Characterisation of macrophage iron metabolism in interstitial lung disease
Item statusRestricted Access
Embargo end date20/06/2023
Boz, Cecilia Isotta
INTRODUCTION: Interstitial lung disease (ILD) is used to describe a heterogeneous group of disorders, characterised by inflammation and fibrosis of the pulmonary interstitium. Previous work has shown alveolar macrophage (AM) CD71 expression, which is also known as the transferrin receptor 1 (Tfr1), was able to stratify lung fibrosis patients into progressors (CD71high) and nonprogressors (CD71low), implying a role for iron metabolism in fibrotic ILDs. This led to the initial hypothesis that the labile iron in AM is associated with lung fibrosis and progression of disease. The overall thesis aim was to investigate iron metabolism and ferroptosis pathways in relevant cell lines and bronchoalveolar lavage cells from patients with lung fibrosis. METHODS: Alveolar macrophages derived from bronchoalveolar lavages were assayed for iron through Prussian blue staining (Golde Score), ICP-MS and two fluorescent probes. The phenotype of alveolar macrophages was characterised through qPCR and flow cytometry. Clinical samples were derived from patients diagnosed with IPF, unclassifiable ILD (UNC) with or without fibrosis and unexplained cough with or without haemoptysis. Multiple regression analysis was performed to assess association of Golde score and other clinical variable on fibrosis and progression of disease in ILD. The BAL cell transcriptome, through RNAseq, was compared in patients with progressive versus non-progressive fibrotic ILD. Ferroptosis was assessed through RSL3 and ferrous ammonium sulphate (FAS) treatment in macrophages (RAW 264.7, THP1 and AM) and epithelial cells (HT29). RESULTS: Iron accumulation in AM was significantly increased in patients with fibrosis (IPF and UNC with fibrosis) compared to those without fibrosis. Higher levels of iron were shown to be associated with fibrosis and IPF diagnosis. Patients with IPF had a comparatively higher level of the genes encoding the iron export protein ferroportin, and the iron transporter DMT1, compared to UNC with fibrosis patients, perhaps reflecting a cellular response to high iron content. Higher Golde score was not associated with progression of ILD (defined as >10% decline in FVC over 12 months) and indeed high Golde score was associated with slightly lower risk of progression. RNAseq was performed on a cohort of progressors (N=4) and non-progressors (N=6). No differentially expressed genes (DEGs) were identified having a false-discovery rate (FDR)- adjusted P-value of <0.1. When a more relaxed threshold of unadjusted P<0.05 was considered, 215 genes were upregulated and 400 downregulated in progressors compared to non-progressors. None of the genes encoding the iron and macrophage polarisation proteins previously discussed were statistically different. Due to the increased iron level of the alveolar macrophages of fibrotic ILD patients, their ability to undergo ferroptosis, an iron regulated form of cell death, was tested. The susceptibility of macrophages to ferroptosis was assessed using a murine macrophage cell line (RAW), and a human monocytic cell line differentiated to macrophages (THP-1), and compared to HT29, a human colon cancer cell line known to undergo ferroptosis. Macrophages were less sensitive to drugs that induce and rescue ferroptosis compared to HT29 cells. RAW and THP1 cells were however more sensitive to iron loading as shown by increased reactive oxygen species (ROS) compared to HT29. Limited study on alveolar macrophages, suggested similar results to THP1 when using pharmacological induction of ferroptosis. Iron-loading significantly reduced viability in all cell lines, but not proportionate to the ROS increase suggesting macrophages are relatively resistant to iron loading and ferroptosis. CONCLUSIONS: In this single centre study, Golde score was independently associated with the presence of fibrosis in ILD. At the gene expression level, the phenotype of these cells was not significantly different between IPF, UNC with fibrosis and UNC without fibrosis, but some differences were shown between the UNC with fibrosis and IPF groups when looking at the macrophage inflammatory phenotype. Golde score was not associated with disease progression and indeed higher Golde score was associated with a slightly lower risk of progression. BAL cell transcriptome of patients with progressive versus nonprogressive fibrotic ILD was not significantly different but the sample size was small. Macrophages (RAW, THP1 and alveolar macrophages) were relatively insensitive to pharmacologically induced ferroptosis compared to an epithelial cell line (HT29). In contrast to HT29 cells, RSL3 induced significant apoptosis as well as ferroptosis in human macrophages. Macrophages produced a higher level of ROS upon iron-loading, but their viability was relatively maintained compared to the HT29 cell line. These findings suggest that macrophages were relatively insensitive to iron loading and was consistent with the observation that alveolar macrophages remain viable even if they have high iron levels. Future studies could elucidate whether iron loading in macrophages leads to a protective or injurious phenotype, thus leading to potential treatments.