Healthy repair of the alveoli requires alveolar stem cells to differentiate into cells designed for gas exchange. In chronic lung fibrotic disease like idiopathic pulmonary fibrosis (IPF), alveolar epithelial cells regenerate abnormally. The cause of this is unknown but its highly cellular, inflamed and structurally altered regenerating niche is likely to be relevant. Here, in unique sets of human lung tissues capturing advancing fibrosis, and with a 33-plex single cell imaging mass cytometry (IMC), we provide a high resolution and comprehensive temporo-spatial cell atlas of the regenerating alveolar niches. Using a suite of mathematical tools, we expose an organized immune network and identify CD206hi alveolar macrophages as a central immune cell in the immune-alveolar epithelial interactome. A spatially-directed receptor-ligand analysis offers an in-silico mechanism by which these macrophages influenced alveolar regeneration. Our study unravels a complex cellular environment and identifies key interactions that influence alveolar regeneration in a fibrotic lung.

Andrew Fisher declares the following: Grant Award to Newcastle University from Mallinckrodt Pharmaceuticals, Chiesi and Eurofins/TGI Consultancy via Newcastle University with Fibrofind, Mallinckrodt and Sanofi James Shaw declares the following: Scientific Advisory Board for Mogrify (consultancy fee paid to Newcastle University).

The study is funded by combination of grants from MRC UKRMP Grant MR/S020918/1; NIHR Oxford Biomedical Research Centre grant; University of Oxford Medical Science Division and Newcastle University Flow Core Facility. LPH is supported by MRC (grant CFR01480) and the NIHR Oxford Biomedical Research Centre. AJF is supported by the National Institute for Health and Care Research (NIHR) Blood and Transplant Research Unit in Organ Donation and Transplantation (NIHR203332), a partnership between NHS Blood and Transplant, University of Cambridge and Newcastle University. The views expressed are those of the author(s) and not necessarily those of the NIHR, NHS Blood and Transplant or the Department of Health and Social Care.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

All patients provided informed consent for use of their tissue via the Cellular and Molecular Mechanisms in Chronic Lung Diseases (EXPLANT) study which was approved by the NHS Research Ethics Service (11/NE/0291) and was sponsored by Newcastle Upon Tyne Hospitals NHS Foundation Trust ( R&D ref 5885).

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

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I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

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Data availability The analysis results are presented as a dynamic online resource in Multi-Dimensional Viewer (MDV) (https://mdv.molbiol.ox.ac.uk/projects/mdv_project/7430). All source data for the figures are found within the project 7430 in the MDV link. Code availability The complete code for the Spatial Omics Oxford (SpOOx) pipeline and the SpOOx v2.0 (as an update) is available as a GitHub repository under the GPL license: https://github.com/Taylor-CCB-Group/SpOOx. The Multi-Dimensional Viewer code is available under the GPL license: https://github.com/Taylor-CCB-Group/MDV.