natural and antigen-induced antibody production, B1 cells participate in the early defense against bacteria. The G protein-coupled receptor 183 (GPR183), also known as Epstein-Barr virus-induced gene 2 (EBI2), is an oxysterol-activated chemotactic receptor that regulates migration of B cells. We investigated the role of GPR183 in B1 cells in the peritoneal cavity and omentum. B1 cells expressed GPR183 at the mRNA level and migrated towards the GPR183 ligand 7α,25-dihydroxycholesterol (7α,25-OHC). GPR183 knock-out (KO) mice had smaller omenta, but with normal numbers of B1 cells, whereas they had fewer B2 cells in the omentum and peritoneal cavity than wildtype (WT) mice. GPR183 was not responsible for B1 cell accumulation in the omentum in response to i.p. lipopolysaccharide (LPS)-injection, in spite of a massive increase in 7α,25-OHC levels. Lack of GPR183 also did not affect B1a- or B1b cell-specific antibody responses after vaccination. In conclusion, we found that GPR183 is non-essential for the accumulation and function of B1 cells in the omentum and peritoneal cavity, but that it influences the abundance of B2 cells in these compartments.

Oxysterols in the Immune Response to Bacterial and Viral Infections.

Oxidized cholesterols, the so-called oxysterols, are widely known to regulate cholesterol homeostasis. However, more recently oxysterols have emerged as important lipid mediators in the response to both bacterial and viral infections. This review summarizes our current knowledge of selected oxysterols and their receptors in the control of intracellular bacterial growth as well as viral entry into the host cell and viral replication. Lastly, we briefly discuss the potential of oxysterols and their receptors as drug targets for infectious and inflammatory diseases.

Discovery of GPR183 Agonists Based on an Antagonist Scaffold.

The G protein-coupled receptor GPR183/EBI2, which is activated by oxysterols, is a therapeutic target for inflammatory and metabolic diseases where both antagonists and agonists are of potential interest. Using the piperazine diamide core of the known GPR183 antagonist (E)-3-(4-bromophenyl)-1-(4-(4-methoxybenzoyl)piperazin-1-yl)prop-2-en-1-one (NIBR189) as starting point, we identified and sourced 79 structurally related compounds that were commercially available. In vitro screening of this compound collection using a Ca2+ mobilization assay resulted in the identification of 10 compounds with agonist properties. To enable establishment of initial structure-activity relationship trends, these were supplemented with five in-house compounds, two of which were also shown to be GPR183 agonists. Taken together, our findings suggest that the agonist activity of this compound series is dictated by the substitution pattern of one of the two distal phenyl rings, which functions as a molecular efficacy-switch.

EBI2 is expressed in glial cells in multiple sclerosis lesions, and its knock-out modulates remyelination in the cuprizone model.

EBI2 receptor regulates the immune system, and in multiple, sclerosis is upregulated in the central nervous system infiltrating lymphocytes. In newborn EBI2-deficient mice, myelin development is delayed, and its persistent antagonism inhibits remyelination in chemically demyelinated organotypic cerebellar slices. We used the cuprizone model of multiple sclerosis to elucidate the role of central nervous system-expressed EBI2 in de- and remyelination. The wild-type and EBI2 knock-out mice were fed 0.2% cuprizone in chow for 5 weeks and allowed to recover on a normal diet for 2 weeks. The data showed less efficient recovery of myelin, attenuated oligodendrocyte loss, fewer astrocytes and increased total cholesterol levels in the EBI2 knock-out mice after recovery. Moreover, the wild-type mice upregulated EBI2 expression after recovery confirming the involvement of EBI2 signalling during recovery from demyelination in the cuprizone model. The pro-inflammatory cytokine levels were at comparable levels in the wild-type and EBI2 knock-out mice, with only minor differences in TNFα and IL1ß levels either at peak or during recovery. The neuroinflammatory signalling molecules, Abl1 kinase and NFКB1 (p105/p50) subunit, were significantly downregulated in the EBI2 knock-out mice at peak of disease. Immunohistochemical investigations of EBI2 receptor distribution in the central nervous system (CNS) cells in multiple sclerosis (MS) brain revealed strong expression of EBI2 in astrocytes and microglia inside the plaques implicating glia-expressed EBI2 in multiple sclerosis pathophysiology. Taken together, these findings demonstrate the involvement of EBI2 signalling in the recovery from demyelination rather than in demyelination and as such warrant further research into the role of EBI2 in remyelination.

The oxysterol receptor GPR183 in inflammatory bowel diseases.

