Activation of Mast-Cell-Expressed Mas-Related G-Protein-Coupled Receptors Drives Non-histaminergic Itch

Abstract

Classical itch studies have focused on immunoglobulin E (IgE)-mediated mast cell activation and histamine release. Recently, members of the Mas-related G-protein-coupled receptor (Mrgpr) family have been identified as mast cell receptors, but their role in itch is unclear. Here, we report that mast cell activation via Mrgprb2 evoked non-histaminergic itch in mice independently of the IgE-Fc epsilon RI (FcεRI)-histamine axis. Compared with IgE-FcεRI stimulation, Mrgprb2 activation of mast cells was distinct in both released substances (histamine, serotonin, and tryptase) and the pattern of activated itch-sensory neurons. Mrgprb2 deficiency decreased itch in multiple preclinical models of allergic contact dermatitis (ACD), a pruritic inflammatory skin disorder, and both mast cell number and PAMP1-20 concentrations (agonist of the human Mrgprb2 homolog, MRGPRX2) were increased in human ACD skin. These findings suggest that this pathway may represent a therapeutic target for treating ACD and mast-cell-associated itch disorders in which antihistamines are ineffective.

Keywords: GPCR; MRGPRX2; Mrgpr; Mrgprb2; histamine; itch; mast cell; pruritus; receptor.

Figure 1.. Compared with FcεRI Activation, Mrgprb2 Agonist PAMP9–20 Elicits Non-histaminergic Itch and Differential Pruritogen Release

(A–H) Scratching bouts from injection of pruritogen at either the nape or the cheek (mean plus SEM depicted). Each open circle represents an independent mouse data point. *p < 0.05; ***p < 0.001; n.s., not significant by two-tailed unpaired Student’s t test. (A and B) PAMP9–20 injection into either the nape (50 μL) (A) or cheek (10 μL) (B) of WT and Mrgprb2^−/− (B2 −/−) animals. (A) 8 μg: WT, n = 6; B2 −/−, n = 7; 24 μg: WT, n = 13, B2 −/−, n = 12; 40 μg: WT, n = 5, B2 −/−, n = 5. (B) 4.8 μg: WT, n = 4, B2 −/−, n = 4; 8 μg: WT, n = 5, B2 −/−, n = 4. (C) Injection of 24 μg PAMP9–20 (50 μL of 300 μM) at the nape in the presence of either vehicle (Veh) or cromolyn. Vehicle, n = 9; cromolyn n = 8. (D) Injection of 24 μg PAMP9–20 (50 μL of 300 μM) into the nape of WT and SASH (cKit^W-sh) mice. For both, n = 8. (E) 1 μg anti-IgE injection into WT and B2 −/− mice. WT, n = 7; B2 −/−, n = 6. (F) 50 μg ovalbumin injection into sensitized WT and B2 −/− animals. WT, n = 6; B2 −/−, n = 9. (G) 1 μg anti-IgE injection at the cheek with the indicated antagonist. Vehicle, n = 13; H1 blocker, n = 9; H4 blocker, n = 14; ritanserin, n = 10; mianserin, n = 6; cyproheptadine, n = 8; ondansetron, n = 5. (H) 24 μg PAMP9–20 injection at the nape with the indicated antagonist. Vehicle, n = 15; H1 blocker, n = 11; H4 blocker, n = 16; ritanserin, n = 14; mianserin, n = 6; cyproheptadine, n = 8; ondansetron, n = 5. (I–K) In vitro release of histamine (I), serotonin (J), and tryptase beta 2 (K) from mouse peritoneal mast cells upon stimulation by various concentrations of either PAMP9–20 or anti-IgE. Open circles depict independent biological replicates from peritoneal mast cells isolated from >4 animals (mean plus SEM depicted). *p < 0.05; ***p < 0.001 by two-tailed unpaired Student’s t test. (I) PAMP9–20: 2.5, n = 8; 25, n = 5; 125, n = 7. For anti-IgE: 2.5, n = 5; 8, n = 7; 25, n = 8. (J and K) All concentrations n = 3. See also Figures S1 and S2.

Figure 2.. Differential Mast Cell Agonism Exhibits Varied Sensory Neuron Activation Profiles

(A and A′) Graphical depiction (A) and experimental flowchart (A′) of in vivo Ca²⁺ imaging of Pirt-Cre-GCaMP6s sensory neurons. White arrowheads indicate activated neurons. (B) Total number of activated neurons for the labeled compound (mean plus SEM depicted). Open circles represent imaging trials from independent mice. **p < 0.01; ***p < 0.001; n.s., not significant by two-tailed unpaired Student’s t test. PAMP9–20: WT, n = 12, B2 −/−, n = 5; compound 48/80: WT, n = 5, B2 −/−, n = 5; anti-IgE: WT, n = 3, B2 −/−, n = 4. (C, D, C′, and D′) Averaged Ca²⁺ imaging traces with ±95% confidence intervals (CIs) (C and D) and heatmaps (C′ and D′) of individual neurons activated by 300 mM PAMP9–20 (C and C′; n = 72) and 100 μg/mL anti-IgE (D and D′; n = 72).<p/>(E–G) Box (25^th and 75^th percentiles) and whisker (max and min) plots. **p < 0.01; ***p < 0.001 by two-tailed unpaired Student’s t test. (E) Time to first detected activation within trial period for neurons activated by the indicated test compounds. PAMP9–20, n = 95; CQ, n = 79; anti-IgE, n = 79. (F) Number of Ca²⁺ signal peaks (>20% increase over baseline) within imaging trial period. PAMP9–20, n = 74; anti-IgE, n = 85. (G) Positive peak area (amplitude over baseline 3 duration) within the imaging trial period. PAMP9–20, n = 74; anti-IgE, n = 85. See also Figure S3.

