Asthma, accompanied by lung inflammation, bronchoconstriction and airway hyper-responsiveness, is a significant public health burden. Here we report that Mas-related G protein-coupled receptors (Mrgprs) are expressed in a subset of vagal sensory neurons innervating the airway and mediates cholinergic bronchoconstriction and airway hyper-responsiveness. These findings provide insights into the neural mechanisms underlying the pathogenesis of asthma.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
We thank M. Anderson at Johns Hopkins University Pain Imaging Core for assisting with ex vivo vagal GCaMP imaging. We thank S. Klein at Johns Hopkins University Department of Molecular Microbiology and Immunology for providing the mouse-adapted influenza A/California/04/2009 H1N1 virus. We thank R. Rabold and L. Zhen for providing technical support for Flexivent experiments. This study was supported by grants from the NIH (NS054791 to X.D., NS087088 to L.H., HL010342 to W.M., HL112919 to B.U., DK110366 to M.K. and HL122228 to B.C.) and by the American Asthma Foundation (to X.D.).
Integrated supplementary information
. (a) RT-PCR analysis of Mrgprc11 expression in various tissues from wild-type mice. Mrgprc11 was only detected in the DRG and vagal ganglia. (b) RT-PCR analysis of hMrgprx1 expression in various tissues. hMrgprx1 was detected in the jugular ganglion, but not in the nodose ganglion or lung. The hMrgprx1 gene sequence only contains one exon; therefore, primers for RT-PCR are not intron-spanning. “–RT” controls were used to verify the complete digestion of genomic DNA. hTrpv1 expression was detected in both jugular and nodose ganglia, but not in the lung. The results were repeated with tissues collected from 5 different donors. All the PCR results were confirmed by DNA sequencing. (c-e) Sections of vagal ganglia from Wnt-1Cre; ROSA26lacZ mice were stained with antibodies against MrgprC11 (red) and LacZ (green). Mice usually have a fused nodose-jugular complex with most jugular neurons situated on the rostral side and most nodose neurons situated on the caudal side. Arrowheads mark MrgprC11+ neurons. MrgprC11 was expressed in the jugular sensory neurons, which are located in the rostral part of the vagal ganglia. Multiple images were stitched together to show the whole section. Scale bar represents 50 μm. All the immunostaining and RT-PCR experiments were repeated independently three times with similar results.
. Rabbit polyclonal MrgprC11 antibody was custom-made by Proteintech Group, Inc. The specificity of the antibody was verified by immunostaining of DRG sections (a) and heterologous cells (b). (a) A subpopulation of small-diameter DRG sensory neurons from WT, but not Mrgpr-clusterΔ-/- mice, was recognized by the antibody, demonstrating that the antibody detects one (or several) of the 12 Mrgprs deleted in the Mrgpr-clusterΔ-/- mice. (b) The 12 Mrgprs that were deleted in Mrgpr-clusterΔ-/- mice were cloned into a mammalian expression vector and transfected individually into human embryonic kidney (HEK) 293 cells. Only HEK293 cells expressing MrgprC11 were stained by the antibody, whereas cells expressing the other 11 Mrgprs were not recognized by the antibody. These results demonstrate that the antibody we generated specifically detects MrgprC11. Scale bar represents 50 μm. The experiments were repeated independently three times with similar results.
Supplementary Figure 3 Itch receptor MrgprC11 is expressed in a specific subset of vagal sensory neurons.
. (a-i) Characterization of MrgprC11+ jugular sensory neurons. Vagal ganglia sections derived from wild-type mice were stained with antibodies against MrgprC11 (red) and various sensory neuron markers (green). MrgprC11+ jugular sensory neurons do not express the myelinated neuron marker neurofilament 200kD (a-c). The majority of MrgprC11+ jugular sensory neurons (82.8%) were labeled by IB4 (d-f), and 33.8% of MrgprC11+ neurons expressed neuropeptide substance P (g-i). Arrowheads mark representative double-labeled neurons. Scale bar represents 50 μm. The experiments were repeated independently three times with similar results.
. (a) The percentage of total vagal sensory neurons from wild-type and Mrgpr-clusterΔ-/-mice responding to Bam8-22 (2 μM). (n=4 per genotype, p=0.003). (b) Bam8-22 (2 μM) induced action potentials in vagal sensory neurons. All Bam8-22-sensitive neurons (as determined by calcium imaging, n = 6 neurons) elicited a train of action potentials evoked by subsequent Bam8-22 treatment. (c-d) Calcium responses of vagal sensory neurons to Bam8-22 (2 μM), capsaicin (1 μM) and α,β-methylene ATP (10 μM). Bam8-22-responsive vagal sensory neurons also responded to capsaicin (d), but did not respond to α,β-methylene ATP (c), a selective agonist for nodose sensory neurons (n=20 cells). (e) siRNA knockdown of MrgprC11 abolished the Bam8-22-induced calcium responses in vagal sensory neurons from WT mice (n=4 per genotype, p=0.003). (f) Electroporation of expression construct for MrgprC11 rescued the Bam8-22-induced calcium responses in vagal sensory neurons from Mrgpr-clusterΔ-/-mice. (n=4, p=0.006) ***P < 0.005, two-tailed unpaired Student’s t test. Data are reported as mean ± s.e.m. All population data for fluorescence ratio (340/380) (average from ≥ 20 cells) are presented as mean ± s.e.m.
