Publications by authors named "Leo A H Zeef"

40 Publications

Memory CD8 T cells exhibit tissue imprinting and non-stable exposure-dependent reactivation characteristics following blood-stage Plasmodium berghei ANKA infections.

Immunology 2021 Aug 18. Epub 2021 Aug 18.

Faculty of Biology, Medicine and Health, The Lydia Becker Institute of Immunology and Inflammation, University of Manchester, Manchester, UK.

Experimental cerebral malaria (ECM) is a severe complication of Plasmodium berghei ANKA (PbA) infection in mice, characterized by CD8 T-cell accumulation within the brain. Whilst the dynamics of CD8 T-cell activation and migration during extant primary PbA infection have been extensively researched, the fate of the parasite-specific CD8 T cells upon resolution of ECM is not understood. In this study, we show that memory OT-I cells persist systemically within the spleen, lung and brain following recovery from ECM after primary PbA-OVA infection. Whereas memory OT-I cells within the spleen and lung exhibited canonical central memory (Tcm) and effector memory (Tem) phenotypes, respectively, memory OT-I cells within the brain post-PbA-OVA infection displayed an enriched CD69 CD103 profile and expressed low levels of T-bet. OT-I cells within the brain were excluded from short-term intravascular antibody labelling but were targeted effectively by longer-term systemically administered antibodies. Thus, the memory OT-I cells were extravascular within the brain post-ECM but were potentially not resident memory cells. Importantly, whilst memory OT-I cells exhibited strong reactivation during secondary PbA-OVA infection, preventing activation of new primary effector T cells, they had dampened reactivation during a fourth PbA-OVA infection. Overall, our results demonstrate that memory CD8 T cells are systemically distributed but exhibit a unique phenotype within the brain post-ECM, and that their reactivation characteristics are shaped by infection history. Our results raise important questions regarding the role of distinct memory CD8 T-cell populations within the brain and other tissues during repeat Plasmodium infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/imm.13405DOI Listing
August 2021

Experimental long-term diabetes mellitus alters the transcriptome and biomechanical properties of the rat urinary bladder.

Sci Rep 2021 07 30;11(1):15529. Epub 2021 Jul 30.

School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Oxford Road, Manchester, M13 9PT, UK.

Diabetes mellitus (DM) is the leading cause of chronic kidney disease and diabetic nephropathy is widely studied. In contrast, the pathobiology of diabetic urinary bladder disease is less understood despite dysfunctional voiding being common in DM. We hypothesised that diabetic cystopathy has a characteristic molecular signature. We therefore studied bladders of hyperglycaemic and polyuric rats with streptozotocin (STZ)-induced DM. Sixteen weeks after induction of DM, as assessed by RNA arrays, wide-ranging changes of gene expression occurred in DM bladders over and above those induced in bladders of non-hyperglycaemic rats with sucrose-induced polyuria. The altered transcripts included those coding for extracellular matrix regulators and neural molecules. Changes in key genes deregulated in DM rat bladders were also detected in db/db mouse bladders. In DM rat bladders there was reduced birefringent collagen between detrusor muscle bundles, and atomic force microscopy showed a significant reduction in tissue stiffness; neither change was found in bladders of sucrose-treated rats. Thus, altered extracellular matrix with reduced tissue rigidity may contribute to voiding dysfunction in people with long-term DM. These results serve as an informative stepping stone towards understanding the complex pathobiology of diabetic cystopathy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-94532-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8324824PMC
July 2021

Zebrafish IL-4-like Cytokines and IL-10 Suppress Inflammation but Only IL-10 Is Essential for Gill Homeostasis.

J Immunol 2020 08 8;205(4):994-1008. Epub 2020 Jul 8.

Lydia Becker Institute of Immunology and Inflammation, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M13 9PT, United Kingdom; and

Mucosal surfaces such as fish gills interface between the organism and the external environment and as such are major sites of foreign Ag encounter. In the gills, the balance between inflammatory responses to waterborne pathogens and regulatory responses toward commensal microbes is critical for effective barrier function and overall fish health. In mammals, IL-4 and IL-13 in concert with IL-10 are essential for balancing immune responses to pathogens and suppressing inflammation. Although considerable progress has been made in the field of fish immunology in recent years, whether the fish counterparts of these key mammalian cytokines perform similar roles is still an open question. In this study, we have generated IL-4/13A and IL-4/13B mutant zebrafish () and, together with an existing IL-10 mutant line, characterized the consequences of loss of function of these cytokines. We demonstrate that IL-4/13A and IL-4/13B are required for the maintenance of a Th2-like phenotype in the gills and the suppression of type 1 immune responses. As in mammals, IL-10 appears to have a more striking anti-inflammatory function than IL-4-like cytokines and is essential for gill homeostasis. Thus, both IL-4/13 and IL-10 paralogs in zebrafish exhibit aspects of conserved function with their mammalian counterparts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.2000372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416321PMC
August 2020

Plasticity of Mitochondrial DNA Inheritance and its Impact on Nuclear Gene Transcription in Yeast Hybrids.

Microorganisms 2020 Mar 31;8(4). Epub 2020 Mar 31.

Manchester Institute of Biotechnology, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M1 7DN, UK.

