To characterize the impact of butyrate exposure on the mast cell transcriptome, we measured gene expression profiles of two independent primary human mast cell cultures upon 24 h of 5mM butyrate treatment using RNA-Sequencing (RNA-Seq). Across all genes detected, 551 were upregulated by butyrate whereas 864 genes were downregulated (FDR<0.05; Fig. 1A,B). Correlation of gene expression values between the two biological replicates was high, both before and after butyrate treatment (R²>0.97, Supplementary Fig. 1A,B). Pathway enrichment analyses indicated that downregulated genes were mainly associated with leukocyte and mast cell activation (Fig. 1C,D, upper panels). Indeed, expression of genes coding for proteins involved in FcεRI- and MRGPRX2-mediated mast cell activation was significantly downregulated (including BTK, SYK, LAT and MRGPRX2, Supplementary Table 1), supporting our earlier findings that butyrate inhibits mast cell activation induced via IgE and substance P (an MRGPRX2 ligand). Upregulated genes were enriched in more diverse biological pathways (Fig. 1C,D, lower panels), including genes involved in responses to metal ions (i.e., MT1 family proteins), cellular responses to external stimuli and Ras/Rab GTPase signaling (Fig. 1C,D, lower panels, Supplementary Table 1).
The downregulated genes - in particular canonical mast cell genes - showed substantially higher median basal expression values than unaffected or upregulated genes (Fig. 1E). This is in line with aprevious study in cancer cells demonstrating that HDAC inhibitors are more likely to repress highly expressed genes 26. Conversely, upregulated genes displayed slightly lower basal expression levels as compared to unaffected genes (Fig. 1E). Nevertheless, high basal expression levels were not solely predictive for responsiveness to butyrate treatment, as the 50 most highly expressed genes did not respond to butyrate treatment (Fig. 1F).
Together, these results show that butyrate, in primary human mast cells, selectively downregulates gene expression associated with leukocyte/mast cell activation and upregulates a more diverse group of genes. Basal expression levels alone only partially predict responsiveness to butyrate treatment, indicating a more selective mechanism of action through which butyrate exerts it effects.