Publications by authors named "Andreas W Sailer"

43 Publications

The oxysterol receptor GPR183 in inflammatory bowel diseases.

Br J Pharmacol 2020 Nov 4. Epub 2020 Nov 4.

Institute of Molecular Medicine, RWTH Aachen University, Aachen, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/bph.15311DOI Listing
November 2020

Elevated oxysterol levels in human and mouse livers reflect nonalcoholic steatohepatitis.

J Lipid Res 2019 07 21;60(7):1270-1283. Epub 2019 May 21.

Department of Gastroenterology and Hepatology, University Hospital Zurich and University of Zurich, Zurich, Switzerland

Nonalcoholic steatohepatitis (NASH), a primary cause of liver disease, leads to complications such as fibrosis, cirrhosis, and carcinoma, but the pathophysiology of NASH is incompletely understood. Epstein-Barr virus-induced G protein-coupled receptor 2 (EBI2) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-diHC) are recently discovered immune regulators. Several lines of evidence suggest a role of oxysterols in NASH pathogenesis, but rigorous testing has not been performed. We measured oxysterol levels in the livers of NASH patients by LC-MS and tested the role of the EBI2-7α,25-diHC system in a murine feeding model of NASH. Free oxysterol profiling in livers from NASH patients revealed a pronounced increase in 24- and 7-hydroxylated oxysterols in NASH compared with controls. Levels of 24- and 7-hydroxylated oxysterols correlated with histological NASH activity. Histological analysis of murine liver samples demonstrated ballooning and liver inflammation. No significant genotype-related differences were observed in mice and mice with defects in the 7α,25-diHC synthesizing enzymes CH25H and CYP7B1 compared with wild-type littermate controls, arguing against an essential role of these genes in NASH pathogenesis. Elevated 24- and 7-hydroxylated oxysterol levels were confirmed in murine NASH liver samples. Our results suggest increased bile acid synthesis in NASH samples, as judged by the enhanced level of 7α-hydroxycholest-4-en-3-one and impaired 24-hydroxycholesterol metabolism as characteristic biochemical changes in livers affected by NASH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1194/jlr.M093229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6602130PMC
July 2019

YAP, but Not RSPO-LGR4/5, Signaling in Biliary Epithelial Cells Promotes a Ductular Reaction in Response to Liver Injury.

Cell Stem Cell 2019 07 9;25(1):39-53.e10. Epub 2019 May 9.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland. Electronic address:

Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/β-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/β-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2019.04.005DOI Listing
July 2019

Enzymatic interconversion of the oxysterols 7β,25-dihydroxycholesterol and 7-keto,25-hydroxycholesterol by 11β-hydroxysteroid dehydrogenase type 1 and 2.

J Steroid Biochem Mol Biol 2019 06 19;190:19-28. Epub 2019 Mar 19.

Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland. Electronic address:

Oxysterols are cholesterol metabolites derived through either autoxidation or enzymatic processes. They consist of a large family of bioactive lipids that have been associated with the progression of multiple pathologies. In order to unravel (patho-)physiological mechanisms involving oxysterols, it is crucial to elucidate the underlying formation and degradation of oxysterols. A role of 11β-hydroxysteroid dehydrogenases (11β-HSDs) in oxysterol metabolism by catalyzing the interconversion of 7-ketocholesterol (7kC) and 7β-hydroxycholesterol (7βOHC) has already been reported. The present study addresses a function of 11β-HSD1 in the enzymatic generation of 7β,25-dihydroxycholesterol (7β25OHC) from 7-keto,25-hydroxycholesterol (7k25OHC) and tested whether 11β-HSD2 is able to catalyze the reverse reaction. For the first time, using recombinant enzymes, the formation of 7k25OHC from 7kC by cholesterol 25-hydroxylase (CH25H) and further stereospecific oxoreduction to 7β25OHC by human and mouse 11β-HSD1 could be demonstrated. Additionally, experiments using human 11β-HSD2 showed the oxidation of 7β25OHC to 7k25OHC. Molecular modeling provided an explanation for the stereospecific interconversion of 7β25OHC and 7k25OHC. Production of the Epstein-Barr virus-induced gene 2 (EBI2) ligand 7β25OHC from 7k25OHC in challenged tissue by 11β-HSD1 may be important in inflammation. In conclusion, these results demonstrate a novel glucocorticoid-independent pre-receptor regulation mediated by 11β-HSDs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jsbmb.2019.03.011DOI Listing
June 2019

The EBI2-oxysterol axis promotes the development of intestinal lymphoid structures and colitis.

Mucosal Immunol 2019 05 11;12(3):733-745. Epub 2019 Feb 11.

Department of Gastroenterology and Hepatology, University Hospital Zurich, University of Zurich, Zurich, Switzerland.

The gene encoding for Epstein-Barr virus-induced G-protein-coupled receptor 2 (EBI2) is a risk gene for inflammatory bowel disease (IBD). Together with its oxysterol ligand 7α,25-dihydroxycholesterol, EBI2 mediates migration and differentiation of immune cells. However, the role of EBI2 in the colonic immune system remains insufficiently studied. We found increased mRNA expression of EBI2 and oxysterol-synthesizing enzymes (CH25H, CYP7B1) in the inflamed colon of patients with ulcerative colitis and mice with acute or chronic dextran sulfate sodium (DSS) colitis. Accordingly, we detected elevated levels of 25-hydroxylated oxysterols, including 7α,25-dihydroxycholesterol in mice with acute colonic inflammation. Knockout of EBI2 or CH25H did not affect severity of DSS colitis; however, inflammation was decreased in male EBI2 mice in the IL-10 colitis model. The colonic immune system comprises mucosal lymphoid structures, which accumulate upon chronic inflammation in IL-10-deficient mice and in chronic DSS colitis. However, EBI2 mice formed significantly less colonic lymphoid structures at baseline and showed defects in inflammation-induced accumulation of lymphoid structures. In summary, we report induction of the EBI2-7α,25-dihydroxycholesterol axis in colitis and a role of EBI2 for the accumulation of lymphoid tissue during homeostasis and inflammation. These data implicate the EBI2-7α,25-dihydroxycholesterol axis in IBD pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41385-019-0140-xDOI Listing
May 2019

A novel modelling mechanism of PAEL receptor and GABARAPL2 interaction involved in Parkinson's disease.

Neurosci Lett 2018 04 26;673:12-18. Epub 2018 Feb 26.

Drug Development, School of Medicine, Trinity College Dublin, Ireland. Electronic address:

Parkin associated endothelin like receptor (PAELR) is G-protein coupled and ubiquitinated by parkin, promoting its degradation. In autosomal recessive Parkinson's disease, mutations in parkin lead to PAELR aggregation in the endoplasmic reticulum (ER), ER stress, neurotoxicity and cell death. We have identified previously that the protein kinase C interacting protein (PICK1) interacts with and regulates the expression and cell toxicity of PAELR. Here, we experimentally identify and provide in-silico modelling of a novel interaction between PAELR and GABARAPL2 (γ-aminobutyrate type A receptor associated protein like 2), which is an autophagosome-specific Ub-like protein implicated in vesicle trafficking and autophagy. We show that the family of GABARAPs interact with the carboxy terminal (ct) of PAELR and find the cysteine rich region (-CCCCCC-EEC) of ct-PAELR interacts with the GABA binding site of GABARAPL2. This interaction is modelled by in-slico analysis and confirmed using affinity chromatography, showing Myc-tagged GABARAPL2 is retained by a GST fusion of the ct-PAELR. We also demonstrate that transient transfection of GABARAPL2 in HEK293 cells reduces PAELR expression. This study supports the idea that protein levels of PAELR are likely regulated by a multitude of proteins including parkin, PICK1 and GABARAPL2 via mechanisms that include ubiquitination, proteasomal degradagtion and autophagy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neulet.2018.02.055DOI Listing
April 2018

Preferential amplification of a human mitochondrial DNA deletion in vitro and in vivo.

Sci Rep 2018 01 29;8(1):1799. Epub 2018 Jan 29.

Novartis Institutes for BioMedical Research, Novartis Campus, Basel, CH-4056, Switzerland.

We generated induced pluripotent stem cells (iPSCs) from patient fibroblasts to yield cell lines containing varying degrees of heteroplasmy for a m.13514 A > G mtDNA point mutation (2 lines) and for a ~6 kb single, large scale mtDNA deletion (3 lines). Long term culture of the iPSCs containing a single, large-scale mtDNA deletion showed consistent increase in mtDNA deletion levels with time. Higher levels of mtDNA heteroplasmy correlated with increased respiratory deficiency. To determine what changes occurred in deletion level during differentiation, teratomas comprising all three embryonic germ layers were generated from low (20%) and intermediate heteroplasmy (55%) mtDNA deletion clones. Regardless of whether iPSCs harbouring low or intermediate mtDNA heteroplasmy were used, the final levels of heteroplasmy in all teratoma germ layers increased to a similar high level (>60%). Thus, during human stem cell division, cells not only tolerate high mtDNA deletion loads but seem to preferentially replicate deleted mtDNA genomes. This has implications for the involvement of mtDNA deletions in both disease and ageing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-20064-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789095PMC
January 2018

An Activating Janus Kinase-3 Mutation Is Associated with Cytotoxic T Lymphocyte Antigen-4-Dependent Immune Dysregulation Syndrome.

Front Immunol 2017 15;8:1824. Epub 2017 Dec 15.

Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany.

