Publications by authors named "Adam P Cribbs"

26 Publications

  • Page 1 of 1

Nanopore sequencing of single-cell transcriptomes with scCOLOR-seq.

Nat Biotechnol 2021 Jul 1. Epub 2021 Jul 1.

Botnar Research Centre, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, National Institute of Health Research Oxford Biomedical Research Unit (BRU), University of Oxford, Oxford, UK.

Here we describe single-cell corrected long-read sequencing (scCOLOR-seq), which enables error correction of barcode and unique molecular identifier oligonucleotide sequences and permits standalone cDNA nanopore sequencing of single cells. Barcodes and unique molecular identifiers are synthesized using dimeric nucleotide building blocks that allow error detection. We illustrate the use of the method for evaluating barcode assignment accuracy, differential isoform usage in myeloma cell lines, and fusion transcript detection in a sarcoma cell line.
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http://dx.doi.org/10.1038/s41587-021-00965-wDOI Listing
July 2021

Syncytiotrophoblast Extracellular Vesicles From Late-Onset Preeclampsia Placentae Suppress Pro-Inflammatory Immune Response in THP-1 Macrophages.

Front Immunol 2021 7;12:676056. Epub 2021 Jun 7.

Nuffield Department of Women's and Reproductive Health, University of Oxford, Oxford, United Kingdom.

Syncytiotrophoblast derived Extracellular Vesicles (STBEV) from normal pregnancy (NP) have previously been shown to interact with circulating monocytes and B cells and induce pro-inflammatory cytokine release. Early-onset preeclampsia (EOPE) is associated with an exacerbated inflammatory response, yet there is little data regarding late-onset PE (LOPE) and immune function. Here, using a macrophage/monocyte cell line THP-1, we investigated the inflammatory potential of STBEV, comprising medium/large-STBEV (>200nm) and small-STBEV (<200nm), isolated from LOPE (n=6) and normal (NP) (n=6) placentae via dual-lobe placental perfusion and differential centrifugation. THP-1 cells bound and internalised STBEV isolated from NP and LOPE placentae, as revealed by flow cytometry, confocal microscopy, and ELISA. STBEV-treated THP-1 cells were examined for cytokine gene expression by RT-qPCR and the cell culture media examined for secreted cytokines/chemokines. As expected, NP medium/large-STBEV significantly upregulated the transcriptional expression of TNF-α, IL-10, IL-6, IL-12, IL-8 and TGF-β compared to PE medium/large-STBEV. However, there was no significant difference in the small STBEV population between the two groups, although in general, NP small STBEVs slightly upregulated the same cytokines. In contrast, LOPE STBEV (medium and large) did not induce pro-inflammatory responses by differentiated THP-1 macrophages. This decreased effect of LOPE STBEV was echoed in cytokine/chemokine release. Our results appear to suggest that STBEV from LOPE placentae do not have a major immune-modulatory effect on macrophages. In contrast, NP STBEV caused THP-1 cells to release pro-inflammatory cytokines. Thus, syncytiotrophoblast extracellular vesicles from LOPE dampen immune functions of THP-1 macrophages, suggesting an alternative mechanism leading to the pro-inflammatory environment observed in LOPE.
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http://dx.doi.org/10.3389/fimmu.2021.676056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8215361PMC
June 2021

Interleukin-17A Causes Osteoarthritis-Like Transcriptional Changes in Human Osteoarthritis-Derived Chondrocytes and Synovial Fibroblasts .

Front Immunol 2021 12;12:676173. Epub 2021 May 12.

The Botnar Research Centre, Nuffield Department of Orthopaedics Rheumatology and Musculoskeletal Science, University of Oxford, Oxford, United Kingdom.

Increased interleukin (IL)-17A has been identified in joints affected by osteoarthritis (OA), but it is unclear how IL-17A, and its family members IL-17AF and IL-17F, can contribute to human OA pathophysiology. Therefore, we aimed to evaluate the gene expression and signalling pathway activation effects of the different IL-17 family members in chondrocytes and synovial fibroblasts derived from cartilage and synovium of patients with end-stage knee OA. Immunohistochemistry staining confirmed that IL-17 receptor A (IL-17RA) and IL-17RC are expressed in end-stage OA-derived cartilage and synovium. Chondrocytes and synovial fibroblasts derived from end-stage OA patients were treated with IL-17A, IL-17AF, or IL-17F, and gene expression was assessed with bulk RNA-Seq. Hallmark pathway analysis showed that IL-17 cytokines regulated several OA pathophysiology-related pathways including immune-, angiogenesis-, and complement-pathways in both chondrocytes and synovial fibroblasts derived from end-stage OA patients. While overall IL-17A induced the strongest transcriptional response, followed by IL-17AF and IL-17F, not all genes followed this pattern. Disease-Gene Network analysis revealed that IL-17A-related changes in gene expression in these cells are associated with experimental arthritis, knee arthritis, and musculoskeletal disease gene-sets. Western blot analysis confirmed that IL-17A significantly activates p38 and p65 NF-κB. Incubation of chondrocytes and synovial fibroblasts with anti-IL-17A monoclonal antibody secukinumab significantly inhibited IL-17A-induced gene expression. In conclusion, the association of IL-17-induced transcriptional changes with arthritic gene-sets supports a role for IL-17A in OA pathophysiology. Future studies should further investigate the role of IL-17A in the OA joint to establish whether anti-IL-17 treatment could be a potential therapeutic option in OA patients with an inflammatory phenotype.
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http://dx.doi.org/10.3389/fimmu.2021.676173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8153485PMC
May 2021

