Anti-TNP IgE (Cat: 557079, 0.5 mg/mL) and Rat Anti-Substance-P (556312) were purchased from BD Pharmingen (Franklin Lakes, NJ, USA). TNP-Ova (sc-396493) was purchased from ChemCruz (Dallas, TX, USA). Hank’s balanced salt solution (HBSS), 0.5 M ethylenediaminetetraacetic acid (EDTA), and type IV collagenase (17104-019) were purchased from Life Technologies Corporation (Grand Island, NY, USA). DNase I (10104159001) was purchased from Roche Diagnostics (Mannheim, Germany). Dispase II (D4693-1G), periodic acid–Schiff reagent (395B-1KT), and toluidine blue (T3260-5G) were purchased from Sigma Aldrich (St. Louis, MO, USA). ACK lysing buffer (#118-156-721) was purchased from Quality Biological (Gaithersburg, MD, USA). All flow cytometry antibodies including Zombie Yellow (77168), CD16/32 (clone 93, 101320), CD45-pacific blue (clone 30-F11, 103126), CD11b-per CP-cy5.5 (clone M1/70, 101228), Ly6G-FITC (clone 1A8, 127606), CD170-PE (Siglec-F, clone S17007L, 155506), and Fixation Buffer (#420801) were purchased from BioLegend (San Diego, CA, USA). UltraComp eBeads (01-3333-42), Alexa Fluor 647-conjugated donkey anti-mouse IgG (A31571), Alexa Fluor 647-conjugated donkey anti-rat IgG (A48272), and Alexa Fluor 488-conjugated goat anti-rabbit IgG (A11008) secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA). Protein Gene Product (PGP) 9.5 rabbit polyclonal antibody (GTX109637) was purchased from GeneTex (Irvine, CA, USA). Human/mouse myeloperoxidase (MPO) and polyclonal goat IgG antibody (AF-3667-SP) were purchased from R&D Systems (Minneapolis, MN, USA). Anti-mouse monoclonal eosinophil peroxidase (EPx) antibody (clone MM25-8.2.2) was obtained from Mayo Clinic, Phoenix, AZ, USA. Alexa Fluor 647-conjugated donkey anti-goat IgG (705-605-003) and Alexa Fluor 488-conjugated donkey anti-rat (712-545-150) antibodies were purchased from Jackson ImmunoResearch Laboratories (West Groove, PA, USA). Rodent block M (RBM961g) was purchased from BioCare Medical (Pacheco, CA, USA). DAPI and ProLong Gold antifade reagent were purchased from Molecular Probes (Eugene, OR, USA). RNeasy Fibrous Tissue Kit was purchased from Qiagen (Hilden, Germany). High-Capacity RNA-to-cDNA Kit (#4368814) and Fast SYBR Green Master mix (#4385612) were purchased from Applied Biosystems (Vilnius, Lithuania).

Mice were kept in a pathogen-free environment and on hardwood bedding that had been autoclaved. All investigations were carried out on 8–10-week-old mice of both genders. CRISPR/Cas9 technology was used by the CRISPR core at the University of Pennsylvania to develop MrgprB2^−/− mice as described previously (40), and C57BL/6 [wild-type (WT)] mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). β-arr2 ^flox/flox (β-arr2^fl/fl ) mice were a kind gift from Robert Lefkowitz and Julia Walker (Duke University Medical Center, Durham, NC, USA); Cpa3-Cre (Cre is expressed under the promoter of Cpa3, expressed only in MCs) mice were generously provided by Stephen J. Galli (Stanford University, Stanford, CA, USA). As previously described, animals with an MC-specific deletion of β-arr2 (Cpa3^Cre+ /β-arr2^fl/fl ) were produced by mating Cpa3Cre mice with β-arr2^fl/fl mice (41). The University of Pennsylvania’s Institutional Animal Care and Use Committee granted approval (Protocol Number 803883) for the use of mice.

Lung inflammation was induced in the experimental model by modifying the protocol of Jin et al. (6). Mice were anesthetized using 50 µL of ketamine–xylazine intraperitoneally and were passively sensitized with 10 µg of anti-TNP IgE in 100 µL phosphate-buffered saline (PBS) through retro-orbital injection. After 24 hours, mice were anesthetized, and 200 µg of antigen (TNP-Ova) or PBS was administered intranasally. After 24 hours of intranasal challenge, mice were sacrificed and used for experiments, as described below.

The tracheas of individual mice were exposed and cannulated. To obtain BAL, lungs were instilled with 1 mL of 1× HBSS with 100 µM EDTA three times (6). The first flush was kept separately to collect cell-free supernatant for ELISA and stored at − 80°C.