Mitochondrial DNA (mtDNA) in yeast is biparentally inherited, but colonies rapidly lose one type of parental mtDNA, thus becoming homoplasmic. Therefore, hybrids between the yeast species possess two homologous nuclear genomes, but only one type of mitochondrial DNA. We hypothesise that the choice of mtDNA retention is influenced by its contribution to hybrid fitness in different environments, and the allelic expression of the two nuclear sub-genomes is affected by the presence of different mtDNAs in hybrids. hybrids preferentially retained mtDNA when formed on rich media at colder temperatures, while mtDNA was primarily retained on non-fermentable carbon source, at any temperature. Transcriptome data for hybrids harbouring different mtDNA showed a strong environmentally dependent allele preference, which was more important in respiratory conditions. Co-expression analysis for specific biological functions revealed a clear pattern of concerted allelic transcription within the same allele type, which supports the notion that the hybrid cell works preferentially with one set of parental alleles (or the other) for different cellular functions. Given that the type of mtDNA retained in hybrids affects both nuclear expression and fitness, it might play a role in driving hybrid genome evolution in terms of gene retention and loss.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/microorganisms8040494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232527PMC
March 2020

Severe type I interferonopathy and unrestrained interferon signaling due to a homozygous germline mutation in .

Sci Immunol 2019 12;4(42)

Department of Pathology, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK.

Excessive type I interferon (IFNα/β) activity is implicated in a spectrum of human disease, yet its direct role remains to be conclusively proven. We investigated two siblings with severe early-onset autoinflammatory disease and an elevated IFN signature. Whole-exome sequencing revealed a shared homozygous missense Arg148Trp variant in , a transcription factor that functions exclusively downstream of innate IFNs. Cells bearing STAT2 in homozygosity (but not heterozygosity) were hypersensitive to IFNα/β, which manifest as prolonged Janus kinase-signal transducers and activators of transcription (STAT) signaling and transcriptional activation. We show that this gain of IFN activity results from the failure of mutant STAT2 to interact with ubiquitin-specific protease 18, a key STAT2-dependent negative regulator of IFNα/β signaling. These observations reveal an essential in vivo function of STAT2 in the regulation of human IFNα/β signaling, providing concrete evidence of the serious pathological consequences of unrestrained IFNα/β activity and supporting efforts to target this pathway therapeutically in IFN-associated disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciimmunol.aav7501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115903PMC
December 2019

Cognitive dysfunction in diabetic rats is prevented by pyridoxamine treatment. A multidisciplinary investigation.

Mol Metab 2019 10 5;28:107-119. Epub 2019 Aug 5.

Faculty of Biology, Medicine and Health, University of Manchester, UK. Electronic address:

Objective: The impact of diabetes mellitus on the central nervous system is less widely studied than in the peripheral nervous system, but there is increasing evidence that it elevates the risk of developing cognitive deficits. The aim of this study was to characterize the impact of experimental diabetes on the proteome and metabolome of the hippocampus. We tested the hypothesis that the vitamin B6 isoform pyridoxamine is protective against functional and molecular changes in diabetes.

Methods: We tested recognition memory using the novel object recognition (NOR) test in streptozotocin (STZ)-induced diabetic, age-matched control, and pyridoxamine- or insulin-treated diabetic male Wistar rats. Comprehensive untargeted metabolomic and proteomic analyses, using gas chromatography-mass spectrometry and iTRAQ-enabled protein quantitation respectively, were utilized to characterize the molecular changes in the hippocampus in diabetes.

Results: We demonstrated diabetes-specific, long-term (but not short-term) recognition memory impairment and that this deficit was prevented by insulin or pyridoxamine treatment. Metabolomic analysis showed diabetes-associated changes in 13/82 identified metabolites including polyol pathway intermediates glucose (9.2-fold), fructose (4.9-fold) and sorbitol (5.2-fold). We identified and quantified 4807 hippocampal proteins; 806 were significantly altered in diabetes. Pathway analysis revealed significant alterations in cytoskeletal components associated with synaptic plasticity, glutamatergic signaling, oxidative stress, DNA damage and FXR/RXR activation pathways in the diabetic rat hippocampus.

Conclusions: Our data indicate a protective effect of pyridoxamine against diabetes-induced cognitive deficits, and our comprehensive 'omics datasets provide insight into the pathogenesis of cognitive dysfunction enabling development of further mechanistic and therapeutic studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molmet.2019.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6822151PMC
October 2019

Publisher Correction: SOX9 regulated matrix proteins are increased in patients serum and correlate with severity of liver fibrosis.

Sci Rep 2019 Aug 6;9(1):11547. Epub 2019 Aug 6.

Wellcome Centre for Cell-Matrix Research, Faculty of Biology, Medicine & Health, Manchester Academic Health Science Centre, University of Manchester, Oxford Road, Manchester, M13 9PT, UK.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-47715-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683292PMC
August 2019

Infection-Induced Resistance to Experimental Cerebral Malaria Is Dependent Upon Secreted Antibody-Mediated Inhibition of Pathogenic CD8 T Cell Responses.

Front Immunol 2019 19;10:248. Epub 2019 Feb 19.

Faculty of Biology, Medicine and Health, The Lydia Becker Institute of Immunology and Inflammation, University of Manchester, Manchester, United Kingdom.

