A systems-genetics approach to dissect inflammation in fibrosis

Lead Research Organisation: Imperial College London
Department Name: Dept of Medicine

Abstract

Fibrosis is the formation of excess connective tissue in an organ or tissue as a result of a reparative or reactive process and could therefore be regarded as an aberrant or excessive wound healing response. Fibrosis develops as a consequence of various underlying diseases and presents a major unsolved therapeutic problem as there is still no cure for fibrosis. In almost all forms of fibrosis, inflammatory-immunological reactions take place in earliest stages and there is now mounting evidence that fibrosis does not occur in the absence of a primary inflammatory insult. Wound healing represents a paradigm for progression of inflammation to fibrosis. In wound healing and various pathologies associated with inflammatory fibrosis (kidney and pulmonary fibrosis), innate immune cells are highly represented, and among the most abundant of these are macrophages.

This project aims to characterise the molecular mechanisms involved in macrophage-mediated wound healing. This will allow understanding the mechanisms underlying inflammatory fibrosis as dysregulation in wound healing results in fibrosis.

Genetic variation among individuals could affect the activation of their macrophages and cause quantitative variability in wound healing. We investigated this hypothesis by showing that genetic variation in a genetically heterogeneous mice population affects the expression of key wound healing genes in their macrophages and their susceptibility to wound healing. In our proposal, we will ask two fundamental questions: What is the full spectrum of the genetic determinants of macrophage activation in mice and how does this relate to genetic variability associated with wound healing? To answer this, we will use an approach called systems-genetics which integrates genetic variation between individuals with gene expression in macrophages to build biological networks and identify the genetic control points of networks associated with wound healing. Using the systems-genetics approach, we will find novel genetic factors for wound healing by identifying novel candidates that could be studied in diseases characterised by inflammatory fibrosis.

Technical Summary

The overall aim of this project is the identification of the genetic determinants of inflammation in wound healing and more generally in fibrosis. We will undertake a systems-genetics approach where genetic determinants of complete transcriptome in primary bone marrow derived macrophages of an outbred mice population will be integrated with a genome-wide association study (GWAS) for wound healing in the same population. To achieve this, we have cultured bone marrow derived macrophages from 1370 outbred mice and also collected the wound healing phenotype by using the ear punch model. Our preliminary GWAS for wound healing using the genotype/haplotype data (7 million single nucleotide polymorphisms) in these mice showed that it is a heritable and polygenic trait. We also showed that Tnfa, Ccl2 and Tgfb1, Vegfa, four major wound healing-associated genes known to be expressed in macrophages correlated positively and negatively respectively with the ear wound area in BMDMs from 92 outbred mice. We will perform mRNA sequencing by high throughput sequencing (RNA-seq) in 250 outbred mice. This will generate a comprehensive catalogue of the genetic determinants of macrophage gene expression (expression Quantitative Trait Loci; eQTLs), splicing QTLs (sQTLs) and gene co-expression networks. We will use the complete macrophage transcriptome for correlation with the wound healing phenotype and integrate the results with the GWAS for wound healing to identify master genetic regulators (mGR) of networks associated with wound healing. We will functionally validate these mGR in mice macrophages and establish whether they are present in human tissues from scleroderma patients where macrophage-mediated inflammatory fibrosis is well known. We will finally test whether the wound healing-associated networks in mice are also enriched for genetic susceptibility signals in 3 inflammatory fibrotic conditions: idiopathic pulmonary fibrosis, scleroderma and Crohn's disease.