The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection.

bioRxiv 2021 Mar 26. Epub 2021 Mar 26.

We identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone , a compound in clinical trials for anti-fibrotic and anti-inflammatory applications , as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry . We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir . Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginone's translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.
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http://dx.doi.org/10.1101/2021.03.22.436522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010724PMC
March 2021

Dissecting the Role of BET Bromodomain Proteins BRD2 and BRD4 in Human NK Cell Function.

Front Immunol 2021 26;12:626255. Epub 2021 Feb 26.

Botnar Research Center, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, National Institute of Health Research Oxford Biomedical Research Unit (BRU), University of Oxford, Oxford, United Kingdom.

Natural killer (NK) cells are innate lymphocytes that play a pivotal role in the immune surveillance and elimination of transformed or virally infected cells. Using a chemo-genetic approach, we identify BET bromodomain containing proteins BRD2 and BRD4 as central regulators of NK cell functions, including direct cytokine secretion, NK cell contact-dependent inflammatory cytokine secretion from monocytes as well as NK cell cytolytic functions. We show that both BRD2 and BRD4 control inflammatory cytokine production in NK cells isolated from healthy volunteers and from rheumatoid arthritis patients. In contrast, knockdown of BRD4 but not of BRD2 impairs NK cell cytolytic responses, suggesting BRD4 as critical regulator of NK cell mediated tumor cell elimination. This is supported by pharmacological targeting where the first-generation pan-BET bromodomain inhibitor JQ1(+) displays anti-inflammatory effects and inhibit tumor cell eradication, while the novel bivalent BET bromodomain inhibitor AZD5153, which shows differential activity towards BET family members, does not. Given the important role of both cytokine-mediated inflammatory microenvironment and cytolytic NK cell activities in immune-oncology therapies, our findings present a compelling argument for further clinical investigation.
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http://dx.doi.org/10.3389/fimmu.2021.626255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953504PMC
February 2021

Mapping the musculoskeletal system one cell at a time.

Nat Rev Rheumatol 2021 05;17(5):247-248

The Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Science, University of Oxford, Oxford, UK.

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http://dx.doi.org/10.1038/s41584-021-00600-7DOI Listing
May 2021

Hypoxia Induces Transcriptional and Translational Downregulation of the Type I IFN Pathway in Multiple Cancer Cell Types.

Cancer Res 2020 12 28;80(23):5245-5256. Epub 2020 Oct 28.

Department of Medical Oncology, Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.

Hypoxia is a common phenomenon in solid tumors and is strongly linked to hallmarks of cancer. Recent evidence has shown that hypoxia promotes local immune suppression. Type I IFN supports cytotoxic T lymphocytes by stimulating the maturation of dendritic cells and enhancing their capacity to process and present antigens. However, little is known about the relationship between hypoxia and the type I IFN pathway, which comprises the sensing of double-stranded RNA and DNA (dsRNA/dsDNA) followed by IFNα/β secretion and transcriptional activation of IFN-stimulated genes (ISG). In this study, we determined the effects of hypoxia on the type I IFN pathway in breast cancer and the mechanisms involved. In cancer cell lines and xenograft models, mRNA and protein expressions of the type I IFN pathway were downregulated under hypoxic conditions. This pathway was suppressed at each level of signaling, from the dsRNA sensors RIG-I and MDA5, the adaptor MAVS, transcription factors IRF3, IRF7, and STAT1, and several ISG including RIG-I, IRF7, STAT1, and ADAR-p150. Importantly, IFN secretion was reduced under hypoxic conditions. HIF1α- and HIF2α-mediated regulation of gene expression did not explain most of the effects. However, ATAC-seq data revealed in hypoxia that peaks with STAT1 and IRF3 motifs had decreased accessibility. Collectively, these results indicate that hypoxia leads to an overall downregulation of the type I IFN pathway due to repressed transcription and lower chromatin accessibility in an HIF1/2α-independent manner, which could contribute to immunosuppression in hypoxic tumors. SIGNIFICANCE: These findings characterize a new mechanism of immunosuppression by hypoxia via downregulation of the type I IFN pathway and its autocrine/paracrine effects on tumor growth.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-2306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611234PMC
December 2020

Inhibition of Histone H3K27 Demethylases Inactivates Brachyury (TBXT) and Promotes Chordoma Cell Death.

