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Phagocytosis-independent antimicrobial activity of mast cells by means of extracellular trap formation

Maren von Köckritz-Blickwede, Oliver Goldmann, Pontus Thulin, Katja Heinemann, Anna Norrby-Teglund, Manfred Rohde and Eva Medina

Data supplements

  • Supplemental materials for: von Kockritz-Blickwede et al

    Files in this Data Supplement:

    • Figure S1. Granule componets are critical for the production of MCETs (JPG, 41.3 KB) -
      Immunofluorescence photographs showing viable (green) versus dead (red) MCs: (Ai) Stimulation of MCs with 100mU/ml H2O2-producing enzyme glucose oxidase results in strong production of MCETs. (Aii) Degranulated MCs after 6 h of treatment with 10 µg/ml compound 48/80. Compound 48/80 has been shown to induce degranulation of MCs (Liz GZ, Chai OH, Song CH. Inhibitory effects of epigallocatechin gallate on compound 48/80-induced mast cell activation and passive cutaneous anaphylaxis. Exp Mol Med. 2005;31:290-196.). Notice that degranulation per se does not induce MCETs formation. (Aiii) Compound 48/80-treated degranulated MCs (1 h, 10 µg/ml) stimulated with H2O2-producing glucose oxidase for additional 5 h failed to produce MCETs. All bars represent 50 µm.





    • Figure S2. Presence of mast cells (MCs) in biopsy samples collected from patients with streptococcal soft-tissue infections (JPG, 60.1 KB) -
      Snap-frozen tissue biopsies (n=10) collected at the site of infection from patients were sectioned, fixed and immunostained for mast cell tryptase or S. pyogenesS. pyogenes (stained green) in Dapi-stained blue tissue biopsy (bar, 50 µm). (D) High magnification confocal microscopy showing colocalization of S. pyogenes and MCs tryptase (bars, 10 µm): (Di) immunostaining of S. pyogenes with Alexa green-labelled antibodies; (Dii) immunostaining of MCs tryptase with Alexa-red labelled antibodies; (Diii) overlay of A1-2 (colocalization of tryptase and bacteria results in a yellow staining (arrows)).

Article Figures & Data

  • Figure 1

    In vitro interactions of bacteria with human MCs (HMC-1). (A) Double immunofluorescence staining for determination of extracellular/intracellular location of S pyogenes associated with MCs (bar, 3.5 μm). Extracellular bacteria were stained with polyclonal rabbit anti–S pyogenes antibodies, followed by Alexa green–conjugated goat antirabbit antibodies (Sigma-Aldrich). After several washes, the cells were permeabilized by 0.025% Triton X-100 in PBS and washed again, and intracellular (as well as extracellular) bacteria were stained by anti–S pyogenes antibodies, followed by Alexa red–conjugated goat antirabbit antibodies (Sigma-Aldrich). Exclusively extracellular bacteria are labeled in green (i), extracellular plus intracellular bacteria are labeled in red (ii), and MC nuclei are labeled in blue (iii). An overlaid merged image where extracellular bacteria are labeled yellow and intracellular bacteria in red is shown (iv). (B) Transmission electron microscopic examination of cross-sections of HMC-1 cells cocultured with S pyogenes (bar, 2.5 μm). Bacteria are indicated by black arrows. Notice that all streptococcal microorganisms are extracellularly located. (C) Immunofluorescence staining for determination of extracellular (yellow) and intracellular (red) location of S aureus associated with MCs (bar, 3.5 μm). (D) Transmissionelectron microscopic examination of cross-sections of HMC-1 cells showing internalized S aureus (i,ii; white arrows). Extracellular bacteria (i) are indicated by a black arrow (bars, 1 μm). (E) Growth of S pyogenes in medium alone, in coculture with MCs, in coculture with cytochalasin D–treated MCs, or in coculture with MCs separated by a transwell system. Data are expressed as x-fold increase in bacterial growth with respect to the original inoculum. Each point represents the mean plus or minus SD of 3 independent experiments. *P < .05 by F-test for S pyogenes growth in medium control versus S pyogenes growth in the presence of either untreated or cytochalasin D–treated HMC-1 cells.

  • Figure 2

    Immunofluorescence and FESEM examination of human MCETs. MCs were seeded on poly-L-lysine–coated glass slides, stimulated with 25 nM PMA for 10 minutes, then infected with FITC-labeled S pyogenes (MOI 25:1) for 1 hour and fixed with 4% paraformaldehyde. (A) Colocalization of DNA, S pyogenes, and LL-37 in MCETs (bars, 10 μm): (i) Dapi-stained DNA; (ii) FITC-labeled S pyogenes; (iii) immunostaining of LL-37 with Alexa-red–labeled antibodies against human LL-37; and (iv) overlay of A1-3. (B) Immunostaining of MCETs with Alexa-red-labeled antibodies against histones (bar, 10 μm). Insert in the lower-left corner (ii) shows a higher magnification of S pyogenes (green) entrapped in the MCETs (red; bar, 3.5 μm). (C) Immunostaining of MCETs with Alexa-red–labeled antibodies against tryptase. S pyogenes are labeled green and DNA are labeled blue (bar, 8 μm). (D) FESEM image of MCETs produced by human MCs during coculture with S pyogenes. Microorganisms entrapped in the MCETs structures are indicated by white arrows (bar, 5 μm). (E) Higher magnification showing S pyogenes entrapped in the fibers of the MCETs (white arrows; bar, 1 μm).

