Pulmonary Macrophage Subsets Associated with Lung Allograft Dysfunction Revealed by Single-Cell RNA Sequencing

Purpose Lung transplant (LT) recipients experience a low survival rate compared with other solid organ transplant recipients mainly due to chronic lung allograft dysfunction (CLAD). Acute lung allograft dysfunction (ALAD) episodes is a risk factor of subsequent CLAD. The contribution of lung macrophages (Macs) to ALAD and CLAD is not clear. We applied single-cell RNA sequencing (scRNAseq) to profile cells during quiescence, ALAD, and CLAD, in order to determine the role of Macs in dysfunctional lung allografts. Methods Fresh bronchoalveolar lavage (BAL) cells from 6 LT patients, 3 with stable lung function (SLF) and 3 undergoing an episode of ALAD, as well as cells from 3 explanted CLAD lungs were used for scRNAseq. R Bioconductor and Seurat were used to perform QC, dimensionality reduction, annotation, and trajectory. Donor and recipient deconvolution was performed using single nucleotide variations. Results Our data revealed that Macs are highly heterogeneous (∼12 transcriptionally distinct subsets from all 3 SLF). We identified four Mac subsets more prominent in ALAD BAL compared to stable (Fig 1A). Of the four, three populations were uniquely present in the CLAD lung samples compared to a control donor lung sample (Fig 1B). Based on pathway analysis and the top differentially expressed genes in BAL (Fig 1C) and CLAD lung (Fig 1D), we annotated them as proinflammatory (CXCL10+), Ig-regulated (FcγRIIb+), and metallothioneins-mediated inflammatory (MT) Macs. Pseudotime analysis suggested that CXCL10+ and FcγRIIb+ Macs represent an earlier stage of differentiation than other Macs (Fig 1E). Deconvolution demonstrated that donor Macs are lost with time post-transplant (Fig 1F) and absent from CLAD lungs (Fig 1G). Conclusion Using scRNAseq, we observed Mac heterogeneity and identified specific subsets of Macs that may be associated with allograft dysfunction. Further exploration with scRNAseq will shed light on LT immunobiology and the role of Macs in allograft injury and dysfunction.