Cancer Res 2020 10 27;80(20):4540-4551. Epub 2020 Aug 27.

Department of Pathology, UCL Cancer Institute, University College London, London, United Kingdom.

Expression of the transcription factor brachyury (TBXT) is normally restricted to the embryo, and its silencing is epigenetically regulated. TBXT promotes mesenchymal transition in a subset of common carcinomas, and in chordoma, a rare cancer showing notochordal differentiation, TBXT acts as a putative oncogene. We hypothesized that TBXT expression is controlled through epigenetic inhibition to promote chordoma cell death. Screening of five human chordoma cell lines revealed that pharmacologic inhibition of the histone 3 lysine 27 demethylases KDM6A (UTX) and KDM6B (JMJD3) leads to cell death. This effect was phenocopied by dual genetic inactivation of KDM6A/B using CRISPR/Cas9. Inhibition of KDM6A/B with a novel compound KDOBA67 led to a genome-wide increase in repressive H3K27me3 marks with concomitant reduction in active H3K27ac, H3K9ac, and H3K4me3 marks. was a KDM6A/B target gene, and chromatin changes at following KDOBA67 treatment were associated with a reduction in TBXT protein levels in all models tested, including primary patient-derived cultures. In all models tested, KDOBA67 treatment downregulated expression of a network of transcription factors critical for chordoma survival and upregulated pathways dominated by ATF4-driven stress and proapoptotic responses. Blocking the AFT4 stress response did not prevent suppression of TBXT and induction of cell death, but ectopic overexpression of increased viability, therefore implicating TBXT as a potential therapeutic target of H3K27 demethylase inhibitors in chordoma. Our work highlights how knowledge of normal processes in fetal development can provide insight into tumorigenesis and identify novel therapeutic approaches. SIGNIFICANCE: Pharmacologic inhibition of H3K27-demethylases in human chordoma cells promotes epigenetic silencing of oncogenic TBXT, alters gene networks critical to survival, and represents a potential novel therapy.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-1387DOI Listing
October 2020

Novel biomarkers of a peripheral blood interferon signature associated with drug-naïve early arthritis patients distinguish persistent from self-limiting disease course.

Sci Rep 2020 06 1;10(1):8830. Epub 2020 Jun 1.

Botnar Research Centre, Windmill Road, Headington, Oxford, OX3 7LD, United Kingdom.

We profiled gene expression signatures to distinguish rheumatoid arthritis (RA) from non-inflammatory arthralgia (NIA), self-limiting arthritis (SLA), and undifferentiated arthritis (UA) as compared to healthy controls as novel potential biomarkers for therapeutic responsiveness. Global gene expression profiles of PBMCs from 43 drug-naïve patients presenting with joint symptoms were evaluated and differentially expressed genes identified by comparative analysis with 24 healthy volunteers. Patients were assessed at presentation with follow up at 6 and 12 months. Gene ontology and network pathway analysis were performed using DAVID Bioinformatics Resources v6.7. Gene expression profiles were also determined after disease-modifying anti-rheumatic drug (DMARD) treatment in the inflammatory arthritis groups (i.e. RA and UA) and confirmed by qRT-PCR. Receiver operating characteristic (ROC) curves analysis and Area Under the Curve (AUC) estimation were performed to assess the diagnostic value of candidate gene expression signatures. A type I interferon (IFN) gene signature distinguished DMARD-naïve patients who will subsequently develop persistent inflammatory arthritis (i.e. RA and UA) from those with NIA. In patients with RA, the IFN signature is characterised by up-regulation of SIGLEC1 (p = 0.00597) and MS4A4A (p = 0.00000904). We also identified, EPHB2 (p = 0.000542) and PDZK1IP1 (p = 0.0206) with RA-specific gene expression profiles and elevated expression of the ST6GALNAC1 (p = 0.0023) gene in UA. ROC and AUC risk score analysis suggested that MSA4A (AUC: 0.894, 0.644, 0.720), PDZK1IP1 (AUC: 0.785, 0.806, 0.977), and EPHB2 (AUC: 0.794, 0.723, 0.620) at 0, 6, and 12 months follow-up can accurately discriminate patients with RA from healthy controls and may have practical value for RA diagnosis. In patients with early inflammatory arthritis, ST6GALNAC1 is a potential biomarker for UA as compared with healthy controls whereas EPHB2, MS4A4A, and particularly PDZK1IP1 may discriminate RA patients. SIGLEC1 may also be a useful marker of disease activity in UA.
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http://dx.doi.org/10.1038/s41598-020-63757-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264129PMC
June 2020

Advances and challenges in epigenomic single-cell sequencing applications.