Cerebral malaria (CM) is one of the most severe complications of infection. There is evidence that repeated parasite exposure promotes resistance against CM. However, the immunological basis of this infection-induced resistance remains poorly understood. Here, utilizing the ANKA (PbA) model of experimental cerebral malaria (ECM), we show that three rounds of infection and drug-cure protects against the development of ECM during a subsequent fourth (4X) infection. Exposure-induced resistance was associated with specific suppression of CD8 T cell activation and CTL-related pathways, which corresponded with the development of heterogeneous atypical B cell populations as well as the gradual infection-induced generation and maintenance of high levels of anti-parasite IgG. Mechanistically, transfer of high-titer anti-parasite IgG did not protect 1X infected mice against ECM and depletion of atypical and regulatory B cells during 4X infection failed to abrogate infection-induced resistance to ECM. However, IgMi mice that were unable to produce secreted antibody, or undergo class switching, during the repeated rounds of infection failed to develop resistance against ECM. The failure of infection-induced protection in IgMi mice was associated with impaired development of atypical B cell populations and the inability to suppress pathogenic CD8 T cell responses. Our results, therefore, suggest the importance of anti-parasite antibody responses, gradually acquired, and maintained through repeated infections, for modulating the B cell compartment and eventually suppressing memory CD8 T cell reactivation to establish infection-induced resistance to ECM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.00248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6394254PMC
July 2020

SOX9 regulated matrix proteins are increased in patients serum and correlate with severity of liver fibrosis.

Sci Rep 2018 12 17;8(1):17905. Epub 2018 Dec 17.

Wellcome Centre for Cell-Matrix Research, Faculty of Biology, Medicine & Health, Manchester Academic Health Science Centre, University of Manchester, Oxford Road, Manchester, M13 9PT, UK.

Extracellular matrix (ECM) deposition and resultant scar play a major role in the pathogenesis and progression of liver fibrosis. Identifying core regulators of ECM deposition may lead to urgently needed diagnostic and therapetic strategies for the disease. The transcription factor Sex determining region Y box 9 (SOX9) is actively involved in scar formation and its prevalence in patients with liver fibrosis predicts progression. In this study, transcriptomic approaches of Sox9-abrogated myofibroblasts identified >30% of genes regulated by SOX9 relate to the ECM. Further scrutiny of these data identified a panel of highly expressed ECM proteins, including Osteopontin (OPN), Osteoactivin (GPNMB), Fibronectin (FN1), Osteonectin (SPARC) and Vimentin (VIM) as SOX9 targets amenable to assay in patient serum. In vivo all SOX-regulated targets were increased in human disease and mouse models of fibrosis and decreased following Sox9-loss in mice with parenchymal and biliary fibrosis. In patient serum samples, SOX9-regulated ECM proteins were altered in response to fibrosis severity, whereas comparison with established clinical biomarkers demonstrated superiority for OPN and VIM at detecting early stages of fibrosis. These data support SOX9 in the mechanisms underlying fibrosis and highlight SOX9 and its downstream targets as new measures to stratify patients with liver fibrosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-36037-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297163PMC
December 2018

Human notochordal cell transcriptome unveils potential regulators of cell function in the developing intervertebral disc.

Sci Rep 2018 08 27;8(1):12866. Epub 2018 Aug 27.

Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Stopford Building, Oxford Road, Manchester, M13 9PT, United Kingdom.

The adult nucleus pulposus originates from the embryonic notochord, but loss of notochordal cells with skeletal maturity in humans is thought to contribute to the onset of intervertebral disc degeneration. Thus, defining the phenotype of human embryonic/fetal notochordal cells is essential for understanding their roles and for development of novel therapies. However, a detailed transcriptomic profiling of human notochordal cells has never been achieved. In this study, the notochord-specific marker CD24 was used to specifically label and isolate (using FACS) notochordal cells from human embryonic and fetal spines (7.5-14 weeks post-conception). Microarray analysis and qPCR validation identified CD24, STMN2, RTN1, PRPH, CXCL12, IGF1, MAP1B, ISL1, CLDN1 and THBS2 as notochord-specific markers. Expression of these markers was confirmed in nucleus pulposus cells from aged and degenerate discs. Ingenuity pathway analysis revealed molecules involved in inhibition of vascularisation (WISP2, Noggin and EDN2) and inflammation (IL1-RN) to be master regulators of notochordal genes. Importantly, this study has, for the first time, defined the human notochordal cell transcriptome and suggests inhibition of inflammation and vascularisation may be key roles for notochordal cells during intervertebral disc development. The molecules and pathways identified in this study have potential for use in developing strategies to retard/prevent disc degeneration, or regenerate tissue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-31172-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110784PMC
August 2018

Changes in S100 Proteins Identified in Healthy Skin following Electrical Stimulation: Relevance for Wound Healing.

Adv Skin Wound Care 2018 Jul;31(7):322-327

Chloe Lallyett, PhD, is a Science Teacher, Parrs Wood High School, Manchester, United Kingdom; Ching-Yan Chloé Yeung, PhD, is a Postdoctorate Fellow, Institute of Sports Medicine Copenhagen, Denmark; Rie Harboe Nielson, MD, PhD, is a Physician, Department of Rheumatology, Aarhus University Hospital, Denmark; Leo A. H. Zeef, PhD, is Experimental Officer, University of Manchester, United Kingdom; David Chapman-Jones, LLM(Med), PhD, is Professor of Healthcare and Director, Institute of Healthcare Policy and Practice, University of the West of Scotland, United Kingdom; Michael Kjaer, MD, PhD, is Professor of Sports Medicine, University of Copenhagen, Denmark; and Karl E. Kadler, PhD, is Professor of Biochemistry, University of Manchester, United Kingdom.

