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Fibroproliferative Invasive Fibroblasts in Idiopathic Pulmonary Fibrosis

Periodic Reporting for period 1 - FIBROSIS (Fibroproliferative Invasive Fibroblasts in Idiopathic Pulmonary Fibrosis)

Reporting period: 2018-09-03 to 2020-09-02

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease that kills more people each year than most cancers. Scarring occurs in IPF patients' lungs as the disease progresses make it increasingly difficult to breath and carry out normal tasks. There are limited therapies for patients with IPF and none that can cure the disease fully. This makes IPF an important disease to research.

In a healthy lung, epithelial cells line the surfaces in contact with the air and a cell type called a fibroblast make the glue and creates the scaffolding, composed of extracellular matrix (ECM) proteins, for other cells. In IPF patients it is believed that the airway lining cells become injured. For an unknown reason the normal repair response, led by fibroblasts, becomes dysfunctional. Instead of a neat repair to the damaged area the fibroblasts become highly active, increase in number, and produce large amounts of ECM glue and scaffolding leading to the spread of thick and stiff scar tissue. This scarring makes it difficult for the lung to function normally and can ultimately be fatal. Lung scarring can occur in other lung diseases like Systemic Sclerosis, and most recently in some patients recovering from COVID-19 infection.

To understand why the normal repair process goes wrong and results in progressive scarring of the lung in diseases like IPF we must first understand the cells involved. The latest research suggests that many types of the key repair cell type, fibroblasts, exist in our lungs. Studying this fibroblasts and how they contribute to scarring will benefits not just patients with IPF but also patients with others conditions in which scarring occurs. This project tackled the question of whether specific fibroblast sub-types might be more involved in the abnormal repair process than others and examined the role of key, known, fibroblast sub-types.

The objectives of this project were:
(1) Study the characteristics, genetics and behavior of key fibroblast sub-types to identify ways in which they contribute to disease.
(2) Use the very latest technologies to definitively identify the different fibroblast sub-types in the lung.
(3) Study disease models of lung scarring to try and understand what fibroblast sub-type, if any, is the major driver of disease and lung tissue damage.
Objective 1: In experimental models some fibroblasts have the ability to move through scar tissue to sites of injury and appear to be a major producers of ECM glue and scaffolding that creates scar tissue in a diseased lung. These fibroblasts were called invasive fibroblasts and may be centrally involved in the scarring process. We used advanced technologies to investigate the genes, information carrying copies of specific pieces of DNA, expressed by these fibroblasts. We identified markers with which to definitively identify invasive fibroblasts in the mouse and human lung. This allowed us to collect these specific cells for further study. In experimental models, fibroblasts collected from animal models of disease using these new markers behaved like invasive fibroblasts; migrating through scar mimicking matrix. This suggests we had successfully found new ways to isolate this cell type. Fibroblasts isolated from IPF patient lung tissue using the novel markers were added to the lungs of an experimental mouse model. They caused much greater scarring than control fibroblasts that did not express these cell surface markers. Therapeutic interventions to block signalling pathways we had identified reduced the ability of these fibroblasts to migrate, invade, and create scar tissue.

We also investigated a fibroblast subtype known as a lipofibroblast, an important cell during lung development, that might help lung tissue regeneration after injury. We undertook a detailed study of this cell type and demonstrated that they support airway lining cells to grow and divide, a key process in lung repair. This cell type has been commonly identified in rodent lungs but not often in the human lung leading to confusion about whether this cell type really exists in humans. Using genetic sequencing of all lung cells, we definitively demonstrated that this cell type is present in the human lung but that markers reported most often reported in scientific papers are not good at identifying them. We found new markers that better identified this cell type in the lung at all developmental stages.

Objective 2 & 3: To identify the fibroblast subtypes in the lungs of healthy people and patients with IPF we used a technique called single cell RNA sequencing (scRNA-seq). This allows us to investigate the individual cells that make up the lung. With scRNA-seq we can study all of the genes in each lung cell and identify cells that express similar genes. By doing this we can identify known cell types using markers reported in scientific papers and also new cell types. We undertook scRNA-seq analysis of mouse lungs at different stages of development and identified up to eight different fibroblast sub-types as the developed. This included sub-types identified previously, like myofibroblasts, but also a new type we named Ebf1+ fibroblasts. We checked all markers commonly reported in scientific papers used to identify the different known fibroblast types. We found that in general these markers were not unique to a single sub-type or that often they were not expressed by a sub type at a high enough level to be easily identified. This means that researchers did not have effective tools with which to study these different populations. We identified new markers that researchers could use to isolate all of the different fibroblast sub-types in the future. We showed that each subtype becomes distinct during lung development and can be identified clearly at each developmental stage. We found that mice and humans had very similar types of fibroblasts and that they expressed similar makers and signalling molecules.
This is a first-of-its-kind study and the most comprehensive analysis of lung fibroblasts ever performed. We discovered new ways to identify and study fibroblasts in the lung and identified a new fibroblast sub-type. Our analysis of fibroblasts in the developing and adult mouse and human lung has helped us to solve many controversies that existed in the scientific literature. Our study shows that myofibroblasts, long believed to increase in number in the IPF lung and be the key scar producer, may not be as central to IPF as previously thought. In our analysis many fibroblast sub types likely contribute to scarring in the lung and due to this study we can now target each type individually and/or identify a therapeutic target common to all subtypes. This is an important change in our understanding of the scarring process in the lung. We have shown that a fibroblast sub type called a lipofibroblast does exist in the human lung and we have provided new ways to identify and study this cell type. We have shown that fibroblast sub type is “fixed” in early lung development and that these sub types don’t turn into other sub types in disease. In our ongoing work we will investigate the contribution of different cell types to disease and identify what communication pathways are central to each type. We will next investigate communication between different cell types in the healthy and diseased lung to try and understand who the key driver of disease is.