Planned Impact

The proposed research aims to generate new knowledge about the genetic determinants of inflammation in fibrosis. The immediate users of the research outputs will be academic researchers in the field of innate immunity through our first aim: generation of a comprehensive catalog of the genetic determinants of macrophage gene expression (eQTLs) and gene co-expression networks by RNA-seq in a high-resolution mapping outbred mice population. This will generate a unique resource which will facilitate understanding one of the most challenging questions in the field of innate immunity: What are the genetic determinants of the full spectrum of macrophage gene expression and how does it relate to inflammatory disease? Our second aim is to use macrophage gene expression as an intermediate phenotype to explain genetic variation in wound healing measured by genome-wide association (GWA). Complex trait geneticists will be the direct immediate academic beneficiaries of this approach. One emerging view is that the substantial amount of data generated by GWA studies is not optimally used (or understood) to explain how genes, as part of cellular networks/pathways are associated with complex traits. It is now recognised that a systems-genetics approach is needed to understand the genetic architecture of complex disorders by multi-level data integration (genotype + cellular mRNA levels + phenotype or clinical trait) to unravel cellular mechanisms leading to optimal drug target identification that will contribute to nation's health. Systems-genetics is a newly emerging field that asks a fundamental question that is required to understand, complement and optimally use the GWA data generated for complex diseases: How is the common genomic variation regulate gene expression in a cell type clinically relevant for the disease? We believe our integrative approaches using RNA-seq in an effector cell type (macrophages) will explain the missing heritability observed in GWA studies for various fibrotic disorders and influence future genetic, diagnostic and pharmacological studies on inflammatory fibrosis aiming to enhance the quality of life and health.

The longer term beneficiaries of our proposed programme will be general public, students, the commercial private sector and potential patients. Public awareness on discovery-oriented translational research is crucial. We will communicate research activities to the general public within special initiatives at Imperial College London. This latter and MRC Clinical Sciences Centre are highly active in educational activities and engaging with the general public, including the recent 10-day exhibition at Imperial College or Fabrics of Life workshops, encompassing a series of projects that involve collaborations between scientific, artistic and creative industries. The MRes programme at Imperial College London provides graduate students from the life sciences, engineering and physical sciences with a platform to overcome traditional barriers and collaboratively work on the general health problems and applications in systems biology. Both the PI and co-investigator have active roles in engaging with students within this MRes programme to teach and discuss the advantages of systems-genetics with the students. Throughout the project, we will also be pro-active in developing links with industry to seek potential collaboration partners developing therapies targeting specifically macrophages in fibrotic disorders. Identification of new drug targets for treatment of patients suffering from inflammatory fibrosis is the the ultimate goal but we are aware that the time-frame for such an ambitious impact is beyond the measurable objectives of the current proposal. However we are confident in delivering significant progress in general public awareness, student and the commercial private sector engagement throughout the lifetime of this grant application.

Publications

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Behmoaras J (2015) Macrophage epoxygenase determines a profibrotic transcriptome signature. in Journal of immunology (Baltimore, Md. : 1950)

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Moreno-Moral A (2017) Systems Genetics as a Tool to Identify Master Genetic Regulators in Complex Disease. in Methods in molecular biology (Clifton, N.J.)

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Moreno-Moral A (2017) Systems Genetics as a Tool to Identify Master Genetic Regulators in Complex Disease. in Methods in molecular biology (Clifton, N.J.)

 
Description Training of pathologist
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Training/educational developments (including courses and course material)
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
Impact The integrative aspect of multi-level data analysis (i.e. systems-genetics approaches) had an impact on the education of researchers at Imperial College London. This field is constantly progressing with new methodologies and the educational aspect of it is crucial to understand how genetics can bring answers for complex disorders.
 
Description Macrophage multinucleation in health and disease
Amount £886,970 (GBP)
Funding ID MR/N01121X/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 04/2016 
End 03/2020
 
Title Delivery of siRNA to rodent wounds to modify healing properties 
Description This method is the result of a collaboration between the PI's group and Ben Almquist (Imperial College London, Bioengineering). It consists of the delivery of siRNA agains single or a set of genes (i.e. network) from chemically modified bandages directly to the wound. The unique expertise of Ben Almquist allows a progressive delivery of siRNA which is taken up by the cells involved in the healing (inflammatory and resident) to modify the kinetics of the process. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact -Direct in vivo confirmation of the genes identified by this grant (A systems-genetics approach to dissect inflammation in fibrosis). 
 
