Dissecting the molecular pathogenesis of Legionella spp. in human lung models

Project Information

OPTIMISE

Grant agreement ID: 844197

DOI

10.3030/844197

Project closed

EC signature date 10 April 2019

Start date 1 August 2019

End date 31 July 2021

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 212 933,76

EU contribution

€ 212 933,76

Coordinated by

THE QUEEN'S UNIVERSITY OF BELFAST
United Kingdom

Periodic Reporting for period 1 - OPTIMISE (Dissecting the molecular pathogenesis of Legionella spp. in human lung models)

Reporting period: 2019-08-01 to 2021-07-31

Summary of the context and overall objectives of the project

The Marie Skłodowska Curie Action (MSCA) OPTIMISE “Dissecting the molecular pathogenesis of Legionella spp. in human lung models” aimed to establish human Precision-Cut Lung Slices (hPCLS) as physiologically relevant model to study infection by the human bacterial pathogen Legionella pneumophila and related species.
Legionella spp. are environmental bacteria that can infect susceptible individuals and cause a lethal pneumonia known as Legionnaires’ Disease. Aging populations and the increasing use of immune modulatory therapies have given rise to a growing incidence of opportunistic infections caused by pathogens such as Legionella spp..
As for many other infectious diseases, most of our knowledge about the mechanisms driving the outcome of the Legionella-host interaction, originates from studies in cellular and surrogate animal models, which often fail to recapitulate key aspects of human disease. The mechanisms underlying the susceptibility to disease and the virulence of different Legionella isolates in the human lung remain largely elusive. However, as also strikingly highlighted by the current COVID-19 pandemic, understanding disease mechanisms and rapid translation from mechanistic research into new therapies, requires information about the processes occurring in human tissue.
The MSCA OPTIMISE aimed to address this gap in knowledge by 1) optimizing the generation of hPCLS, small uniform slices of human lung tissue from donated human lungs, 2) characterizing their viability, architecture and cellular composition and 3) measuring tissue invasion, growth and pathology induced by different Legionella isolates. Hundreds of these slices can be generated from one human lung, allowing comparison of many bacteria or treatments at the same time in the laboratory. The specific objectives of OPTIMISE were to reveal more about the mechanisms driving 1) host-susceptibility to Legionnaire’s Disease and 2) the virulence of different strains of these environmental bacteria by analyzing the response of the whole tissue, but also of individual lung cells to infection with Legionella.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Work for this action was distributed into 5 work packages (WPs).
Work package 1 focused on the establishment of hPCLS for Legionella infection and on its characterization. This work package has revealed that Legionella infects and replicates in the hPCLS. Moreover, as previously observed in other models, infection and proliferation in human lung tissue is governed by the activity of a secretion system used by the pathogen to inject a broad range of effector proteins into host cells that allow to manipulate the physiology of these cells. We also completed a literature review on the use of human and other animal PCLS to study respiratory infectious diseases, which was in the final stage of the production and publication process in the journal Molecular Microbiology, when this report was submitted.
WP2 focused on analyzing the response to Legionella infection of each cell present in the hPCLS. Using 10x Genomics single cell transcriptomics and mapping of the results to gene expression fingerprints of different cell types we identified 43 cell types in hPCLS and determined that infection induces a strong inflammatory response in many most cells in the tissue. These data are currently prepared for publication.
WP3 aimed at taking this infection model to the next level by modelling infection of human lungs with Legionella through ex vivo lung perfusion (EVLP). However, due to COVID-19 pandemic the human lung tissue supply ceased, preventing us from working on this WP. We then focused on setting up new cell-based assays to complement the data obtained in WP2.
WP4 and 5 focused on complementary researcher training and dissemination and communication of the results. The fellow attended a different range of training activities and participated in different events that allowed her to develop and expand on her skills as e.g. mentoring training and writing workshops. The fellow has also acquired the qualification of Associated Fellow of the Higher Education Teaching Academy, served as reviewer for different journals and as judge for research events, and as co-supervised 5 early career scientists during this MSCA. Even though the COVID-19 pandemic has strongly impacted the OPTIMISE project, the results obtained in this MSCA have been presented at 5 national and international events, reaching a diverse audience of scientists and clinicians. Additionally, the fellow has also communicated the project to the general public at the Northern Ireland Science Festival outreach event.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The action OPTIMISE established hPCLS as an effective platform to study Legionella infection, allowing to dissect differences in virulence of clinical isolates. Moreover, the action also delivered one of, to date, first single cell transcriptomics analyses of hPCLS, demonstrating the presence of an extraordinary cellular complexity in the slices and the capacity to dissect responses of individual cells to infection. Combined, this illustrates the potential of hPCLS to serve as a physiological model for human respiratory disease, promising to not only overcome limitations of surrogate animal models and provide more accurate, exploitable data, but also contribute to the reduction animal use for research.
OPTIMISE provides proof-of-concept and this methodology can now be adopted for different pathogens, in order to shed light on key cellular and molecular events that occur during the warfare between host and pathogen. In addition, scalability of the hCPLS model for screening many conditions places it also a powerful tool for the discovery of new antimicrobials in the future.

Filling human lung tissue with low-melting agarose.

hPCLS (green) infected with Legionella (red)

hPCLS in culture in a 24 well plate after slicing