Immune cell trafficking is an important mechanism for the pathogenesis of inflammatory bowel disease (IBD). The oxysterol receptor GPR183 and its ligands, dihydroxylated oxysterols, can mediate positioning of immune cells including innate lymphoid cells. GPR183 has been mapped to an IBD risk locus, however another gene, Ubac2 is encoded on the reverse strand and associated with Behçet's disease, therefore the role of GPR183 as a genetic risk factor requires validation. GPR183 and production of its oxysterol ligands are up-regulated in human IBD and murine colitis. Gpr183 inactivation reduced severity of colitis in group 3 innate lymphoid cells-dependent colitis and in IL-10 colitis but not in dextran sodium sulphate colitis. Irrespectively, Gpr183 knockout strongly reduced accumulation of intestinal lymphoid tissue in health and all colitis models. In conclusion, genetic, translational and experimental studies implicate GPR183 in IBD pathogenesis and GPR183-dependent cell migration might be a therapeutic drug target for IBD. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.

Prognostic Value of Genes and Immune Infiltration in Prostate Tumor Microenvironment.

BACKGROUND: Prostate cancer (PCa) is one of the most common cancers and the fifth leading cause of cancer-related death in men. Immune responses in the tumor microenvironment are hypothesized to be related to the prognosis of PCa patients; however, no studies are available to confirm the same. In this study, we aimed to explore the potential link between these two factors and identify new biomarkers to estimate the survival rate of PCa patients. METHODS: A total of 490 cases were obtained from The Cancer Genome Atlas (TCGA) database. The gene expression data were analyzed by the ESTIMATE algorithm to evaluate the immune and stromal scores. The survival rate was calculated according to the case-specific clinical data. Enrichment analysis was performed to discover the main biological processes and signaling pathways of immune responses. We further identified and analyzed hub genes in the protein-protein interaction (PPI) network and evaluated their prognostic values. RESULTS: Immune score significantly correlated with immune cell infiltration and overall survival of PCa patients. The genes CXCR4 and GPR183, identified as hub genes in the PPI network, correlated with immune cell infiltration and prognosis of PCa patients. CONCLUSION: CXCR4 and GPR183 participate in immune cell infiltration and function in PCa patients. The immune score, as well as the expression of CXCR4 and GPR183 in prostate cancer tissues, could be potential indexes for the prognosis of prostate cancer.

GPR183 Regulates Interferons, Autophagy, and Bacterial Growth During Mycobacterium tuberculosis Infection and Is Associated With TB Disease Severity.

Oxidized cholesterols have emerged as important signaling molecules of immune function, but little is known about the role of these oxysterols during mycobacterial infections. We found that expression of the oxysterol-receptor GPR183 was reduced in blood from patients with tuberculosis (TB) and type 2 diabetes (T2D) compared to TB patients without T2D and was associated with TB disease severity on chest x-ray. GPR183 activation by 7α,25-dihydroxycholesterol (7α,25-OHC) reduced growth of Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis BCG in primary human monocytes, an effect abrogated by the GPR183 antagonist GSK682753. Growth inhibition was associated with reduced IFN-ß and IL-10 expression and enhanced autophagy. Mice lacking GPR183 had significantly increased lung Mtb burden and dysregulated IFNs during early infection. Together, our data demonstrate that GPR183 is an important regulator of intracellular mycobacterial growth and interferons during mycobacterial infection.

Diverse Immunoregulatory Roles of Oxysterols-The Oxidized Cholesterol Metabolites.

Intermediates of both cholesterol synthesis and cholesterol metabolism can have diverse roles in the control of cellular processes that go beyond the control of cholesterol homeostasis. For example, oxidized forms of cholesterol, called oxysterols have functions ranging from the control of gene expression, signal transduction and cell migration. This is of particular interest in the context of immunology and immunometabolism where we now know that metabolic processes are key towards shaping the nature of immune responses. Equally, aberrant metabolic processes including altered cholesterol homeostasis contribute to immune dysregulation and dysfunction in pathological situations. This review article brings together our current understanding of how oxysterols affect the control of immune responses in diverse immunological settings.

Downregulation of GPR183 on infection restricts the early infection and intracellular replication of mycobacterium tuberculosis in macrophage.

GPR183/EBI2 is a key chemotactic receptor for the positioning of B cells in lymphoid organs, and also for the migration of T cells and other immune cells. Here, we demonstrate that the downregulation of GPR183 in macrophage induced during Mtb infection restrains the bacterial early infection and intracellular replication. Overexpression of GPR183 or stimulation with its natural ligand favors Mtb replication in macrophage, while treatment with its antagonist represses both Mtb early infection and intracellular replication. With mutational analysis, we find that substitution of Asp-73, Arg-83, Tyr-112, Tyr-256 abolished the promotive effect of GPR183 on Mtb early infection and replication in macrophage. In conclusion, we demonstrated that beside the known role of chemotaxis receptor, GPR183 also functions directly in the interaction between macrophage and Mtb in a cell-autonomous way.