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. 2012 May;80(5):1891-9.
doi: 10.1128/IAI.00050-12. Epub 2012 Feb 27.

Afa/Dr diffusely adhering Escherichia coli strain C1845 induces neutrophil extracellular traps that kill bacteria and damage human enterocyte-like cells

Viviana Marin-Esteban  1 , Isabelle Turbica, Guillaume Dufour, Nicolas Semiramoth, Aude Gleizes, Roseline Gorges, Isabelle Beau, Alain L Servin, Vanessa Lievin-Le Moal, Catherine Sandré, Sylvie Chollet-Martin
Affiliations

Afa/Dr diffusely adhering Escherichia coli strain C1845 induces neutrophil extracellular traps that kill bacteria and damage human enterocyte-like cells

Viviana Marin-Esteban et al. Infect Immun. 2012 May.

Abstract

We recently documented the neutrophil response to enterovirulent diffusely adherent Escherichia coli expressing Afa/Dr fimbriae (Afa/Dr DAEC), using the human myeloid cell line PLB-985 differentiated into fully mature neutrophils. Upon activation, particularly during infections, neutrophils release neutrophil extracellular traps (NETs), composed of a nuclear DNA backbone associated with antimicrobial peptides, histones, and proteases, which entrap and kill pathogens. Here, using fluorescence microscopy and field emission scanning electron microscopy, we observed NET production by PLB-985 cells infected with the Afa/Dr wild-type (WT) E. coli strain C1845 VSports手机版. We found that these NETs were able to capture, immobilize, and kill WT C1845 bacteria. We also developed a coculture model of human enterocyte-like Caco-2/TC7 cells and PLB-985 cells previously treated with WT C1845 and found, for the first time, that the F-actin cytoskeleton of enterocyte-like cells is damaged in the presence of bacterium-induced NETs and that this deleterious effect is prevented by inhibition of protease release. These findings provide new insights into the neutrophil response to bacterial infection via the production of bactericidal NETs and suggest that NETs may damage the intestinal epithelium, particularly in situations such as inflammatory bowel diseases. .

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Fig 1
Fig 1
Characterization of PLB-985-derived NETs by immunofluorescence and FESEM. Cells were seeded on poly-l-lysine-coated coverslips and stimulated for 4 h with PMA in 0.5% FCS-supplemented HBSS medium. (A) Hoechst-stained DNA from cells stimulated with PMA exhibiting a web-like aspect (left) or a fiber-like aspect (right); (B) colocalization of Hoechst-stained DNA with MPO (left) or with β2-integrin (CD11b/CD18) (right); (C) FESEM images of NETs. Bars on immunofluorescence pictures, 10 μm.
Fig 2
Fig 2
Differentiated PLB-985 cells produce NETs in response to PMA via a ROS-dependent mechanism. Extracellular DNA was detected using Sytox green, a non-cell-permeant DNA dye. The time course and concentration effect of PMA were examined on freshly isolated blood PMNs (A), differentiated PLB-985 cells (B), and differentiated X-CGD PLB-985 cells deficient in gp91-phox NOX2 subunits (C). Cells were incubated with 5 μM Sytox green and with PMA at the indicated concentrations (0 to 250 nM). In some experiments, wild-type (D) and X-CGD PLB-985 (E) cells were also directly stimulated with hydrogen peroxide (0 to 2,500 nM). Sytox green fluorescence was recorded in arbitrary units (AU) at increasing incubation times (from 0 to 300 min). The data are means ± SEMs and are representative of at least 3 separate experiments.
Fig 3
Fig 3
NET induction by enterovirulent Afa/Dr DAEC. PLB-985 cells were seeded on poly-l-lysine-coated glass slides and incubated for 4 h with WT C1845 at an MOI of 10 in 0.5% FCS-supplemented HBSS medium at 37°C. (A) Hoechst-stained DNA and phase-contrast micrography of C1845 bacteria; (B and C) FESEM images showing C1845 bacterial cells associated with NETs; (D and E) for quantitative experiments, PLB-985 cells were incubated for 4 h with or without WT C1845, LF-82, CFA-I, or CFA-III (MOI, 60 bacteria per cell). Extracellular DNA was quantified with Sytox green as described in the text. Data from six different experiments are represented. In panel D, the boxes extend from the first to the third quartiles, the median is indicated by a horizontal line, and maximal and minimal values are represented at the end of the whiskers. In panel E, the data are means ± SEMs. *, P < 0.05 compared with noninfected control cells.
Fig 4
Fig 4
Extracellular bacterial killing is associated with NET formation independently of phagocytosis-mediated killing. PLB-985 cells were seeded in 24-well tissue culture plates and prestimulated for 4 h with 20 nM PMA. Before bacterial challenge, PMA-stimulated cells were treated with DNase (to dismantle NETs), whereas unstimulated cells were treated with cytochalasin D (phagocytosis inhibitor). After coincubation of PLB-985 cells and C1845 bacteria (MOI, 0.1) for 45 min, the number of surviving bacteria was evaluated as the number of CFU recovered after 24 h of culture. (A) Percent bacterial killing by netosis was calculated from the CFU values obtained with PMA-stimulated cells subsequently treated with DNase. (B) Percent bacterial killing by phagocytosis was calculated from the CFU values obtained with unstimulated cells treated with cytochalasin D and was compared to those obtained after netosis killing. The graph indicates mean bacterial killing ± SEM in five separate experiments.
Fig 5
Fig 5
C1845-induced NETs affect F-actin cytoskeleton organization in Caco-2/TC7 enterocyte-like cells. PLB-985 cells were infected with gentamicin-killed C1845 prior to incubation with Caco-2/TC7 cell monolayers for 4 h. (A to G) After washing, the cells were labeled for F actin or β2-integrin. Preparations were examined by epifluorescence microscopy with a confocal laser scanning microscope. Optical sectioning was used to collect 20 en face images 0.1 nm apart. Horizontal views were obtained by integrating images obtained with a step position of 1, using LSM510 (version 2.5) software (Zeiss, Germany). (A) F-actin staining of Caco-2/TC7 cells alone; (B) F-actin staining of Caco-2/TC7 cells cocultured with PLB-985 alone; (C) F-actin staining of Caco-2/TC7 cells cultured with killed C1845 cells; (D and E) Caco-2/TC7 cells cocultured with killed C1845-treated PLB-985 cells and stained for F-actin (red) and β2-integrin (green), respectively; (F) F-actin staining of Caco-2/TC7 cells cocultured with PLB-985 cells treated with pentoxifylline prior to contact with killed WT C1845 cells; (G) evidence of bacteria trapped by extracellular DNA (Hoechst blue staining), which colocalized with β2-integrin (green). The images are representative of three different experiments. Magnifications (A to G), ×63. (H) The coculture model was adapted to the 96-well plate format, and extracellular DNA was quantified by fluorimetry using Sytox green; results are means ± SEMs of at least three experiments. *, P < 0.05 compared with the other two conditions.
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References

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