Figure 3.. Mrgprb2 Agonism Excites Multiple Itch Sensory Neuron Subtypes in an Activation Pattern Distinct from FcεRI

(A) Graphical depiction of itch neuron subtypes. (A′) Experimental flowchart for in vivo Ca²⁺ imaging of mast cell agonists plus neuronal activators. (B) Percent overlap of activated sensory neurons from a peripheral injection of 300 mM PAMP9–20 and a repeat injection of either 300 μM PAMP9–20 (n = 4) or 10 μg/mL compound 48/80 (n = 3). The percentages for these and subsequent data were normalized to repeated PAMP 9–20 injection. (C) Normalized percentage overlap between 300 μM PAMP9–20 and 10 mM chloroquine (CQ; n = 5), 1 mM 5-HT (n = 6), or 10 mM β-alanine (n = 5) and between anti-IgE and CQ (n = 3), 5-HT (n = 4), or β-alanine (n = 4). (D) Normalized percentage overlap between PAMP9–20 and either 9 mM histamine (n = 3) or 3 mM capsaicin (n = 3) and between anti-IgE and either histamine (n = 4) or capsaicin (n = 4). (E–G and E′–G′) Averaged Ca²⁺ imaging traces with ±95% CIs (E–G) and heatmaps (E′–G′) of individual neurons activated by both PAMP9–20 and CQ (n = 42; E and E′), 5-HT (n = 42; F and F′), or β-alanine (n = 38; G and G′). (H) Total number of neurons activated by the indicated test compound in the presence of vehicle, cetirizine (H1R antagonist), or JNJ7777120 (H4R antagonist). For anti-IgE: vehicle (veh), n = 4; cetirizine, n = 3; JNJ7777120, n = 3. For PAMP9–20: vehicle, n = 5; cetirizine, n = 3; JNJ7777120, n = 3. For histamine (Hist): vehicle, n = 5; cetirizine, n = 3; JNJ7777120, n = 3. For 5-HT: vehicle, n = 3; cetirizine, n = 3; JNJ7777120, n = 3. (C–H) Mean plus SEM is depicted. Open circles represent individual, imaged mouse DRGs. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant; two-tailed unpaired Student’s t test. See also Figure S3.

Figure 4.. Mast Cell Mrgprs Mediate a Component of Murine ACD Itch, and PAMP1–20, an MRGPRX2 Agonist, Is Upregulated in Human ACD Skin

(A–C) Scratching bouts in WT and Mrgprb2^−/− animals with ACD (mean plus SEM depicted). Open circles represent independent mice. *p < 0.05; ***p < 0.001 by two-tailed unpaired Student’s t test. For all compounds, vehicle was acetone: olive oil (4:1 v/v). (A) ACD elicited by squaric acid dibutyl ester (SADBE). Vehicle: WT, n = 8, B2 −/−, n = 6; SADBE: WT, n = 13, B2 −/−, n = 12. (B) 2-Phenyl-5-oxazolone (oxazolone). WT, n = 11, B2 −/−, n = 13. (C) 1-Chloro-2,4-dinitrobenzene (DNCB): WT, n = 9, B2 −/−, n = 8. (D) Representative flow cytometry plots of DNCB-treated ear skin and vehicle treated ear skin from WT and B2 −/− animals. Numbers indicate the percentage of cells within boxes. (E and E′) Representative images of control (E) and ACD (E′) human skin stained with avidin-fluorescein isothiocyanate (FITC). White arrows indicate positive staining. Scale bar, 100 μM. (F) Number of avidin-stained cells per square millimeter (mean plus SEM depicted). Open circles represent separate sections. *p < 0.05 by two-tailed unpaired Student’s t test. Control (CTRL), n = 5; contact dermatitis, n = 5. (G) Number of CD45⁺ leukocytes per biopsy from DNCB- and vehicle-treated WT and B2 −/− mouse ear skin (mean plus SEM depicted). Open circles represent independent samples from separate mice. **p < 0.01 by two-tailed unpaired Student’s t test. For vehicle: WT, n = 3, B2 −/−, n = 3. For DNCB: WT, n = 12, B2 −/−, n = 19. (H–H″) Representative images of control (H) and ACD human skin (H′ and H″) stained with either anti-PAMP (1–20) (human) antiserum (H and H′) or antiserum preabsorbed with excess PAMP1–20 peptide (H″). H′ and H″ are from adjacent sections from the same patient. (I) Number of PAMP1–20-positive cells as a percentage of DAPI. Control, 0/1,616; ACD, 1,669/2,071; ACD preabsorbed, 63/2,097. ***p < 0.001 by chi-square test. Samples from at least 5 different ACD patients were tested. See also Figure S4.