Murepavadin, a Small Molecule Host Defense Peptide Mimetic, Activates Mast Cells via MRGPRX2 and MrgprB2

Abstract

Pseudomonas aeruginosa is a frequent cause of hospital-acquired wound infection and is difficult to treat because it forms biofilms and displays antibiotic resistance. Previous studies in mice demonstrated that mast cells (MCs) not only contribute to P. aeruginosa eradication but also promote wound healing via an unknown mechanism. We recently reported that host defense peptides (HDPs) induce human MC degranulation via Mas-related G protein-coupled receptor-X2 (MRGPRX2). Small molecule HDP mimetics have distinct advantages over HDPs because they are inexpensive to synthesize and display high stability, bioavailability, and low toxicity. Murepavadin is a lipidated HDP mimetic, (also known as POL7080), which displays antibacterial activity against a broad panel of multi-drug-resistant P. aeruginosa. We found that murepavadin induces Ca²⁺ mobilization, degranulation, chemokine IL-8 and CCL3 production in a human MC line (LAD2 cells) endogenously expressing MRGPRX2. Murepavadin also caused degranulation in RBL-2H3 cells expressing MRGPRX2 but this response was significantly reduced in cells expressing missense variants within the receptor's ligand binding (G165E) or G protein coupling (V282M) domains. Compound 48/80 induced β-arrestin recruitment and promoted receptor internalization, which resulted in substantial decrease in the subsequent responsiveness to the MRGPRX2 agonist. By contrast, murepavadin did not cause β-arrestin-mediated MRGPRX2 regulation. Murepavadin induced degranulation in mouse peritoneal MCs via MrgprB2 (ortholog of human MRGPRX2) and caused increased vascular permeability in wild-type mice but not in MrgprB2^-/- mice. The data presented herein demonstrate that murepavadin activates human MCs via MRGPRX2 and murine MCs via MrgprB2 and that MRGPRX2 is resistant to β-arrestin-mediated receptor regulation. Thus, besides its direct activity against P. aeruginosa, murepavadin may contribute to bacterial clearance and promote wound healing by harnessing MC's immunomodulatory property via the activation of MRGPRX2.

Keywords: MRGPRX2; MrgprB2; antimicrobial peptides; host defense peptides; mast cells; murepavadin.

Figure 1

Murepavadin induces intracellular Ca²⁺ mobilization, degranulation and causes chemokine production in human MCs. (A) Ca²⁺ mobilization measurement following murepavadin (10 μM) stimulation of Fura-2-loaded LAD2 cells. (B) LAD2 cells were exposed to indicated concentrations of murepavadin (30 min) and degranulation was assayed by measuring the release of β-hexosaminidase. (C, D) LAD2 cells were exposed to 10 μM of murepavadin for 24 h and the production of IL-8 and CCL3 were determined by ELISA. Data presented are the mean ± SEM of at least three experiments. Statistical significance was determined by t-test or one-way ANOVA with Dunnett’s multiple comparisons at a value **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 2

Murepavadin induces intracellular Ca²⁺ mobilization, degranulation and chemokine production in MCs through MRGPRX2. (A) Calcium mobilization measurement following murepavadin (10 μM) stimulation of Fura-2 loaded WT-RBL and RBL-MRGPRX2 cells. (B) WT-RBL and RBL-MRGPRX2 cells were exposed to 10 μM murepavadin (30 min), and degranulation was assayed by measuring the release of β-hexosaminidase. (C) RBL-MRGPRX2 cells were incubated in the presence or absence of pertussis toxin (PTx), at a concentration of 100 ng/mL for 16 h and exposed to indicated concentrations of murepavadin (30 min) and degranulation was assayed by measuring the release of β-hexosaminidase. (D, E) WT-RBL and RBL-MRGPRX2 cells were incubated in the presence or absence of pertussis toxin (PTx) at a concentration of 100 ng/mL for 16 h and exposed to 10 μM of murepavadin (24 h) and the production of cytokine TNF-α and chemokine CCL2 were quantified by ELISA. Data presented are the mean ± SEM of at least three experiments. Statistical significance was determined by two-way ANOVA with Šídák’s or Tukey’s multiple comparisons at a value *p < 0.05, ***p < 0.001, ****p < 0.0001, and ns denotes “not significant”.