Curr Opin Chem Biol 2020 08 15;57:17-26. Epub 2020 Apr 15.

Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, NIHR Oxford BRU, University of Oxford, OX3 7LD, UK. Electronic address:

Understanding multicellular physiology and pathobiology requires analysis of the relationship between genotype, chromatin organisation and phenotype. In the multi-omics era, many methods exist to investigate biological processes across the genome, transcriptome, epigenome, proteome and metabolome. Until recently, this was only possible for populations of cells or complex tissues, creating an averaging effect that may obscure direct correlations between multiple layers of data. Single-cell sequencing methods have removed this averaging effect, but computational integration after profiling distinct modalities separately may still not completely reflect underlying biology. Multiplexed assays resolving multiple modalities in the same cell are required to overcome these shortcomings and have the potential to deliver unprecedented understanding of biology and disease.
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http://dx.doi.org/10.1016/j.cbpa.2020.01.013DOI Listing
August 2020

Histone H3K27me3 demethylases regulate human Th17 cell development and effector functions by impacting on metabolism.

Proc Natl Acad Sci U S A 2020 03 2;117(11):6056-6066. Epub 2020 Mar 2.

Botnar Research Center, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, National Institute for Health Research Oxford Biomedical Research Unit, University of Oxford, OX3 7LD Oxford, United Kingdom;

T helper (Th) cells are CD4 effector T cells that play a critical role in immunity by shaping the inflammatory cytokine environment in a variety of physiological and pathological situations. Using a combined chemico-genetic approach, we identify histone H3K27 demethylases KDM6A and KDM6B as central regulators of human Th subsets. The prototypic KDM6 inhibitor GSK-J4 increases genome-wide levels of the repressive H3K27me3 chromatin mark and leads to suppression of the key transcription factor RORγt during Th17 differentiation. In mature Th17 cells, GSK-J4 induces an altered transcriptional program with a profound metabolic reprogramming and concomitant suppression of IL-17 cytokine levels and reduced proliferation. Single-cell analysis reveals a specific shift from highly inflammatory cell subsets toward a resting state upon demethylase inhibition. The root cause of the observed antiinflammatory phenotype in stimulated Th17 cells is reduced expression of key metabolic transcription factors, such as PPRC1. Overall, this leads to reduced mitochondrial biogenesis, resulting in a metabolic switch with concomitant antiinflammatory effects. These data are consistent with an effect of GSK-J4 on Th17 T cell differentiation pathways directly related to proliferation and include regulation of effector cytokine profiles. This suggests that inhibiting KDM6 demethylases may be an effective, even in the short term, therapeutic target for autoimmune diseases, including ankylosing spondylitis.
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http://dx.doi.org/10.1073/pnas.1919893117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084125PMC
March 2020

An ontogenetic switch drives the positive and negative selection of B cells.

Proc Natl Acad Sci U S A 2020 02 4;117(7):3718-3727. Epub 2020 Feb 4.

Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS Oxford, United Kingdom;

Developing B cells can be positively or negatively selected by self-antigens, but the mechanisms that determine these outcomes are incompletely understood. Here, we show that a B cell intrinsic switch between positive and negative selection during ontogeny is determined by a change from Lin28b to let-7 gene expression. Ectopic expression of a Lin28b transgene in murine B cells restored the positive selection of autoreactive B-1 B cells by self-antigen in adult bone marrow. Analysis of antigen-specific immature B cells in early and late ontogeny identified Lin28b-dependent genes associated with B-1 B cell development, including and , and Lin28b-independent effects are associated with the presence or absence of self-antigen. These findings identify cell intrinsic and extrinsic determinants of B cell fate during ontogeny and reconcile lineage and selection theories of B cell development. They explain how changes in the balance of positive and negative selection may be able to adapt to meet the immunological needs of an individual during its lifetime.
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http://dx.doi.org/10.1073/pnas.1915247117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035474PMC
February 2020

Mass cytometry analysis reveals a distinct immune environment in peritoneal fluid in endometriosis: a characterisation study.

BMC Med 2020 01 7;18(1). Epub 2020 Jan 7.

Botnar Research Centre, NIHR Biomedical Research Unit Oxford, Nuffield Department of Musculoskeletal Sciences, University of Oxford, Oxford, UK.

Background: Endometriosis is a gynaecological condition characterised by immune cell infiltration and distinct inflammatory signatures found in the peritoneal cavity. In this study, we aim to characterise the immune microenvironment in samples isolated from the peritoneal cavity in patients with endometriosis.

Methods: We applied mass cytometry (CyTOF), a recently developed multiparameter single-cell technique, in order to characterise and quantify the immune cells found in peritoneal fluid and peripheral blood from endometriosis and control patients.

