Human mast cell degranulation and preformed TNF secretion require mitochondrial translocation to exocytosis sites: Relevance to atopic dermatitis Bodi.

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Human mast cell degranulation and preformed TNF secretion require mitochondrial translocation to exocytosis sites: Relevance to atopic dermatitis Bodi Zhang, MD, MPH, PhD, Konstantinos-Dionysios Alysandratos, MD, Asimenia Angelidou, MD, Shahrzad Asadi, PharmD, Nikolaos Sismanopoulos, MD, Danae-Anastasia Delivanis, MD, Zuyi Weng, MS, Alexandra Miniati, MD, Magdalini Vasiadi, BS, Alexandra Katsarou-Katsari, MD, PhD, Benchun Miao, PhD, Susan E. Leeman, PhD, Dimitrios Kalogeromitros, MD, PhD, Theoharis C. Theoharides, MS, PhD, MD Journal of Allergy and Clinical Immunology Volume 127, Issue 6, Pages 1522-1531.e8 (June 2011) DOI: 10.1016/j.jaci.2011.02.005 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Mitochondrial translocation and degranulation in hCBMCs observed by confocal microscopy. A, Mitochondrial distribution in resting (panels 1-3), IgE-incubated (1 μg/mL) (panels 4-6), and IgE (1 μg/mL) + streptavidin (125 ng/mL)–stimulated (panels 7-9) cells. Cells were stained with LysoTracker (a, green) and MitoTracker (b, red). B, β-hex release from hCBMCs treated with IgE or IgE + streptavidin (Strep) for 30 minutes. n = 3; ∗P < .05 compared with control. Bars equal to 5 μm. Journal of Allergy and Clinical Immunology 2011 127, 1522-1531.e8DOI: (10.1016/j.jaci.2011.02.005) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Mitochondrial translocation observed by differential interference contrast, and degranulation and TNF secretion in LAD2 mast cells. A, Mitochondrial distribution of control (upper panels) and degranulated (bottom panels) mast cells stimulated with SP (2 μmol/L) for 30 minutes. Cells were stained with MitoTracker (red). Mast cell degranulation by SP was confirmed by β-hex release (B) and TNF secretion (C). n = 3; ∗P < .05 compared with control. Journal of Allergy and Clinical Immunology 2011 127, 1522-1531.e8DOI: (10.1016/j.jaci.2011.02.005) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Quantification of mitochondrial translocation during degranulation in human LAD2 cells. A, Control (n = 19; panels 1-4) and degranulated (n = 20; panels 5-8) mast cells (SP 2 μmol/L, 30 minutes). B, Percentage of translocated mitochondria in resting and stimulated cells (n = 3). C, Number of mitochondria per cell within 1 μm from the cell surface. Cells were randomly selected for analysis (∗P < .05; ∗∗P < .01; horizontal bars indicate the means). Bars equal to 5 μm. Journal of Allergy and Clinical Immunology 2011 127, 1522-1531.e8DOI: (10.1016/j.jaci.2011.02.005) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Z-stack mitochondrial fluorescence projection in control and stimulated human LAD2 mast cells. Control (A) and degranulated (B) LAD2 cell. White dashed lines represent the nuclear region. C, The percentage of the cellular area with detectable mitochondrial fluorescence indicating mitochondrial distribution was calculated from 20 cells (∗P < .05). Journal of Allergy and Clinical Immunology 2011 127, 1522-1531.e8DOI: (10.1016/j.jaci.2011.02.005) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Electron photomicrographs showing mitochondrial translocation in human skin mast cells from patients with AD. Human skin mast cells from control (3 subjects, 9 mast cells; A) and lesional skin from patients with AD (5 subjects, 14 mast cells; magnification ×13,800; B). Mitochondria are shown within white rectangles. White asterisks represent secreted granular material. C, Number of mitochondria per cell. D, Percentage of mitochondria close to the cell surface in each cell (∗∗P < .01; horizontal bars indicate the means). Journal of Allergy and Clinical Immunology 2011 127, 1522-1531.e8DOI: (10.1016/j.jaci.2011.02.005) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Mdivi-1 and Drp1 siRNA inhibit mast cell mitochondrial translocation, degranulation, and preformed TNF secretion. A, Mitochondrial morphology before or after stimulation with SP and pretreatment with mdivi-1. Cells were stained with LysoTracker (green) and MitoTracker (red). B, Percentage of translocated mitochondria in resting, stimulated, and mdivi-1-treated cells. C, TNF secretion. D, β-hex release. E, β-hex release after treatment with Drp1 siRNA before SP stimulation (n = 3; ∗P < .05). Bars equal to 5 μm. Journal of Allergy and Clinical Immunology 2011 127, 1522-1531.e8DOI: (10.1016/j.jaci.2011.02.005) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 Intracellular calcium increase and Drp1 phosphorylation at Ser-616 during LAD2 mast cell degranulation. A, Intracellular calcium level was measured after SP stimulation. B, LAD2 cell degranulation with or without extracellular calcium, as measured by β-hex release. C, Western blot of Drp1 phosphorylation at Ser-616 after SP (2 μmol/L) stimulation with or without extracellular calcium. D, Drp1 protein density was normalized against β-actin (n = 3; ∗P < .05). There was no statistical difference in Drp1 phosphorylation at Ser-616 between control and SP stimulation in the absence of calcium. Journal of Allergy and Clinical Immunology 2011 127, 1522-1531.e8DOI: (10.1016/j.jaci.2011.02.005) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 8 Drp1 and calcineurin gene expression in skin from patients with AD compared with healthy controls. Gene expression of Drp1 (controls, n = 10; patients, n = 10; A) and calcineurin (controls, n = 10; patients, n = 9; B). The calcineurin patient samples were 1 fewer because the cDNA was exhausted. Relative quantities of mRNA expression were measured by quantitative real-time-PCR and normalized to GAPDH (horizontal bars indicate the means). Journal of Allergy and Clinical Immunology 2011 127, 1522-1531.e8DOI: (10.1016/j.jaci.2011.02.005) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Viability testing during human mast cell degranulation Viability testing during human mast cell degranulation. LAD2 cells were stimulated with different concentrations of SP for 30 minutes. Cell viability was tested by using Trypan blue exclusion. Journal of Allergy and Clinical Immunology 2011 127, 1522-1531.e8DOI: (10.1016/j.jaci.2011.02.005) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Detection of preformed TNF in unstimulated LAD2 mast cells Detection of preformed TNF in unstimulated LAD2 mast cells. Unstimulated LAD2 cells were lysed and sonicated to release all intracellular components. TNF concentration was determined by ELISA (n = 3). Journal of Allergy and Clinical Immunology 2011 127, 1522-1531.e8DOI: (10.1016/j.jaci.2011.02.005) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Decrease in Drp1 mRNA (A) and protein (B) after siRNA treatment of LAD2 cells. LAD2 cells were transfected with Drp1 siRNA for 5 hours and incubated in growth medium for 6 hours. A, TaqMan PCR was performed (n = 3). B, Western blot analysis of the Drp1 protein 48 hours after siRNA treatment. Journal of Allergy and Clinical Immunology 2011 127, 1522-1531.e8DOI: (10.1016/j.jaci.2011.02.005) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Effect of the cation ionophore A23187 on LAD2 mitochondrial translocation. LAD2 cells were either unstimulated (upper panels) or stimulated with calcium ionophore A23187 (1 μmol/L) for 30 minutes (lower panels). Cells were stained with LysoTracker (a, green) and MitoTracker (b, red). Bars equal 5 μm. Journal of Allergy and Clinical Immunology 2011 127, 1522-1531.e8DOI: (10.1016/j.jaci.2011.02.005) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Increased gene expression of Drp1 (A) and calcineurin activity (B) after SP stimulation of LAD2 cells for 6 hours. A, Drp1 gene expression in SP-stimulated LAD2 cells for 6 hours. B, LAD2 cells were stimulated by SP for 30 minutes in either calcium or calcium-free medium. Calcineurin activity was measured as phosphate release following the protocol provided (n = 3; ∗P < .05). There was no statistical difference in calcineurin activity between control and SP stimulation in the absence of calcium. Journal of Allergy and Clinical Immunology 2011 127, 1522-1531.e8DOI: (10.1016/j.jaci.2011.02.005) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Increased TAC1 gene expression in lesional skin from patients with AD compared with controls. Gene expression of TAC1 (controls, n = 10; patients, n = 10). Relative quantities of mRNA expression were measured by quantitative real-time PCR and normalized to GAPDH (∗P < .05; horizontal bars indicate the means). Journal of Allergy and Clinical Immunology 2011 127, 1522-1531.e8DOI: (10.1016/j.jaci.2011.02.005) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Diagrammatic representation of the proposed intracellular steps involved in mitochondrial fission and translocation during human mast cell degranulation. deP, Dephosphorylated. Journal of Allergy and Clinical Immunology 2011 127, 1522-1531.e8DOI: (10.1016/j.jaci.2011.02.005) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions