Leucocyte and Pathogen Trafficking through Lymphatics in Inflammation and Immunity

Lead Research Organisation: University of Oxford

Abstract

We are investigating how cells enter and move within lymphatic vessels so that in the future we can develop new therapies to block inflammation, prevent tumour spread, and make the delivery of vaccines to emerging infections more efficient. The lymphatic vessels form a dense network that intertwines the blood circulation and whose main functions are to maintain fluid balance and to transport cells carrying foreign antigen to the lymph nodes where they meet T and B cells and stimulate the immune response. Importantly, the lymphatics also contribute to diseases such as cancer by allowing metastatic tumours (e.g. skin and breast cancer) to spread to other parts of the body, and they act as a reservoir for malignant cells. Even though the involvement of lymphatics in disease have been known for a long time, their biology is still not well understood and so research in my group is dedicated to understanding how immune cells and tumours enter and navigate within the lymphatics. Thus far we have discovered that in inflammation, entry of immune cells carrying proteins from infectious agents depends critically on recognition of inflammation-induced protein receptors on the surface of lymphatic vessels, and that this is obligatory for the generation of the immune response to infection. We have also identified a separate mechanism that controls tumour cell entry to the lymphatics during lymph node metastasis in murine tumours and humans. We are also particularly interested in researching a molecule in lymphatics called LYVE-1 and we anticipate that further research in this area will reveal new ways to block inflammation, and to promote protective immunity to emerging infectious agents by enhancing vaccine delivery.

Technical Summary

Goals To investigate mechanisms of lymphatic trafficking in inflammation, immunity and cancer. In addition to maintaining fluid balance and tissue homeostasis the lymphatics also provide a conduit for trafficking of antigen presenting cells to lymph nodes for activation of nave T cells, and in so doing they allow a route for tumour metastasis. Recent work has focused on understanding the key molecular mechanisms regulating entry of leukocytes and tumour cells to lymphatics and their subsequent trafficking to lymph nodes. We have discovered a separate new integrin and chemokine dependent pathway for translymphatic migration of dendritic cells (DC) that functions in inflammation (1) and demonstrated its importance for dermal antigen delivery during T cell responses - identifying a new potential target for intervention (2). We have focused on two lectin-like receptors of the Link superfamily, LYVE-1 and CD44. LYVE-1 is the major HA receptor of lymphatic endothelial cells and was originally cloned and characterized in my laboratory (3). We have shown that in addition to mediation of lymphatic trafficking of DC, LYVE-1 also plays a role in lymphatic invasion in tumour metastasis and in the resolution of inflammation in a model of acute lung injury. We have identified tumour lymphatic endotherlial cell markers (TLEM) and have shown these to be important in nodal metastasis in a range of murine and human cancers (4). The crystal structure and activation mechanism of CD44, an inflammatory leukocyte HA receptor closely related to LYVE-1 and which plays a pivotal role in transmigration of activated leukocytes across inflamed vascular endothelium have been determined (5). Future research plans Future work will concentrate on the definition of the lymphatic entry points and molecular mechanisms for transmigration of different leukocyte populations. The use of microscopic imaging and structure/function analyses will allow us to determine how receptors such as LYVE-1 mediate cell trafficking in animal models of inflammation. Moreover, we will pursue the 3D structure of the LYVE-1 ectodomain and elucidate the mechanisms regulating it's interaction with hyaluronan, search for additional LYVE-1 ligands mediating leukocyte trafficking, define downstream signalling pathways mediating transendothelial migration and investigate function blocking antibodies of potential therapeutic value for inflammation and cancer. References: (1) Johnson et al. 2006 J Exp Med 203: 2763; (2) Teoh et al. 2009 J Immunol 182: 2425; (3) Gale et al 2007 Mol Cell Biol 27: 595; (4) Clasper et al. 2008 Cancer Res 68:7293; (5) Banerji et al. 2007 Nature Struct Mol Biol 14:234

Publications

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Bauer J (2018) TGFß counteracts LYVE-1-mediated induction of lymphangiogenesis by small hyaluronan oligosaccharides. in Journal of molecular medicine (Berlin, Germany)

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Brown M (2018) Lymphatic exosomes promote dendritic cell migration along guidance cues. in The Journal of cell biology

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Jackson DG (2019) Hyaluronan in the lymphatics: The key role of the hyaluronan receptor LYVE-1 in leucocyte trafficking. in Matrix biology : journal of the International Society for Matrix Biology

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Sun M (2019) Hyaluronan Derived From the Limbus is a Key Regulator of Corneal Lymphangiogenesis. in Investigative ophthalmology & visual science

 
Description MRC Research Grant Streptococcal dissemination
Amount £592,795 (GBP)
Funding ID MR/L008610/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2014 
End 03/2017
 
