Exosome secretion by eosinophils: A possible role in asthma pathogenesis Carla Mazzeo, PhD, José Antonio Cañas, MSc, Maria Paz Zafra, MSc, Ainara Rojas.

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Exosome secretion by eosinophils: A possible role in asthma pathogenesis Carla Mazzeo, PhD, José Antonio Cañas, MSc, Maria Paz Zafra, MSc, Ainara Rojas Marco, MD, Mar Fernández-Nieto, MD, PhD, Veronica Sanz, VET, María Mittelbrunn, PhD, Manuel Izquierdo, PhD, Francesc Baixaulli, MSc, Joaquín Sastre, MD, PhD, Victoria del Pozo, PhD Journal of Allergy and Clinical Immunology Volume 135, Issue 6, Pages 1603-1613 (June 2015) DOI: 10.1016/j.jaci.2014.11.026 Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Detection of MVBs in eosinophils by using confocal microscopy. The upper panels show the z axis maximum-intensity projection of sections of LBPA- and CD63-labeled eosinophils, and the bottom panels show a stack of merged images, with coincident labeling appearing in yellow and indicating the presence of MVBs. Analysis with the JACoP plugin for ImageJ software indicated that in section 7 colocalization is partial, with different intensities (Pearson coefficient, r = 0.62). In this experiment 6 human donors and 20 cells per donor were analyzed. Journal of Allergy and Clinical Immunology 2015 135, 1603-1613DOI: (10.1016/j.jaci.2014.11.026) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Detection of MVBs in eosinophils by using TEM. A, 8,000× magnification of eosinophils. Arrows indicate the presence of MVBs. The rectangle indicates 1 selected MVB (80,000× magnification). Arrowheads show the presence of LBPA. B, 8,000× magnification of eosinophils. The rectangle indicates the presence of 1 mature MVB (80,000× magnification). Arrowheads show the process of inward budding of MVBs and also some ILVs. C, 8,000× magnification of 1 eosinophil. The rectangle indicates the fusion of 1 MVB with the plasma membrane and concomitant secretion on its content. 40,000× magnification of the MVB fusion is shown. The arrow indicates the presence of one extracellular vesicle with the features of an exosome. This image is of a representative eosinophil of 44 analyzed from 10 different human donors. Journal of Allergy and Clinical Immunology 2015 135, 1603-1613DOI: (10.1016/j.jaci.2014.11.026) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Time-course study of IFN-γ induction degranulation of eosinophil vesicles expressing GFP-CD63. Time-lapse acquisition (1 frame per minute; exposure, 1 second) and analysis were performed in transfected eosinophils (75% efficiency). A and B, Results of viable eosinophils (n = 3; I, II, and III) stimulated with IFN-γ. C, Unstimulated eosinophils were also analyzed (n = 7). The figure shows a representation of the intensity of fluorescence with respect to time in 2 defined regions of interest (ROI 1, total cell area; ROI 2, plasma membrane area) from Video E1. A switch in the fluorescence intensity from cytoplasm to the plasma membrane was observed after 8 minutes in stimulated eosinophils. Journal of Allergy and Clinical Immunology 2015 135, 1603-1613DOI: (10.1016/j.jaci.2014.11.026) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 MVB traffic after IFN-γ stimulation of eosinophils. A, Eosinophils (2 × 106) from 2 different donors were cultured with or without IFN-γ. At different times, cells were collected, fixed, permeabilized, and labeled with LBPA–Alexa Fluor 647. Epifluorescent images were taken, and the LBPA fluorescence intensity was measured in 50 cells in each condition. The sum intensity of LBPA fluorescence (mean ± SD per cell area) was obtained at 10 minutes, 4 hours, and 16 hours after stimulation and compared with that seen in cells without stimuli. The difference was significant at every time point of treatment (P < .05). B, Eosinophils (2 × 106) from 3 different donors were cultured with or without IFN-γ. After 10 minutes, cells were collected, fixed, permeabilized, and labeled with CD63 and LBPA. The fluorescence intensity was measured by using flow cytometry; typically, 50,000 to 100,000 total events were acquired. Data represent MFI ± SD (n = 3). A representative histogram showing CD63+ and LBPA+ cells and isotype labeling is displayed. Journal of Allergy and Clinical Immunology 2015 135, 1603-1613DOI: (10.1016/j.jaci.2014.11.026) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Characterization of eosinophil-derived exosomes. A, WB analysis from exosomes was achieved by using antibodies against Alix, CD9, and CD63 on IFN-γ induction. α-Tubulin was used as a loading control in eosinophils to show that exosomes come from the same number of eosinophils. M, Medium. Exosome-depleted culture medium was used as a control for the cell-free condition. B, WB analysis from exosomes was achieved by using antibodies against Alix, CD9, and CD63 on eotaxin induction. α-Tubulin was used as a loading control in eosinophils. C, Alix and CD9 WB analysis was performed from exosomes on IFN-γ induction in the presence (+) or absence (−) of PKC inhibitor (PKCi; 5 μmol/L). β-Actin was used as a loading control in eosinophils. Fig 5, A-C, are representative examples of several WBs, all displaying similar results. Bands were quantified by densitometry, and histograms with means ± SDs (n = 3) are shown. D, Exosomes were measured by using NanoSight LM10 in the supernatant from cultures of 2 × 106 eosinophils (n = 18). The histogram represents particle size distribution (exosome × 108/mL vs size in nanometers). E, TEM of eosinophil exosomes showing their ultrastructure. Vesicles display the characteristic cup-shape morphology of exosomes. Arrowheads indicate positive immunogold labeling: Alix (i) or CD63 (ii). This image is a representative of 22 exosomes tested from 6 different donors. (iii), Representative image of exosome preparation from eosinophils under low magnification. Journal of Allergy and Clinical Immunology 2015 135, 1603-1613DOI: (10.1016/j.jaci.2014.11.026) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Eosinophil exosome secretion from healthy subjects and asthmatic patients. Exosomes isolated from the culture medium of 2 × 106 eosinophils from healthy subjects and asthmatic patients induced or not with IFN-γ for 10 minutes were quantified by using NanoSight LM10 analysis. A, The bar graph represents means ± SDs of exosome concentrations from 7 asthmatic patients and 5 healthy subjects. B, Screen shots of video from NanoSight LM10 of eosinophil exosome light scatter showing the difference in secretion between healthy subjects and asthmatic patients. C, WB of exosome lysates from healthy subjects (H) and asthmatic patients (A) was performed with EPO, MBP, and ECP antibodies. M, Medium. Exosome-depleted culture medium was used as a control for cell-free condition. β-Actin was assayed as a loading control to show that exosomes come from the same number of eosinophils. Journal of Allergy and Clinical Immunology 2015 135, 1603-1613DOI: (10.1016/j.jaci.2014.11.026) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions