A mast cell receptor mediates post-stroke brain inflammation via a dural-brain axis

Abstract

The immune environment surrounding the brain plays a fundamental role in monitoring signs of injury. Insults, including ischemic stroke, can disrupt this balance and incite an exaggerated inflammatory response, yet the underlying mechanism remains unclear. Here, we show that the mast-cell-specific receptor Mrgprb2 regulates post-stroke brain inflammation from the meninges. Mrgprb2 causes meningeal mast cell degranulation after stroke, releasing immune mediators. This process recruits skull bone marrow neutrophils into the dura and further promotes neutrophil migration from the dura into the brain by cleaving the chemorepellent semaphorin 3a. We demonstrate that the human ortholog, MRGPRX2, is expressed in human meningeal mast cells and is activated by upregulation of the neuropeptide substance P following stroke. Pharmacologically inhibiting Mrgprb2 reduces post-stroke inflammation and improves neurological outcomes in mice, providing a druggable target. Collectively, our study identifies Mrgprb2 as a critical meningeal gatekeeper for immune migration from skull bone marrow reservoirs into the brain.

Keywords: Mrgpr receptor; inflammation; ischemic stroke; mast cell; meninges; semaphorin; skull bone marrow; substance P.

Figure 1.. Mrgprb2^−/− mice are protected from ischemic stroke injury.

(A) Schematic of transient middle cerebral artery occlusion (tMCAO) model. Black and green dotted line indicates filament and white vessel indicates MCA that is occluded (BioRender). (B) Representative 2,3,5-triphenyltetrazolium (TTC) staining of WT/Mrgprb2^−/− mice brains. Each column represents one brain from anterior to posterior. Infarct is denoted by region of brain with no red dye uptake, separated by dotted line. (C) Quantification of WT/Mrgprb2^−/− brain stroke volumes using TTC stain 48h post-tMCAO (WT n=33, Mrgprb2^−/− n=27). (D) Representative T2-weighted MRIs of brains of WT/Mrgprb2^−/− mice 48h post-tMCAO. Each column represents one brain from anterior to posterior. Infarct is denoted by brighter signal in the right hemisphere. Signal intensity (in arbitrary units) is denoted by color bar. (E) Quantification of stroke volume in WT/Mrgprb2^−/− mice 48h post-tMCAO (WT n=11, Mrgprb2^−/− n=8). (F) WT/Mrgprb2^−/− mice neurological scores pre- and post-tMCAO. Higher score indicates better overall sensorimotor function (WT n=16, Mrgprb2^−/− n=12). (G) Latency to fall on rotarod for WT/Mrgprb2^−/− mice pre- and post-tMCAO. Higher latency to fall indicates better motor function (WT n=6, Mrgprb2^−/− n=10). (H) left, Left front paw and right, left hind paw toe spread pre- and post-tMCAO. (WT n=4, Mrgprb2^−/− n=4). (I) Kaplan-Meier survival curve of WT/Mrgprb2^−/− mice post-tMCAO. (WT n=10, Mrgprb2^−/− n=10). Statistical analyses: two-sided Student’s t-test (C and E), two-way ANOVA with Sidak’s multiple comparisons test (F and H), Kruskal-Wallis test (G), and log-rank test (I). Bar graphs indicate mean ± SEM. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.. Mrgprb2 is expressed in meningeal mast cells and is activated after tMCAO.

(A) Whole mount dura from Mrgprb2-Cre;tdT mice. left, tdT signal correlates to Mrgprb2 expression. middle, CD31 counterstain identifies vasculature. right, Merged channels with DAPI. Scale bar=500 μm. (B) Magnified whole mount dura. Scale bar=25 μm. (C) Flow cytometry of Mrgprb2-Cre;tdT peritoneal fluid, dura, and whole brain. left, Live CD45-positive cells gated for mast cells using CD117 and FcER1α. Number is frequency of mast cells among CD45-positive cells. right, Mast cells gated for tdT fluorescence represented as cell count versus intensity. Number is percentage of mast cells that are tdT-positive. (D) Representative immunofluorescence images of WT/Mrgprb2^−/− right hemispheric dura 6h post-tMCAO. Avidin denotes mast cell stain and DAPI identifies nuclei. White arrows indicate degranulated cells. Scale bar=25 μm. (E) Percent of degranulated mast cells in WT/Mrgprb2^−/− meninges in dura overlaying the contralateral/stroke brain hemispheres (WT n=7, Mrgprb2^−/− n=8). (F) Relative mRNA expression in WT/Mrgprb2^−/− dura 48h post-tMCAO, normalized to WT sham expression (WT sham n=4, Mrgprb2^−/− sham n=4, WT MCAO n=3, Mrgprb2^−/− MCAO n=4). (G) Neutrophil count/frame in WT/Mrgprb2^−/− dura overlaying the contralateral/stroke brain hemisphere post-tMCAO. (left, WT n=4, Mrgprb2^−/− n=5, middle, WT n=3, Mrgprb2^−/− n=4, right, WT n=5, Mrgprb2^−/− n=5). Statistical analyses: two-way ANOVA with Sidak’s multiple comparisons test (E-G), and Kruskal-Wallis test (F, Tpsb2). Bar graphs indicate mean ± SEM. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.. Mrgprb2^−/− mice exhibit reduced brain inflammation after tMCAO, which is rescued by meningeal mast cell engraftment.