Results: Our results demonstrate the presence of more than 40 different distinct immune cell types within the peritoneal cavity. This suggests that there is a complex and highly heterogeneous inflammatory microenvironment underpinning the pathology of endometriosis. Stratification by clinical disease stages reveals a dynamic spectrum of cell signatures suggesting that adaptations in the inflammatory system occur due to the severity of the disease. Notably, among the inflammatory microenvironment in peritoneal fluid (PF), the presence of CD69 T cell subsets is increased in endometriosis when compared to control patient samples. On these CD69 cells, the expression of markers associated with T cell function are reduced in PF samples compared to blood. Comparisons between CD69 and CD69 populations reveal distinct phenotypes across peritoneal T cell lineages. Taken together, our results suggest that both the innate and the adaptive immune system play roles in endometriosis.

Conclusions: This study provides a systematic characterisation of the specific immune environment in the peritoneal cavity and identifies cell immune signatures associated with endometriosis. Overall, our results provide novel insights into the specific cell phenotypes governing inflammation in patients with endometriosis. This prospective study offers a useful resource for understanding disease pathology and opportunities for identifying therapeutic targets.
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http://dx.doi.org/10.1186/s12916-019-1470-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6945609PMC
January 2020

A Chemical Probe for Tudor Domain Protein Spindlin1 to Investigate Chromatin Function.

J Med Chem 2019 10 15;62(20):9008-9025. Epub 2019 Oct 15.

Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences , Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai , New York , New York 10029 , United States.

Modifications of histone tails, including lysine/arginine methylation, provide the basis of a "chromatin or histone code". Proteins that contain "reader" domains can bind to these modifications and form specific effector complexes, which ultimately mediate chromatin function. The spindlin1 (SPIN1) protein contains three Tudor methyllysine/arginine reader domains and was identified as a putative oncogene and transcriptional coactivator. Here we report a SPIN1 chemical probe inhibitor with low nanomolar in vitro activity, exquisite selectivity on a panel of methyl reader and writer proteins, and with submicromolar cellular activity. X-ray crystallography showed that this Tudor domain chemical probe simultaneously engages Tudor domains 1 and 2 via a bidentate binding mode. Small molecule inhibition and siRNA knockdown of SPIN1, as well as chemoproteomic studies, identified genes which are transcriptionally regulated by SPIN1 in squamous cell carcinoma and suggest that SPIN1 may have a role in cancer related inflammation and/or cancer metastasis.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00562DOI Listing
October 2019

Bromodomain inhibition of the coactivators CBP/EP300 facilitate cellular reprogramming.

Nat Chem Biol 2019 05 8;15(5):519-528. Epub 2019 Apr 8.

School of Medicine, Koç University, Istanbul, Turkey.

Silencing of the somatic cell type-specific genes is a critical yet poorly understood step in reprogramming. To uncover pathways that maintain cell identity, we performed a reprogramming screen using inhibitors of chromatin factors. Here, we identify acetyl-lysine competitive inhibitors targeting the bromodomains of coactivators CREB (cyclic-AMP response element binding protein) binding protein (CBP) and E1A binding protein of 300 kDa (EP300) as potent enhancers of reprogramming. These inhibitors accelerate reprogramming, are critical during its early stages and, when combined with DOT1L inhibition, enable efficient derivation of human induced pluripotent stem cells (iPSCs) with OCT4 and SOX2. In contrast, catalytic inhibition of CBP/EP300 prevents iPSC formation, suggesting distinct functions for different coactivator domains in reprogramming. CBP/EP300 bromodomain inhibition decreases somatic-specific gene expression, histone H3 lysine 27 acetylation (H3K27Ac) and chromatin accessibility at target promoters and enhancers. The master mesenchymal transcription factor PRRX1 is one such functionally important target of CBP/EP300 bromodomain inhibition. Collectively, these results show that CBP/EP300 bromodomains sustain cell-type-specific gene expression and maintain cell identity.
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http://dx.doi.org/10.1038/s41589-019-0264-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504645PMC
May 2019

An essential role for the Zn transporter ZIP7 in B cell development.

Nat Immunol 2019 03 4;20(3):350-361. Epub 2019 Feb 4.

Midwest Immunology Clinic, Plymouth, MN, USA.

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn in modulating B cell receptor signal strength and positive selection.
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http://dx.doi.org/10.1038/s41590-018-0295-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561116PMC
March 2019

Design, Synthesis and Characterization of Covalent KDM5 Inhibitors.

Angew Chem Int Ed Engl 2019 01 7;58(2):515-519. Epub 2018 Dec 7.

Structural Genomics Consortium & Target Discovery Institute, University of Oxford, NDM Research Building, Roosevelt Drive, Oxford, OX3 7DQ and OX3 7FZ, UK.