Objective: Targeted electrical energy applied to wounds has been shown to improve wound-healing rates. However, the mechanisms are poorly understood. The aim of this study was to identify genes that are responsive to electrical stimulation (ES) in healthy subjects with undamaged skin.

Methods: To achieve this objective, study authors used a small, noninvasive ES medical device to deliver a continuous, specific, set sequence of electrical energy impulses over a 48-hour period to the skin of healthy volunteers and compared resultant gene expression by microarray analysis.

Main Results: Application of this specific ES resulted in differential expression of 105 genes, the majority of which were down-regulated. Postmicroarray analyses revealed there was commonality with a small number of genes that have previously been shown to be up-regulated in skin wounds, including venous leg ulcers.

Conclusions: The specific sequence of ES applied continuously for 48 hours to the skin of healthy patients has the effect of modifying expression in a number of identified genes. The identification of the differential expression in this subset of genes in healthy subjects provides new potential lines of scientific inquiry for identifying similar responses in subjects with slow or poorly healing wounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/01.ASW.0000533722.06780.03DOI Listing
July 2018

Type I interferon-mediated autoinflammation due to DNase II deficiency.

Nat Commun 2017 12 19;8(1):2176. Epub 2017 Dec 19.

Pathology Department, Hôpital Necker-Enfants Malades, Assistance Publique-Hôpitaux de Paris, Paris, 75015, France.

Microbial nucleic acid recognition serves as the major stimulus to an antiviral response, implying a requirement to limit the misrepresentation of self nucleic acids as non-self and the induction of autoinflammation. By systematic screening using a panel of interferon-stimulated genes we identify two siblings and a singleton variably demonstrating severe neonatal anemia, membranoproliferative glomerulonephritis, liver fibrosis, deforming arthropathy and increased anti-DNA antibodies. In both families we identify biallelic mutations in DNASE2, associated with a loss of DNase II endonuclease activity. We record increased interferon alpha protein levels using digital ELISA, enhanced interferon signaling by RNA-Seq analysis and constitutive upregulation of phosphorylated STAT1 and STAT3 in patient lymphocytes and monocytes. A hematological disease transcriptomic signature and increased numbers of erythroblasts are recorded in patient peripheral blood, suggesting that interferon might have a particular effect on hematopoiesis. These data define a type I interferonopathy due to DNase II deficiency in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-017-01932-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736616PMC
December 2017

Glucocorticoid therapy regulates podocyte motility by inhibition of Rac1.

Sci Rep 2017 07 27;7(1):6725. Epub 2017 Jul 27.

Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, Faculty of Biology Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester, England.

Nephrotic syndrome (NS) occurs when the glomerular filtration barrier becomes excessively permeable leading to massive proteinuria. In childhood NS, immune system dysregulation has been implicated and increasing evidence points to the central role of podocytes in the pathogenesis. Children with NS are typically treated with an empiric course of glucocorticoid (Gc) therapy; a class of steroids that are activating ligands for the glucocorticoid receptor (GR) transcription factor. Although Gc-therapy has been the cornerstone of NS management for decades, the mechanism of action, and target cell, remain poorly understood. We tested the hypothesis that Gc acts directly on the podocyte to produce clinically useful effects without involvement of the immune system. In human podocytes, we demonstrated that the basic GR-signalling mechanism is intact and that Gc induced an increase in podocyte barrier function. Defining the GR-cistrome identified Gc regulation of motility genes. These findings were functionally validated with live-cell imaging. We demonstrated that treatment with Gc reduced the activity of the pro-migratory small GTPase regulator Rac1. Furthermore, Rac1 inhibition had a direct, protective effect on podocyte barrier function. Our studies reveal a new mechanism for Gc action directly on the podocyte, with translational relevance to designing new selective synthetic Gc molecules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-06810-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5532274PMC
July 2017

Whole Transcriptome Analysis of Pre-invasive and Invasive Early Squamous Lung Carcinoma in Archival Laser Microdissected Samples.

Respir Res 2017 01 10;18(1):12. Epub 2017 Jan 10.

Manchester Thoracic Oncology Centre, University Hospital of South Manchester, Manchester, England, M23 9LT, UK.

Background: Preinvasive squamous cell cancer (PSCC) are local transformations of bronchial epithelia that are frequently observed in current or former smokers. Their different grades and sizes suggest a continuum of dysplastic change with increasing severity, which may culminate in invasive squamous cell carcinoma (ISCC). As a consequence of the difficulty in isolating cancerous cells from biopsies, the molecular pathology that underlies their histological variability remains largely unknown.

Method: To address this issue, we have employed microdissection to isolate normal bronchial epithelia and cancerous cells from low- and high-grade PSCC and ISCC, from paraffin embedded (FFPE) biopsies and determined gene expression using Affymetric Human Exon 1.0 ST arrays. Tests for differential gene expression were performed using the Bioconductor package limma followed by functional analyses of differentially expressed genes in IPA.

Results: Examination of differential gene expression showed small differences between low- and high-grade PSCC but substantial changes between PSCC and ISCC samples (184 vs 1200 p-value <0.05, fc ±1.75). However, the majority of the differentially expressed PSCC genes (142 genes: 77%) were shared with those in ISCC samples. Pathway analysis showed that these shared genes are associated with DNA damage response, DNA/RNA metabolism and inflammation as major biological themes. Cluster analysis identified 12 distinct patterns of gene expression including progressive up or down-regulation across PSCC and ISCC. Pathway analysis of incrementally up-regulated genes revealed again significant enrichment of terms related to DNA damage response, DNA/RNA metabolism, inflammation, survival and proliferation. Altered expression of selected genes was confirmed using RT-PCR, as well as immunohistochemistry in an independent set of 45 ISCCs.