Heterozygous mutations in the cytotoxic T lymphocyte antigen-4 (CTLA-4) are associated with lymphadenopathy, autoimmunity, immune dysregulation, and hypogammaglobulinemia in about 70% of the carriers. So far, the incomplete penetrance of CTLA-4 haploinsufficiency has been attributed to unknown genetic modifiers, epigenetic changes, or environmental effects. We sought to identify potential genetic modifiers in a family with differential clinical penetrance of CTLA-4 haploinsufficiency. Here, we report on a rare heterozygous gain-of-function mutation in Janus kinase-3 (JAK3) (p.R840C), which is associated with the clinical manifestation of CTLA-4 haploinsufficiency in a patient carrying a novel loss-of-function mutation in CTLA-4 (p.Y139C). While the asymptomatic parents carry either the CTLA-4 mutation or the JAK3 variant, their son has inherited both heterozygous mutations and suffers from hypogammaglobulinemia combined with autoimmunity and lymphoid hyperplasia. Although the patient's lymph node and spleen contained many hyperplastic germinal centers with follicular helper T (T) cells and immunoglobulin (Ig) G-positive B cells, plasma cell, and memory B cell development was impaired. CXCR5PD-1TIGIT T cells contributed to a large part of circulating T cells, but they produced only very low amounts of interleukin (IL)-4, IL-10, and IL-21 required for the development of memory B cells and plasma cells. We, therefore, suggest that the combination of the loss-of-function mutation in CTLA-4 with the gain-of-function mutation in JAK3 directs the differentiation of CD4 T cells into dysfunctional T cells supporting the development of lymphadenopathy, hypogammaglobulinemia, and immunodeficiency. Thus, the combination of rare genetic heterozygous variants that remain clinically unnoticed individually may lead to T cell hyperactivity, impaired memory B cell, and plasma cell development resulting finally in combined immunodeficiency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2017.01824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5770691PMC
December 2017

EBI2 regulates pro-inflammatory signalling and cytokine release in astrocytes.

Neuropharmacology 2018 05 31;133:121-128. Epub 2018 Jan 31.

Drug Development, School of Medicine, Trinity College, Dublin, Ireland.

The endogenous oxysterol 7α, 25-dihydroxycholesterol (7α25HC) ligand activates the G protein-coupled receptor EBI2 to regulate T cell-dependant antibody response and B cell migration. We have demonstrated that EBI2 is expressed in human and mouse astrocytes, that 7α25HC induces intracellular signalling and astrocyte migration, and that EBI2 plays a role in the crosstalk between astrocytes and macrophages. Recently, we demonstrate that EBI2 regulates myelin development and inhibits LPC-induced demyelination. Here, we show that 7α25HC inhibits LPS- and IL17/TNF-induced pro-inflammatory cytokine release in astrocytes. We observe the following: 1. Human astrocytes treated with IL17/TNF increases the nuclear translocation of NFκB, which is attenuated by pre-treatment with 7α25HC; 2. IL17/TNF increases cell impedance in human astrocytes, which is also attenuated by pre-treatment with 7α25HC; 3. The EBI2 antagonist NIBR189 inhibits these effects of 7α25HC, supporting the role of EBI2; 4. in vivo data corroborate these in vitro findings, showing that EBI2 knock-out (KO) animals display enhanced pro-inflammatory cytokine in response to LPS challenge, in the brain. These results demonstrate a role for oxysterol/EBI2 signalling in attenuating the response of astrocytes to pro-inflammatory signals as well as limiting the levels of pro-inflammatory cytokines in the brain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuropharm.2018.01.029DOI Listing
May 2018

Genome-wide CRISPR screen for PARKIN regulators reveals transcriptional repression as a determinant of mitophagy.

Proc Natl Acad Sci U S A 2018 01 21;115(2):E180-E189. Epub 2017 Dec 21.

Novartis Institutes for BioMedical Research, Basel CH 4002, Switzerland;

PARKIN, an E3 ligase mutated in familial Parkinson's disease, promotes mitophagy by ubiquitinating mitochondrial proteins for efficient engagement of the autophagy machinery. Specifically, PARKIN-synthesized ubiquitin chains represent targets for the PINK1 kinase generating phosphoS65-ubiquitin (pUb), which constitutes the mitophagy signal. Physiological regulation of PARKIN abundance, however, and the impact on pUb accumulation are poorly understood. Using cells designed to discover physiological regulators of PARKIN abundance, we performed a pooled genome-wide CRISPR/Cas9 knockout screen. Testing identified genes individually resulted in a list of 53 positive and negative regulators. A transcriptional repressor network including THAP11 was identified and negatively regulates endogenous PARKIN abundance. RNAseq analysis revealed the PARKIN-encoding locus as a prime THAP11 target, and CRISPR knockout in multiple cell types enhanced pUb accumulation. Thus, our work demonstrates the critical role of PARKIN abundance, identifies regulating genes, and reveals a link between transcriptional repression and mitophagy, which is also apparent in human induced pluripotent stem cell-derived neurons, a disease-relevant cell type.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1711023115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777035PMC
January 2018

EBI2 receptor regulates myelin development and inhibits LPC-induced demyelination.