Lung samples from experimental mice were fixed in 10% formalin overnight. Samples were dehydrated through a series of increasing ethanol washes and embedded in paraffin. Paraffin-embedded samples were sliced into 5 μm cross sections and stained with toluidine blue as described previously (42). Quantitation of MC was conducted by counting the number in a total of five fields from different lung tissue sections of both control and challenged mice, and the mean was calculated in each mouse. The lung sections were also stained with H&E.

Periodic acid–Schiff (PAS) staining was performed according to the manufacturer’s protocol. Briefly, sections were deparaffinized and hydrated. The slides were immersed in periodic acid solution for 5 min, rinsed and immersed in Schiff’s reagent for 15 min at room temperature (RT), washed and counterstained with hematoxylin, and washed. Finally, the slides were dehydrated and mounted in xylene-based mounting media. Images were captured by Nikon Eclipse Ni microscope (Minato City, Tokyo, Japan) using ×20 magnification.

Single-cell suspension was prepared by digesting lung tissue in RPMI10 media containing type IV collagenase (600 U/mL), dispase II (1 mg/mL), and DNase I (25 μg/mL) for 30 min at 37°C. The digested tissue was filtered through a 70-μm filter, washed, and suspended in FACS buffer [PBS containing 2% fetal bovine serum (FBS)]. Each sample was Fc blocked with anti-CD16/32 antibody (1:100) for 15 min at 4°C. Samples were then stained with respective antibody cocktail (neutrophils —Zombie Yellow, CD45, CD11b, and Ly6G; eosinophils —Zombie Yellow, CD45, CD11b, and CD170) for 30 min at 4°C. Samples were washed and then fixed with Fixation Buffer. Data were acquired using a BD LSR II flow cytometer (San Jose, CA, USA) and analyzed using the FlowJo software version 10.8.1 (Tree Star Inc., Ashland, OR, USA). For single stain compensation, beads (UltraComp eBeads) were used (43).

Immunofluorescence experiments were performed using the same tissue blocks as used in histological studies. EPx staining was conducted following the protocol as described previously (44). Briefly, tissue sections were deparaffinized, hydrated, antigen retrieved, and incubated with pepsin for 30 min at room temperature. The sections were then washed, blocked with rodent block M, and incubated with mouse anti-EPx (1:50) primary antibody overnight at 4°C. Sections were then washed three times with PBS containing 0.1% Tween 20 and incubated with Alexa Fluor 647-conjugated donkey anti-mouse IgG secondary antibodies (1:750). The tissue sections were then washed and mounted with DAPI.

Neutrophil (MPO), substance P, and PGP9.5 staining were performed as described previously, with some modifications (45–47). Briefly, tissue sections were deparaffinized, hydrated, antigen retrieved, and blocked with 1% bovine serum albumin (BSA) in PBS-T (PBS containing 0.2% Triton X-100). Tissue sections were incubated with human/mouse MPO polyclonal goat IgG (1:250), rat anti-SP (1:200), and PGP9.5 rabbit polyclonal (1:250) primary antibodies overnight at 4°C. Sections were then washed three times with PBS and incubated with Alexa Fluor 647-conjugated donkey anti-goat IgG (1:1,500), Alexa Fluor 488-conjugated donkey anti-rat IgG (1:500), Alexa Fluor 647-conjugated donkey anti-rat IgG (1:1,500), and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:750) for 1 hour at room temperature in the dark. The tissue sections were then washed and mounted with DAPI. Images were acquired using a Nikon Eclipse Ni microscope.

Lung samples were homogenized, and total RNA was extracted and purified by RNeasy Fibrous Tissue Kit. A total of 1 µg of RNA was used to synthesize cDNA using the High-Capacity RNA-to-cDNA Kit according to the manufacturer’s instructions. All the qRT-PCRs were performed using Fast SYBR Green Master Mix. Using specific primers, the mRNA expressions of mouse MrgprB2 (forward 5′-TACTTCTGCAGAGAGCCATGC-3′, reverse 5′-GCTGCAGCTCTGAACAGTTTC-3′), TNF-α (forward 5′-TCTCATGCACCACCATCAAGGACT-3′, reverse 5′-ACCACTCTCCCTTTGCAGAACTCA-3′), IL-4 (forward 5′-TCCTCACAGCAACGAAGAACACCA-3′, reverse 5′-GCAGCTTATCGATGAATCCAGGCA-3′), IL-5 (forward 5′-CTCTGTTGACAAGCAATGAGACG 3′, reverse 5′-TCTTCAGTATGTCTAGCCCCTG-3′), IL-13 (forward 5′-AGACCAGACTCCCCTGTGCA-3′, reverse 5′-TGGGTCCTGTAGATGGCATTG-3′), Tac1 (forward 5′-GGCCAAGGAGAGCAAAGA-3′, reverse 5′-CGAGGATTTTCATGTTCGATT-3′), Tac4 (forward 5’-GTAGCTTCCTCAGCCATGCAG-3, reverse 5’-CCGCCCCCAAATACAATACA-3’), and Actin (forward 5′-GATTACTGCTCTGGCTCCTAGC-3′, reverse 5′-GACTCATCGTACTCCTGCTTGC-3′) were analyzed using QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The analysis was performed in triplicate, and Actin was used as an internal control. All data presented as fold changes of target genes were determined using the 2^−ΔΔCt method with actin as a stable reference gene.