. (a) Since the Mrgprc11 locus in the mouse genome has an extremely high level of repetitive sequences, multiple attempts to generate Mrgprc11 single gene knockout mice by homologous recombination and CRISPR approaches have failed. We have generated an Mrgpr-clusterΔ-/- mouse line in which 12 Mrgpr coding sequences including Mrgprc11 were deleted1. We performed RT-PCR to detect expression of the 12 Mrgpr genes in WT vagal ganglia. The gene expression of Mrgpra1, a3, a10, a19, b4, b5 and c11 were detected in WT vagal ganglia. (b) HEK293 cells were transfected with expression constructs for the 12 Mrgpr genes individually and their responses to Bam8-22 were tested via calcium imaging. Only HEK293 cells expressing MrgprC11 responded to Bam8-22. The black line on the graph indicates the application of Bam8-22 (1 μM). All population data for fluorescence ratio (340/380) (n=20 cells) are presented as mean ± s.e.m. The experiments were repeated independently three times with similar results.
. (a) Perfusion chamber and diagraph showing the ex vivo vagal ganglia GCaMP imaging. The trachea, lung and the intact vagus nerves and vagal ganglia were dissected from Pirt-cre; R26-GCaMP6 mice and were pinned in a two-compartment tissue chamber. The trachea and lung were placed in the airway chamber to receive chemical stimuli. The left vagal ganglia were placed in the VG chamber. There is no fluid exchange between the two chambers. This assay allowed us to monitor the calcium transients in the cell bodies of vagal sensory neurons while stimuli were applied onto the airway innervated by the vagal sensory nerves. Bam8-22 (10 μM) and reversed Bam (10 μM) were administered onto the airway through the cannulated trachea and the surface of the rostral vagal ganglia were imaged by a multiphoton microscope. (b) Representative ex vivo vagal ganglia calcium images showing [Ca2+]in increase in vagal sensory neurons. Neurons activated by Bam8-22 were indicated by the arrowheads. Bam8-22 induced robust Ca2+ increase in 2.67 % (33/1237, 10 mice) of vagal sensory neurons, while reversed Bam8-22 peptide only induced minimal activity of the vagal sensory neurons (0.3%, 3/921, 5 mice). (c) Time course of the average amplitude of the calcium transient evoked by Bam8-22 (33 neurons from 10 mice). The data are expressed as the percentage of baseline calcium transient (ΔF/F0) and are presented as mean ± s.e.m. Black bars indicate when stimuli were applied. Scale bar represents 20 μm. The experiments were repeated independently three times with similar results.
Supplementary Figure 7 Activation of MrgprC11+ jugular sensory neurons changes the respiratory pattern in mice.
. (a-b) Bam8-22 evoked an increase in the amplitude of the respiratory waveform (a) and an increase in the respiratory rate (b) in WT mice, but not in Mrgpr-clusterΔ-/- mice. The respiratory changes reached their peak at 1 min and gradually returned to baseline at 3 min. (WT-Saline, n=9; WT-Bam8-22, n=11; KO-Saline, n=7; KO-Bam8-22, n=8; for (a), WT-Saline vs WT-Bam8-22, p=0.0022; WT-Bam8-22 vs KO-Bam8-22, p=0.029; for (b) WT-Saline vs WT-Bam8-22, p=0.0003; WT-Bam8-22 vs KO-Bam8-22, p=0.0038). Changes in respiration were calculated over a 3-minute time period. (c) Baseline respiratory rate was comparable between wild-type (n=12) and Mrgpr-clusterΔ-/-mice, suggesting that the loss of Mrgprs does not affect the normal functions of the respiratory system (n=8). (P=0.72). (d-e) Ipratropium bromide inhibited the increase in the amplitude of the respiratory waveform (d) but not in the respiratory rate (e) induced by Bam8-22. All methacholine-induced respiratory effects were blocked by ipratropium bromide (d-e). (Naive-Saline, n=6; Ipra-Saline, n=7; Naive-MCh, n=7; Ipra-MCh, n=8; Naive-Bam8-22, n=8; Ipra-Bam8-22, n=8; for (d), Naive-MCh vs Ipra-MCh; p=0.0003; Naive-Bam8-22 vs Ipra-Bam, p=0.0001; for (e), Naive-MCh vs Ipra-MCh, p=0.039; Naive Bam vs Ipra-Bam, p=0.509). ***p< 0.005, *p< 0.05, two-tailed unpaired Student’s t test. Data are reported as mean ± s.e.m.
