Epigenetic alterations underlie airway macrophage differentiation and phenotype during lung fibrosis

Airway macrophages (AMs) are key regulators of the lung environment and are implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a fatal respiratory disease with no cure. However, the epigenetics of AMs development and function in IPF are limited. Here, we characterised the DNA-methylation (DNAm) profile of AMs from IPF (n=30) and healthy (n=14) donors. Our analysis revealed epigenetic heterogeneity was a key characteristic of IPF AMs. DNAm ‘clock’ analysis indicated epigenetic alterations in IPF-AMs was not associated with accelerated ageing. In differential DNAm analysis, we identified numerous differentially methylated positions (DMPs, n=11) and regions (DMRs, n=49) between healthy and IPF AMs respectively. DMPs and DMRs encompassed genes involved in lipid ( LPCAT1 ) and glucose ( PFKB3 ) metabolism and importantly, DNAm status was associated with disease severity in IPF. Collectively, our data identify that profound changes in the epigenome underpin the development and function of AMs in the IPF lung. ### Competing Interest Statement A.J.B. received, unrelated to this work, consultancy fees from Ammax, Devpro, and Ionis pharmaceuticals, via his institution. Unrelated to the current work, T.M.M. has, via his institution, received industry- academic funding from GlaxoSmithKline R&D and UCB and has received consultancy or speakers fees from Apellis, Astra Zeneca, Bayer, Blade Therapeutics, Boehringer Ingelheim, Bristol Myers Squibb, Galapagos, GlaxoSmithKline R&D, Indalo, Novartis, Pliant, ProMetic, Respivnat, Roche, Samumed, and UCB. P.L.M. received industry-academic funding from AstraZeneca and has received speaker and consultancy fees from Boehringer Ingelheim and Hoffman La Roche outside the submitted work via his institution. * Abbreviations : AMs : Airway macrophages CpG : Cytosine-guanine dinucleotides FVC : Forced vital capacity HC : Healthy control DHS : DNase-I hypersensitivity sites DNAm : DNA methylation DMPs : Differentially methylated positions DMRs : Differentially methylated regions IPF : Idiopathic pulmonary fibrosis Myld-CpGs : Myeloid marker CpG dinucleotides pcHiC : Promoter capture HiC scRNA-Seq : Single-cell RNA sequencing WGBS : Whole genome bisulphite sequencing