Mouse BAL TNF-α and IL-13 were assessed using immunoassay kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. The limits of detection for each enzyme-linked immunosorbent assay (ELISA) were 5–10 pg/mL.

Data shown are mean ± SEM values derived from at least three independent experiments. Statistical significance was determined by two-way ANOVA. Error bars represent mean ± SEM from at least three independent experiments. Significant differences were set at *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001 and analyzed using GraphPad Prism, version 6.07.

Jin et al. (6) recently utilized IgE sensitization and antigen challenge to determine the role of MC-derived cytokines in AAI in vivo. We used a similar approach to determine if MrgprB2 contributes to IgE-mediated responses. For this, we passively sensitized WT and MrgprB2^−/− mice with TNP-specific IgE, challenged with antigen (TNP-Ova), and sacrificed the mice the following day. As expected, no transcript for MrgprB2 could be detected in the lung of MrgprB2^−/− mice before or after IgE sensitization and antigen challenge ( Figure 1A ). However, MrgprB2 transcript was present in the lung of WT mice, and this was significantly increased in the lung following IgE-sensitized and antigen challenge. To determine if this correlated with increased MC recruitment, we performed toluidine blue staining of lung sections. As shown in Figures 1B, C , IgE/antigen exposure resulted in a significant increase in MC numbers in the lung when compared to the control challenge. However, this increase was significantly reduced in the lung of MrgprB2^−/− mice. These findings suggest that following IgE/antigen exposure, MCs are recruited to the lung through signaling via MrgprB2.

Airway sensory neurons express FcεRI, and its activation results in increased expression of Tac1 and the release of its gene product SP, which serves to amplify type 2 (AAI) in mice (12). In addition, FcεRI stimulation of MCs promotes autocrine secretion of the neuropeptide hemokinin-1 (HK-1), which provides an adjuvant effect for the development of MC-mediated AAI (48). We therefore sought to determine if IgE/antigen exposure results in the upregulation of Tac1 and Tac4 transcripts (encoding SP and HK-1, respectively). As shown in Figure 1D , IgE/antigen exposure resulted in no significant increase in the expression of Tac4 transcript in either WT or MrgprB2^−/− mice. By contrast, there was a ~6-fold increase in Tac1 expression in WT mice, but this response was abolished in MrgprB2^−/− ( Figure 1E ). We also performed immunofluorescence staining of the lung sections to determine if the changes in Tac1 expression correlated with the generation of SP. As shown in Figures 1F, G , IgE/antigen exposure resulted in increased SP expression in WT mice, but this response was substantially reduced in MrgprB2^−/− mice.

Serhan et al. (5) recently showed that SP expression is restricted to PGP9.5+ mouse cutaneous neuronal fibers. An immunofluorescence study using a PGP9.5 antibody has recently been used to characterize the presence of nerve fibers in whole-mount human airway biopsies (49). Furthermore, in guinea pigs, PGP9.5+ and SP immunoreactive fibers are increased during respiratory syncytial virus infection (47). We therefore used a double immunofluorescence staining to determine the expression of PGP9.5+ nerve fibers (Green) and SP (Red) in mouse lungs following IgE sensitization and antigen challenge ( Figure 1H ). We found that compared to control, IgE/antigen caused a substantial increase in PGP9.5+ immunofluorescence, which colocalized with SP. Furthermore, this increase in double immunofluorescence was substantially reduced in MrgprB2^−/− mice ( Figure 1H ).