Figure 3

Naturally occurring MRGPRX2 missense variants are hypo-responsive to murepavadin for MC degranulation. (A) Snake diagram of MRGPRX2 indicating the two amino acid residues to be investigated. (B) Single amino acid substitution for each of the MRGPRX2 variant is shown in the table. (C) Cell surface receptor expression of the wild-type MRGPRX2 (WT) and its missense variants (G165E and V282M) was confirmed using flow cytometry. (D) RBL cells expressing MRGPRX2 (WT) and its variants (G165E and V282M) were exposed to 10 μM murepavadin (30 min) and degranulation was assayed by measuring the release of β-hexosaminidase. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons at a value **p < 0.01 and ****p < 0.0001.

Figure 4

Murepavadin does not promote β-arrestin recruitment following MRGPRX2 activation. (A, B) HTLA-MRGPRX2 cells were exposed to indicated concentrations of C48/80 or murepavadin for 16 h. Medium was removed and Bright-Glo solution (100 µL) was added into each well (96-well plate) and β-arrestin gene expression (in relative luminescence unit) was measured. (C, D) HTLA-MRGPRX2 cells were exposed to MRGPRX2 ligand (C48/80 or murepavadin, 16 h) and cell surface receptor expression of MRGPRX2 was confirmed using flow cytometry and quantified using a mean fluorescent intensity (MFI) in comparison to the untreated control. (E, F) Representative histograms of MRGPRX2 cell surface receptor expression of HTLA-MRGPRX2 cells. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons at a value ****p < 0.0001 and ns denotes “not significant”.

Figure 5

Murepavadin does not induce MRGPRX2 internalization or desensitization. (A) RBL-MRGPRX2 cells were exposed to MRGPRX2 ligand (C48/80 3 μg/mL or murepavadin 10 μM, 16 h) and cell surface receptor expression of MRGPRX2 was confirmed using flow cytometry. A representative histogram of MRGPRX2 cell surface receptor expression is shown. (B) Quantitative analysis of cell surface receptor expression was calculated using a mean fluorescent intensity (MFI) in comparison to the untreated control. (C, D) Calcium mobilization measurements of RBL-MRGPRX2 cells exposed to MRGPRX2 ligand (C48/80 3 μg/mL or murepavadin 10 μM, 16 h) following C48/80 (3 μg/mL) or murepavadin (10 μM) stimulation, respectively. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons and two-way ANOVA with Šídák’s multiple comparisons at a value *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns denotes “not significant”.

Figure 6

Murepavadin activates murine MCs in vitro and in vivo via MrgprB2. (A) WT and MrgprB2^-/- PMCs were exposed to 10 μM murepavadin (30 min) and degranulation was assayed by measuring the release of β-hexosaminidase. (B) Representative images of Evan’s blue dye extravasation in WT and MrgprB2^-/- mice in vivo. Mice were intradermally injected with 20 μL of murepavadin (30 μM) or PBS, and extravasation of Evan’s blue dye was determined after 30 min. (C) Quantification of extravasation of Evan’s blue dye in WT and MrgprB2^-/- mice (n = 7-8). Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons at a value **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns denotes “not significant”.

Figure 7

Proposed mechanism of action of murepavadin/MRGPRX2-induced bacterial clearance and wound healing. Murepavadin activates MCs via MRGPRX2. An increase in intracellular calcium following murepavadin stimulation triggers MC degranulation and chemokine production (IL-8, CCL3, CCL2, and TNF-α). These MC mediators induce immune cell recruitment and subsequently contribute to host defense and wound healing.