Histone lysine demethylases (KDMs) are involved in the dynamic regulation of gene expression and they play a critical role in several biological processes. Achieving selectivity over the different KDMs has been a major challenge for KDM inhibitor development. Here we report potent and selective KDM5 covalent inhibitors designed to target cysteine residues only present in the KDM5 sub-family. The covalent binding to the targeted proteins was confirmed by MS and time-dependent inhibition. Additional competition assays show that compounds were non 2-OG competitive. Target engagement and ChIP-seq analysis showed that the compounds inhibited the KDM5 members in cells at nano- to micromolar levels and induce a global increase of the H3K4me3 mark at transcriptional start sites.
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http://dx.doi.org/10.1002/anie.201810179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391970PMC
January 2019

Science and Bioethics of CRISPR-Cas9 Gene Editing: An Analysis Towards Separating Facts and Fiction.

Yale J Biol Med 2017 12 19;90(4):625-634. Epub 2017 Dec 19.

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.

Since its emergence in 2012, the genome editing technique known as CRISPR-Cas9 and its scientific use have rapidly expanded globally within a very short period of time. The technique consists of using an RNA guide molecule to bind to complementary DNA sequences, which simultaneously recruits the endonuclease Cas9 to introduce double-stranded breaks in the target DNA. The resulting double-stranded break is then repaired, allowing modification or removal of specific DNA bases. The technique has gained momentum in the laboratory because it is cheap, quick, and easy to use. Moreover, it is also being applied to generate more complex animal model systems. Such use of genome editing has proven to be highly effective and warrants a potential therapy for both genetic and non-genetic diseases. Although genome editing has the potential to be a transformative therapy for patients it is still in its infancy. Consequently, the legal and ethical frameworks are yet to be fully discussed and will be an increasingly important topic as the technology moves towards more contentious issues such as modification of the germline. Here, we review a number of scientific and ethical issues which may potentially influence the development of both the technology and its use in the clinical setting.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733851PMC
December 2017

Production and Concentration of Lentivirus for Transduction of Primary Human T Cells.

Methods Mol Biol 2016 ;1448:85-93

Kennedy Institute of Rheumatology, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7FY, UK.

Lentiviral vectors have emerged as efficient tools for investigating T cell biology through their ability to efficiently deliver transgene expression into both dividing and nondividing cells. Such lentiviral vectors have the potential to infect a wide variety of cell types. However, despite this advantage, the ability to transduce primary human T cells remains challenging and methods to achieve efficient gene transfer are often time consuming and expensive. We describe a method for generating lentivirus that is simple to perform and does not require the purchase of non-standard equipment to transduce primary human T cells. Therefore, we provide an optimized protocol that is easy to implement and allow transduction with high efficiency and reproducibility.
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http://dx.doi.org/10.1007/978-1-4939-3753-0_7DOI Listing
January 2018

Indoleamine 2,3-dioxygenase-1 is protective in atherosclerosis and its metabolites provide new opportunities for drug development.

Proc Natl Acad Sci U S A 2015 Oct 5;112(42):13033-8. Epub 2015 Oct 5.

Nuffield Department of Orthopedics, Rheumatology, and Musculoskeletal Sciences, Kennedy Institute of Rheumatology, University of Oxford, Headington, Oxford OX3 7FY, United Kingdom;

Atherosclerosis is the major cause of cardiovascular disease (CVD), the leading cause of death worldwide. Despite much focus on lipid abnormalities in atherosclerosis, it is clear that the immune system also has important pro- and antiatherogenic functions. The enzyme indoleamine-2,3-dioxygenase (IDO) catalyses degradation of the essential amino acid tryptophan into immunomodulatory metabolites. How IDO deficiency affects immune responses during atherogenesis is unknown and we explored potential mechanisms in models of murine and human atherosclerosis. IDO deficiency in hypercholesterolemic ApoE(-/-) mice caused a significant increase in lesion size and surrogate markers of plaque vulnerability. No significant changes in cholesterol levels were observed but decreases in IL-10 production were found in the peripheral blood, spleen and lymph node B cells of IDO-deficient compared with IDO-competent ApoE(-/-) mice. 3,4,-Dimethoxycinnamoyl anthranilic acid (3,4-DAA), an orally active synthetic derivative of the tryptophan metabolite anthranilic acid, but not l-kynurenine, enhanced production of IL-10 in cultured splenic B cells. Finally, 3,4-DAA treatment reduced lesion formation and inflammation after collar-induced arterial injury in ApoE(-/-) mice, and reduced cytokine and chemokine production in ex vivo human atheroma cell cultures. Our data demonstrate that endogenous production of tryptophan metabolites via IDO is an essential feedback loop that controls atherogenesis and athero-inflammation. We show that the IDO pathway induces production of IL-10 in B cells in vivo and in vitro, suggesting that IDO may induce immunoregulatory functions of B cells in atherosclerosis. The favorable effects of anthranilic acid derivatives in atherosclerosis indicate a novel approach toward therapy of CVD.
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http://dx.doi.org/10.1073/pnas.1517820112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4620898PMC
October 2015

Reply: To PMID 24891289.