J Neuroinflammation 2017 Dec 16;14(1):250. Epub 2017 Dec 16.

Drug Development, School of Medicine, Trinity College, Dublin, Ireland.

Background: The G protein-coupled receptor EBI2 (Epstein-Barr virus-induced gene 2) is activated by 7α, 25-dihydroxycholesterol (7α25HC) and plays a role in T cell-dependant antibody response and B cell migration. Abnormal EBI2 signaling is implicated in a range of autoimmune disorders; however, its role in the CNS remains poorly understood.

Methods: Here we characterize the role of EBI2 in myelination under normal and pathophysiological conditions using organotypic cerebellar slice cultures and EBI2 knock-out (KO) animals.

Results: We find that MBP expression in brains taken from EBI2 KO mice is delayed compared to those taken from wild type (WT) mice. In agreement with these in vivo findings, we show that antagonism of EBI2 reduces MBP expression in vitro. Importantly, we demonstrate that EBI2 activation attenuates lysolecithin (LPC)-induced demyelination in mouse organotypic slice cultures. Moreover, EBI2 activation also inhibits LPC-mediated release of pro-inflammatory cytokines such as IL6 and IL1β in cerebellar slices.

Conclusions: These results, for the first time, display a role for EBI2 in myelin development and protection from demyelination under pathophysiological conditions and suggest that modulation of this receptor may be beneficial in neuroinflammatory and demyelinating disorders such as multiple sclerosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12974-017-1025-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5732472PMC
December 2017

EBI2 Is Highly Expressed in Multiple Sclerosis Lesions and Promotes Early CNS Migration of Encephalitogenic CD4 T Cells.

Cell Rep 2017 01;18(5):1270-1284

Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, 55131 Mainz, Germany. Electronic address:

Arrival of encephalitogenic T cells at inflammatory foci represents a critical step in development of experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. EBI2 and its ligand, 7α,25-OHC, direct immune cell localization in secondary lymphoid organs. CH25H and CYP7B1 hydroxylate cholesterol to 7α,25-OHC. During EAE, we found increased expression of CH25H by microglia and CYP7B1 by CNS-infiltrating immune cells elevating the ligand concentration in the CNS. Two critical pro-inflammatory cytokines, interleukin-23 (IL-23) and interleukin-1 beta (IL-1β), maintained expression of EBI2 in differentiating Th17 cells. In line with this, EBI2 enhanced early migration of encephalitogenic T cells into the CNS in a transfer EAE model. Nonetheless, EBI2 was dispensable in active EAE. Human Th17 cells do also express EBI2, and EBI2 expressing cells are abundant within multiple sclerosis (MS) white matter lesions. These findings implicate EBI2 as a mediator of CNS autoimmunity and describe mechanistically its contribution to the migration of autoreactive T cells into inflamed organs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2017.01.020DOI Listing
January 2017

Mutations in MAPKBP1 Cause Juvenile or Late-Onset Cilia-Independent Nephronophthisis.

Am J Hum Genet 2017 02 12;100(2):323-333. Epub 2017 Jan 12.

INSERM, UMR-1163, Laboratory of Inherited Kidney Diseases, 75015 Paris, France; Paris Descartes - Sorbonne Paris Cité University, Imagine Institute, 75015 Paris, France.

Nephronophthisis (NPH), an autosomal-recessive tubulointerstitial nephritis, is the most common cause of hereditary end-stage renal disease in the first three decades of life. Since most NPH gene products (NPHP) function at the primary cilium, NPH is classified as a ciliopathy. We identified mutations in a candidate gene in eight individuals from five families presenting late-onset NPH with massive renal fibrosis. This gene encodes MAPKBP1, a poorly characterized scaffolding protein for JNK signaling. Immunofluorescence analyses showed that MAPKBP1 is not present at the primary cilium and that fibroblasts from affected individuals did not display ciliogenesis defects, indicating that MAPKBP1 may represent a new family of NPHP not involved in cilia-associated functions. Instead, MAPKBP1 is recruited to mitotic spindle poles (MSPs) during the early phases of mitosis where it colocalizes with its paralog WDR62, which plays a key role at MSP. Detected mutations compromise recruitment of MAPKBP1 to the MSP and/or its interaction with JNK2 or WDR62. Additionally, we show increased DNA damage response signaling in fibroblasts from affected individuals and upon knockdown of Mapkbp1 in murine cell lines, a phenotype previously associated with NPH. In conclusion, we identified mutations in MAPKBP1 as a genetic cause of juvenile or late-onset and cilia-independent NPH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajhg.2016.12.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5294754PMC
February 2017

EBI2 Expression and Function: Robust in Memory Lymphocytes and Increased by Natalizumab in Multiple Sclerosis.

