Butyrate inhibits human mast cell activation via epigenetic regulation of FcεRI-mediated signaling

Abstract

Background: Short-chain fatty acids (SCFAs) are fermented dietary components that regulate immune responses, promote colonic health, and suppress mast cell-mediated diseases. However, the effects of SCFAs on human mast cell function, including the underlying mechanisms, remain unclear. Here, we investigated the effects of the SCFAs (acetate, propionate, and butyrate) on mast cell-mediated pathology and human mast cell activation, including the molecular mechanisms involved.

Method: Precision-cut lung slices (PCLS) of allergen-exposed guinea pigs were used to assess the effects of butyrate on allergic airway contraction. Human and mouse mast cells were co-cultured with SCFAs and assessed for degranulation after IgE- or non-IgE-mediated stimulation. The underlying mechanisms involved were investigated using knockout mice, small molecule inhibitors/agonists, and genomics assays.

Results: Butyrate treatment inhibited allergen-induced histamine release and airway contraction in guinea pig PCLS. Propionate and butyrate, but not acetate, inhibited IgE- and non-IgE-mediated human or mouse mast cell degranulation in a concentration-dependent manner. Notably, these effects were independent of the stimulation of SCFA receptors GPR41, GPR43, or PPAR, but instead were associated with inhibition of histone deacetylases. Transcriptome analyses revealed butyrate-induced downregulation of the tyrosine kinases BTK, SYK, and LAT, critical transducers of FcεRI-mediated signals that are essential for mast cell activation. Epigenome analyses indicated that butyrate redistributed global histone acetylation in human mast cells, including significantly decreased acetylation at the BTK, SYK, and LAT promoter regions.

Conclusion: Known health benefits of SCFAs in allergic disease can, at least in part, be explained by epigenetic suppression of human mast cell activation.

Keywords: FcεRI signaling; butyrate; histone deacetylase; mast cells; short-chain fatty acids.

Figure 1.. Butyrate inhibits OVA-induced airway contraction in an ex vivo model of bronchoconstriction.

A, OVA-induced reduction of airway luminal area in precision cut lung slices (PCLS) of OVA-sensitized guinea pigs either untreated or treated with butyrate for 24 h. B, Effect of different concentrations of butyrate on the airway luminal area after the final OVA challenge. As a control, slices were stimulated with histamine to induce strong contraction (1.84 mg/ml). C, Video stills depicting the effects of butyrate on OVA-induced airway contraction in PCLS. The white scale bar indicates 500 μm. OVA stimulation in vehicle treated PCLS induced strong airway contraction (‘control’). Note that the airway, indicated by a yellow arrow, is located close to a blood vessel. Pre-treatment of 5 and 25 mM butyrate inhibits OVA-induced airway contraction. Data represent mean ± SEM, statistical significance was tested using a (A) two-way ANOVA test or (B) one-way ANOVA test: Results in A and B are pooled from 2-3 independent experiments performed with PCLS from different animals (n=2-3).

Figure 2.. Butyrate and propionate, but not acetate, inhibit IgE and non-IgE mediated mast cell activation.

A, Percentage of mast cell degranulation (as measured by beta-hexosaminidase release) after IgE-mediated activation using either DNP-HSA or anti-IgE stimulation in mouse and human mast cells treated with increasing concentrations of acetate (left), propionate (middle) and butyrate (right). B, Percentage of degranulation after IgE- and non-IgE (C48/80 or Substance P) mediated mast cell activation in untreated or treated (5 mM butyrate, 24 h) human mast cells. C, Effects of butyrate and propionate on IL-6 cytokine production in mouse mast cells after IgE-mediated activation. Results are pooled from 3 independent experiments performed with mouse or human mast cells from 3 different donors (n=3/group). Data represent mean ± SEM, statistical significance was tested using a one-way ANOVA test: #, significantly increased compared to non-stimulated; *, Significantly decreased compared to control (P < 0.05). * P < 0.05; ** P < 0.01; ***, ^### P < 0.001. NS = not significant, DNP-HSA = Dinitrophenyl - Human Serum Albumins, Sub. P. = Substance P.

