An interorgan neuroimmune circuit promotes visceral hypersensitivity

Abstract

Visceral pain disorders such as interstitial cystitis/bladder pain syndrome (IC/BPS) and irritable bowel syndrome (IBS) often manifest concurrently in the bladder and colon. Yet, the mechanistic basis of such comorbidities and the transmission of neural hypersensitivity across organ systems has remained a mystery. Here, we identify a mast cell-sensory neuron circuit that initiates bladder inflammation and simultaneously propagates neural hypersensitivity to the colon in a murine model of IC/BPS. We unveil anatomic heterogeneity of mast cells in relation to nociceptors in the bladder and their critical dependence on Mas-related G protein-coupled receptor B2 (MrgprB2) to promote visceral hypersensitivity. Employing retrograde neuronal tracing, in vivo calcium imaging, and intersectional genetics, we uncover a population of polyorganic sensory neurons that simultaneously innervate multiple organs and exhibit functional convergence. Importantly, using humanized mice, we demonstrate that pharmacological blockade of mast cell-expressed MRGPRX2, the human ortholog of MrgprB2, attenuates both bladder pathology and colonic hypersensitivity. Our studies reveal evolutionarily conserved neuroimmune mechanisms by which immune cells can directly convey signals from one organ to another through sensory neurons, in the absence of physical proximity, representing a new therapeutic paradigm.

Figure 1. Bladder inflammation induces colon hypersensitivity.

a, Schematic of the experimental protocol for murine IC/BPS-like disease induction followed by behavioral and functional assays targeting the bladder or colon. b, Response frequencies to suprapubic mechanical stimuli by a 0.04 g von Frey filament of mice treated with vehicle (n = 11) or LL-37 (n = 14). c, Representative bladder histology (left) and histological scores (right) at day 3 post-instillation in vehicle- or LL-37-treated mice (n = 7 per group). Box indicates representative edema and infiltration of leukocytes including PMNs. Scale bar, 500 μm. d, Urination patterns (left), small void numbers (middle), and center void percentages (right) of vehicle and LL-37-treated mice (n = 10 per group). e, VMR to urinary bladder distention as measured by the area under the curve (AUC) in mice treated intravesically with vehicle (n = 8) or LL-37 (n = 11); normalized to baseline (see Methods). Biological replicates are indicated in graphs (left), and representative traces are shown on the right. Scale bars, 300 μV (vertical) and 10 s (horizontal). f, Representative distal colon histology (left) and histological scores (right) at day 3 post-instillation in vehicle- or LL-37-treated mice (n = 4 and 6, respectively). Scale bar, 500 μm. g, Average transepithelial currents (I_SC; left) and quantification of baseline I_SC (right) recorded in distal colonic mucosa obtained from vehicle- or LL-37-treated mice (n = 10 per group). h, Expulsion time following the insertion of a 3 mm glass bead into the colon of vehicle- or LL-37-treated mice (n = 10 per group). i, VMR to colorectal distention in vehicle- or LL-37-treated mice (n = 8 per group). Biological replicates are indicated in graphs (left), and representative traces are shown on the right. Scale bars, 300 μV (vertical) and 10 s (horizontal). Data are presented as mean ± s.e.m. P values are shown in plots. b, e, i, Two-way repeated ANOVA with Sidak’s multiple comparisons test. c, d, f, g, h, Two-sided Student’s t-tests with Welch’s correction. The diagram in a was created by J. Gregory.

Figure 2. Polyorganic sensory neurons promote bladder-colon sensitization.

a, Schematic of dual-color CTB retrograde labeling of DRG sensory neurons innervating the urinary bladder (CTB-647, purple) and the distal colon (CTB-488, green). b, Representative whole-mount images of DRG (L6) after tracing. Scale bar, 200 μm. c, Venn diagram of the numbers of L6 DRG sensory neurons with single (purple or green) or dual (gray) retrograde labeling from the bladder and distal colon of mice (n = 3). d, Schematic of in vivo calcium imaging of L6 DRG neurons. Graded mechanical stimulation of the bladder and colon were performed through an intravesical catheter or intracolonic balloon, respectively. The insets represent calcium traces from individual neurons and are shown and color coded according to the categories shown in (e). e, Heatmap of calcium responses of L6 DRG neurons obtained after sequential bladder-colon graded stimulation of Pirt^Cre;GCaMP6f^flox/+ mice. Neurons were functionally classified in three categories based on their response to stimuli and sorted by ΔF/F: Bladder (bladder responsive only), Colon (colon responsive only), and Convergent (dual bladder and colon responsive). Stimulations are shown on top of the heatmap. Scale bars, 20 cells (vertical) and 15 s (horizontal). f, Quantification of recorded cells per category shown on (e), purple: bladder responsive only, green: colon responsive only, and gray: dual bladder-colon responsive neurons. N = 144 cells; from 4 mice. g, Intersectional genetic strategy to selectively express an inhibitory GPCR (Gi-DREADD) in polyorganic sensory neurons using a microinjection of AAV9-hSyn-Flpo into the bladder and AAV9-hSyn-Cre into the colon of the RC::FPGi dual-recombinase responsive allele. The insets represent Flpo recombinase resulting in mCherry fluorescence, and further exposure to Cre recombinase resulting in Gi-DREADD expression in the overlapping populations. h, Timeline of administration of the Gi-DREADD ligand DCZ (100 μg kg⁻¹), twice a day) for 5 d followed by a 2 d washout. i, j, k, VMR to bladder distension (i; n = 4 per group), colon distension (j; n = 10 AAV-Control, 9 AAV-Cre+AAV-Flpo), and colonic motility (k; n = 14 AAV-Control, 13 AAV-Cre+AAV-Flpo) in mice whose polyorganic sensory neurons were inhibited compared to controls following DCZ treatment upon IC/BPS induction. Data are presented as mean ± s.e.m. P values are shown in plots. i, j, Two-way repeated ANOVA with Sidak’s multiple comparisons test. k, Two-sided Student’s t-tests with Welch’s correction. The diagrams in a, d, g were created by J. Gregory.

