Activation of ryanodine-sensitive calcium store drives pseudo-allergic dermatitis via Mas-related G protein-coupled receptor X2 in mast cells

Abstract

Mast cell (MC) activation is implicated in the pathogenesis of multiple immunodysregulatory skin disorders. Activation of an IgE-independent pseudo-allergic route has been recently found to be mainly mediated via Mas-Related G protein-coupled receptor X2 (MRGPRX2). Ryanodine receptor (RYR) regulates intracellular calcium liberation. Calcium mobilization is critical in the regulation of MC functional programs. However, the role of RYR in MRGPRX2-mediated pseudo-allergic skin reaction has not been fully addressed. To study the role of RYR in vivo, we established a murine skin pseudo-allergic reaction model. RYR inhibitor attenuated MRGPRX2 ligand substance P (SP)-induced vascular permeability and neutrophil recruitment. Then, we confirmed the role of RYR in an MC line (LAD2 cells) and primary human skin-derived MCs. In LAD2 cells, RYR inhibitor pretreatment dampened MC degranulation (detected by β-hexosaminidase retlease), calcium mobilization, IL-13, TNF-α, CCL-1, CCL-2 mRNA, and protein expression activated by MRGPRX2 ligands, namely, compound 48/80 (c48/80) and SP. Moreover, the inhibition effect of c48/80 by RYR inhibitor was verified in skin MCs. After the confirmation of RYR2 and RYR3 expression, the isoforms were silenced by siRNA-mediated knockdown. MRGPRX2-induced LAD2 cell exocytosis and cytokine generation were substantially inhibited by RYR3 knockdown, while RYR2 had less contribution. Collectively, our finding suggests that RYR activation contributes to MRGPRX2-triggered pseudo-allergic dermatitis, and provides a potential approach for MRGPRX2-mediated disorders.

Keywords: MRGPRX2; calcium; degranulation; mast cell; pseudo-allergy; ryanodine receptor.

Figure 1

Dantrolene attenuates SP-induced in vivo murine skin anaphylaxis model. Dantrolene or saline was i.p. injected for 2 consecutive days, and then SP (50 µM) or saline was intradermally injected into one side of the ear. (A–C) Vascular permeability was quantified by Evans blue extravasation, and the thickness of the ear was determined by a vernier caliper (n = 6). (A) Representative photo of Evans blue extravasation in the murine skin anaphylaxis model. (B) Quantification of Evans blue extravasation. (C) Ear tissue thickness after the injection of SP or saline. (D, E) Ear tissue was collected and neutrophil infiltration (CD11b+ Ly6G+ live cells) was determined by flow cytometry (n = 5–7). (D) Representative flow cytometry images. (E) Percentage of neutrophils in the live skin cell population. Data shown are mean ± SEM. Ctrl.: control, inh.: inhibitor. ns: not significant, *p < 0.05, **p < 0.01.

Figure 2

RYR inhibitor perturbs MRGPRX2-mediated mast cell degranulation and calcium mobilization. LAD2 cells were treated with RYR inhibitor (dantrolene, 100 µM) for 15 min and then the cells were stimulated with c48/80 (5 µg/ml) or SP (30 µM). (A, B) β‐Hexosaminidase release and (C, D) calcium mobilization was determined, respectively. The data are from 7 to 15 independent experiments. ns: not significant, Ctrl.: control, inh.: inhibitor. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3

MRGPRX2 activation-induced cytokine mRNA generation relies on the activation of RYR. Cells were preincubated with or without RYR inhibitor (dantrolene, 100 µM) for 15 min and stimulated with (A) SP (60 µM) or (B) c48/80 (5 µg/ml). Cytokine mRNA was determined by RT-qPCR. The data were normalized against the cell receiving no inhibitor and stimuli. Data shown are mean ± SEM of n = 7–11. inh.: inhibitor. *p < 0.05, **p < 0.01.

Figure 4

MRGPRX2-activated cytokine protein release is inhibited by RYR inhibitor. LAD2 cells were preincubated with or without RYR inhibitor (dantrolene, 100 µM) for 15 min and stimulated with (A) SP (60 µM) or (B) c48/80 (5 µg/ml) for 24 h. Supernatants were collected and IL-13, TNF-α, CCL-1, and CCL-2 protein release was quantified by ELISA. Data shown are mean ± SEM of n = 8–12. Ctrl.: control, inh.: inhibitor. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

RYR3 contributes to the degranulation and calcium mobilization activated by MRGPRX2. LAD2 cells were treated with RYR2- and RYR3-specific siRNA or non-target siRNA, respectively, then (A, B) β‐hexosaminidase release and (C, D) calcium mobilization was detected. The data are from seven to nine independent experiments. ns: not significant, *p < 0.05.

Figure 6

Cytokine mRNA generation via MRGPRX2 relies predominantly on the activation of RYR3. LAD2 cells were treated with RYR2- and RYR3-specific siRNA or non-target siRNA, then stimulated with (A) c48/80 (5 µg/ml) or (B) SP (60 µM). TNF-α, CCL-1, and CCL-2 mRNA expression were determined by RT-qPCR. The data were normalized against the cell receiving no inhibitor and stimuli. Data shown are mean ± SEM of n = 7. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, **** p<0.0001..