Single-Cell RNA Sequencing Identifies Candidate Renal Resident Macrophage Gene Expression Signatures across Species

Abstract

Background: Resident macrophages regulate homeostatic and disease processes in multiple tissues, including the kidney. Despite having well defined markers to identify these cells in mice, technical limitations have prevented identification of a similar cell type across species. The inability to identify resident macrophage populations across species hinders the translation of data obtained from animal model to human patients.

Methods: As an entry point to determine novel markers that could identify resident macrophages across species, we performed single-cell RNA sequencing (scRNAseq) analysis of all T and B cell-negative CD45⁺ innate immune cells in mouse, rat, pig, and human kidney tissue.

Results: We identified genes with enriched expression in mouse renal resident macrophages that were also present in candidate resident macrophage populations across species. Using the scRNAseq data, we defined a novel set of possible cell surface markers (Cd74 and Cd81) for these candidate kidney resident macrophages. We confirmed, using parabiosis and flow cytometry, that these proteins are indeed enriched in mouse resident macrophages. Flow cytometry data also indicated the existence of a defined population of innate immune cells in rat and human kidney tissue that coexpress CD74 and CD81, suggesting the presence of renal resident macrophages in multiple species.

Conclusions: Based on transcriptional signatures, our data indicate that there is a conserved population of innate immune cells across multiple species that have been defined as resident macrophages in the mouse. Further, we identified potential cell surface markers to allow for future identification and characterization of this candidate resident macrophage population in mouse, rat, and pig translational studies.

Keywords: immunology; macrophages.; transcriptional profiling.

Graphical abstract

Figure 1.

scRNAseq identifies clusters of cells with unique gene expression patterns in the renal innate immune compartment. (A) Schematic of experimental design. (B) tSNE plot and (C) heatmap of innate immune cells in mouse, rat, pig, and human kidneys. The heatmap depicts the top five DEGs in each cluster of innate immune cells.

Figure 2.

Canonical markers can be used to identify distinct clusters of innate immune cells in the mouse kidney. tSNE projections and accompanying violin plots depicting genes used to identify (A) neutrophils (Lcn2), (B) NK cells (Gzma), (C) ILCs (Il7r), (D) infiltrating macrophages (Itgam, Ccr2), (E) resident macrophages (Adgre1, Cd64), and (F) dendritic cells (Snx22, Batf3).

Figure 3.

scRNAseq can be used to identify novel markers of innate immune cells in the mouse kidney. (A) Single cells from the mouse kidney were manually annotated according to expression of the canonical genes identified in Figure 2. (B) Heatmap identifying novel gene expression signatures in each innate immune compartment. A more detailed list of genes is included in Supplemental Table 2. (C) tSNE and violin plots depicting novel genes used to identify Ly6c^hi infiltrating macrophages (Chil3, Plac8), Ly6c^lo infiltrating macrophages (Fabp4, Ear2), and resident macrophages (C1qc, Cd81).

Figure 4.

Fluidigm CI scRNAseq confirms several of the top DEGs in mouse resident macrophages. Mouse resident macrophages were flow-sorted using the canonical CD11b and F4/80 markers and RNAseq was performed using the Fluidigm CI platform. (A) PCA plot, (B) violin plot, and (C) heatmap identifying the top 100 DEGs between infiltrating and resident macrophages using Fluidigm C1 scRNAseq.

Figure 5.

scRNAseq data identifies a unique cluster of cells in multiple species that share the same gene expression pattern as mouse resident macrophages. (A–D) tSNE and violin plots showing the top DEGs (C1qc, Cd81, Cd74, Apoe) that were used to identify candidate resident macrophages in scRNAseq data from mouse, rat, pig, and human kidneys.

Figure 6.

scRNAseq identifies distinct renal innate immune cells. (A) tSNE plot of manually annotated mouse, rat, pig, and human innate immune compartments. (B) tSNE and violin plots showing expression of commonly used human macrophage markers (CD68, CD163, CD14, FCGR3A) in scRNAseq data from human kidney tissue.

Figure 7.

Genes identified by scRNAseq are enriched in mouse resident macrophages at the protein level. (A) Representative histograms depicting C1q, CD81, CD74, and Apoe expression in mouse infiltrating and resident macrophages that were gated on CD11b and F/480 (Supplemental Figure 2). FMO, fluorescence minus one antibody of interest; Inf., Infiltrating macrophage; Res., resident macrophage. (B) Quantification of the percentage of resident and infiltrating macrophages that express each marker. Data are shown as mean±SEM; ****P<0.001. (C) Representative flow cytometry plots showing expression of CD81 and CD74 in innate immune cells from mouse, rat, and human kidney tissue. (D) Representative histograms depicting protein expression of C1q, CD81, and CD74 in candidate resident (red) and nonresident macrophages (blue) identified using the CD81 antibody. (E) Quantification of the percentage of CD81⁺ candidate resident macrophages and CD81⁻ nonresident macrophages that express C1q and CD74 in mouse, rat, and human kidney tissue. Each point represents an individual kidney sample that was harvested from mice, rats, or humans. Data are shown as mean±SEM. *P<0.01; ****P<0.001. mouse n=5, rat n=3 or 6, and human n=3.

Figure 8.

Candidate resident macrophages identified using novel markers have minimal exchange with the peripheral blood. Quantification showing the percent chimerism of B/T cells, resident macrophages (canonical markers), resident macrophages (novel markers), infiltrating macrophages, neutrophils, and NK cells. Each dot represents data collected from a single kidney harvested from individual mice. **P<0.01; ****P<0.001. B/T cells, B and T cells; Resident (Canonical), resident macrophages identified using CD11b and F4/80; Resident (Novel), resident macrophages identified using CD81 and C1q; Infiltrating, infiltrating macrophages.