Reconstruction of Transcriptional Programs of Monocyte to Macrophage Sequential Cell Fate Transition Using Single Cell RNA Sequencing in Sex Mis-Matched Lung Transplantation

Abstract

Purpose Alveolar macrophages are key regulators of the innate immune response and participate in the stem cell niche to organise tissue repair and lung homeostasis . The fate of donor alveolar macrophages is poorly understood as existing studies have relied on sex chromosome labelling or HLA typing methodologies. We sought to confirm the existence of a long-lived pool of donor-derived alveolar macrophages using single cell RNA sequencing in sex mismatched lung transplant patients. Methods BAL cells from 17 individuals, including 11 non-transplant (7 female) and 6 lung transplant recipients (3 male->female, 1 female->male) were isolated and sorted for live cells. Single cells were captured and barcoded using the 10X Genomics Single Cell 3′ Gene Expression platform. Sequencing data was filtered and aligned to human reference genome and count tables analysed in Seurat and Monocle packages. Results Our analysis revealed 2 transcripts specific to female and male cells respectively; XIST (X-inactive specific transcript) and RPS4Y1 (Ribosomal Protein S4 Y-Linked 1) with 70% of cells able to be sex matched (Table1). Of 4 sex mismatched lung transplant patients, only 1 male 8.4 months post-transplant, had alveolar macrophages of donor origin (17.5%). Engrafting monocytes acquired a near identical phenotype to donor macrophages, with trajectory inference of recipient cells revealing arrested expression of genes associated with chemotaxis (CCL2) and tissue remodelling (TIMP1) and upregulation of scavenger receptor (MSR1) and lipid metabolism (FABP4 and SCD) genes (Figure 1). Conclusion Our work questions the existence of long-lived self-renewing populations of alveolar macrophages. Knowledge of the transcriptional programs associated with cell fate transition from peripheral blood derived monocyte to alveolar macrophage will aid in deciphering the pathogenesis of lung diseases involving monocyte/macrophage lineages.