Title Single cell RNA-sequencing 
Description Single cell RNA-sequencing (ScRNA-seq) was optimised succesfully on the 10X Genomics platform using peripheral blood cells from a healthy donor 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact The use of scRNA-seq will have a considerable impact for all the research projects. Our preliminary results showed that the technique can allow clustering of a heterogenous cell population based on their transcriptome 
 
Title in vitro methods or advances in experimental design which can reduce the number of animals used 
Description We have generated a unique database in human scleroderma macrophages. We have RNA-seq data in the monocyte-derived macrophages from their peripheral blood and this data is in the process of getting integrated with the clinical phenotypes. The genes identified using this high-throughput methodology will generate hypotheses without using animal models, a critical aspect in the refinement of experimental designs. 
Type Of Material Model of mechanisms or symptoms - human 
Year Produced 2014 
Provided To Others? Yes  
Impact The systems-genetics approaches in human tissues are powerful methods to understand gene regulation in disease. We focus on an intermediate phenotype (macrophage transcriptome) and establish the link between genotype-macrophage transcriptome-phenotype. The advantage over traditional GWAS approaches are considerable: (i) the focus on an effector cell, providing insights into mechanisms in humans (ii) generation/identification of co-expression networks and their genetic control, providing insights into the pathology (iii) genetic architecture of the expression QTLs (cis vs. trans) 
 
Title orthogonal organic phase separation (OOPS) 
Description unbiased detection of cross-linked protein-RNA and free protein, or protein-bound RNA and free RNA. (TRIZOL-based methodology) 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact N/A 
 
Title High throughput sequencing datasets in fibrosis 
Description RNA-seq data from 150 outbred mice bone marrow derived macrophages. These mice show extreme phenotypes in terms of wound healing (fast vs. slow healers) RNA-seq from 50 scleroderma patient macrophages and 12 controls 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact Comparative inter-specie analysis of wound healing/fibrosis Genetic control of gene expression in macrophages during fibrosis Gene co-expression networks in macrophages during fibrosis Correlation between genome-wide transcription levels and clinical (scleroderma) or physiological (wound healing) phenotypes 
 
Title Lipidomics profiling in healthy macrophages 
Description Lipid quantification (>200 lipid species) during a TLR4 activation timecourse 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? No  
Impact This is a preliminary dataset and an ongoing collaboration 
 
Title Scleroderma eQTL dataset 
Description Genome-wide SNPs found to be associated in -cis with genome-wide RNA-seq expression data in Scleroderma patients and healthy controls 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact N/A 
 
Title Scleroderma macrophage dataset 
Description RNA-sequencing from scleroderma patient and healthy individual macrophages. The cells were differentiated from the peripheral blood monocytes. 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
Impact A recent publication in Annals of the Rheumatic Diseases where the Pi is the corresponding author (PMID: 29348297) 
 
Title time-course of RNA-sequencing in human macrophages 
Description RNA-seq from human macrophages stimulated with LPS (30min, 3h, 8h and 16h) or left unstimulated (basal) 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact N/A 
 
Description Collaboration with Federico Torta 
Organisation National University of Singapore
Department Department of Biochemistry
Country Singapore 
Sector Academic/University 
PI Contribution Analyse LPS-mediated transcriptome in human macrophages
Collaborator Contribution analyse LPS-mediated lipidome in human macrophages
Impact 1. LPS-mediated transcriptome in healthy human macrophages; 2. LPS-mediated lipidome in healthy human macrophages; 3. Integrative analyses; 4. Publication writing. Multidisciplinary (Immunity and lipid biology)
Start Year 2016
 
Description Collaboration with Jesus Gil 
Organisation Imperial College London
Department MRC London Institute of Medical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have shared our wound healing model in vivo with Jesus Gil's group at the MRC LMS for senescence-related projects
Collaborator Contribution Sharing material, intellectual contribution and grant application (Life ARC)
Impact Grant application to Development Gap Fund (LifeArc, MRC). The collaboration is multi-disciplinary.
Start Year 2017
 