Cell Rep 2017 01;18(1):213-224

Laboratories of Neuroimmunology, Neuroscience Research Center, Department of Clinical Neurosciences, Lausanne University Hospital, Chemin des Boveresses 155, 1066 Epalinges, Switzerland; Department of Pathology and Immunology, Geneva University Medical Center, Rue Michel-Servet 1, 1211 Geneva 4, Switzerland; Division of Neurology, Department of Clinical Neurosciences, Lausanne University Hospital, Rue du Bugnon 46, 1011 Lausanne, Switzerland. Electronic address:

The interaction between oxysterols and the G protein-coupled receptor Epstein-Barr virus-induced gene 2 (EBI2) fine-tunes immune cell migration, a mechanism efficiently targeted by several disease-modifying treatments developed to treat multiple sclerosis (MS), such as natalizumab. We previously showed that memory CD4 T lymphocytes migrate specifically in response to 7α,25-dihydroxycholesterol (7α,25-OHC) via EBI2 in the MS murine model experimental autoimmune encephalomyelitis. However, the EBI2 expression profile in human lymphocytes in both healthy and MS donors is unknown. Here, we characterize EBI2 biology in human lymphocytes. We observed that EBI2 is functionally expressed on memory CD4 T cells and is enhanced under natalizumab treatment. These data suggest a significant role for EBI2 in human CD4 T cell migration, notably in patients with MS. Better knowledge of EBI2 involvement in autoimmunity may therefore lead to an improved understanding of the physiopathology of MS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2016.12.006DOI Listing
January 2017

Reduced Plasma Levels of 25-Hydroxycholesterol and Increased Cerebrospinal Fluid Levels of Bile Acid Precursors in Multiple Sclerosis Patients.

Mol Neurobiol 2017 12 23;54(10):8009-8020. Epub 2016 Nov 23.

Swansea University Medical School, Singleton Park, Swansea, SA2 8PP, UK.

Multiple sclerosis (MS) is an autoimmune, inflammatory disease of the central nervous system (CNS). We have measured the levels of over 20 non-esterified sterols in plasma and cerebrospinal fluid (CSF) from patients suffering from MS, inflammatory CNS disease, neurodegenerative disease and control patients. Analysis was performed following enzyme-assisted derivatisation by liquid chromatography-mass spectrometry (LC-MS) exploiting multistage fragmentation (MS ). We found increased concentrations of bile acid precursors in CSF from each of the disease states and that patients with inflammatory CNS disease classified as suspected autoimmune disease or of unknown aetiology also showed elevated concentrations of 25-hydroxycholestertol (25-HC, P < 0.05) in CSF. Cholesterol concentrations in CSF were not changed except for patients diagnosed with amyotrophic lateral sclerosis (P < 0.01) or pathogen-based infections of the CNS (P < 0.05) where they were elevated. In plasma, we found that 25-HC (P < 0.01), (25R)26-hydroxycholesterol ((25R)26-HC, P < 0.05) and 7α-hydroxy-3-oxocholest-4-enoic acid (7αH,3O-CA, P < 0.05) were reduced in relapsing-remitting MS (RRMS) patients compared to controls. The pattern of reduced plasma levels of 25-HC, (25R)26-HC and 7αH,3O-CA was unique to RRMS. In summary, in plasma, we find that the concentration of 25-HC in RRMS patients is significantly lower than in controls. This is consistent with the hypothesis that a lower propensity of macrophages to synthesise 25-HC will result in reduced negative feedback by 25-HC on IL-1 family cytokine production and exacerbated MS. In CSF, we find that the dominating metabolites reflect the acidic pathway of bile acid biosynthesis and the elevated levels of these in CNS disease is likely to reflect cholesterol release as a result of demyelination or neuronal death. 25-HC is elevated in patients with inflammatory CNS disease probably as a consequence of up-regulation of the type 1 interferon-stimulated gene cholesterol 25-hydroxylase in macrophages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12035-016-0281-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5684259PMC
December 2017

The EBI2 signalling pathway plays a role in cellular crosstalk between astrocytes and macrophages.

Sci Rep 2016 05 11;6:25520. Epub 2016 May 11.

Drug Development, School of Medicine, Trinity College, Dublin, Ireland.

EBI2 is a G protein-coupled receptor activated by oxysterol 7α, 25-dihydroxycholesterol (7α25HC) and regulates T cell-dependant antibody response and B cell migration. We recently found EBI2 is expressed in human astrocytes, regulates intracellular signalling and modulates astrocyte migration. Here, we report that LPS treatment of mouse astrocytes alters mRNA levels of EBI2 and oxysterols suggesting that the EBI2 signalling pathway is sensitive to LPS-mediated immune challenge. We also find that conditioned media obtained from LPS-stimulated mouse astrocytes induces macrophage migration, which is inhibited by the EBI2 antagonist NIBR189. These results demonstrate a role for the EBI2 signalling pathway in astrocytes as a sensor for immune challenge and for communication with innate immune cells such as macrophages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep25520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4863252PMC
May 2016

The Role of the Oxysterol/EBI2 Pathway in the Immune and Central Nervous Systems.

