Dynamics of human monocytes and airway macrophages during healthy aging and after transplant

Abstract

The ontogeny of airway macrophages (AMs) in human lung and their contribution to disease are poorly mapped out. In mice, aging is associated with an increasing proportion of peripherally, as opposed to perinatally derived AMs. We sought to understand AM ontogeny in human lung during healthy aging and after transplant. We characterized monocyte/macrophage populations from the peripheral blood and airways of healthy volunteers across infancy/childhood (2-12 yr), maturity (20-50 yr), and older adulthood (>50 yr). Single-cell RNA sequencing (scRNA-seq) was performed on airway inflammatory cells isolated from sex-mismatched lung transplant recipients. During healthy aging, the proportions of blood bronchoalveolar lavage (BAL) classical monocytes peak in adulthood and decline in older adults. scRNA-seq of BAL cells from lung transplant recipients indicates that after transplant, the majority of AMs are recipient derived. These data show that during aging, the peripheral monocyte phenotype is consistent with that found in the airways and, furthermore, that the majority of human AMs after transplant are derived from circulating monocytes.

Figure 1.

CM populations in the periphery are similar to those found in the airway. (A) Gating strategy for polychromatic flow cytometry analysis of blood monocyte subsets. After exclusion of debris and selecting live, CD45⁺ cells, peripheral blood mononuclear phagocytes were identified as Lin⁻ (CD3, CD4, CD8, CD19, CD20, CD34, and FCεRI), HLA-DR⁺cells which were either CD14⁺CD16⁻ (classical monocytes), CD14⁺CD16⁺ (intermediate monocytes), or CD14⁻CD16⁺ (non-classical monocytes). (B) Proportions of classical circulating monocytes in children (n = 17), younger adults (n = 7), and older adults (n = 8). (C) Gating strategy for flow cytometry analysis of BAL monocytes. (D) Total proportions of CMs in the BAL of children (n = 5), younger adults (n = 5), and older adults (n = 9).(E) Proportions of AMs in children (n = 6), younger adults (n = 5), and older adults (n = 8). Values shown are mean ± SEM. *, P < 0.05; **, P < 0.01; Mann–Whitney U test.

Figure S1.

Gating strategy for polychromatic flow cytometry analysis of peripheral blood CMs in children. NK, natural killer; SSC, side scatter.

Figure 2.

Comparable patterns of surface protein expression on monocytes found in the airways and periphery. (A) Back-gating of monocyte phenotypic markers in peripheral blood of healthy controls; blue indicates high expression and red low expression. RNAseq, RNA sequencing. (B) Proportions of circulating CMs expressing CD11b in younger adults (n = 7) and older adults (n = 8), CD11c in younger adults (n = 6) and older adults (n = 9), and CD163 in younger adults (n = 7) and older adults (n = 8). (C) Proportions of CMs in the BAL of healthy volunteers expressing CD11b, CD11c, and CD163 in younger adults (n = 5) and older adults (n = 9). Values shown are mean ± SEM. *, P < 0.05; **, P < 0.01, Mann–Whitney U test.

Figure S2.

Flow cytometry analysis of BAL CMs in children. (A) Gating strategy for polychromatic flow cytometry analysis of BAL CMs in children. (B) Fluorescence minus one controls for CD11b, CD11c, or CD163 staining. NK, natural killer; SSC, side scatter.

Figure 3.

AMs in the adult human lung after transplant are peripherally derived. (A) Schematic for sex-mismatched transplant model. Representative t-SNE plots showing gene expression levels in single cells derived from sex-mismatched lung transplant patients. (B) Male donor to female recipient expressing RPS4Y1. (C) Male donor to female recipient expressing XIST. (D) Female donor to male recipient expressing RPS4Y1. (E) Female donor to male recipient expressing XIST. (F and G) Female donor to male recipient expressing CD68 (F) and macrophage receptor with collagenous structure (MARCO; G). (H) Quantification of donor- or recipient-derived AMs in lung transplant patients (n = 4). Values shown are mean ± SEM. **, P < 0.01, Mann–Whitney U test.