Mechanistic basis of antifungal potency of the airway epithelium

Human beings inhale many thousands of toxic or infectious particles daily, which represent a continuous risk to respiratory health. To remain healthy, our lungs must eliminate these noxious particles and maintain a sterile environment. Airborne spores of the most prominent fungal pathogen of human lungs, Aspergillus fumigatus, are a major component of the air we breathe and initiate more than 3 million chronic and 200,000 invasive diseases annually, worldwide. In European alone aspergillus-related diseases are likely to exceed 2 million in number per year. Some groups of severely immunocompromised patients, such those undergoing bone marrow transplants have just a 10% survival rate once a fungal infection is contracted. Remarkably, while fungal diseases cause more deaths annually than tuberculosis or malaria, we still lack effective drugs to treat them.

I have previously found that the lung epithelium can grab fungal spores, swallow them up, and kill them and that this process is altered in lung cells from patients having a higher risk of fungal lung disease, such as patients with chronic obstructive pulmonary disease (COPD). Using state-of-the-art technologies to study the interaction of genetically-engineered fluorescent fungal mutant strains and mutant lungs cells, I aim to determine how healthy epithelial cells of the human lung recognise fungal spores and kill them, how this process might influence communication between immune cells in the lung environment, and how this process is altered in cells from patients that have a higher risk of fungal disease.

A detailed understanding of how epithelial cells contribute to clear inhaled A. fumigatus and maintain a healthy lung environment will enable us to design new antifungal therapies, and potentially lead to better ways of preventing dangerous responses to other airborne pathogens and pollutants causing lung diseases.

Technical Summary
I have demonstrated that phagocytic activities of the respiratory epithelium play a crucial role in host defence by killing ingested A. fumigatus spores, and that this defence is radically altered in human airway epithelial cells (AECs) from COPD patients. I thus hypothesise that AECs provide a critical antimicrobial defence against everyday spore exposure, and that aberrant spore uptake and killing promote Aspergillus-related lung disease.

By exploiting my single-cell platforms to perform molecular and cellular studies of A. fumigatus-AEC interactions in vitro, in vivo and in primary AECs, this work aims to define, for the first time, the mechanistic basis of effective and dysfunctional A. fumigatus clearance by AECs. In particular, it aims to:

1)Identify the fungal cell wall components driving effective A. fumigatus clearance by AECs. This will be achieved measuring A. fumigatus uptake and intracellular killing during morphotype-specific in vitro challenges of AECs in the presence of selective fungal cell wall inhibitors and during in vitro and in vivo challenges with an extant panel of A. fumigatus cell wall mutants.

2)Define the epithelial components directing effective A. fumigatus clearance by AECs. This will be achieved analysing A. fumigatus uptake and intracellular killing in AECs, subjected to targeted and global CRISPR/Cas9 genome editing of plasma membrane epithelial proteins.

3) Characterise the molecular basis of dysfunctional AEC activities in COPD patients. This will be achieved testing the role of the identified fungal and epithelial components in comparison of commercially-acquired primary human AECs from donors with and without COPD.

Understanding how the lung coordinates mucosal homeostasis and maintenance of airway sterility is of major clinical importance and will aid the identification of immunomodulators to facilitate treatment and limit respiratory damage following exposure to this and other respiratory pathogens.