Arthritis Rheumatol 2015 May;67(5):1406-7

Kennedy Institute of Rheumatology and Botnar Research Centre, University of Oxford; Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK.

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http://dx.doi.org/10.1002/art.39042DOI Listing
May 2015

Methotrexate Restores Regulatory T Cell Function Through Demethylation of the FoxP3 Upstream Enhancer in Patients With Rheumatoid Arthritis.

Arthritis Rheumatol 2015 May;67(5):1182-92

Kennedy Institute of Rheumatology and University of Oxford, Oxford, UK.

Objective: We have previously shown, in a cohort of untreated rheumatoid arthritis (RA) patients, that the suppressive function of Treg cells is defective. However, other studies in cohorts of patients with established RA have shown that Treg cell function is normal. We hypothesized that treatment may restore Treg cell function and lead to reduced disease activity. The aim of this study was to investigate whether treatment with methotrexate (MTX) can result in epigenetic changes that lead to restoration of the Treg cell suppressive function in RA.

Methods: Peripheral blood samples from RA patients were assessed using (3) H-thymidine incorporation to measure Treg cell suppression of T cell proliferation, and by enzyme-linked immunosorbent assay to determine Treg cell suppression of interferon-γ production. CTLA-4 and FoxP3 expression was measured by flow cytometry and quantitative polymerase chain reaction (qPCR) in Treg cells from healthy individuals and RA patients. CD4+ T cells isolated from healthy individuals were cultured with interleukin-2 (IL-2), IL-6, and tumor necrosis factor α in the presence or absence of MTX, and FoxP3 expression was determined using qPCR and flow cytometry. Methylation of the FOXP3 upstream enhancer was analyzed by bisulfite sequencing PCR.

Results: Defective Treg cell function was observed only in RA patients who had not been treated with MTX, whereas Treg cells from MTX-exposed RA patients had restored suppressive function. This restored suppression was associated with increased expression of FoxP3 and CTLA-4 in Treg cells. Bisulfite sequencing PCR of Treg cells cultured in MTX revealed a significant reduction in methylation of the FOXP3 upstream enhancer.

Conclusion: This study identifies a novel mechanism of action of MTX, in which treatment of RA patients with MTX restores defective Treg cell function through demethylation of the FOXP3 locus, leading to a subsequent increase in FoxP3 and CTLA-4 expression.
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http://dx.doi.org/10.1002/art.39031DOI Listing
May 2015

A novel upstream enhancer of FOXP3, sensitive to methylation-induced silencing, exhibits dysregulated methylation in rheumatoid arthritis Treg cells.

Eur J Immunol 2014 Oct 18;44(10):2968-78. Epub 2014 Aug 18.

Kennedy Institute of Rheumatology, University of Oxford, Oxford, United Kingdom.

Treg-cell function is compromised in rheumatoid arthritis (RA). As the master regulator of Treg cells, FOXP3 controls development and suppressive function. Stable Treg-cell FOXP3 expression is epigenetically regulated; constitutive expression requires a demethylated Treg-specific demethylated region. Here, we hypothesised that methylation of the FOXP3 locus is altered in Treg cells of established RA patients. Methylation analysis of key regulatory regions in the FOXP3 locus was performed on Treg cells from RA patients and healthy controls. The FOXP3 Treg-specific demethylated region and proximal promoter displayed comparable methylation profiles in RA and healthy-donor Treg cells. We identified a novel differentially methylated region (DMR) upstream of the FOXP3 promoter, with enhancer activity sensitive to methylation-induced silencing. In RA Treg cells we observed significantly reduced DMR methylation and lower DNA methyltransferase (DNMT1/3A) expression compared with healthy Treg cells. Furthermore, DMR methylation negatively correlated with FOXP3 mRNA expression, and Treg cells isolated from rheumatoid factor negative RA patients were found to express significantly higher levels of FOXP3 than Treg cells from RhF-positive patients, with an associated decrease in DMR methylation. In conclusion, the novel DMR is involved in the regulation of Treg-cell FOXP3 expression, but this regulation is lost post-transcriptionally in RA Treg cells.
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http://dx.doi.org/10.1002/eji.201444453DOI Listing
October 2014

Treg cell function in rheumatoid arthritis is compromised by ctla-4 promoter methylation resulting in a failure to activate the indoleamine 2,3-dioxygenase pathway.

