IL-33 Sensitizes Mast Cells to PIEZO1 Stimulation Leading to Degranulation

PIEZO1 is a mechanosensitive calcium-permeable ion channel that converts mechanical stimuli into biological signals [1]. PIEZO1 plays important roles in innate immune cells including monocytes and natural killer cells [2, 3]. However, the role of Piezo1 in mast cells remains unexplored. Mast cells release histamine in response to mechanical stimuli such as pressure, which triggers itching in a subset of patients with chronic inducible urticaria [4]. We therefore investigated the connection between PIEZO1 and mast cells.

First, we examined Piezo1 mRNA expression in mouse bone marrow–derived mast cells (BMMCs) in the presence or absence of various stimuli. We found that IL-33, SCF, and IgE significantly upregulated Piezo1 mRNA expression compared with that in the control (Figure 1A). Notably, IL-33 caused an approximately 20-fold increase in Piezo1 mRNA levels. Consistent with these findings, analysis of IL-33–induced gene expression patterns in BMMCs from a Gene Expression Omnibus dataset (GSE96695) identified Piezo1 as one of the top upregulated genes (Figure S1). Piezo1 mRNA levels also increased in mouse connective tissue–type mast cells (CTMCs) stimulated with IL-33 (Figure S2A), and IL-33 stimulation upregulated PIEZO1 protein expression in BMMCs (Figure 1B). These findings suggest that IL-33 induces PIEZO1 expression in mouse mast cells.

We then assessed the functional role of IL-33–induced PIEZO1 in mast cells. We measured intracellular Ca²⁺ levels in response to stimulation with Yoda1, a specific PIEZO1 activator [5], in IL-33–pretreated or IL-33–untreated BMMCs. Yoda1 increased intracellular Ca²⁺ levels in IL-33–pretreated, but not in IL-33–untreated BMMCs (Figure 1C). Consistently, Yoda1 stimulation induced degranulation responses in IL-33–pretreated, but not in IL-33–untreated BMMCs, as determined by measuring β-hexosaminidase release and CD63 expression, which were reduced by siRNA-mediated Piezo1 knockdown (Figure 1D–F and Figure S3). In addition, Yoda1 stimulation induced the release of histamine, prostaglandin D2 (PGD2), leukotrienes (LTC4/D4/E4), and cytokines (IL-4 and IL-13) in IL-33–pretreated, but not in IL-33–untreated BMMCs (Figure 1G and Figure S4). The release of both β-hexosaminidase and histamine was suppressed by EGTA, a calcium chelator (Figure 1D,G), suggesting that Ca²⁺ influx via PIEZO1 plays a role in these responses. Similar results were obtained in CTMCs (Figure S2B,C). IL-33 also upregulated PIEZO1 in human mast cells (huMCs) derived from CD34⁺ stem cells isolated from human peripheral blood (Figure 1H and Figure S5A). Furthermore, IL-33–pretreated, but not IL-33–untreated huMCs increased CD63 expression upon Yoda1 stimulation (Figure 1I and Figure S5B). These findings suggest that IL-33 induction of PIEZO1 in mast cells leads to degranulation and the release of lipid mediators and cytokines upon PIEZO1 stimulation in mice and humans.

The in vitro findings in mast cells were recapitulated in an in vivo model (Figure 2A). Wild-type (WT) mice pretreated with IL-33 followed by PIEZO1 activation with Yoda1 (IL-33/Yoda1 mice) showed increased Evans blue cutaneous leakage (Figure 2B). WT mice pretreated with vehicle followed by Yoda1 stimulation (PBS/Yoda1 mice) did not show increased Evans blue leakage. WT mice pretreated with IL-33 followed by vehicle stimulation (IL-33/PBS mice) showed slightly increased Evans blue leakage, which might be due to histamine release upon IL-33 stimulation alone [6]. Serum MCP-1 levels showed similar patterns to the Evans blue cutaneous leakage (Figure 2C). Treatment with olopatadine, an H1 receptor blocker, before Yoda1 administration suppressed the increase in Evans blue cutaneous leakage in IL-33/Yoda1 mice (Figure S6), suggesting that histamine was, at least in part, responsible for the increase in vascular permeability. Measurement of innocuous mechanically evoked itch behaviors known as alloknesis in mice [7] using von Frey filaments (0.07 g) showed that alloknesis scores were higher in IL-33/Yoda1 mice than in IL-33/PBS, PBS/Yoda1, or PBS/PBS mice (Figure 2D). Importantly, these findings were not observed in mast cell–deficient Kit^w-sh/w-sh mice (Figure 2E–G), suggesting that PIEZO1 expressed on mast cells plays an important role in the regulation of vascular permeability and alloknesis at least under the current experimental conditions. Given that PIEZO1 expressed on sensory neurons is crucial for the induction of alloknesis [5], both mast cells and sensory neurons may play an important role in the induction of alloknesis, probably by interacting with each other.

Finally, we determined whether IL-33 sensitized mast cells to nocuous mechanical pressure in the mouse skin in a PIEZO1-dependent manner. WT mice pretreated with IL-33 followed by exposure to mechanical pressure using von Frey filament (26 g) (IL-33/26 g mice) showed higher levels of Evans blue cutaneous leakage than WT mice with no IL-33 pretreatment (Figure S7A). Administration of a Piezo1-specific antagonist, Dooku1 [8], decreased Evans blue cutaneous leakage in IL-33/26 g mice (Figure S7B). Importantly, the increased Evan blue cutaneous leakage was not observed in mast cell–deficient Kit^w-sh/w-sh mice pretreated with IL-33 followed by mechanical pressure using von Frey filament (26 g) (Figure S7C). These findings indicate that nocuous mechanical pressure increased vascular permeability in IL-33–pretreated mice and that this response was mediated by PIEZO1 and mast cells.

In summary, we suggest that IL-33 upregulates PIEZO1 expression in mast cells, thereby sensitizing mast cells to PIEZO1 stimulation or mechanical pressure. The present findings suggest a novel role of IL-33 in sensitizing mast cells to mechanical stimuli, which might be involved in the pathophysiology of pressure-related chronic inducible urticaria.

Y.K. and A.N. designed the study. Y.K., K.S., N.T., K.I., and Y.N. performed the experiments and analyzed the data. T.S., D.N., S.T., and S.K. supervised experimental procedures and study concepts. Y.K. and N.T. prepared the figures. Y.K., N.T., and A.N. wrote the manuscript. S.K. and A.N. finalized the manuscript. All authors approved the final version of the manuscript.

The authors declare no conflicts of interest.