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FULL: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0051727
Subversion of Autophagy in Adherent Invasive Escherichia coli-Infected Neutrophils Induces Inflammation and Cell Death
Abderrahman Chargui, Annabelle Cesaro, Sanda Mimouna, Mohamed Fareh, Patrick Brest, Philippe Naquet, Arlette Darfeuille-Michaud, Xavier Hébuterne, Baharia Mograbi , Valérie Vouret-Craviari , Paul Hofman
Faculté de Médecine, Nice, France
December 14, 2012
Abstract
Invading bacteria are recognized, captured and killed by a specialized form of autophagy, called xenophagy. Recently, defects in xenophagy in Crohn’s disease (CD) have been implicated in the pathogenesis of human chronic inflammatory diseases of uncertain etiology of the gastrointestinal tract. We show here that pathogenic adherent-invasive Escherichia coli (AIEC) isolated from CD patients are able to adhere and invade neutrophils, which represent the first line of defense against bacteria. Of particular interest, AIEC infection of neutrophil-like PLB-985 cells blocked autophagy at the autolysosomal step, which allowed intracellular survival of bacteria and exacerbated interleukin-8 (IL-8) production. Interestingly, this block in autophagy correlated with the induction of autophagic cell death. Likewise, stimulation of autophagy by nutrient starvation or rapamycin treatment reduced intracellular AIEC survival and IL-8 production. Finally, treatment with an inhibitor of autophagy decreased cell death of AIEC-infected neutrophil-like PLB-985 cells. In conclusion, excessive autophagy in AIEC infection triggered cell death of neutrophils.
figure 2. Inhibition of autophagic flux by AIEC LF82 infection.
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 (A, left panel, B), quantitative RT-PCR (A rigth panel), immunofluorescence (C, D) and ultrastructural TEM analysis (E).
(A) 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
(A right panel). Data are means ± SEM of three experiments. ** p<0.001.
(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.
(C) 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).
(D) 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).
(E) 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.
Subversion of Autophagy in Adherent Invasive Escherichia coli-Infected Neutrophils Induces Inflammation and Cell Death
Abderrahman Chargui, Annabelle Cesaro, Sanda Mimouna, Mohamed Fareh, Patrick Brest, Philippe Naquet, Arlette Darfeuille-Michaud, Xavier Hébuterne, Baharia Mograbi , Valérie Vouret-Craviari , Paul Hofman
Faculté de Médecine, Nice, France
December 14, 2012
Abstract
Invading bacteria are recognized, captured and killed by a specialized form of autophagy, called xenophagy. Recently, defects in xenophagy in Crohn’s disease (CD) have been implicated in the pathogenesis of human chronic inflammatory diseases of uncertain etiology of the gastrointestinal tract. We show here that pathogenic adherent-invasive Escherichia coli (AIEC) isolated from CD patients are able to adhere and invade neutrophils, which represent the first line of defense against bacteria. Of particular interest, AIEC infection of neutrophil-like PLB-985 cells blocked autophagy at the autolysosomal step, which allowed intracellular survival of bacteria and exacerbated interleukin-8 (IL-8) production. Interestingly, this block in autophagy correlated with the induction of autophagic cell death. Likewise, stimulation of autophagy by nutrient starvation or rapamycin treatment reduced intracellular AIEC survival and IL-8 production. Finally, treatment with an inhibitor of autophagy decreased cell death of AIEC-infected neutrophil-like PLB-985 cells. In conclusion, excessive autophagy in AIEC infection triggered cell death of neutrophils.
figure 2. Inhibition of autophagic flux by AIEC LF82 infection.
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 (A, left panel, B), quantitative RT-PCR (A rigth panel), immunofluorescence (C, D) and ultrastructural TEM analysis (E).
(A) 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
(A right panel). Data are means ± SEM of three experiments. ** p<0.001.
(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.
(C) 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).
(D) 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).
(E) 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.
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