Conclusions: Gene expression profiles in PSCC and ISCC differ greatly in terms of numbers of genes with altered transcriptional activity. However, altered gene expression in PSCC affects canonical pathways and cellular and biological processes, such as inflammation and DNA damage response, which are highly consistent with hallmarks of cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12931-016-0496-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223343PMC
January 2017

Regulation of the BMP Signaling-Responsive Transcriptional Network in the Drosophila Embryo.

PLoS Genet 2016 07 5;12(7):e1006164. Epub 2016 Jul 5.

Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

The BMP signaling pathway has a conserved role in dorsal-ventral axis patterning during embryonic development. In Drosophila, graded BMP signaling is transduced by the Mad transcription factor and opposed by the Brinker repressor. In this study, using the Drosophila embryo as a model, we combine RNA-seq with Mad and Brinker ChIP-seq to decipher the BMP-responsive transcriptional network underpinning differentiation of the dorsal ectoderm during dorsal-ventral axis patterning. We identify multiple new BMP target genes, including positive and negative regulators of EGF signaling. Manipulation of EGF signaling levels by loss- and gain-of-function studies reveals that EGF signaling negatively regulates embryonic BMP-responsive transcription. Therefore, the BMP gene network has a self-regulating property in that it establishes a balance between its activity and that of the antagonistic EGF signaling pathway to facilitate correct patterning. In terms of BMP-dependent transcription, we identify key roles for the Zelda and Zerknüllt transcription factors in establishing the resulting expression domain, and find widespread binding of insulator proteins to the Mad and Brinker-bound genomic regions. Analysis of embryos lacking the BEAF-32 insulator protein shows reduced transcription of a peak BMP target gene and a reduction in the number of amnioserosa cells, the fate specified by peak BMP signaling. We incorporate our findings into a model for Mad-dependent activation, and discuss its relevance to BMP signal interpretation in vertebrates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1006164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4933369PMC
July 2016

PSR1 Is a Global Transcriptional Regulator of Phosphorus Deficiency Responses and Carbon Storage Metabolism in Chlamydomonas reinhardtii.

Plant Physiol 2016 Mar 24;170(3):1216-34. Epub 2015 Dec 24.

Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom

Many eukaryotic microalgae modify their metabolism in response to nutrient stresses such as phosphorus (P) starvation, which substantially induces storage metabolite biosynthesis, but the genetic mechanisms regulating this response are poorly understood. Here, we show that P starvation-induced lipid and starch accumulation is inhibited in a Chlamydomonas reinhardtii mutant lacking the transcription factor Pi Starvation Response1 (PSR1). Transcriptomic analysis identified specific metabolism transcripts that are induced by P starvation but misregulated in the psr1 mutant. These include transcripts for starch and triacylglycerol synthesis but also transcripts for photosynthesis-, redox-, and stress signaling-related proteins. To further examine the role of PSR1 in regulating lipid and starch metabolism, PSR1 complementation lines in the psr1 strain and PSR1 overexpression lines in a cell wall-deficient strain were generated. PSR1 expression in the psr1 lines was shown to be functional due to rescue of the psr1 phenotype. PSR1 overexpression lines exhibited increased starch content and number of starch granules per cell, which correlated with a higher expression of specific starch metabolism genes but reduced neutral lipid content. Furthermore, this phenotype was consistent in the presence and absence of acetate. Together, these results identify a key transcriptional regulator in global metabolism and demonstrate transcriptional engineering in microalgae to modulate starch biosynthesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1104/pp.15.01907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4775146PMC
March 2016

Chick tendon fibroblast transcriptome and shape depend on whether the cell has made its own collagen matrix.

Sci Rep 2015 Sep 4;5:13555. Epub 2015 Sep 4.

Wellcome Trust Centre for Cell-Matrix Research, Oxford Road, Manchester M13 9PT United Kingdom.

Collagen- and fibrin-based gels are extensively used to study cell behaviour. However, 2D-3D and collagen-fibrin comparisons of gene expression, cell shape and mechanotransduction, with an in vivo reference, have not been reported. Here we compared chick tendon fibroblasts (CTFs) at three stages of embryonic development with CTFs cultured in collagen- or fibrin-based tissue engineered constructs (TECs). CTFs synthesised their own collagen matrix in fibrin-based TECs and better recapitulated the gene expression, collagen fibril alignment and cell shape seen in vivo. In contrast, cells in 3D collagen gels exhibited a 2D-like morphology and expressed fewer of the genes expressed in vivo. Analysis of YAP/TAZ target genes showed that collagen gels desensitise mechanotransduction pathways. In conclusion, gene expression and cell shape are similar on plastic and 3D collagen whereas cells in 3D fibrin have a shape and transcriptome better resembling the in vivo situation. Implications for wound healing are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep13555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4559659PMC
September 2015

Characterization of samhd1 morphant zebrafish recapitulates features of the human type I interferonopathy Aicardi-Goutières syndrome.

J Immunol 2015 Mar 11;194(6):2819-25. Epub 2015 Feb 11.

Manchester Centre for Genomic Medicine, Institute of Human Development, Faculty of Medical and Human Sciences, University of Manchester, Manchester M13 9WL, United Kingdom; Laboratory of Neurogenetics and Neuroinflammation, Imagine Institute, Necker Hospital for Sick Children, 75015 Paris, France.