Figure 3.. The effects of SCFAs are independent of GPR41, GPR43 and PPAR stimulation, but dependent on HDAC activity.

A, Percentage of mast cell degranulation after IgE-mediated activation in mouse mast cells from WT, GPR41^−/− or GPR43^−/− mice (left panel). Effects of a GPR43 agonist on mast cell degranulation (right panel). B, Effect of different PPAR agonists on the percentage of degranulation via IgE-mediated activation in mouse mast cells. C, The effect of butyrate or TSA (10 μM) on HDAC activity in mouse and human mast cells. HDAC activity was measured by adding cell permeable HDAC substrate containing an acetylated lysine side chain; subsequent deacetylation by HDAC releases a detectable fluorophore. D, Percentage of degranulation after TSA treatment (left panel: 10 – 1000 nM, right panel: 1000 nM) in mouse and human mast cells. Results are pooled from 3 independent experiments performed with mouse and human mast cells from 3 different donors (n=3/group). Data represent mean ± SEM, statistical significance was tested using a one-way ANOVA test or an unpaired Student’s t test: *P < 0.05; **P < 0.01; ***P < 0.001. TSA = Trichostatin A, Sub. P. = Substance P.

Figure 4.. Butyrate induces human mast cell gene expression changes associated with cytokine signaling and mast cell activation.

A, Scatterplot comparison of gene expression levels measured by microarray analysis in unstimulated human mast cells either untreated or exposed to 5 mM butyrate for 24 h. Upregulated genes (>1.0 log2 fold change) are indicated in green, downregulated genes (<−1.0 log2 fold change) in red. B, Selected pathways that were strongly affected by butyrate treatment in unstimulated human mast cells. Y-axis denotes P-values on a −log10 scale. C, D, Butyrate-induced upregulation or downregulation of genes associated with the GO:0045576 – ‘Mast Cell Activation’ pathway represented as a Venn diagram (C) or bar graph (D) in unstimulated human mast cells treated with or without butyrate for 24 h. E, Butyrate-induced downregulation of BTK, SYK and LAT was validated using qPCR in human mast cells from 3 additional independent donors. F, Genes upregulated in response to 24h butyrate treatment (compared to vehicle treated cells, indicated as 0 h) showed overall low expression levels in vehicle treated mast cells, while downregulated genes showed significantly higher expression levels prior to treatment. Downregulated mast cell activation genes in particular are highly expressed before butyrate treatment (right boxplot). A-D, F, Results are from a single culture of human mast cells. E, Results are pooled from 3 independent experiments performed in human mast cells from 3 different donors. Data represent mean ± SEM, statistical significance was tested using a one-way ANOVA test: *P < 0.05; **P < 0.01; ***P < 0.001. GO = Gene ontology.

Figure 5.. Butyrate induces a loss of histone acetylation at the transcription start site of key genes involved in FcεRI-mediated signaling.

A, Venn diagram depicting the overlap in H3K27Ac enrichment peaks (reproducibly detected in both donors) detected at 0h and 3h of butyrate treatment. Only a minority (~1%) of regions lose all H3K27Ac signal, in contrast to de novo enrichment of 14292 unique peaks following butyrate treatment. B, Butyrate treatment induces elevated megabases (Mb) of genome covered by H3K27Ac. C, Genome browser view of histone 3 lysine 27 acetylation (H3K27Ac) levels at the KIT locus, as measured by ChIP-Seq before and after 3h of 5 mM butyrate exposure. D, Average H3K27Ac levels around the TSS of mast cell activation genes downregulated by 24 hours of butyrate exposure (see Fig.4D). E-G, Genome browser views depicting H3K27ac levels across loci encoding key signaling molecules involved in FcεRI-mediated signaling. TSS regions show reduced acetylation levels for BTK (64% decrease), SYK (43% decrease) and LAT (70% decrease), correlating with their loss of expression upon butyrate treatment. Similar results were obtained using mast cells from 2 independent cultures of 2 different donors; data obtained from donor 2 is shown. TSS = transcription start site.