Quantitative In-Depth Transcriptome Analysis Implicates Peritoneal Macrophages as Important Players in the Complement and Coagulation Systems

Abstract

To obtain a more detailed picture of macrophage (MΦ) biology, in the current study, we analyzed the transcriptome of mouse peritoneal MΦs by RNA-seq and PCR-based transcriptomics. The results show that peritoneal MΦs, based on mRNA content, under non-inflammatory conditions produce large amounts of a number of antimicrobial proteins such as lysozyme and several complement components. They were also found to be potent producers of several chemokines, including platelet factor 4 (PF4), Ccl6, Ccl9, Cxcl13, and Ccl24, and to express high levels of both TGF-β1 and TGF-β2. The liver is considered to be the main producer of most complement and coagulation components. However, we can now show that MΦs are also important sources of such compounds including C1qA, C1qB, C1qC, properdin, C4a, factor H, ficolin, and coagulation factor FV. In addition, FX, FVII, and complement factor B were expressed by the MΦs, altogether indicating that MΦs are important local players in both the complement and coagulation systems. For comparison, we analyzed human peripheral blood monocytes. We show that the human monocytes shared many characteristics with the mouse peritoneal MΦs but that there were also many major differences. Similar to the mouse peritoneal MΦs, the most highly expressed transcript in the monocytes was lysozyme, and high levels of both properdin and ficolin were observed. However, with regard to connective tissue components, such as fibronectin, lubricin, syndecan 3, and extracellular matrix protein 1, which were highly expressed by the peritoneal MΦs, the monocytes almost totally lacked transcripts. In contrast, monocytes expressed high levels of MHC Class II, whereas the peritoneal MΦs showed very low levels of these antigen-presenting molecules. Altogether, the present study provides a novel view of the phenotype of the major MΦ subpopulation in the mouse peritoneum and the large peritoneal MΦs and places the transcriptome profile of the peritoneal MΦs in a broader context, including a comparison of the peritoneal MΦ transcriptome with that of human peripheral blood monocytes and the liver.

Keywords: coagulation system; complement system; liver; mRNA; macrophage; monocyte; transcriptome.

Figure 1

Gating strategy used for identification and sorting of peritoneal macrophages (i.e., CD19⁻, CD11b⁺, and F4/80⁺) and B cells (CD19⁺ and low FSC-A).

Figure 2

Purity of human peripheral blood monocytes obtained from PBMCs by magnetic cell sorting using CD14 microbeads. Separated cells were stained with anti-human CD14 PE antibody and analyzed by flow cytometry. Representative flow cytometry histograms show PBMCs before sorting (A) and cells after sorting (B).

Figure 3

Signature gene sets identified for different MΦ subpopulations by previous single cell analysis. Transcript levels in the peritoneal MQs for a number of genes previously identified as signature transcripts for a few different MQ, monocyte, and dendritic cell populations by single cell analysis as summarized by Summers et al. [19]. The number of reads for each of the different genes are given in actual numbers we obtained from the Thermo Fisher Ampliseq analysis. The numbers in brackets are the numbers obtained from the GATC RNA-seq analysis. When the number or reads in one of these two studies are higher than 50 the transcript is marked in orange. When there is a major difference between the two analysis methods the RNA-seq value is marked by red text.