In humans, loss of function mutations in the SAMHD1 (AGS5) gene cause a severe form of Aicardi-Goutières syndrome (AGS), an inherited inflammatory-mediated encephalopathy characterized by increased type I IFN activity and upregulation of IFN-stimulated genes (ISGs). In particular, SAMHD1-related AGS is associated with a distinctive cerebrovascular pathology that commonly leads to stroke. Although inflammatory responses are observed in immune cells cultured from Samhd1 null mouse models, these mice are physically healthy, specifically lacking a brain phenotype. We have investigated the use of zebrafish as an alternative system for generating a clinically relevant model of SAMHD1-related AGS. Using temporal gene knockdown of zebrafish samhd1, we observe hindbrain ventricular swelling and brain hemorrhage. Furthermore, loss of samhd1 or of another AGS-associated gene, adar, leads to a significant upregulation of innate immune-related genes and an increase in the number of cells expressing the zebrafish type I IFN ifnphi1. To our knowledge, this is the first example of an in vivo model of AGS that recapitulates features of both the innate immune and neurological characteristics of the disease. The phenotypes associated with loss of samhd1 and adar suggest a function of these genes in controlling innate immune processes conserved to zebrafish, thereby also contributing to our understanding of antiviral signaling in this model organism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1403157DOI Listing
March 2015

The effect of type 1 IFN on human aortic endothelial cell function in vitro: relevance to systemic lupus erythematosus.

J Interferon Cytokine Res 2014 May 20;34(5):404-12. Epub 2014 Jan 20.

1 Arthritis Research UK Epidemiology Unit, Institute of Inflammation and Repair, Manchester Academic Health Sciences Centre, The University of Manchester , Manchester, United Kingdom .

Cardiovascular disease (CVD) is an important cause of morbidity and mortality in patients with systemic lupus erythematosus. The etiopathogenesis of premature CVD is not fully understood, but recently interferon-alpha (IFNα) has been implicated as a contributing factor. Since IFNα has been associated with both disease activity and endothelial dysfunction in lupus patients, we aimed to determine whether IFNα has direct effects on human aortic endothelial cell (HAoEC) function in vitro. We studied the function of IFNα2b-treated HAoECs in terms of cell proliferation, capillary-like network formation, and nitric oxide (NO) generation. Changes in gene expression were also analyzed using an exon gene array. IFNα2b regulated the expression of 198 genes, including recognized interferon-stimulated genes (ISGs). Gene ontology analysis showed over-representation of genes involved in antigen presentation and host response to virus but no significant changes in clusters of genes recognized as important in endothelial cell activation or dysfunction. HAoEC proliferation, tubule formation, and NO bioavailability were unchanged, suggesting that IFNα in isolation does not have a direct impact on aortic endothelial cell function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/jir.2013.0016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4015474PMC
May 2014

Certolizumab pegol attenuates the pro-inflammatory state in endothelial cells in a manner that is atheroprotective.

Clin Exp Rheumatol 2013 Mar-Apr;31(2):225-33. Epub 2012 Dec 13.

University of Manchester, UK.

Objectives: Rheumatoid arthritis (RA) is associated with accelerated atherosclerosis and premature cardiovascular death. Anti-TNF therapy is thought to reduce clinical cardiovascular disease risk and improve vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to explore the effects of certolizumab pegol (CZP) on TNF-activated human aortic endothelial cells (HAoECs).

Methods: HAoECs were cultured in vitro and exposed to i) TNF alone, ii) TNF plus CZP, or iii) neither agent. Microarray analysis and quantitative polymerase chain reaction were used to analyse gene expression. Activation of NF-κB was investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was performed to detect microparticle release from HAoECs.

Results: TNF alone had strong effects on endothelial gene expression, while TNF and CZP together produced a global gene expression pattern similar to untreated controls. In particular, genes for E-selectin, VCAM-1 and ICAM-1 were significantly up-regulated by TNF treatment. Notably, the TNF/CZP cocktail prevented the up-regulation of these genes. TNF-induced nuclear translocation of NF-κB was abolished by treatment with CZP. In addition the increased production of endothelial microparticles in TNF-activated HAoECs was prevented by treatment with CZP.

Conclusions: We have found at cellular level, that a clinically available TNF inhibitor, CZP i) reduces adhesion molecule expression; ii) prevents TNF-induced activation of the NF-κB pathway and iii) prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
June 2013

A conserved function for the H2A.Z C terminus.

J Biol Chem 2012 Jun 9;287(23):19148-57. Epub 2012 Apr 9.

Faculty of Life Sciences, University of Manchester M13 9PT, United Kingdom.

Histone H2A variants generate diversity in chromatin structure and functions, as nucleosomes containing variant H2A histones have altered physical, chemical, and biological properties. H2A.Z is an evolutionarily ancient and highly conserved H2A variant that regulates processes ranging from gene expression to the DNA damage response. Here we find that the unstructured portion of the C-terminal tail of H2A.Z is required for the normal functions of this histone variant in budding yeast. We have also identified a novel splice isoform of the human H2A.Z-2 gene that encodes a C-terminally truncated H2A.Z protein that is similar to the truncation mutants we identified in yeast. The short forms of H2A.Z in both yeast and human cells are more loosely associated with chromatin than the full-length proteins, indicating a conserved function for the H2A.Z C-terminal tail in regulating the association of H2A.Z with nucleosomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M111.317990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365947PMC
June 2012

Genome-wide analysis of gene expression during Xenopus tropicalis tadpole tail regeneration.

