Fig 2: CD203c+-EVs derived from basophils were significantly increased in HR patients. Basophils from control and HR patients were isolated by negative selection method. Non-basophil cells in PBMCs were labeled with biotin-conjugated antibodies(CD3, CD4, CD7, CD14, CD15,CD16, CD36, CD45RA, HLA-DR and CD235a) and anti-biotin microbeads, then non-basophils were depleted by a BeyoMag™ separator.Then the pre-enriched basophils were labeled with CD123, and eluted from column after magnetic separation. After incubation using serum-free medium, EVs were isolated using ultracentrifugation from culture supernatant for further analysis. (A–C) The purity of pre-enriched basophils and isolated basophils were identified using flow cytometry, which expressed CD123 over 95%. (D, E) The percentage of CD203c+-EVs in control and HR groups were determined by flow cytometry. The percentage of CD203c+-EVs increased in HR-0H and HR-treated-2H group, which decreased after treatment in HR-treated-24H group. All values represent the mean ± standard deviation. Difference to control: **p < 0,01; #Difference between groups: # p < 0,05; 1 way ANOVA with Newman-Keuls Multiple Comparison Test; n = 4.

Fig 3: SG induced HR promoted the production of mast cell/basophil specific EVs. The aim of this study was to evaluate the diagnostic sensitivity of mast cell/basophil specific EVs in SG induced HR. HR triggered mast cell/basophil degranulation and activation, which leaded releasing EVs and inflammatory mediators into serum. Serum was obtained from SG induced HR patients at different time point. The mast cell/basophil specific EVs and inflammatory mediators in serum were determined and analyzed. EVs derived from HR groups enriched specific proteins (CD63, CD203c, FcϵRI and tryptase) of mast cell/basophil, which were related with the inflammatory levels induced by SG. We hypothesize that mast cell/basophil specific EVs could serves as a novel diagnostic and prognostic biomarker for SG induced HR. Abbreviations: SG, Succinylated gelatin; MVB, multivesicular body.

Fig 4: HR promoted the concentration of EVs and CD203c+-EVs and CD63+-EVs derived from serum. 500μl serum from control and different time point HR groups were used to pellet EVs, finally, the EVs were resuspended in 200μl PBS. The total concentration of EVs was determined by a BCA assay. The protein expression levels of CD63, CD203c, CD9 and CD81 in EVs/serum were identified using western blotting. (A) Total concentration of EVs isolated from same volume serum of control and different time point HR groups, n = 10. (B–F) Relative CD63, CD203c, CD9 and CD81 protein expression levels of EVs derived from same volume of serum in control and HR groups, 30μl EVs/lane, n = 5 (G–I) Relative CD63 and CD203c expression levels in same volume of serum in the control and HR groups, 30μl serum/lane, n = 5. All values represent the mean ± standard deviation. Difference to control: **p < 0,01; ***p < 0,001; #Difference between groups: # p < 0,05; ## p < 0,01; 1 way ANOVA with Newman-Keuls Multiple Comparison Test.

Fig 5: The protein expression and concentration of CD203c+-EVs positively correlated with inflammatory mediators. (A–F) Correlation analysis between CD203c expression levels(flow cytometry) in EVs and inflammatory mediators in serum. R: Pearson correlation coefficient, Correlation coefficient |R|>0.5, p < 0.05 was considered statistically significant. (G–L) Correlation analysis between the concentration of EVs and inflammatory mediators. R: Pearson correlation coefficient, Correlation coefficient |R|>0.5, p < 0.05 was considered statistically significant. (M, N) ROC curve of EVs concentration and CD203c expression in different timepoint groups for predicting the diagnosis of HR.