. (a) Representative trace showing the change of RL after retro-orbital I.V. injection of Bam8-22 (10 mg/ml, 50 μl) in mice. (b) Retro-orbital I.V. injection of MCh (30 μg/ml, 50 μl) induced comparable bronchoconstriction in wild-type (n=6) and Mrgpr-clusterΔ-/-mice (n=6), (p=0.67), suggesting that the loss of Mrgprs did not affect the normal physiological responses of airway smooth muscle. (c) WT (n=5), TRPA1 knockout (n=4), TRPV1 knockout (n=5), and TRPV1/TRPA1 double knockout mice (n=4) exhibited similar bronchoconstriction after Bam8-22 injection. (WT vs TRPA1KO, p=0.66; WT vs TRPV1 KO, p=0.27; WT vs DKO, p=0.15). (d) The deletion of Mrgpr genes in the Mrgpr-clusterΔ-/- did not affect the development of the allergic model since the OVA specific IgE level was comparable in WT and Mrgpr-clusterΔ-/- mice after OVA administration. (WT-PBS, n=5; WT-OVA, n=6; KO-PBS, n=5; KO-OVA; n=5; p=0.375). (e) Representative trace showing the change of RL after retro-orbital I.V. injection of NPFF (10 mg/ml, 50 μl) in mice. Two-tailed unpaired Student’s t test. Data are reported as mean ± s.e.m. The experiments in (a) and (e) were repeated independently three times with similar results.
Supplementary Figure 9 Bam8-22 activates gpMrgprX1-expressing vagal sensory neurons and induces bronchoconstriction in guinea pigs.
. (a) RT-PCR analysis of gpMrgprx1 (XM_003462608) expression in various guinea pig tissues. gpMrgprx1 was detected in guinea pig jugular ganglion, nodose ganglion and DRG, but not in the lung. (b) RT-PCR analysis of airway-innervating guinea pig vagal sensory neurons. Airway-innervating vagal sensory neurons were retrogradely labelled by DiI. Dissociated vagal sensory neurons were cultured on coverslips and DiI-labelled neurons were harvested by glass-pipette into PCR tubes to generate cell lysate for RT-PCR. Three tubes of DiI-labelled jugular neurons (jugular-1, jugular-2 and jugular-3) and three tubes of DiI labelled nodose neurons (nodose-1, nodose-2, nodose-3) were collected with each tube containing 25 neurons. The expression of gpMrgprx1 was detected from all six tubes of airway-innervating vagal sensory neurons. gpTrpv1 is also expressed in all six tubes of airway-labeled neurons. Whole VG, cDNA generated from whole vagal ganglia. All the PCR results were confirmed by DNA sequencing. All the RT-PCR experiments were repeated independently three times with similar results. (c) The percentage of guinea pig vagal sensory neurons responding to Bam8-22 after electroporation of scramble siRNA (n=4) or gpMrgprx1 siRNA (n=4), (p=0.0011). (d-e) Calcium responses of guinea pig vagal sensory neurons to Bam8-22 (10 μM). All population data for fluorescence ratio (340/380) (n= 20 cells) are presented as mean ± s.e.m. (f) Representative trace showing the change of RL after retro-orbital I.V. injection of Bam8-22 (10 mg/ml, 50 μl) in guinea pigs. The experiments were repeated independently three times with similar results. ***p< 0.005, two-tailed unpaired Student’s t test. Data are reported as mean ± s.e.m.
. Wild-type and Mrgpr-clusterΔ-/-mice exhibited comparable inflammatory cell counts (a) and cytokine levels (b) in bronchoalveolar lavage fluid (BALF) 5 days after IAV inoculation. (WT-Ctrl, n=5; WT-IAV, n=6; KO-Ctrl, n=5; KO-IAV, n=6; WT-IAV vs KO-IAV, total cell p=0.66; macrophage/monocyte p=0.57; neutrophil p=0.72; lymphocyte p=0.77; TNFα p=0.21; IFNγ p=0.36; IL-6 p=0.16). two-tailed unpaired Student’s t test. Data are reported as mean ± s.e.m.
Supplementary Figure 11 Mrgprs do not mediate allergic inflammation-induced airway hyper-responsiveness.
. Wild-type and Mrgpr-clusterΔ-/- mice exhibited comparable airway hyperresponsiveness in house dust mite-induced allergic lung inflammation. (WT-Naïve, n=8; KO-naïve, n=8; WT-HDM, n=8; KO-HDM, n=9; WT-HDM vs KO-HDM, p=0.49). two-tailed unpaired Student’s t test. Data are reported as mean ± s.e.m.