Distinct Transcriptional and Functional Differences of Lung Resident and Monocyte-Derived Alveolar Macrophages During the Recovery Period of Acute Lung Injury

Abstract

In acute lung injury, two subsets of lung macrophages exist in the alveoli: tissue-resident alveolar macrophages (AMs) and monocyte-derived alveolar macrophages (MDMs). However, it is unclear whether these 2 subsets of macrophages have different functions and characteristics during the recovery phase. RNA-sequencing of AMs and MDMs from the recovery period of LPS-induced lung injury mice revealed their differences in proliferation, cell death, phagocytosis, inflammation and tissue repair. Using flow cytometry, we found that AMs showed a higher ability to proliferate, whereas MDMs expressed a larger amount of cell death. We also compared the ability of phagocytosing apoptotic cells and activating adaptive immunity and found that AMs have a stronger ability to phagocytose, while MDMs are the cells that activate lymphocytes during the resolving phase. By testing surface markers, we found that MDMs were more prone to the M1 phenotype, but expressed a higher level of pro-repairing genes. Finally, analysis of a publicly available set of single-cell RNA-sequencing data on bronchoalveolar lavage cells from patients with SARS-CoV-2 infection validated the double-sided role of MDMs. Blockade of inflammatory MDM recruitment using CCR2^-/- mice effectively attenuates lung injury. Therefore, AMs and MDMs exhibited large differences during recovery. AMs are long-lived M2-like tissue-resident macrophages that have a strong ability to proliferate and phagocytose. MDMs are a paradoxical group of macrophages that promote the repair of tissue damage despite being strongly pro-inflammatory early in infection, and they may undergo cell death as inflammation fades. Preventing the massive recruitment of inflammatory MDMs or promoting their transition to pro-repairing phenotype may be a new direction for the treatment of acute lung injury.

Keywords: Acute Lung Injury; Alveolar Macrophage; Inflammation; Monocyte-Derived Macrophage.

Figure 1. AMs and MDMs in LPS-induced ALI. (A) Gating strategy of AMs and MDMs in BALF cells. AMs were identified as CD45⁺Ly6G⁻CD64⁺MerTK⁺SiglecF⁺CD11b⁻ cells; MDMs were identified as CD45⁺Ly6G⁻CD64⁺MerTK⁺SiglecF⁻CD11b⁺ cells. (B, C) Total cell number and protein level dynamics in ALI mice BALF (n=3 per time point). (D) Wet/dry weight ratio changes of lung tissue in ALI mice. (E) Dynamic changes of AMs and MDMs in mice BALF (n=3 per time point).

Figure 2. Gene expression differences between AMs and MDMs. (A) Experiment design for sorting and sequencing of AMs and MDMs. (B) PCA of AMs and MDMs that sorted from ALI mice BALF on day 6 (each point represents 5 mice). (C) Volcano plot of differentially expressed genes in AMs and MDMs. (D) Clustering heatmap of the top 50 differentially expressed genes in AMs and MDMs.

Figure 3. Transcriptional level differences between AMs and MDMs. (A, B) Top 10 BP, CC, and MF enrichments of MDMs and AMs that sorted from ALI mice BALF on day 6. (C) Top 20 upregulated and downregulated KEGG pathways of MDMs that sorted from ALI mice BALF on day 6. (D) Heatmap of genes that represent phagocytosis, proliferation, inflammation, and cell death of AMs and MDMs that sorted from ALI mice BALF on day 6.

BP, biological function; CC, cellular component; MF, molecular function.

Figure 4. Viability and functional differences between AMs and MDMs. (A-C) Ki67, Edu and 7-AAD staining of AMs and MDMs on day 6 post LPS (n=4). Experiment was repeated three times. (D) Study design for phagocytosis analysis. (E) Percentage of CFSE+cells of AMs and MDMs (n=4). Experiment was repeated three times. (F) GSEA of biological functions that represent cell adhesion, cell motility, cell migration, and positive regulation of lymphocyte activation, proliferation, and differentiation of AMs and MDMs. (G) Representative FACS plot and quantification of T cell activation marker CD25 and CD69 expression after co-cultured with AMs or MDMs overnight (n=4). (H) Migration rates of AMs and MDMs 6, 12, 24 h after scratch (n=4).

^*p<0.05; ^**p<0.01; ^***p<0.001; ^****p<0.0001.

Figure 5. Phenotype differences of AMs and MDMs. (A) CD86, iNOS, RELM-α and CD206 expression levels of AMs and MDMs on day 6 post LPS (n=4). Experiment was repeated three times. (B) GSEA of inflammatory response and cytokine production functions of AMs and MDMs. (C) Heatmap of genes that represent tissue-repairing of AMs and MDMs. (D) GSEA of functions that represent regulating endothelial cell proliferation of AMs and MDMs. (E) Representative flow cytometry plots of Ly6c^lo and Ly6c^hi MDMs among Siglec-F⁻CD11b⁺ MDMs from BALF on day 3 and day 7. (F) Percentages of Ly6c^hi and Ly6c^lo MDMs at different time points of ALI (n=3) per time point.

^*p<0.05; ^**p<0.01; ^***p<0.001; ^****p<0.0001.

Figure 6. Analysis of scRNA-seq data on BAL cells from patients with SARS-CoV-2 infection. (A) The heatmaps of hierarchically clustered top 30 DEGs across 3 groups of macrophages. The gene names were listed to the left. (B) UMAP projection of 2 and 3 macrophage groups among controls (n=4) and patients (moderate, n=3; severe, n=6). (C) The GO and KEGG analysis of up-regulated DEGs showing some highlighted pathways in MDMs. (D) Representative flow cytometry plots of AMs (Siglec-F⁺CD11b⁻) and MDMs (Siglec-F⁻CD11b⁺) among CD64⁺MerTK⁺ cells from BALF and quantification of cell numbers (±SEM) on the right (n=3–4). Experiment was repeated three times. (E) Lung tissue and BALF of WT and CCR2^−/− mice on day3 post LPS. (F) Pulmonary pathology of WT and CCR2^−/− mice on day 3 post LPS (bar=100 µm). (G, H): Lung wet/dry ratio and BALF total protein of WT and CCR2^−/− mice on day 3 post LPS (n=3–4). Experiment was repeated three times.

^*p<0.05; ^**p<0.01.