To the Editor:
Mouse mast cells (MCs) have been classified as connective tissue MCs (CTMCs) and mucosal MCs (MMCs)1. Mouse bone marrow-derived cultured MCs (BMCMCs) generated with IL-3 (IL-3-MCs) have been considered “immature”, with partial maturation induced by changing to SCF (stem cell factor)-containing media (SCF-MCs)2. We investigated the molecular mechanisms of SCF-induced BMCMC maturation using transcriptome profiling.
SCF-MCs were larger than IL-3-MCs, with ~60% reduced surface expression of c-kit (SCF receptor). IL-3-MCs were exclusively alcian blue(+)safranin(−). SCF-MCs contained a proportion of safranin(+)/alcian blue(+) cells2 (Online Fig 1A-1C).
Principle component analysis (PCA) revealed three populations: peritoneal MCs (PMCs, green), IL-3-MCs (red) and SCF-MCs (blue) (Fig 1A). Euclidean distance measurements and hierarchical clustering confirmed this (Online Fig 2A; Fig 1B). We identified 308 upregulated and 307 downregulated differentially expressed genes (DEGs) in PMCs vs. IL-3-MCs, and 80 upregulated and 178 downregulated DEGs in SCF-MCs vs. IL-3-MCs (adjusted P value <0.05, mean of normalized counts ≥500 and fold change >4) (Online Fig 2B).
Figure 1.
Transcriptome profiling of peritoneal mast cells (PMCs) vs. cultured SCF-MCs and IL-3-MCs. (A) Population principal component analysis (PCA). (B) MC population hierarchical clustering (top margin). (C-E) Heatmaps of MC-enriched protease-encoding transcripts (C), MAS related GPR (MRGPR) family-encoding transcripts (D) and bioamine biosynthesis transcripts (E). Genes are ordered by log2 fold changes between PMC and IL-3-MCs, and DE genes between PMC and. IL-3-MCs with an adjusted P value <0.05 are in red. Data: PMCs from four mice vs. IL-3- and SCF-MCs from two mice.
Functional analysis of upregulated DEGs with PANTHER pathway-classification system3 revealed significant enrichment for genes in ‘heparin metabolic process’ (Online Table 1). Heparin sulfate (HS) is the dominant CTMC proteoglycan and chondroitin sulfate (CS) is predominant in MMCs4. Transcripts involved in HS chain polymerization/sulfation (e.g., Ext1, Ext2, Ndst2, Glce and Hs2st1) were remarkably upregulated in PMCs vs. IL-3-MCs. Transcripts of enzymes involved in CS chain polymerization/sulfation (e.g., Chsy1, Chst15, C4st-1[Chst11] or C4st-2), were decreased or comparable between PMCs and IL-3-MCs (Fig 1C; Online Fig 3).
Genes for MC-enriched proteases, including tryptases (Tpsb2, Tpsg1, Tpsab1), chymases (Mcpt4, Cma1, Cma2) and others (Plau, Ctsg, C2) were expressed at significantly higher levels in PMCs vs. IL-3-MCs (Fig 1D). IL-3-MCs expressed higher levels of Mcpt2, the signature protease in esophageal and tracheal MCs.1 Transcripts of histidine decarboxylase (Hdc), the rate-limiting enzyme for histamine biosynthesis, were ~4.8 fold higher in PMC vs. IL-3-MCs (Fig 1E).
Eight weeks after changing IL-3-MCs to SCF partially restored expression of HS-related genes (Fig 1C; Online Fig 3), but at levels lower than in PMCs. Similarly, SCF-MCs significantly upregulated Mcpt4 (a chymase) transcripts vs. IL-3-MCs, but at levels lower than in PMCs. SCF exposure did not alter expression of MC tryptases (Tpsg1, Tpsb2) or other MC chymases (Cma1, Cma2) (Fig 1D; Online Table 2). Finally, SCF did not restore expression of enzymes involved in biological amines synthesis, including Hdc (Fig 1E), or increase histamine content (Online Fig 1D).
Three MC-specific MAS-related G protein-coupled receptor (MRGPR) family members and PMC signature genes, Mrgprb1, Mrgprb2 and Mrgprx2 (Mrgprb10), were markedly upregulated in PMCs vs. IL-3-MCs. SCF exposure restored expression of PMC signature Mrgpr family members to levels comparable to those in PMCs (Fig 2A-2B; Online Table 3). Indeed, unlike IL-3-MCs (Fig 2C-2E, black curves), SCF-MCs were activated by substance P (SP) or compound 48/80 (Fig 2C-2E, red curves). PMCs with levels of Mrgprb2 similar to SCF-MCs were also activated by SP, but not MCs maintained in IL-3+SCF (Online Fig 4). Because the human homolog of Mrgprb2 mediates SP-induced MC activation, upregulation of Mrgprb2 probably mediates SP- and compound 48/80-induced MC activation in SCF-MCs5 and also contributes to SP-induced PMC activation.
Figure 2.
Mrgprb2 transcript upregulation in SCF-MCs is associated with enhanced responsiveness to substance P (SP). (A) Heatmaps and (B) RT-qPCR of Mrgpr-transcripts. Data presented as relative expression of transcripts vs. β-actin. Data represent MCs derived from two mice for RT-qPCR. * P<0.05 between the two groups. (C) Substance P (SP) or (D) compound 48/80 activation of IL-3- or SCF-MCs quantified as mean fluorescence intensity (MFI) of Alexa 488-conjugated avidin (details in online Methods). (E) Histamine release; stimulation for 1 h. * P <0.05 at certain time points or with certain drug concentrations for 30 min. Error bars represent SEM.
16 transcription factors (TFs) were upregulated in PMCs vs. IL-3-MCs (> 4 fold), and 9 TFs were significantly restored by SCF exposure (Online Table 4). Marked upregulation of HES and its downstream molecule GATA3, and downregulation of C/EBP alpha in PMCs and SCF-MCs, are all necessary for MC fate determination6.
In conclusion, we have shown that switching from IL-3 to SCF partially programs immature BMCMCs toward CTMCs through transcriptional upregulation of HS-biosynthesis enzymes, certain MC-specific proteases, MRGPR family members and TFs required for MC lineage determination.
Supplementary Material
Acknowledgements
This study was supported by NIH T32 training award to Y.W. (T32 AI 7290-33); an award from MSD Life Science Foundation and Public Interest Incorporated Foundation (Japan) to K.M.; and NIH grants (NIAMS R01 AR067145 and NIAID R01 AI132494) and United States-Israel Binational Science Foundation (Grant 2017182) to S.J.G.
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