Development and Characterisation of a New In Vitro Murine Mucosal Mast Cell Model

Abstract

Mouse mast cells (MCs) fall into two subpopulations with well-defined roles and characteristics: connective tissue mast cells (CTMCs) and mucosal mast cells (MMCs) [1, 2]. While in vitro models of CTMCs exist in humans and mice, a reliable and relevant model of MMCs is still lacking. While a few previously described protocols have used the addition of TGF-β1 and IL-9 alongside IL-3 and SCF from the onset of bone marrow cell culture to generate MMC-like cells, these approaches often vary in culture duration and in the nature of the resulting cells [3, 4]. Here, we propose a two-step protocol that more faithfully reflects the two major stages of MMC differentiation, enabling the generation of mouse bone marrow-derived mucosal mast cells (BM-MMCs) in vitro. Bone marrow cells were cultured in a complete Opti-MEM medium supplemented with IL-3 and SCF for 4 weeks to induce MC commitment before adding for one additional week both IL-9 and TGF-β1 to promote MMC proliferation and maturation [2, 3, 5] (Figures 1A and S1). This delayed addition of IL-9 and TGF-β1 resulted in a higher percentage of cells showing an MMC phenotype compared with its addition at the start of culture (Figure S2). BM-MMCs were phenotypically and functionally compared to the previously established CTMC model (PCMCs) [6]. In 5 weeks, this two-step differentiation protocol produces approximately 35 million MCs (36 ± 9 million, n = 7 mice) from 1 million bone marrow cells (Figure S3A). After 7 weeks in culture, the BM-MMC showed more than 95% viability (Figure S3B). BM-MMCs expressed the MC markers FcεRI, CD117 and ST2 (IL-33 receptor) together with the typical MMC markers CD103 and MCPT1 (Figure 1B–D) and showed IL-3 dependency (Figure S4). Furthermore, these cells did not stain positively for avidin (which binds to heparin contained in the granules of CTMCs) (Figure 1D) and exhibited a reduced granular mass, histamine, and MCPT6 contents as compared to their PCMC counterparts (Figure 1E–G). BM-MMCs degranulated in response to FcεRI aggregation but not to the 48/80 compound secretagogue, as expected for MMCs which do not express its receptor Mrgprb2 (Figure 1H–J).

Transcriptomic analysis showed clear differences in gene expression between the two cell types. Among the 40 most variable genes, the typical MMC genes Mcpt1, Mcpt2, Mcpt8 and Itgae, and the typical CTMC genes Mrgprb2 and Mrgprb1 were clearly clustered in BM-MMCs and PCMCs, respectively (Figure S5A). We found 5174 differentially expressed genes (DEGs, Padj < 0.01 and Fold change > 2) (Figure 2A and Table S1) between BM-MMCs and PCMCs. Further analysis of the expression of the prototypical MC genes substantiated that BM-MMCs displayed MMC features, including a diminished ability to produce histamine and the expression of genes involved in chondroitin sulfate synthesis (Figure 2B–D) (5). This analysis also revealed that receptors relevant to MC biology were differentially regulated between BM-MMC and PCMC (Figure 2E).

We next investigated whether the genetic signature of BM-MMCs could be found in MMCs described in the literature. Based on a transcriptional analysis of β7 integrin^High (MMCs) versus β7 integrin^Low (CTMCs) lung MCs from HDM-challenged mice [2], gene set enrichment analysis (GSEA) indicated that genes overexpressed in β7^High MCs were significantly enriched in BM-MMCs (Figure 2F). Likewise, GSEA analysis of sc-RNA-Seq dataset from mouse MCs isolated from different tissues, which exhibited a clear clustering of MMCs (Mrgprb2⁻) and CTMCs (Mrgprb2⁺) [1] showed that the MMC gene signature was significantly enriched in BM-MMCs (Figure 2G). Reciprocally, the enrichment score of the 100 upregulated genes in BM-MMCs revealed a transcriptomic signature similar to that of mouse MMCs identified by Tauber et al. [1] (Figure 2H–J). Moreover, the RNA-Seq analysis of the genes coding pattern recognition receptors and antimicrobial molecules underlined the distinct functional roles of BM-MMCs and PCMCs and the obvious antimicrobial capacity of BM-MMCs (Figure S5B,C).

Thus, BM-MMCs, which have transcriptomic characteristics similar to MMCs, can be an effective tool for studying a significant number of viable MMCs during a 15-day period in vitro.

Experimental design: E.E., L.B.; Conducting experiments: L.B., N.S.; Statistical analysis: E.E., L.B.; Writing (original draft): E.E., L.B., G.D.; Writing (review and editing): all authors.

The authors declare no conflicts of interest.