BMC Dev Biol 2011 Nov 15;11:70. Epub 2011 Nov 15.

Faculty of Life Sciences, University of Manchester, UK.

Background: The molecular mechanisms governing vertebrate appendage regeneration remain poorly understood. Uncovering these mechanisms may lead to novel therapies aimed at alleviating human disfigurement and visible loss of function following injury. Here, we explore tadpole tail regeneration in Xenopus tropicalis, a diploid frog with a sequenced genome.

Results: We found that, like the traditionally used Xenopus laevis, the Xenopus tropicalis tadpole has the capacity to regenerate its tail following amputation, including its spinal cord, muscle, and major blood vessels. We examined gene expression using the Xenopus tropicalis Affymetrix genome array during three phases of regeneration, uncovering more than 1,000 genes that are significantly modulated during tail regeneration. Target validation, using RT-qPCR followed by gene ontology (GO) analysis, revealed a dynamic regulation of genes involved in the inflammatory response, intracellular metabolism, and energy regulation. Meta-analyses of the array data and validation by RT-qPCR and in situ hybridization uncovered a subset of genes upregulated during the early and intermediate phases of regeneration that are involved in the generation of NADP/H, suggesting that these pathways may be important for proper tail regeneration.

Conclusions: The Xenopus tropicalis tadpole is a powerful model to elucidate the genetic mechanisms of vertebrate appendage regeneration. We have produced a novel and substantial microarray data set examining gene expression during vertebrate appendage regeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-213X-11-70DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3247858PMC
November 2011

The forkhead transcription factor FOXK2 promotes AP-1-mediated transcriptional regulation.

Mol Cell Biol 2012 Jan 14;32(2):385-98. Epub 2011 Nov 14.

University of Manchester, Manchester, United Kingdom.

The transcriptional control circuitry in eukaryotic cells is complex and is orchestrated by combinatorially acting transcription factors. Forkhead transcription factors often function in concert with heterotypic transcription factors to specify distinct transcriptional programs. Here, we demonstrate that FOXK2 participates in combinatorial transcriptional control with the AP-1 transcription factor. FOXK2 binding regions are widespread throughout the genome and are often coassociated with AP-1 binding motifs. FOXK2 acts to promote AP-1-dependent gene expression changes in response to activation of the AP-1 pathway. In this context, FOXK2 is required for the efficient recruitment of AP-1 to chromatin. Thus, we have uncovered an important new molecular mechanism that controls AP-1-dependent gene expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.05504-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255788PMC
January 2012

Knock-in reconstitution studies reveal an unexpected role of Cys-65 in regulating APE1/Ref-1 subcellular trafficking and function.

Mol Biol Cell 2011 Oct 24;22(20):3887-901. Epub 2011 Aug 24.

Department of Medical and Biological Sciences, University of Udine, 33100 Udine, Italy.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1) protects cells from oxidative stress via the base excision repair pathway and as a redox transcriptional coactivator. It is required for tumor progression/metastasis, and its up-regulation is associated with cancer resistance. Loss of APE1 expression causes cell growth arrest, mitochondrial impairment, apoptosis, and alterations of the intracellular redox state and cytoskeletal structure. A detailed knowledge of the molecular mechanisms regulating its different activities is required to understand the APE1 function associated with cancer development and for targeting this protein in cancer therapy. To dissect these activities, we performed reconstitution experiments by using wild-type and various APE1 mutants. Our results suggest that the redox function is responsible for cell proliferation through the involvement of Cys-65 in mediating APE1 localization within mitochondria. C65S behaves as a loss-of-function mutation by affecting the in vivo folding of the protein and by causing a reduced accumulation in the intermembrane space of mitochondria, where the import protein Mia40 specifically interacts with APE1. Treatment of cells with (E)-3-(2-[5,6-dimethoxy-3-methyl-1,4-benzoquinonyl])-2-nonyl propenoic acid, a specific inhibitor of APE1 redox function through increased Cys-65 oxidation, confirm that Cys-65 controls APE1 subcellular trafficking and provides the basis for a new role for this residue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E11-05-0391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3192867PMC
October 2011

Absolute and relative quantification of mRNA expression (transcript analysis).

Methods Mol Biol 2011 ;759:73-86

Faculty of Life Sciences, The University of Manchester, Manchester, UK.

In this protocol, we describe a pipeline for transcript analysis in yeast via the quantification of mRNA expression levels. In the first section, we consider the well-established, proprietary Affymetrix GeneChip® approach to generating transcriptomics data. In the next section, we concentrate on providing a detailed protocol for the validation of these data using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The protocol provides suggested examples of hardware, software, and consumables/reagents required to perform these experiments. There are of course many other options available using alternative approaches (or indeed suppliers), but this protocol is intended to provide an approach that is flexible, inexpensive, sensitive, and easy to use.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-61779-173-4_5DOI Listing
December 2011

Glucose depletion inhibits translation initiation via eIF4A loss and subsequent 48S preinitiation complex accumulation, while the pentose phosphate pathway is coordinately up-regulated.

Mol Biol Cell 2011 Sep 27;22(18):3379-93. Epub 2011 Jul 27.

University of Manchester, Manchester M13 9PT, UK.

