PMNs undergo autophagic death and NETosis on infection with AIEC LF82.

<p>Differentiated PLB-985 cells or PMN were infected (MOI 50) with K12 or AIEC LF82 bacteria for 1 h and gentamycin was added for the following 5 h, cells were then analysed. (<b>A</b>) Control or K12- or AIEC LF82-infected PLB-985 cells, treated or not with 3-methyladenine (3-MA, 5 mM, autophagy inhibitor) or Zvad (5 µM, a pancaspase inhibitor), were incubated with propidium iodide (PI) (5 µg/ml) and cell death was analysed by flow cytometric analysis as described in the materials and methods section. The average ± S.D. is shown for three independent experiments, *p<0.003. Inset: immunoblot analysis of LC3-II testifying to the induction of autophagy in infected cells. (<b>B,</b> left panel) PMNs were infected with AIEC LF82 and cell death was assayed as described in A. Maximal cell death (+) was obtained after treatment with etoposide phosphate (100 µg/ml). To test whether cell death was due to apoptosis, a caspase-3 activity test was performed as described in the materials and methods section (right panel). Maximal cell apoptosis (+) was obtained by treatment with staurosporine (10 µM). (<b>C</b>) Cleavage of PARP and caspase-3 in non-infected or K12- and AIEC LF82-infected differentiated PLB-985 cells were analysed by immunoblot analyses. Etoposide phosphate (100 µg/ml) was used as a positive control. LC3-II was detected in LF82-infected cells and compared to uninfected or K12-infected cells (1+5 h, MOI 50). Actin was used as a loading control. (<b>D</b>) Survival of AIEC LF82 and K12 in differentiated PLB-985 cells was analysed with the gentamycin assay (panel one). To compare the ultrastructural morphology of vesicles and bacteria in PLB-985 cells transmission electron microscopy was performed. Ultrastructural analysis of K12-infected differentiated PLB-985 cells shows bacterial sequestration and degradation in phagocytosis vacuoles (panel two). In contrast, accumulation of autophagic vesicles and bacteria (arrowheads) inside the vacuoles was observed in PLB-985 cells (panel three) and PMNs (panel four). Scale bar = 1 µm. (<b>E</b>) Differentiated PLB-985 were seeded on glass coverslips coated with poly D lysine (5 µg/cm²) and allowed to settle for 1 h. Cells were then infected with AIEC-LF82 (MOI 50) for 4 h (1 h+3 h) and 6 h (1 h+5 h), fixed with 3% paraformaldehyde and stained with Hoechst 33342 (0.5 µg/ml) to visualize DNA. Cells were examined with an epifluorescence Axiophot microscope (Zeiss). Early apoptosis and necrosis were assayed by measuring Annexin-V-fluos (Roche) and propidium iodide (PI). Differentiated non-infected PLB-985 cells or cells infected with AIEC LF82 (MOI 50) for 1 h+5 h were incubated with Annexin-V-fluos and PI and fluorescence was detected on a FACS Calibure.</p

Inhibition of autophagic flux by AIEC LF82 infection.

<p>Human neutrophils or neutrophil-like PLB-985 cells were infected with AIEC LF82 at a MOI of 50 for 1 h than gentamicin (100 µg/ml) was added for 3 h. Cells were and processed for immunoblotting (<b>A,</b> left panel, <b>B</b>), quantitative RT-PCR (<b>A</b> rigth panel), immunofluorescence (<b>C</b>, <b>D</b>) and ultrastructural TEM analysis (<b>E</b>). (<b>A</b>) Time-dependent accumulation (1–8 h) of LC3-II and p62 in infected human neutrophils or neutrophil-like PLB-985 cells compared to uninfected cells analyzed at the end time point. Longer exposure detects the LC3-I band. We checked that AIEC infection did not affect p62 mRNA levels by qRT-PCR analysis (<b>A</b> right panel). Data are means ± SEM of three experiments. ** p<0.001. (<b>B</b>) Autophagic flux was analysed by immunoblot analysis in differentiated PLB-985 cells infected for 3 h with AIEC LF82 bacteria (MOI 50) in the absence or in the presence of E64d/PEPS. Actin was used as a loading control. Control unstimulated cells were analysed at the end time point. (<b>C</b>) Representative confocal images of control (0) or infected cells (LF82) (3 h post infection) showed the colocalization of bacteria with LC3-II and LAMP-1 proteins as indicated by yellow punctiform staining. Insets highlight individual staining of bacteria (DNA staining, blue), LC3-II (Alexa 488, green) and LAMP-1 (Alexa 594, red). (<b>D</b>) Representative confocal micrographs of control (0) and LF82 infected cells (LF82) showing the co-localization of bacteria (DNA staining, blue) within autophagic (LC3-II positive, Alexa 594, red) but not acidic compartments (LysoTracker negative, green). (<b>E</b>) Representative TEM images showing bacteria within endosomes (asterisk), autophagosomes (arrowheads) or free in the cytosol (arrow) in LF82-infected cells (3 h post infection). Bar = 2 µm.</p