Single-Cell RNA Sequencing Identifies Unique Pulmonary Macrophage Subsets Associated with Chronic Lung Allograft Dysfunction

Abstract

<h3>Purpose</h3> Lung transplant recipients have the lowest survival rate compared to all other solid organ transplants, the primary cause being chronic lung allograft dysfunction (CLAD) which manifests as two main clinical phenotypes: bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). Pulmonary macrophages (MΦ) are a significant population of immune cells which serve homeostatic and immunoregulatory roles but may also perform proinflammatory and profibrotic functions when activated. MΦ contribution to CLAD is not clear. We used single-cell RNA sequencing (scRNAseq) to profile MΦ in BOS and RAS explanted lungs and to determine their roles in CLAD. <h3>Methods</h3> 6 fresh lung samples (4 BOS, 2 RAS) from re-transplant cases were collected, processed, subjected to 10X Genomics chemistry for scRNAseq, and compared to 5 donor lung samples. R and Seurat package were used for quality control, dimensionality reduction, and annotation. Slingshot package was utilised for pseudotime trajectory analysis. <h3>Results</h3> Lung tissue showed high MΦ heterogeneity, with at least 20 unique clusters (Fig 1A). CLAD lungs had a higher presence of FCN1⁺ MΦ with high expression of AIF1 activation marker: these were annotated as activated proinflammatory AIF1⁺ MΦ (Fig 1B). In RAS samples, there was a reduction of alveolar FABP4⁺ MΦ (Fig 1C) and an enrichment of MΦ expressing fibrosis-associated genes (SPP1, MERTK, MMP9) that were annotated as pro-fibrotic SPP1⁺ MΦ (Fig 1D). Pseudotime trajectory analysis shows both AIF1⁺ and SPP1⁺ Φ were at an earlier stage of development compared to other clusters. <h3>Conclusion</h3> Using scRNAseq, we have identified novel clusters of MΦ in CLAD lungs. Proinflammatory AIF1⁺ MΦ were found in all CLAD lungs, while a reduction in alveolar FABP4⁺ MΦ and enriched pro-fibrotic SPP1⁺ MΦ may contribute to the parenchymal lung fibrosis found in RAS. <i>In vitro</i> assays on sorted MΦ are under way to further characterise functions of these MΦ subsets and to explore their role in CLAD.