Cellular stress can globally inhibit translation initiation, and glucose removal from yeast causes one of the most dramatic effects in terms of rapidity and scale. Here we show that the same rapid inhibition occurs during yeast growth as glucose levels diminish. We characterize this novel regulation showing that it involves alterations within the 48S preinitiation complex. In particular, the interaction between eIF4A and eIF4G is destabilized, leading to a temporary stabilization of the eIF3-eIF4G interaction on the 48S complex. Under such conditions, specific mRNAs that are important for the adaptation to the new conditions must continue to be translated. We have determined which mRNAs remain translated early after glucose starvation. These experiments enable us to provide a physiological context for this translational regulation by ascribing defined functions that are translationally maintained or up-regulated. Overrepresented in this class of mRNA are those involved in carbohydrate metabolism, including several mRNAs from the pentose phosphate pathway. Our data support a hypothesis that a concerted preemptive activation of the pentose phosphate pathway, which targets both mRNA transcription and translation, is important for the transition from fermentative to respiratory growth in yeast.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E11-02-0153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172263PMC
September 2011

Mutations in PRDM5 in brittle cornea syndrome identify a pathway regulating extracellular matrix development and maintenance.

Am J Hum Genet 2011 Jun;88(6):767-777

Genetic Medicine Research Group, Manchester Biomedical Research Centre, Manchester Academic Health Sciences Centre, University of Manchester and Central Manchester Foundation Trust, St Mary's Hospital, Manchester M13 9WL, UK. Electronic address:

Extreme corneal fragility and thinning, which have a high risk of catastrophic spontaneous rupture, are the cardinal features of brittle cornea syndrome (BCS), an autosomal-recessive generalized connective tissue disorder. Enucleation is frequently the only management option for this condition, resulting in blindness and psychosocial distress. Even when the cornea remains grossly intact, visual function could also be impaired by a high degree of myopia and keratoconus. Deafness is another common feature and results in combined sensory deprivation. Using autozygosity mapping, we identified mutations in PRDM5 in families with BCS. We demonstrate that regulation of expression of extracellular matrix components, particularly fibrillar collagens, by PRDM5 is a key molecular mechanism that underlies corneal fragility in BCS and controls normal corneal development and maintenance. ZNF469, encoding a zinc finger protein of hitherto undefined function, has been identified as a quantitative trait locus for central corneal thickness, and mutations in this gene have been demonstrated in Tunisian Jewish and Palestinian kindreds with BCS. We show that ZNF469 and PRDM5, two genes that when mutated cause BCS, participate in the same regulatory pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajhg.2011.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3113239PMC
June 2011

WT1 and its transcriptional cofactor BASP1 redirect the differentiation pathway of an established blood cell line.

Biochem J 2011 Apr;435(1):113-25

Faculty of Life Sciences, The Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK.

The Wilms' tumour suppressor WT1 (Wilms' tumour 1) is a transcriptional regulator that plays a central role in organogenesis, and is mutated or aberrantly expressed in several childhood and adult malignancies. We previously identified BASP1 (brain acid-soluble protein 1) as a WT1 cofactor that suppresses the transcriptional activation function of WT1. In the present study we have analysed the dynamic between WT1 and BASP1 in the regulation of gene expression in myelogenous leukaemia K562 cells. Our findings reveal that BASP1 is a significant regulator of WT1 that is recruited to WT1-binding sites and suppresses WT1-mediated transcriptional activation at several WT1 target genes. We find that WT1 and BASP1 can divert the differentiation programme of K562 cells to a non-blood cell type following induction by the phorbol ester PMA. WT1 and BASP1 co-operate to induce the differentiation of K562 cells to a neuronal-like morphology that exhibits extensive arborization, and the expression of several genes involved in neurite outgrowth and synapse formation. Functional analysis revealed the relevance of the transcriptional reprogramming and morphological changes, in that the cells elicited a response to the neurotransmitter ATP. Taken together, the results of the present study reveal that WT1 and BASP1 can divert the lineage potential of an established blood cell line towards a cell with neuronal characteristics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20101734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3062854PMC
April 2011

Tartrate-resistant acid phosphatase deficiency causes a bone dysplasia with autoimmunity and a type I interferon expression signature.

Nat Genet 2011 Feb 9;43(2):127-31. Epub 2011 Jan 9.

Manchester Academic Heath Science Centre, University of Manchester, Genetic Medicine, Manchester, UK.

We studied ten individuals from eight families showing features consistent with the immuno-osseous dysplasia spondyloenchondrodysplasia. Of particular note was the diverse spectrum of autoimmune phenotypes observed in these individuals (cases), including systemic lupus erythematosus, Sjögren's syndrome, hemolytic anemia, thrombocytopenia, hypothyroidism, inflammatory myositis, Raynaud's disease and vitiligo. Haplotype data indicated the disease gene to be on chromosome 19p13, and linkage analysis yielded a combined multipoint log(10) odds (LOD) score of 3.6. Sequencing of ACP5, encoding tartrate-resistant acid phosphatase, identified biallelic mutations in each of the cases studied, and in vivo testing confirmed a loss of expressed protein. All eight cases assayed showed elevated serum interferon alpha activity, and gene expression profiling in whole blood defined a type I interferon signature. Our findings reveal a previously unrecognized link between tartrate-resistant acid phosphatase activity and interferon metabolism and highlight the importance of type I interferon in the genesis of autoimmunity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ng.748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3030921PMC
February 2011
-->