Mast cell MrgprB2 in neuroimmune interaction in IgE-mediated airway inflammation and its modulation by β-arrestin2

Figures

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  Figure 1. MrgprB2 contributes to MC recruitment and upregulation of SP in the lungs of IgE-sensitized and antigen-challenged mice. (A) Expression of MrgprB2 transcript in the lungs of WT and MrgprB2−/− experimental mice. (B) Representative images of toluidine blue-stained MCs in the lung sections (scale bar, 50 µm). (C) MC count per field (n = 6–7). mRNA expression of (D) Tac4 and (E) Tac1 in the lungs of WT and MrgprB2−/− mice (n = 5–6). (F) Representative images of immunofluorescence staining of SP expression (anti-SP; green) and nuclear counterstain DAPI (blue) in PBS and IgE-sensitized/antigen-challenged mice. (G) Intensity of anti-SP per field (n = 6). (H) Representative images of immunofluorescence staining of SP expression (anti-SP; red), PGP9.5 (green), and nuclear counterstain DAPI (blue) in PBS and IgE-sensitized and challenged mice. Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons test; error bars are presented as mean ± SEM. Significant differences were set at *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001; ns, non-significant. MC, mast cell; SP, substance P; WT, wild type; PBS, phosphate-buffered saline.

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  Figure 2. MrgprB2 deletion results in reduced cytokine generation in the lungs following IgE sensitization and antigen challenge. Transcripts for (A) TNF-α, (B) IL-4, (C) IL-5, and (D) IL-13 in lung tissue following IgE sensitization/antigen challenge were assessed by RT-PCR (n = 6/group). The production of cytokine (E), TNF-α, and (F) IL-13 from the BAL of the experimental mouse group was quantified by ELISA (n = 6–8/group). Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons test; error bars are presented as mean ± SEM. Significant differences were set at *p < 0.05, **p < 0.01, and ****p < 0.0001. BAL, bronchoalveolar lavage.

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  Figure 3. Absence of MrgprB2 results in reduced inflammatory cell infiltrate and goblet cell hyperplasia in lung tissue of IgE-sensitized and antigen-challenged mice. (A, B) Flow cytometry analysis of eosinophils. Cells from lung digest were stained with fluorescent anti-CD45, anti-CD11b, and anti-CD170 to analyze eosinophil subsets. Representative plots of separate experiments are presented. Percentages of CD11b+CD170+ (eosinophils) within CD45+ cells are shown for lungs from WT and MrgprB2−/− mice. (C, D) Flow cytometry analysis of neutrophils. Cells from lung digest were stained with fluorescent anti-CD45, anti-CD11b, and anti-Ly6G to analyze neutrophil subsets. Percentages of CD11b+Ly6G+ (neutrophils) within CD45+ cells are shown for lungs from WT and MrgprB2−/− mice of different experimental groups. (E) Lung tissue sections were stained with periodic acid–Schiff (PAS) for mucus secretion in the experimental groups. (F) Quantitative mucus-producing (PAS+) cells in the bronchiolar lumen. Magnification, ×20; scale bar, 50 µm. Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons test; error bars are presented as mean ± SEM (n = 6–8 mice/group). Significant differences…

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  Figure 4. β-arr2 contributes to MC recruitment and upregulation of SP in the lungs of IgE-sensitized and antigen-challenged mice. (A) Expression of MrgprB2 transcript in the lungs of Cpa3Cre− /β-arr2fl/fl and Cpa3Cre+ /β-arr2fl/fl experimental mice. (B) Representative images of toluidine blue-stained MCs in the lung sections of experimental mice (scale bar, 50 µm). (C) MC count per field (n = 6–7). (D) Tac1 mRNA expression in the lungs of Cpa3Cre− /β-arr2fl/fl and Cpa3Cre+ /β-arr2fl/fl experimental mice. (E) Representative image of immunofluorescence staining of SP expression (anti-SP; green) and nuclear counterstain DAPI (blue) in PBS and IgE-sensitized and challenged mice. (F) Intensity of anti-SP per field (n = 6). (G) Representative images of immunofluorescence staining of SP expression (anti-SP; red), PGP9.5 (green), and nuclear counterstain DAPI (blue). Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons test; error bars are presented as mean ± SEM. Significant differences were set at **p < 0.01, ***p < 0.001, and ****p < 0.0001. MC, mast cell; SP, substance P; PBS, phosphate-buffered saline.

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  Figure 5. MC-specific deletion of β-arr2 results in reduced IgE-mediated cytokine generation. Expression of (A) TNF-α, (B) IL-4, (C) IL-5, and (D) IL-13 transcripts were assessed by RT-PCR on isolated mRNA samples (n = 5–6/group). Each symbol represents one mouse. The production of cytokine (E), TNF-α, and (F) IL-13 from the BAL of the experimental mouse group was quantified by ELISA. Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons test; error bars are presented as mean ± SEM. Significant differences were set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. MC, mast cell; BAL, bronchoalveolar lavage.

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  Figure 6. Reduced inflammatory cell infiltrate and goblet cell hyperplasia in lung tissue of mice with MC-specific deletion of β-arr2. (A, B) Flow cytometry analysis of eosinophils. Cells from lung digest were stained with fluorescent anti-CD45, anti-CD11b, and anti-CD170 to analyze eosinophil subsets. Representative plots of separate experiments are presented. Percentages of CD11b+CD170+ (eosinophils) within CD45+ cells are shown for lungs from Cpa3Cre− /β-arr2fl/fl and Cpa3Cre+ /β-arr2fl/fl mice of different experimental groups. (C, D) Flow cytometry analysis of neutrophils. Cells from lung digest were stained with fluorescent anti-CD45, anti-CD11b, and anti-Ly6G to analyze neutrophil subsets by flow cytometry. Percentages of CD11b+Ly6G+ (neutrophils) within CD45+ cells are shown for lungs from Cpa3Cre− /β-arr2fl/fl and Cpa3Cre+ /β-arr2fl/fl mice treated with PBS and TNP-Ova. (E) Lung tissue sections were stained with PAS for mucus secretion in the experimental groups. (F) Quantitative mucus-producing (PAS+) cells in the bronchiolar lumen. Magnification, ×20; scale bar, 50 µm. Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons test; error bars are presented as mean…