Network analysis of transcriptomic diversity amongst resident tissue macrophages and dendritic cells in the mouse mononuclear phagocyte system

Abstract

The mononuclear phagocyte system (MPS) is a family of cells including progenitors, circulating blood monocytes, resident tissue macrophages, and dendritic cells (DCs) present in every tissue in the body. To test the relationships between markers and transcriptomic diversity in the MPS, we collected from National Center for Biotechnology Information Gene Expression Omnibus (NCBI-GEO) a total of 466 quality RNA sequencing (RNA-seq) data sets generated from mouse MPS cells isolated from bone marrow, blood, and multiple tissues. The primary data were randomly downsized to a depth of 10 million reads and requantified. The resulting data set was clustered using the network analysis tool BioLayout. A sample-to-sample matrix revealed that MPS populations could be separated based upon tissue of origin. Cells identified as classical DC subsets, cDC1s and cDC2s, and lacking Fcgr1 (encoding the protein CD64) were contained within the MPS cluster, no more distinct than other MPS cells. A gene-to-gene correlation matrix identified large generic coexpression clusters associated with MPS maturation and innate immune function. Smaller coexpression gene clusters, including the transcription factors that drive them, showed higher expression within defined isolated cells, including monocytes, macrophages, and DCs isolated from specific tissues. They include a cluster containing Lyve1 that implies a function in endothelial cell (EC) homeostasis, a cluster of transcripts enriched in intestinal macrophages, and a generic lymphoid tissue cDC cluster associated with Ccr7. However, transcripts encoding Adgre1, Itgax, Itgam, Clec9a, Cd163, Mertk, Mrc1, Retnla, and H2-a/e (encoding class II major histocompatibility complex [MHC] proteins) and many other proposed macrophage subset and DC lineage markers each had idiosyncratic expression profiles. Coexpression of immediate early genes (for example, Egr1, Fos, Dusp1) and inflammatory cytokines and chemokines (tumour necrosis factor [Tnf], Il1b, Ccl3/4) indicated that all tissue disaggregation and separation protocols activate MPS cells. Tissue-specific expression clusters indicated that all cell isolation procedures also co-purify other unrelated cell types that may interact with MPS cells in vivo. Comparative analysis of RNA-seq and single-cell RNA-seq (scRNA-seq) data from the same lung cell populations indicated that MPS

Transcriptomic Analysis of Rat Macrophages

“…In the isolated rat macrophages, Cd4 was highly-expressed by BMDM, MDM and PM but just detectable in AM and ESDM. As in mice ( 5 ), Itgam ( Cd11b ) expression was highest in PM. Itgax ( Cd11c ) is still widely considered a dendritic cell (DC) marker in mice, despite clear expression by many tissue macrophage populations [reviewed in ( 48 )].…”

“…AM = alveolar macrophages, BMDM = bone marrow derived macrophages, MDM = monocyte derived macrophages, PM = peritoneal macrophages, ESDM = embryonic stem cell derived macrophages. (D) Global enrichment of mouse macrophage AM and PM signature gene sets ( 5 ) in rat AM and PM identified by Gene Set Enrichment Analysis. Rat expression data are ranked according to differential expression in AM or PM compared to all other macrophage populations (indicated by red-blue bars), and the murine AM or PM genesets are mapped onto this profile (black bars) to determine enrichment score (green lines).…”

“…We also looked at expression of genes which encode surface markers commonly used to define and separate (sub)populations of mouse macrophages ( 5 , 49 ) (as above). We recently characterised a monoclonal antibody against pig ADGRE1 (F4/80) and found that the antigen was highly-expressed by pig AM ( 50 ).…”

“…For comparative analysis of rat and mouse macrophage signatures, we first analysed the enrichment of 12 mouse macrophage signatures identified from cluster analysis of 466 RNA Sequencing datasets, including microglia, lung, Kupffer cells, CCR7 + DC, monocytes, peritoneum, Lyve1 + macrophages, DC, intestinal macrophages, Langerhans cells, cDC1s, and a cluster common to all mononuclear phagocytes ( 5 ), in each rat macrophage population compared to all the others using Gene Set Enrichment Analysis. The mouse AM and PM signatures showed significant enrichment in the rat AM and PM populations, respectively ( Figure 4D ), whereas no significant enrichment was observed for rat ESDM, BMDM, or MDM.…”

“…The enrichment of Molecular Signature (MSIG) database Gene Ontology sets, including BP, CC and MF (MSIG collection 5) in each macrophage population compared to the other 4 populations combined was analysed using Gene Set Enrichment Analysis (GSEA, Broad Institute) with phenotype permutation and default settings. For comparative analysis of rat and mouse macrophage signatures, we used GSEA to analyse the enrichment of 12 mouse macrophage signatures identified from cluster analysis of 466 RNA Sequencing datasets (cluster 7-microglia, 10-lung, 12-Kupffer cells, 13-CCR7 + DC, 15-monocytes, 21-peritoneum, 22-Lyve1 + , 28-DC, 38-Intestinal macrophages, 43-Langerhans cells, 49-cDC1s, 3-mononuclear phagocytes) ( 5 )in each rat macrophage population compared to all others.…”