Developing model systems to understand the impacts of pollutants on lung health

In parallel data and/or samples (nasal swabs, blood, lung tissue, bronchial alveolar lavage) from human and mouse tissue exposed to defined pollutants (e.g. wood smoke) and/or respiratory infection (e.g. cohorts from Manchester respiratory biobank with RSV, influenza) will be analysed by qPCR, proteomics, mass cytometry, bulk flow cytometry

Objective 1: Assessing In vitro models to investigate the impact of pollution. theaim of this to familiarise with methodologies used throughout the project as well as establish the basic in vitro models used in the project and to develop an understanding of how pollution impacts different cell types. Single cell type (i.e., epithelial cells, macrophages) then advanced cell models (air:liquid interface culture models of the respiratory epithelium with macrophages plus or minus fibroblasts) that have been developed in Manchester and also Immuone will be exposed to defined pollutants using a Vitrocell exposure system.

Aerosols will be generated from real-world emission sources (e.g., biomass burning, cooking, diesel engine), representative of the indoor and/or outdoor environment using previously established procedures at UoM. The generated aerosols will be characterised using a suite of aerosol science instrumentation (e.g., aerosol mass spectrometer, single-particle soot photometer, particle classifiers).

This will enable the linkage of the biological responses with the aerosol physicochemical properties. These air-pollution treated cells will then be challenged with influenza virus or RSV. Prior and after viral challenge, cell supernatants will be analysed for cytokines, matrikines and chemokines and cells will be assessed by qPCR, flow cytometry, colorimetric assays for markers of cell death, inflammation, Epithelial-Mesenchymal Transition and Fibroblast-Myofibroblast Transition, fibrosis, and toxicity

Objective 2: Exploring the in situ response to pollution: Lung slices from healthy mice will be exposed to defined pollutants using the Vitrocell exposure system and impacts analysed as described for Aim 1. Lung slice culture will be performed using published protocols and kept in culture for up to 14 days. In parallel. 'Omic data from in vivo exposed mice and/or human pollution exposures will be used to assess pathways triggered in exposure to defined pollutants to inform the validity of the work in Aim 1.

Objective 3 Assessing the response to pollution following infection challenge aim will explore how changes post-infection in lung architecture impact the response to pollution and vice versa Mice will be exposed to RSV or influenza and the timecourse of infection/pathology analysed to assess changes prior (0d), at the onset of infection (1d), during infection (d4) and post-infection (d8 and 14) using flow cytometry, qPCR and immunohistochemistry. Additionally tissue will be taken from infected mice and used for organ slice cultures which will then be exposed to pollution as described in objectives 1 and 2.