Curr Drug Targets 2016 ;17(16):1851-1860

Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Forum 1, CH-4002, Switzerland.

Oxysterols are pleiotropic messengers interacting with multiple receptor systems. One of the cognate receptors for oxysterols is EBI2, a G protein-coupled receptor highly expressed in the cells of the immune system. Here we discuss the receptor's role in the adapted immunity and inflammation as well as the receptor's expression and function in the CNS with the focus on astrocytes. We also discuss expression and signalling of oxysterol-producing enzymes such as CH25H and CYP7B1 in the CNS and the immune system. These steps will help to elucidate a possible role for this pathway in the physiology of the central and peripheral nervous system and its possible link to human disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1389450117666160217123042DOI Listing
August 2017

Mutations in TRAF3IP1/IFT54 reveal a new role for IFT proteins in microtubule stabilization.

Nat Commun 2015 Oct 21;6:8666. Epub 2015 Oct 21.

Inserm UMR-1163, Laboratory of Hereditary Kidney Diseases, 75015 Paris, France.

Ciliopathies are a large group of clinically and genetically heterogeneous disorders caused by defects in primary cilia. Here we identified mutations in TRAF3IP1 (TNF Receptor-Associated Factor Interacting Protein 1) in eight patients from five families with nephronophthisis (NPH) and retinal degeneration, two of the most common manifestations of ciliopathies. TRAF3IP1 encodes IFT54, a subunit of the IFT-B complex required for ciliogenesis. The identified mutations result in mild ciliary defects in patients but also reveal an unexpected role of IFT54 as a negative regulator of microtubule stability via MAP4 (microtubule-associated protein 4). Microtubule defects are associated with altered epithelialization/polarity in renal cells and with pronephric cysts and microphthalmia in zebrafish embryos. Our findings highlight the regulation of cytoplasmic microtubule dynamics as a role of the IFT54 protein beyond the cilium, contributing to the development of NPH-related ciliopathies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms9666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4617596PMC
October 2015

Detection of dihydroxycholesterols in human plasma using HPLC-ESI-MS/MS.

Steroids 2015 Jul 12;99(Pt B):131-8. Epub 2015 Feb 12.

Analytical Sciences and Imaging, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland. Electronic address:

We report a straightforward sample preparation procedure and a direct liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the analysis of 7alpha,25-dihydroxycholesterol (7α25-OHC) and 7alpha,27-dihydroxycholesterol (7α27-OHC). By applying a slow protein precipitation approach using cold ethanol, we were able to detect and quantify 7α25-OHC and 7α27-OHC in a fast and reliable manner. The average concentrations from 20 healthy individuals were determined to be 0.21±0.05nM for 7α25-OHC and 3.4±0.1nM for 7α27-OHC. In addition, we are the first to report the average degrees of esterification (n=8) to be 73.8% and 82% for 7α25-OHC and 7α27-OHC, respectively. Using the established method, we achieved the sensitivity sufficient for detecting low abundant dihydroxylated oxysterols in healthy individuals. This result should enable extension of these studies towards a comprehensive analysis of oxysterol levels under disease conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.steroids.2015.02.002DOI Listing
July 2015

EBI2 regulates intracellular signaling and migration in human astrocyte.

Glia 2015 Feb 9;63(2):341-51. Epub 2014 Oct 9.

Drug Development, School of Medicine, Trinity College Dublin, Dublin, Ireland.

The G protein-coupled receptor EBI2 (Epstein-Barr virus-induced gene 2) is activated by 7α, 25-dihydroxycholesterol (7α25HC) and plays a role in T cell-dependant antibody response and B cell migration. Aberrant EBI2 signaling is implicated in a range of autoimmune disorders however its role in the CNS remains unknown. Here we characterize the functional role of EBI2 in GLIA cells using primary human astrocytes and EBI2 knockout animals. We find human and mouse astrocytes express EBI2 and the enzymes necessary for synthesis and degradation of 7α25HC. In astrocytes, EBI2 activation stimulates ERK phosphorylation, Ca(2+) signaling and induces cellular migration. These results, for the first time, demonstrate a role for EBI2 in astrocyte function and suggest that modulation of this receptor may be beneficial in neuroinflammatory disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/glia.22757DOI Listing
February 2015

Cholestenoic acids regulate motor neuron survival via liver X receptors.

J Clin Invest 2014 Nov 1;124(11):4829-42. Epub 2014 Oct 1.