Figure 3. The bladder-colon hypersensitivity axis is mast cell- and MrgprB2-dependent.

a, Left: Light sheet image of an optically cleared whole-mount bladder from Trpv1^Cre;Ai14^flox/flox mice stained with the mast cell indicator Avidin. Top right: Selective slice views showing the projection of Trpv1⁺ spinal afferents and the distribution of Avidin⁺ mast cells across the lumen-surface axis of the bladder. Bottom right: Representative slice views showing anatomical proximity between mast cells and nociceptors highlighted by white boxes as lamina propria (LP), muscularis (M), and serosa (S). Scale bars, 500 μm. b, IMARIS 3D rendering of Avidin⁺ mast cells forming physical contact with Trpv1⁺ nociceptor fibers in the LP and S layers, but only being in slight proximity to fibers in the M layers of the bladder. Scale bar, 100 μm. c, IMARIS automated computational analysis of the minimum distance (μm) between modeled Avidin⁺ mast cells and modeled Trpv1⁺ nociceptor fibers across 3 histological layers in the bladder. LP (n = 192 cells), M (n = 235 cells), and S (n = 216 cells); from 4 mice. d, e, f, g, h, Evaluation of bladder pathology (d; n = 5 per group), urinary urgency (e; n = 10 per group), bladder hypersensitivity (f; n = 8 and 10, respectively), colonic hypersensitivity (g; n = 6 and 8, respectively), and motility (h; n = 6 per group) in intravesically LL-37-challenged and diphtheria toxin-injected littermates (LM) controls and Mas-TRECK mice. i, Percentages of tdTomato (MrgprB2⁺) Avidin⁺ cells in all Avidin⁺ mast cells in the LP (n = 393 cells), M (n = 133 cells), and S (n = 222 cells) layers of the bladder in MrgprB2^Cre;Ai9^flox/flox mice (n = 4). j, k, l, m, n, Evaluations in bladder pathology (j; n = 6 and 7, respectively), urgency (k; n = 5 per group), bladder hypersensitivity (l; n = 6 and 7, respectively), colonic hypersensitivity (m; n = 6 per group), and motility (n; n = 6 and 5, respectively) in intravesically LL-37-challenged wild type (MrgprB2^+/+) controls and MrgprB2 mutant mice (MrgprB2^−/−). Data are presented as mean ± s.e.m. P values are shown in plots. f, g, l, m, Two-way repeated ANOVA with Sidak’s multiple comparisons test. d, e, h, j, k, n, Two-sided Student’s t-tests with Welch’s correction.

Figure 4. Pharmacological inhibition of MRGPRX2 alleviates interorgan hypersensitivity.

a, Uniform Manifold Approximation and Projection (UMAP) of individual immune cell clusters in healthy human bladders (11203 cells from n = 3 human donors). b, Feature plots showing the expression of MRGPRX2 genes in the canonical CPA3, FCER1A, KIT, and TPSB2 expressing mast cell UMAP (924 mast cells from n=3 human donors). c, Schematic of humanized huMRGPRX2^KI mouse generation. d, Flow cytometric analysis of MRGPRX2 expression on indicated cell types in the bladder of the huMRGPRX2^KI mice. e, Left, representative Fluo-4 fluorescence heatmap images of huMRGPRX2^KI mouse peritoneal mast cells showing calcium influx induced by the administration of Compound 48/80 (10 μg ml⁻¹) in the setting of vehicle or EP-001 (5 μg ml⁻¹) pre-incubation. Scale bar, 20 μm. Right, representative imaging traces; normalized to baseline (see Methods). Each colored line represents an individual cell. f, g, h, i, j, Evaluations in bladder pathology (f; n = 5 per group), urinary urgency (g; n = 6 per group), bladder hypersensitivity (h; n = 10 and 8, respectively), colonic hypersensitivity (i; n = 13 and 14, respectively), and colonic motility (j; n = 12 and 14, respectively) in intravesically LL-37-challenged and vehicle-treated controls or EP-001-treated huMRGPRX2^KI mic. Data are presented as mean ± s.e.m. P values are shown in plots. h, i, Two-way repeated ANOVA with Sidak’s multiple comparisons test. f, g, j, Two-sided Student’s t-tests with Welch’s correction.