Arthritis Rheumatol 2014 Sep;66(9):2344-54

Kennedy Institute and University of Oxford, Oxford, UK.

Objective: Functionally impaired Treg cells expressing abnormally low levels of CTLA-4 have been well documented in rheumatoid arthritis (RA). However, the molecular defect underlying this reduced expression is unknown. The aims of this study were to assess the role of DNA methylation in regulating CTLA-4 expression in Treg cells isolated from RA patients and to elucidate the mechanism of their reduced suppressor function.

Methods: CTLA-4 expression in Treg cells from RA patients and healthy controls was measured by quantitative polymerase chain reaction (PCR) and flow cytometry. Methylation of the CTLA-4 gene promoter was analyzed by bisulfite-specific PCR, followed by sequencing. Methylation-dependent transcriptional activity of the CTLA-4 gene promoter was measured by luciferase assay, and NF-AT binding to the CTLA-4 gene promoter was determined by chromatin immunoprecipitation. The role of CTLA-4 expression in controlling Teff cells was analyzed using an autologous mixed lymphocyte reaction.

Results: Down-regulation of CTLA-4 expression in Treg cells from RA patients was caused by methylation of a previously unidentified NF-AT binding site within the CTLA-4 gene promoter. As a consequence, Treg cells were unable to induce expression and activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), which in turn resulted in a failure to activate the immunomodulatory kynurenine pathway.

Conclusion: We show for the first time that epigenetic modifications contribute to defective Treg cell function in RA through an inability to activate the IDO pathway. Therefore, this study sets a precedent for investigating potential therapeutic strategies aimed at reinforcing the IDO pathway in RA patients.
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http://dx.doi.org/10.1002/art.38715DOI Listing
September 2014

Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells.

BMC Biotechnol 2013 Nov 12;13:98. Epub 2013 Nov 12.

Kennedy Institute of Rheumatology, Roosevelt Drive, Headington OX3 7FY, Oxford, UK.

Background: Lentiviral vectors have emerged as efficient vehicles for transgene delivery in both dividing and non-dividing cells. A number of different modifications in vector design have increased biosafety and transgene expression. However, despite these advances, the transduction of primary human T cells is still challenging and methods to achieve efficient gene transfer are often expensive and time-consuming.

Results: Here we present a simple optimised protocol for the generation and transduction of lentivirus in primary human CD45RA+ T cells. We show that generation of high-titre lentivirus with improved primary T cell transduction is dependent upon optimised ultracentrifuge speed during viral concentration. Moreover, we demonstrate that transduction efficiency can be increased with simple modifications to the culturing conditions. Overall, a transduction efficiency of up to 89% in primary human CD45RA+ cells is achievable when these modifications are used in conjunction.

Conclusion: The optimised protocol described here is easy to implement and should facilitate the production of high-titre lentivirus with superior transduction efficiency in primary human T cells without the need for further purification methods.
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http://dx.doi.org/10.1186/1472-6750-13-98DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3830501PMC
November 2013

Resistance to regulatory T cell-mediated suppression in rheumatoid arthritis can be bypassed by ectopic foxp3 expression in pathogenic synovial T cells.

Proc Natl Acad Sci U S A 2011 Oct 16;108(40):16717-22. Epub 2011 Sep 16.

Kennedy Institute of Rheumatology, University of Oxford, London W6 8LH, United Kingdom.

Increasing evidence suggests that regulatory T cell (Treg) function is impaired in chronic inflammatory diseases such as rheumatoid arthritis (RA). Here we demonstrate that Tregs are unable to modulate the spontaneous production of TNF-α from RA synovial cells cultured from the diseased synovium site. Cytokine (IL-2, IL-6, TNF-α) activated T cells (Tck), cells we previously demonstrated to mimic the effector function of pathogenic RA synovial T cells, contained Tregs that survived and divided in this cytokine environment; however, the up-regulation of key molecules associated with Treg function (CTLA-4 and LFA-1) was impaired. Furthermore, Tregs were unable to suppress the function of Tcks, including contact-dependent induction of TNF-α from macrophages, supporting the concept that impaired Treg function/responsiveness contributes to chronicity of RA. However, ectopic foxp3 expression in both Tcks and pathogenic RA synovial T cells attenuated their cytokine production and function, including contact-dependent activation of macrophages. This diminished response to cytokine activation after ectopic foxp3 expression involved inhibited NF-κB activity and differed mechanistically from that displayed endogenously in conventional Tregs. These results suggest that diseases such as RA may perpetuate owing to the inability of Tregs to control cytokine-activated T-cell function. Understanding the mechanism whereby foxp3 attenuates the pathogenic function of synovial T cells may provide insight into the mechanisms of chronicity in inflammatory disease and potentially reveal new therapeutic candidates.
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http://dx.doi.org/10.1073/pnas.1112722108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3189031PMC
October 2011
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