Cholestenoic acids are formed as intermediates in metabolism of cholesterol to bile acids, and the biosynthetic enzymes that generate cholestenoic acids are expressed in the mammalian CNS. Here, we evaluated the cholestenoic acid profile of mammalian cerebrospinal fluid (CSF) and determined that specific cholestenoic acids activate the liver X receptors (LXRs), enhance islet-1 expression in zebrafish, and increase the number of oculomotor neurons in the developing mouse in vitro and in vivo. While 3β,7α-dihydroxycholest-5-en-26-oic acid (3β,7α-diHCA) promoted motor neuron survival in an LXR-dependent manner, 3β-hydroxy-7-oxocholest-5-en-26-oic acid (3βH,7O-CA) promoted maturation of precursors into islet-1+ cells. Unlike 3β,7α-diHCA and 3βH,7O-CA, 3β-hydroxycholest-5-en-26-oic acid (3β-HCA) caused motor neuron cell loss in mice. Mutations in CYP7B1 or CYP27A1, which encode enzymes involved in cholestenoic acid metabolism, result in different neurological diseases, hereditary spastic paresis type 5 (SPG5) and cerebrotendinous xanthomatosis (CTX), respectively. SPG5 is characterized by spastic paresis, and similar symptoms may occur in CTX. Analysis of CSF and plasma from patients with SPG5 revealed an excess of the toxic LXR ligand, 3β-HCA, while patients with CTX and SPG5 exhibited low levels of the survival-promoting LXR ligand 3β,7α-diHCA. Moreover, 3β,7α-diHCA prevented the loss of motor neurons induced by 3β-HCA in the developing mouse midbrain in vivo.Our results indicate that specific cholestenoic acids selectively work on motor neurons, via LXR, to regulate the balance between survival and death.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI68506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4347238PMC
November 2014

Oxysterol-EBI2 signaling in immune regulation and viral infection.

Eur J Immunol 2014 Jul 20;44(7):1904-12. Epub 2014 Jun 20.

Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

The seven transmembrane G protein-coupled receptor Epstein-Barr virus (EBV) induced gene 2 (EBI2; also known as GPR183) was identified in 1993 on the basis of its substantial upregulation in EBV-infected cells. It is primarily expressed in lymphoid cells; most abundantly in B cells. EBI2 is central for the positioning of B cells within the lymphoid organs, a process that is regulated in part by a chemotactic gradient formed by the endogenous lipid agonists, and in part by a fine-tuned regulation of EBI2 cell surface expression. The most potent endogenous EBI2 agonist is 7α, 25-dihydroxyxcholesterol (7α,25-OHC), yet many structurally related oxysterols can bind to an EBI2 pocket that is defined by the upper parts of the transmembrane helices and extracellular receptor regions. EBI2 signals via Gαi, as well as via G protein-independent pathways like β-arrestin recruitment. The concerted action of these pathways leads to cell migration. By genetically interfering with its up- and downregulation, EBI2 was also recently shown to induce cell proliferation, an action that could be inhibited by small molecule antagonists. Here, we focus on the oxysterol-EBI2 axis in immune control, including its role in the EBV life cycle. We also summarize the structural and functional properties of EBI2 interaction with oxysterol agonists and small molecule antagonists and discuss EBI2 as therapeutic target for diseases of the immune system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/eji.201444493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4209795PMC
July 2014

The protein interacting with C-kinase (PICK1) interacts with and attenuates parkin-associated endothelial-like (PAEL) receptor-mediated cell death.

J Neurochem 2014 Aug 19;130(3):360-73. Epub 2014 May 19.

Molecular Neuropharmacology, Drug Development, Department of Physiology, School of Medicine, Trinity College Dublin, Dublin, Ireland.

The parkin-associated endothelial-like receptor (PAELR, GPR37) is an orphan G protein-coupled receptor that interacts with and is degraded by parkin-mediated ubiquitination. Mutations in parkin are thought to result in PAELR accumulation and increase neuronal cell death in Parkinson's disease. In this study, we find that the protein interacting with C-kinase (PICK1) interacts with PAELR. Specifically, the Postsynaptic density protein-95/Discs large/ZO-1 (PDZ) domain of PICK1 interacted with the last three residues of the c-terminal (ct) located PDZ motif of PAELR. Pull-down assays indicated that recombinant and native PICK1, obtained from heterologous cells and rat brain tissue, respectively, were retained by a glutathione S-transferase fusion of ct-PAELR. Furthermore, coimmunoprecipitation studies isolated a PAELR-PICK1 complex from transiently transfected cells. PICK1 interacts with parkin and our data showed that PICK1 reduces PAELR expression levels in transiently transfected heterologous cells compared to a PICK1 mutant that does not interact with PAELR. Finally, PICK1 over-expression in HEK293 cells reduced cell death induced by PAEALR over-expression during rotenone treatment and these effects of PICK1 were attenuated during inhibition of the proteasome. These results suggest a role for PICK1 in preventing PAELR-induced cell toxicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jnc.12741DOI Listing
August 2014

Identification and characterization of small molecule modulators of the Epstein-Barr virus-induced gene 2 (EBI2) receptor.