Description Gene therapy in wound healing 
Organisation Imperial College London
Department Department of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution This collaboration with Ben Almquist (Bioengineering) brought together the Systems Genetics knowledge (this grant) and the in vivo application by modulating wound healing in live animals using progressive delivery of siRNA (Ben Almquist). Our contribution is to identify gene networks for in vivo intervention.
Collaborator Contribution Our collborator's contribution is to design special bandages containing siRNA for the gene (or set of genes) identified by our systems-genetics approaches. These bandages are applied to rodent skin wounds and the kinetics of heliang is monitored. This is a collboration that brings in vivo confirmation with the latest bioengineering advances (the bandages have a chemistry to release the siRNA progressivly to the cells involved in the healing process)
Impact This collboration is multi-disciplinary (Genetics, Genomics, Systems Genetics, Bioengineering). 1. We have succesful gene knock-down in vivo using bandages containing siRNA belonging to a specific gene network. These preliminary results form the basis of a set of new experiments where the effect size of each relevant gene is measured in vivo. 2. This collboration is crucial for the in vivo relevance of systems-genetics approaches
Start Year 2016
 
Description Skin/Scar free Healing Group 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution This is an Imperial College Initiative by Prof Anthony Bull, Head of the Department of Bioengineering (Imperial College London). There is a plan to expand the research portfolio focusing on the skin/scar free healing in order to (i) securing funding, (ii) create an internal focus group with a website. This particular expertise is represented by many groups at Imperial and our contribution is our knowledge on macrophage mediated aspects of fibrosis and scarring.
Collaborator Contribution See above. This is a multi-group collaboration focusing on Skin/Scar free Healing.
Impact • The value in forming this group is to explore various avenues for skin/scar free healing research and to provide a framework for further discussions with a view to applying for translational research grants. • Imperial College London is uniquely placed to deliver a translational programme in skin and scar free healing research and in the course of the coming months can build an exceptional portfolio that would strategically position ourselves to apply and be successful in grant application
Start Year 2016
 
Description The genetic determinants of scleroderma 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution The MRC grant project is on systems genetics approaches in macrophages to understand a very basic phenomenon : wound healing in the skin of genetically outbred mice. We have adopted a translational approach in humans by focusing on a very fibrotic complex disorder known as scleroderma (or systemic sclerosis). In collaboration with David Abraham and Chris Dento in UCL, we have collected 50 scleroderma patient monocyte-derived macrophages for RNA-seq analysis (Hiseq 2500, Illumina).
Collaborator Contribution Our collaborators in UCL allowed the Research Technician employed on the project to attend scleroderma clinics and collect the blood samples. Regular meetings with UCL collaborators are held in order to discuss the most recent results. The UCL team (Abraham, Denton) provided insights into the pathology of scleroderma and all results are now being integrated within a general systems-genetics approach
Impact 1. RNA-seq data from 50 scleroderma patient monocyte-derived macrophages 2. Clinical data from 50 scleroderma patients This collaboration is multi-disciplinary as it brings together systems-genetics (Behmoaras, Imperial College London) and clinical data (UCL, Abraham, Denton)
Start Year 2014
 
Title ABBA (A Bayesian Approach for Analysis of Whole-Genome Bisulfite Sequencing Data) 
Description Allows the detection of differentially methylated regions (DMRs) from whole-genome bisulfite sequencing (WGBS). ABBA is implemented as a Perl/R program, which is available with instructions for download at http://abba.systems-genetics.net/ or via http://www.mrc-bsu.cam.ac.uk/software/bioinformatics-and-statistical-genomics/. 
Type Of Technology Webtool/Application 
Year Produced 2017 
Impact Data sharing with the larger academic beneficiaries; a publication in the journal Genetics (PMID:28213474) 
URL http://abba.systems-genetics.net/
 
Description Fibrosis workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact Fibrosis workshop destined to centralize research on different aspects of fibrosis
Year(s) Of Engagement Activity 2018
 
Description Imperial College Press Release 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact An Imperial College Press Release presented the results published in Nature Communications. http://www.imperial.ac.uk/news/180507/ This resulted in public awareness (some online comments allowed the PI to discuss and communicate the findings with a lay audience).
Year(s) Of Engagement Activity 2017
URL http://www.imperial.ac.uk/news/180507/
 
Description Scleroderma paper press release at Duke_NUS, Singapore 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact This is a press release on the latest research published in Annals of the Rheumatic Diseases. The publication had an Altmetric Score of 71 and reached 8 news outlets.
Year(s) Of Engagement Activity 2017
URL https://www.duke-nus.edu.sg/news/genomics-reveals-key-macrophages%E2%80%99-involvement-systemic-scle...