A systems-genetics approach to dissect inflammation in fibrosis

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.

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.

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.