J Med Chem 2014 Apr 15;57(8):3358-68. Epub 2014 Apr 15.

Global Discovery Chemistry; ‡Developmental & Molecular Pathways, §Analytical Sciences, Novartis Institutes for BioMedical Research , Basel 4002, Switzerland.

Oxysterols have recently been identified as natural ligands for a G protein-coupled receptor called EBI2 (aka GPR183) ( Nature 2011 , 475 , 524 ; 519 ). EBI2 is highly expressed in immune cells ( J. Biol. Chem. 2006 , 281 , 13199 ), and its activation has been shown to be critical for the adaptive immune response and has been genetically linked to autoimmune diseases such as type I diabetes ( Nature 2010 , 467 , 460 ). Here we describe the isolation of a potent small molecule antagonist for the EBI2 receptor. First, we identified a small molecule agonist NIBR51 (1), which enabled identification of inhibitors of receptor activation. One antagonist called NIBR127 (2) was used as a starting point for a medicinal chemistry campaign, which yielded NIBR189 (4m). This compound was extensively characterized in binding and various functional signaling assays. Furthermore, we have used 4m to block migration of a monocyte cell line called U937, suggesting a functional role of the oxysterol/EBI2 pathway in these immune cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm4019355DOI Listing
April 2014

Target identification for a Hedgehog pathway inhibitor reveals the receptor GPR39.

Nat Chem Biol 2014 May 16;10(5):343-9. Epub 2014 Mar 16.

Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.

Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nchembio.1481DOI Listing
May 2014

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

Biochem Biophys Res Commun 2014 Apr 27;446(3):663-8. Epub 2014 Jan 27.

Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2014.01.069DOI Listing
April 2014

Identification of the C3a receptor (C3AR1) as the target of the VGF-derived peptide TLQP-21 in rodent cells.

J Biol Chem 2013 Sep 12;288(38):27434-43. Epub 2013 Aug 12.

From Novartis AG, Novartis Campus, CH-4056 Basel, Switzerland and.

TLQP-21, a peptide derived from VGF (non-acronymic) by proteolytic processing, has been shown to modulate energy metabolism, differentiation, and cellular response to stress. Although extensively investigated, the receptor for this endogenous peptide has not previously been described. This study describes the use of a series of studies that show G protein-coupled receptor-mediated biological activity of TLQP-21 signaling in CHO-K1 cells. Unbiased genome-wide sequencing of the transcriptome from responsive CHO-K1 cells identified a prioritized list of possible G protein-coupled receptors bringing about this activity. Further experiments using a series of defined receptor antagonists and siRNAs led to the identification of complement C3a receptor-1 (C3AR1) as a target for TLQP-21 in rodents. We have not been able to demonstrate so far that this finding is translatable to the human receptor. Our results are in line with a large number of physiological observations in rodent models of food intake and metabolic control, where TLQP-21 shows activity. In addition, the sensitivity of TLQP-21 signaling to pertussis toxin is consistent with the known signaling pathway of C3AR1. The binding of TLQP-21 to C3AR1 not only has effects on signaling but also modulates cellular functions, as TLQP-21 was shown to have a role in directing migration of mouse RAW264.7 cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M113.497214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3779738PMC
September 2013

Naive B-cell trafficking is shaped by local chemokine availability and LFA-1-independent stromal interactions.

Blood 2013 May 4;121(20):4101-9. Epub 2013 Apr 4.

Theodor Kocher Institute, University of Bern, Bern, Switzerland.

It is not known how naive B cells compute divergent chemoattractant signals of the T-cell area and B-cell follicles during in vivo migration. Here, we used two-photon microscopy of peripheral lymph nodes (PLNs) to analyze the prototype G-protein-coupled receptors (GPCRs) CXCR4, CXCR5, and CCR7 during B-cell migration, as well as the integrin LFA-1 for stromal guidance. CXCR4 and CCR7 did not influence parenchymal B-cell motility and distribution, despite their role during B-cell arrest in venules. In contrast, CXCR5 played a nonredundant role in B-cell motility in follicles and in the T-cell area. B-cell migration in the T-cell area followed a random guided walk model, arguing against directed migration in vivo. LFA-1, but not α4 integrins, contributed to B-cell motility in PLNs. However, stromal network guidance was LFA-1 independent, uncoupling integrin-dependent migration from stromal attachment. Finally, we observed that despite a 20-fold reduction of chemokine expression in virus-challenged PLNs, CXCR5 remained essential for B-cell screening of antigen-presenting cells. Our data provide an overview of the contribution of prototype GPCRs and integrins during naive B-cell migration and shed light on the local chemokine availability that these cells compute.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2012-10-465336DOI Listing
May 2013

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

Immunity 2012 Sep;37(3):535-48

Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California, San Francisco, CA 94143, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.immuni.2012.06.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3465460PMC
September 2012