Extracellular vesicles and their cargo in the pathogenesis of acute respiratory distress syndrome and pulmonary fibrosis sequelae

When some people have a severe infection, their body's defence systems can over-react and cause damage to their own organs through a process called inflammation. When the lungs are damaged in this way, it is called acute respiratory distress syndrome (ARDS). This can occur due to a variety of insults, the most common being bacterial and viral infections. Recently, the COVID-19 pandemic has been a major cause of ARDS. ARDS causes the lungs to fill up with water, making it very difficult to breathe. These patients therefore need to be looked after in the intensive care unit, where a machine can help support their breathing. The death rate associated with this is 40%. Even those who survive ARDS have considerable recuperation periods and reduced quality of life. 1 in 20 patients who survive develop scarring of lungs (fibrosis).

Cell within the body have the ability to form outpouchings which can break off to form extracellular vesicles (EVs). These EVs can carry various proteins and genetic material between different cell types. If another cell absorbs an EV, the activity and function of that cell may change. EVs are being released and absorbed by cells in our body constantly, when we are healthy and when we are sick. Some EVs are beneficial and can help support the healthy functioning of a cell. However, other EVs can carry damaging contents to cells and in doing so promote organ damage and disease. Studies in mice have shown that damaging EVs are involved in the development of inflammation in ARDS.

My previous work showed that defender cells (alveolar macrophages) in the lungs of patients with ARDS do not work as well as in healthy people. Their ability to clear away dead cells is reduced, and this can lead to increased inflammation in the lungs as a result, thereby contributing to the development of ARDS. The weaker the abilities of the defender cells, the more likely it was that patients with ARDS will remain on a ventilator for longer or die. Further work identified that uptake of EVs by lung defender cells leads to this weaker activity. Mouse studies have shown that during lung injury, EVs can transfer genetic material to defender cells, leading to increased inflammation and potentially an increased risk of lung scarring. My theory is that in ARDS, uptake of EVs and their cargo of genetic material by defender cells leads to weakening of the defender cell function, which contributes to the inflammation seen in ARDS, and may increase the risk of developing lung scarring.

To investigate, I will first aim to determine the size, number and the originating cell of EVs in the airways of patients with ARDS and lung scarring. I will also aim to determine the protein content of these EVs to see if certain inflammatory proteins are increased in ARDS patients. I will then separate the EVs based on their cell of origin, and use these to treat healthy defender cells to determine which type of EV causes the defect in defender cell activity. Once I identify the relevant damaging type of EVs, I will investigate their genetic content, to determine which fragments of genetic material (microRNAs) are more highly expressed in this type of EV. I will then selectively block these fragments of genetic material using mirror-image fragments (antagomirs) before treating defender cells with EVs; if the defender cell function is stronger as a result then transfer of these microRNAs are likely to be the underlying cause of the defender cell defect. I will then aim to block these microRNAs by using antagomirs in models of ARDS in mice and in human lungs donated by deceased patients. If this intervention reduces inflammation in these models, it will support the theory that transfer of microRNAs by EVs causes the defect in defender cell function observed in ARDS. It will also support the theory that blocking these microRNAs is a strategy which could be applied as a treatment for patients with ARDS and post-ARDS lung scarring.

I hypothesise that in Acute Respiratory Distress Syndrome (ARDS) patients, transfer of monocyte-derived extracellular vesicle (EV) cargo to alveolar macrophages (AMs) causes a defect in oxidative metabolism, which impairs efferocytosis and drives alveolar inflammation. Fibrocyte-derived EV transfer to AMs and fibroblasts induces a pro-fibrotic response in a subset of patients.

Objectives:

1) Characterise pulmonary EVs by size, number, cell of origin and proteome in sepsis-related ARDS, post-ARDS pulmonary fibrosis, and control sepsis patients. EV characterisation will be performed using the ExoView platform, proteomics will be performed by liquid chromatography tandem mass spectrometry. EV phenotype will be correlated with clinical outcomes to identify potential biomarkers.

2) Determine the biological effect of pulmonary EVs from ARDS and post-ARDS pulmonary fibrosis patients on primary human AM and fibroblast function, phenotype and metabolic profile. AMs and fibroblasts will be isolated from the lung tissue of never smoking patients undergoing thoracic surgery. Flow cytometry will be used for efferocytosis, phagocytosis and phenotyping assays. Metabolic profiling will be undertaken using the Agilent Seahorse platform.

3) Determine whether the biological effect of ARDS EVs on AMs is mediated by microRNA transfer. MicroRNA sequencing of EV subpopulations will be performed to identify microRNAs enriched in ARDS patients. In vitro antagomir treatment against these enriched microRNAs will determine whether AM function can be rescued, thus identifying potential therapeutic targets.

4) Determine whether antagomir treatment can attenuate inflammation in a murine model of pneumococcal lung injury.

5) Determine whether antagomir treatment can attenuate inflammation in an EV-injured human ex-vivo lung model of ARDS. I will work with Prof Matthay's group at UCSF and injure ex vivo human lungs with EVs isolated from ARDS patients.