MC-derived TNF-α contributes to the pathogenesis of AAI, likely via Th2 cytokine production (50). Studies with human bronchial biopsy samples from subjects with asthma showed that MCs express IL-4 and IL-13 (2). Furthermore, IL-5 contributes to eosinophil activation and bronchial hyperreactivity (51). Jin et al. (6) recently showed that aggregation of FcεRI on mouse bone marrow-derived MCs (BMMCs) results in the generation of TNF-α, IL-4, IL-5, and IL-13. Therefore, we quantitated the transcripts for cytokines in the lung of IgE-sensitized/antigen-challenged WT and MrgprB2^−/− mice. As shown in Figures 2A–D , IgE/antigen exposure resulted in the enhancement of TNF-α, IL-4, IL-5, and IL-13 transcripts, and these responses were significantly reduced in MrgprB2^−/− mice. We also measured the generation of TNF-α and IL-13 in BAL. As shown in Figures 2E, F , IgE/antigen exposure resulted in the secretion of TNF-α and IL-13, and these responses were significantly reduced in MrgprB2^−/− mice.

To determine if the induction of these cytokines is associated with leukocyte recruitment, we first performed H&E staining of lung sections. We found an increased number of inflammatory cells around the bronchioles of WT mice following IgE/antigen exposure, but this response was almost absent in MrgprB2^−/− mice ( Supplementary Figure S1A ). We then used cytospin preparations of BAL cells for immunofluorescence staining of eosinophils. As shown in Supplementary Figures S1B, C , IgE/antigen exposure resulted in eosinophil recruitment, which was substantially reduced in MrgprB2^−/− mice. We also used lung digest for flow cytometry analysis for eosinophil and neutrophil recruitment (gating strategy, Supplementary Figure S5 ). Antigen challenge resulted in both eosinophil and neutrophil recruitment in the lung of WT mice, but these responses were significantly reduced in MrgprB2^−/− mice ( Figures 3A–D ). Reduced eosinophil ( Supplementary Figure S2A ) and neutrophil ( Supplementary Figure S2B ) recruitment were also observed after immunofluorescence staining in paraffin-embedded lung tissue sections of the same experimental mice. The increase in PAS-positive cells among the bronchial epithelial cells indicates goblet cell hyperplasia (52). To detect goblet cells in the bronchiolar epithelium, lung tissue sections were stained with PAS. IgE/antigen exposure resulted in an increased number of PAS-positive cells in WT mice, but this response was significantly reduced in MrgprB2^−/− mice ( Figures 3E, F ).

β-arr2 expressed in hematopoietic cells is required for eosinophil recruitment in the development of AAI in mice through dephosphorylation of the actin-binding protein cofilin (36, 38). We have recently shown that MrgprB2-mediated β-arr2 biased signaling for MC chemotaxis through dephosphorylation of cofilin contributes to rosacea-like inflammation in mice (39). Furthermore, the findings described above that decreased MC number in MrgprB2^−/− mice is associated with reduced cytokine generation, AAI, and mucus secretion raises the interesting possibility that β-arr2-mediated MC recruitment is required to induce these responses. To test this possibility, we utilized Cpa3^Cre+ /β-arr2^fl/fl and Cpa3^Cre− /β-arr2^fl/fl mice for IgE sensitization and challenge studies as described for MrgprB2^−/− mice above. We found that MC-specific deletion of β-arr2 resulted in almost complete inhibition of expression of MrgprB2 transcript ( Figure 4A ), lung MC count ( Figures 4B, C ), Tac1 transcript ( Figure 4D ), and SP generation ( Figures 4E, F ) and its localization with PGP9.5 ( Figure 4G ). The absence of β-arr2 in MCs also resulted in a significant reduction of TNF-α, IL-4, IL-5, and IL-13 transcripts in lung tissue ( Figures 5A–D ) and cytokines in BAL ( Figures 5E, F ).

IgE/antigen exposure caused inflammatory cell recruitment around the bronchioles of control (Cpa3^Cre− /β-arr2^fl/fl ) mice, but this was substantially reduced in Cpa3^Cre+ /β-arr2^fl/fl mice ( Supplementary Figure S3A ). The increased number of eosinophils in BAL ( Supplementary Figure S3B, C ) was also significantly reduced in Cpa3^Cre+ /β-arr2^fl/fl mice. Similarly, neutrophil and eosinophil recruitment in lung digest and PAS staining in the lung tissue, following IgE/antigen challenge, were significantly reduced in the absence of β-arr2 in MCs ( Figures 6A–F ). Reduced eosinophil ( Supplementary Figure S4A ) and neutrophil ( Supplementary Figure S4B ) recruitment were also observed after immunofluorescence staining in paraffin-embedded lung tissue sections of the same experimental mice. These findings suggest that IgE-mediated cytokine generation and AAI require β-arr2-mediated MC recruitment.