(A) Representative flow cytometry gating of WT/Mrgprb2^−/− brains 48h post-tMCAO. left, Live cells are gated using an immune marker (CD45) and myeloid marker (CD11b). right, Myeloid cells are gated using Ly6G and Ly6C to delineate CD45-high neutrophils and monocytes/macrophages, and CD45-low microglia. (B-D) (B) Absolute count of neutrophils, (C) monocytes/macrophages, and (D) CD11b-positive microglia in contralateral/stroke brain hemispheres of WT/Mrgprb2^−/− mice 48h post-tMCAO (WT n=18, Mrgprb2^−/− n=16). (E) Representative immunofluorescence images of WT (left)/Mrgprb2^−/− (right) right brain hemispheres 48h post-tMCAO. top row, CD45-positive immune cells. middle row, GFAP-positive activated astrocytes. Dotted white line separates infarcted tissue without GFAP and live, injured brain tissue with GFAP. bottom row, Merged channels with DAPI. Scale bar=50 μm. (F), left, CCL2 and right, CCL3 protein expression measured by ELISA in contralateral/stroke brain hemispheres of WT/Mrgprb2^−/− mice (CCL2: WT n=6, Mrgprb2^−/− n=6, CCL3: WT n=5, Mrgprb2^−/− n=5). (G-H) (G) IL-6 and (H) neutrophil elastase protein expression measured by ELISA in contralateral/stroke brain hemispheres of WT/Mrgprb2^−/− mice (G, WT n=5, Mrgprb2^−/− n=5, H, WT n=7, Mrgprb2^−/− n=7). (I) Representative immunofluorescence image of Mrgprb2^−/− mouse meninges 8 weeks after engraftment with Mrgprb2-tdT mast cells. left column, tdT denotes engrafted cells. middle column, Avidin denotes all mast cells. right column, Merged channels with DAPI. Scale bar=50 μm. (J) Mast cell count in meninges of Mrgprb2^−/− mice engrafted with saline, WT, or Mrgprb2^−/− mast cells, determined by Avidin-positive cells in the dura, using a 0.408mm² viewing frame (WT saline n=4, WT engraftment n=5, Mrgprb2^−/− saline n=4, Mrgprb2^−/− engraftment n=4). (K) Absolute count of neutrophils and monocytes/macrophages in contralateral/stroke brain hemispheres of WT/Mrgprb2^−/− mast cell engrafted mice (WT n=16, Mrgprb2^−/− n=18). (L). left, Representative T2-weighted MRIs, and right, stroke volume of WT/Mrgprb2^−/− MC-engrafted Mrgprb2^−/− mice 48h post-tMCAO (WT n=9, Mrgprb2^−/− n=8). Statistical analyses: Kruskal-Wallis test (B and G), two-way ANOVA with Sidak’s multiple comparisons test (C-D, F, H, and J-K), and two-sided Student’s t-test (L). Statistical test for C, D, and K was performed on log-transformed data to adjust for non-normality. Bar graphs indicate mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 4.. Mrgprb2 is vital for neutrophil recruitment from the skull bone marrow into the brain.