  • Figure 3

    In vitro interactions of S pyogenes with murine BMMCs. (A) FESEM images of uninfected murine BMMCs after 21 days in culture medium supplemented with recombinant mouse IL-3 (bar, 2 μm). (B) S pyogenes (arrow) attached to the surface of BMMCs (bar, 1 μm). (C) A clump of S pyogenes (arrow) trapped by extracellular fibers produced by BMMCs (bar, 10 μm). Insert in top-right corner shows a higher magnification of an entrapped bacterial clump (bar, 2 μm). (D) MCs in the process of producing MCETs. Some streptococci trapped by incipient MCETs are indicated by white arrows (bar, 5 μm). (E) S pyogenes captured in MCETs (white arrows; bar, 5 μm). (F) Immunofluorescence photograph showing blue labeled DNA (BMMC nuclei and extracellular MCET fibers) and associated red-labeled S pyogenes (bar, 10 μm). (G) Growth of S pyogenes in medium alone, in coculture with MCs, in coculture with cytochalasin D–treated MCs, or in coculture with MCs separated by a transwell system. Data are expressed as x-fold increase in bacterial growth with respect to the original inoculum. Each point represents the mean plus or minus SD of 3 independent experiments. *P < .05 by F-test for S pyogenes growth in medium control versus S pyogenes growth in the presence of either untreated or cytochalasin D–treated BMMCs.

  • Figure 4

    Intact MCs extracellular traps are required for effective growth inhibition of S pyogenes. (A) Immunostaining with Alexa-red–labeled antibodies against histones of (i) intact MCETs or (ii) disrupted MCETs after treatment with DNAse and myeloperoxidase (bars, 10 μm). (iii) Isotype control antibody (bar, 10 μm). The nucleus of MCs was stained with Dapi (blue). (B) Growth of S pyogenes after coculture with MCs treated with DNAse and MPO to dismantle extracellular trap structures, untreated MCs, or in medium without MCs supplemented with DNAse and MPO. Data are expressed as x-fold increase in bacterial growth with respect to the original inoculum. Each point represents the mean plus or minus SD of 3 independent experiments. *P < .05 by F-test for S pyogenes growth in the presence of untreated HMC-1 cells versus growth of S pyogenes in the presence of DNAse/MPO-treated HMC-1 cells. (C) Analysis of viable (green) versus dead (red) bacteria entrapped in MCETs (i) or grown in medium control (ii) as determined by the LIVE/DEAD BacLight Bacterial Viability assay (bars, 3 μm).

  • Figure 5

    Formation of MCETs is dependent of reactive oxygen species (ROS)-induced MCs death. (A) Production of ROS by S pyogenes–infected MCs in the presence or absence of NADPH oxidase inhibitor DPI. Uninfected MCs (i) or S pyogenes–infected MCs cultured in the absence (ii) or presence of DPI (iii) were incubated with NBT for 45 minutes and examined by light microscopy. Precipitation of formazan indicating active production of ROS is indicated by arrows in panel ii (bars, 10 μm). (B) Immunofluorescence photograph showing viable (green) versus dead (red) MCs of uninfected cells (i) or cells after coculture with S pyogenes (ii). Note the release of DNA by dying MCs (arrow) in panel ii. All bars in panel B represent 10 μm. (C) Quantification of MC death in control medium (□), coculture with S pyogenes (■), or coculture with S pyogenes in the presence of DPI (▩). The data are presented as percentage of PI-stained dead cells determined by flow cytometry. Bars represent the means plus or minus SD of 3 independent experiments. *P < .05 by F-test. (D) Quantification of DNA release by MCs after 1 hour of culture in the presence of PMA or S pyogenes and with or without DPI. The amount of DNA was measured as intensity/total flux of red fluorescence (Sytox orange) using the Xenogen Vivo Vision IVIS 200 System with the filter setting of 532 nm excitation/580 nm emission and Igor Pro 4.09A software. A diagram showing the quantitative data are displayed in panel E. (F) FESEM image of MCETs produced by human MCs after 6 hours of either stimulation with 100 mU/mL of glucose oxidase (F) or with S pyogenes (G). Bar, 50 μm.

  • Figure 6

    Structural examination of MCs stimulated with either S pyogenes or glucose oxidase by transmission electron microscopy. MCs were incubated with medium alone (A), with S pyogenes for 6 hours (B), or with 100 mU/mL of glucose oxidase (C). Unstimulated MCs show intact nuclear membrane (A), whereas the external and internal sheets of the nuclear membrane start to separate in S pyogenes–stimulated (B) and glucose oxidase–stimulated (C) MCs. Desintegration of the nuclear membrane can be seen in panels Biii (arrows) and C. Bars are 2 μm (Ai,Bi) and 0.5 μm (Aii,Bii,iii,C).

  • Figure 7

    Induction of MCETs by S aureus and P aeruginosa. HMC-1 cells were seeded on poly-L-lysine–coated glass slides, then infected with S aureus or P aeruginosa (MOI 1:25) for 6 hours and fixed with 4% paraformaldehyde and examined by FESEM. (A) P aeruginosa entrapped by MCETs (white arrows; bars are 10 μm for panel Ai and 2 μm for panel Aii). (B) S aureus entrapped by extracellular fibers produced by MCs (bars are 2 μm for panel Bi and 1 μm for panel Bii).

Supplementary Materials

  • Figure S1

    Supplementary PDF file available online.

  • Figure S2

    Supplementary PDF file available online.