Title Characterisation of leucocyte hyaluronan surface glycocalyx and its role in lymphatic docking and entry 
Description Imaging of hyaluronan on surface of dendritic cells in mouse tissues and in mouse and human tissue-derived dendritic cells using biotinylated VG1. Analaysis of dendritic cell trafficking in mouse inflamed skin lymphatics before/after depletion of surface hyaluronan using 4-MU and hyaluronidase. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2017 
Provided To Others? No  
Impact Publication Nature Immunology paper (Johnson LA et al In Press 2017). Related aspects (HA glycocalyx in mouse macrophages) published in PMID 26823460 
 
Description Aspp1 collaboration 
Organisation University of Oxford
Department Nuffield Department of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise reagents and advice
Collaborator Contribution Expertise reagents and advice
Impact None as yet
Start Year 2016
 
Description Corneal lymphangiogenesis Vivien Coulson-Thomas 
Organisation University of Houston
Department College of Optometry
Country United States 
Sector Academic/University 
PI Contribution Advice expertise and experimental design
Collaborator Contribution Performed analyses of corneal lymphangiogenesis in relationship to local hyaluronan deposition in mouse models of corneal inflammation
Impact Co-author Publication in press in Investigational Ophthalmology and Visual Science 2019
Start Year 2018
 
Description Corneal transplant rejection - Claus Cursiefen, Felix Bock 
Organisation University Clinic Cologne
Department Department of Ophthalmology
Country Germany 
Sector Academic/University 
PI Contribution Provision of blocking mAbs and transgenic mice. Experimental design.
Collaborator Contribution Testing of mAbs and transgenic mice in corneal allograft rejection model. Does disruption of LYVE-1 : hyaluronan interactions in limbal lymphatics ameliorate transplant rejection and/or lymphangiogenesis ?
Impact In progress. Multidisciplinary - clinical (surgical), and microscopic imaging
Start Year 2018
 
Description David Coleman collaboration 
Organisation Oxford University Hospitals NHS Foundation Trust
Country United Kingdom 
Sector Academic/University 
PI Contribution Regular immunoisolation of primary endothelial cells from human surgically excised tissue - generation of materials essential for our research
Collaborator Contribution Provision of human skin samples for isolation of primary cells
Impact Regular supply of primary cells vital for our research and fundamental to most publications coming from my group
Start Year 2016
 
Description David Greaves collaboration 
Organisation University of Oxford
Department Sir William Dunn School of Pathology
Country United Kingdom 
Sector Academic/University 
PI Contribution Intellectual input and experimental design Animal experiments involving cell trafficking
Collaborator Contribution Advice and expertise in measurement of endothelial cell junctional permeability Contribution of transgenic reporter mice
Impact Manuscript in Blood; describing utility of CD68 transgenic reporter mice for macrophage trafficking studies Manuscript in preparation for J Biol Chem
Start Year 2012
 
Description Dontscho Kerjaschki 
Organisation Medical University of Vienna
Department Clinical Institute of Pathology
Country Austria 
Sector Academic/University 
PI Contribution Research collaboration leading to joint publication - currently in final revision at Journal of Cell Biology
Collaborator Contribution Carried out experimental work at host Institute and provided us with reagents to perform related studies in Oxford
Impact This multidisciplinary collaboration has yielded a publication in a prestigious high-impact journal (Journal of Cell Biology) that is currently in its final round of revision with decision expected imminently
Start Year 2017
 
Description Errin Johnson collaboration 
Organisation University of Oxford
Department Sir William Dunn School of Pathology
Country United Kingdom 
Sector Academic/University 
PI Contribution Advice and intellectual contribution
Collaborator Contribution Electron microscopy
Impact Ongoing
Start Year 2015
 
Description Fluorescence and EM imaging of lung 
Organisation Medical University of Vienna
Department Clinical Institute of Pathology
Country Austria 
Sector Academic/University 
PI Contribution Provision of reagents and intellectual input
Collaborator Contribution Performed immunoelectron microscopy and immunofluorescence imaging of thin sections of mouse alveolae
Impact Obtained important data for inclusion in manuscript under preparation for publication in scientific journal
Start Year 2012
 
Description High Resolution confocal Lung Imaging 
Organisation University of California, San Francisco
Department Department of Anatomy
Country United States 
Sector Academic/University 
PI Contribution Provided reagents and intellectual input
Collaborator Contribution Carried out perfusion fixation and inflation of lungs and prepared sections for confocal imaging and z sectioning to identify critical cell population in alveolae
Impact Obtained critical data for inclusion in a manuscript under preparation for submission to scientific journal
Start Year 2012
 
Description Jesus Angulo NMR collaboration 
Organisation University of Seville
Department Department of Bioorganic Chemistry
Country Spain 
Sector Academic/University 
PI Contribution Provision of recombinant soluble CD44 protein for STD NMR experiments
Collaborator Contribution Performed STD NMR analyses
Impact In progress
Start Year 2012
 
Description Lung lymphatics mAbs 
Organisation Medical University of Vienna
Department Clinical Institute of Pathology
Country Austria 
Sector Academic/University 
PI Contribution Generation of mAbs for imaging vasculature in lung sections
Collaborator Contribution EM and confocal imaging of lung vasculature
Impact Manuscript in preparation
Start Year 2012
 
Description Paul Riley collaboration 
Organisation University of Oxford
Department Department of Physiology, Anatomy and Genetics
Country United Kingdom 
Sector Academic/University 
PI Contribution Reagents advice and expertise
Collaborator Contribution Reagents advice and expertise
Impact None as yet
Start Year 2015
 
Description Ralf Richter collaboration 
Organisation Centre for Cooperative Research in Biomaterials (CIC BiomaGUNE)
Department Biosurfaces Research Unit
Country Spain 
Sector Academic/University 
PI Contribution Generation of protein reagents for ligand binding analysis by monitoring changes in mass and viscoelastic properties using accoustic resonance measurements (Quartz Crystal Microbalance with dissipation monitoring - QCM-D) technique and Atomic Force Spectroscopy. Intellectual input experimental design and analysis, co-authoring
Collaborator Contribution Expertise in physical biochemical techniques
Impact 27679982 27332136 Multidisciplinary Biochemistry, Biophysics, Immunology
Start Year 2016
 
Description Regeneron collaboration 
Organisation Regeneron Pharmaceuticals, Inc.
Country United States 
Sector Private 
PI Contribution Characterisation of phenotype of LYVE1 KO mice, generation of potential human function blocking mAbs and authoring of publication
Collaborator Contribution Generated transgenic mice, providing intellectual contributions and technical expertise for mAb production and testing
Impact Generated publication (Johnson et al Nature Immunology In Press 2017 ) Stimulating ongoing research in new areas, identification of possible new targets for anti-inflammatory therapy and future access to further transgenic animals
Start Year 2016
 
Description Robert Gilbert collaboration 
Organisation University of Oxford
Department Wellcome Trust Centre for Human Genetics
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Research using X-Ray crystallography carried out by my Unit postDOC Suneale Banerji with assistance from Robert Gilbert at Wellcome Trust Centre for Human Genomics, Oxford
Collaborator Contribution Expertise in Protein Crystallography and associated physical techniques
Impact Insight into 3D structure of LYVE-1 protein and the mode of its interaction with hyaluronan core to focus of my group research
Start Year 2016
 
Description Sriskandan collaboration 
Organisation Imperial College London
Department Faculty of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Design of experiments and advise on performing experiments to assess interactions between hyaluronan encapsulated Group A strep and lymphatic endothelial cells leading to possible transport to lymph nodes
Collaborator Contribution Provision of wild-type and mutant strains of Strep pyogenes and participation of post-doctoral scientist
Impact Publication in PLoS Pathogens 2015 PMID 26352587 Multidsciplinary Immunology Bacteriology Infectious Diseases
Start Year 2012
 
Description Super resolution microscopy 
Organisation Medical Research Council (MRC)
Department MRC Human Immunology Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution Preparation of primary cells and transfectants, and appropriate mAbs and conjugates for STED microscopic evaluation of receptor surface distribution
Collaborator Contribution Performing STED microscopy and analysis of data
Impact Multidisciplinary collaboration Manuscript submitted to Scientific Reports 2016
Start Year 2014
 
Description Surface Plasmon resonance (Biacore) analysis of receptor ligand binding affinity 
Organisation University of Oxford
Department Sir William Dunn School of Pathology
Country United Kingdom 
Sector Academic/University 
PI Contribution Generated recombinant soluble hyaluronan receptors and mutants thereof and performed Biacore analyses using hyaluronan coated chip to determine binding affinities
Collaborator Contribution Mathematical evaluation of binding data from kinetic analyses and derivation of accurate Kd values
Impact Manuscript that is about to be submitted to J Biol Chem
Start Year 2014
 
Description Tony Day collaboration 
Organisation Wellcome Trust
Department Wellcome Trust Centre for Cell-Matrix Research
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Protein expression and purification, Protein structural analysis, intellectual input, co-authoring
Collaborator Contribution Expertise in Physical techniques including NMR spectroscopy calorimetry, small angle X ray scattering. Production and purification of hyaluronan oligosaccharides and analogues for binding studies and co-crystallisation with receptors.
Impact Ongoing intellectual exchange. Generation of key publications 27733683 26823460 and impending new publication describing crystal structure of ligand binding domain of lymphatic hyaluronan receptor LYVE-1
Start Year 2016
 
Description Hands on Participatory event at Swindon Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Hands on Participatory event to explain the workings of lymph nodes and the lymphatic system for children to learn by play
Year(s) Of Engagement Activity 2020
URL https://www.steam-museum.org.uk/events/event/36/tomorrow_-_swindons_science_festival
 
Description School visit (Oxford) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Approx 30 primary school pupils participated in an interactive session with Suneal Banerji my senior post-doc who gave them an introductory presentation on immunology and the lymphatic system. This was extremely well received and sparked many questions and discussion both during and afterwards.
Year(s) Of Engagement Activity 2016