(A) Outline of UBC-GFP skull transplant experiment (BioRender). (B-C) (B) Absolute GFP-positive neutrophil count and (C) percentage of dural neutrophils that are GFP-positive in WT/Mrgprb2^−/− mice 48h post-tMCAO (WT n=6, Mrgprb2^−/− n=6). (D) Percentage of skull bone marrow neutrophils that are GFP-positive in WT/Mrgprb2^−/− mice 48h post-tMCAO (WT n=5, Mrgprb2^−/− n=6). (E) Representative immunofluorescence image of WT recipient right brain hemisphere 48h post-tMCAO. left, Ly6G denotes neutrophils, middle, GFP denotes cells recruited from skull bone marrow, and right, Merged channels with DAPI. White arrows indicate cells with dual Ly6G/GFP stain. Scale bar=20 μm. (F) Absolute GFP-positive neutrophil count in contralateral/stroke brain hemispheres of WT/Mrgprb2^−/− brains (WT n=5, Mrgprb2^−/− n=6). (G) Percentage of contralateral/stroke brain hemispheres neutrophils that are GFP-positive in WT/Mrgprb2^−/− brains (WT n=5, Mrgprb2^−/− n=6). (H) Semaphorin3a protein levels in WT/Mrgprb2^−/− leptomeninges after sham/tMCAO (WT sham n=7, Mrgprb2^−/− sham n=4, WT tMCAO n=10, Mrgprb2^−/− tMCAO n=9). (I) Neutrophil count/frame in dura overlaying the contralateral/stroke brain hemisphere of vehicle/Sema3-I treated Mrgprb2^−/− mice 48h post-tMCAO. (vehicle n=6, Sema3a-I n=5). (J) Absolute neutrophil count in contralateral/stroke brain hemispheres of vehicle/Sema3a-I treated Mrgprb2^−/− mice 48h post-tMCAO (vehicle n=7, Sema3a-I n=7). (K) left, Representative T2-weighted MRIs and right, stroke volume of vehicle/Sema3a-I treated Mrgprb2^−/− mice 48h post-tMCAO (vehicle n=4, Sema3a-I n=5). (L) Vehicle/Sema3a-I treated Mrgprb2^−/− mice neurological scores pre- and post-tMCAO. Scores normalized to vehicle-treated mice within cohorts (vehicle n=6, Sema3a-I n=6). (M) Recombinant SEMA3A-Fc incubated with dermal fibroblast lysate, WT mast cell lysate, or ADAMTS1 as a positive control. Cleaved, inactive SEMA3A-Fc indicated by smaller 65kDa band. Statistical analyses: Mann-Whitney test (B-C), two-sided Student’s t-test (D, K), two-way ANOVA with Sidak’s multiple comparisons test (F,G,I-J, and L), and two-way ANOVA with Tukey’s multiple comparisons test (H). Statistical test for F, and L was performed on log-transformed data to adjust for non-normality. Bar graphs indicate mean ± SEM. *P < 0.05, **P < 0.01.

Figure 5.. MRGPRX2 mast cells are activated in human stroke dura due in part to substance P.

(A) Immunofluorescence images of human control patient dura. left, Tryptase and middle, Avidin stain mast cells. right, Merged channels with DAPI. Scale bar=10 μm. (B) Immunofluorescence image of human dura. left, MRGPRX2 stain. middle, Avidin co-stain for mast cells. right, Merged channels with DAPI. Scale bar=10 μm. (C) CT-angiogram of patient #1 (patient data in Table S1) presenting with right MCA occlusion. Arrow indicates occlusion. (D) top, Axial/coronal CT of patient at presentation and bottom, after decompressive hemicraniectomy. Dotted lines denote infarcted region. (E) Representative immunofluorescence images of control (top) and stroke (bottom) patient human dural mast cells stained with Avidin and DAPI. Scale bar=10 μm. Each image denotes distinct patient. (F-G) (F) Mast cells/frame and (G) percentage of degranulated mast cells in control/stroke patient dura. Each point represents one patient (control n=3, stroke n=3). (H) Substance P protein levels in control/stroke patient serum (control n=11, stroke n=17). (I) Ratio of WT:MRGPRX2^−/− LAD2 human mast cell beta-hexosaminidase release using control/stroke human patient serum (control n=8, stroke n=8). (J) Ratio of WT:MRGPRX2^−/− LAD2 mast cell beta-hexosaminidase release following incubation with substance P-depleted control/stroke patient serum (control n=3, stroke n=4). Statistical analyses: two-sided Student’s t-test (F-G, and I), Mann-Whitney test (H), and two-way matched ANOVA with Sidak’s multiple comparisons test (J). ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6.. Mrgprb2 inhibition by osthole attenuates post-stroke inflammation and neurologic deficits in mice.

(A) Mast cell activity of WT/Mrgprb2^−/− peritoneal mast cells pre-treated with osthole. Data normalized to vehicle of corresponding genotype (n=3). (B-C) (B) Absolute neutrophil count in contralateral/stroke brain hemispheres of vehicle/osthole treated WT and (C) Mrgprb2^−/− mice (B, vehicle n=12, osthole n=15, C, vehicle n=5, osthole n=7). (D) Representative T2-weighted MRIs of vehicle/osthole treated WT mice. (E-F) Stroke volume in vehicle/osthole treated WT/Mrgprb2^−/− mice 48h post-tMCAO (E, vehicle n=7, osthole n=7, F, vehicle n=5, osthole n=5). (G-H) (G) Vehicle/osthole treated WT and (H) Mrgprb2^−/− mice neurological scores pre- and post-tMCAO. Scores normalized to vehicle-treated mice within cohorts (G, vehicle n=13, osthole n=12, H, vehicle n=6, osthole n=7). (I-J) Kaplan-Meier survival curves of WT/Mrgprb2^−/− vehicle/osthole treated mice post-tMCAO (I, vehicle n=13, osthole n=11, J, vehicle n=8, osthole n=9). Statistical analyses: two-way ANOVA with Tukey’s multiple comparisons test (A), two-way ANOVA with Sidak’s multiple comparisons test (B-C, G-H), two-sided Student’s t-test (E-F), and log-rank test (I-J). Statistical test for B, and C was performed on log-transformed data to adjust for non-normality. Bar graphs indicate mean ± SEM. ns, not significant; *P < 0.05, **P < 0.01, ****P < 0.0001