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Autophagy enhances bacterial clearance during P. aeruginosa lung infection.

Junkins RD, Shen A, Rosen K, McCormick C, Lin TJ - PLoS ONE (2013)

Bottom Line: We further demonstrate that inhibition of autophagy through pharmacological means or protein knockdown inhibits clearance of intracellular P. aeruginosa in vitro, while pharmacologic induction of autophagy significantly increased bacterial clearance.Finally we find that pharmacological manipulation of autophagy in vivo effectively regulates bacterial clearance of P. aeruginosa from the lung.Together our results demonstrate that autophagy is required for an effective immune response against P. aeruginosa infection in vivo, and suggest that pharmacological interventions targeting the autophagy pathway could have considerable therapeutic potential in the treatment of P. aeruginosa lung infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada ; Department of Pediatrics, IWK Health Centre, Halifax, Nova Scotia, Canada ; Beatrice Hunter Cancer Research Institute, Halifax, Nova Scotia, Canada.

ABSTRACT
Pseudomonas aeruginosa is an opportunistic bacterial pathogen which is the leading cause of morbidity and mortality among cystic fibrosis patients. Although P. aeruginosa is primarily considered an extacellular pathogen, recent reports have demonstrated that throughout the course of infection the bacterium acquires the ability to enter and reside within host cells. Normally intracellular pathogens are cleared through a process called autophagy which sequesters and degrades portions of the cytosol, including invading bacteria. However the role of autophagy in host defense against P. aeruginosa in vivo remains unknown. Understanding the role of autophagy during P. aeruginosa infection is of particular importance as mutations leading to cystic fibrosis have recently been shown to cause a blockade in the autophagy pathway, which could increase susceptibility to infection. Here we demonstrate that P. aeruginosa induces autophagy in mast cells, which have been recognized as sentinels in the host defense against bacterial infection. We further demonstrate that inhibition of autophagy through pharmacological means or protein knockdown inhibits clearance of intracellular P. aeruginosa in vitro, while pharmacologic induction of autophagy significantly increased bacterial clearance. Finally we find that pharmacological manipulation of autophagy in vivo effectively regulates bacterial clearance of P. aeruginosa from the lung. Together our results demonstrate that autophagy is required for an effective immune response against P. aeruginosa infection in vivo, and suggest that pharmacological interventions targeting the autophagy pathway could have considerable therapeutic potential in the treatment of P. aeruginosa lung infection.

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P. aeruginosa induces autophagy in primary human and mouse mast cells, and becomes incorporated into autophagosomes.Primary human cord blood derived mast cells (CBMCs) and mouse bone marrow-derived mast cells (BMMCs) were left untreated (NT) or infected with P. aeruginosa strain 8821 at an MOI of 1∶100 for 8 hours. Cells were then fixed and processed for transmission electron microscopic study. Representative images of untreated (A) and P. aeruginosa treated (B) CBMCs are shown. P. aeruginosa could clearly be seen inside double membrane bound vesicles inside the mast cells (insert). The percentage of cytosol encompassed within autophagosomes (C) and the number of autophagosomes per cross section (D) in CBMCs and BMMCs was calculated by area analysis of cross sections containing a portion of the cell nucleus (n = 21± SEM, *p<0.05).
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pone-0072263-g002: P. aeruginosa induces autophagy in primary human and mouse mast cells, and becomes incorporated into autophagosomes.Primary human cord blood derived mast cells (CBMCs) and mouse bone marrow-derived mast cells (BMMCs) were left untreated (NT) or infected with P. aeruginosa strain 8821 at an MOI of 1∶100 for 8 hours. Cells were then fixed and processed for transmission electron microscopic study. Representative images of untreated (A) and P. aeruginosa treated (B) CBMCs are shown. P. aeruginosa could clearly be seen inside double membrane bound vesicles inside the mast cells (insert). The percentage of cytosol encompassed within autophagosomes (C) and the number of autophagosomes per cross section (D) in CBMCs and BMMCs was calculated by area analysis of cross sections containing a portion of the cell nucleus (n = 21± SEM, *p<0.05).

Mentions: We next set out to examine the ultrastructural characteristics of autophagosomes in primary human and murine mast cells. To address this question we infected primary human cord blood derived mast cells (CBMCs) and primary mouse BMMCs with P. aeruginosa strain 8821 at an MOI of 1∶100. Eight hours later cells were fixed and processed for TEM viewing (Figure 2A and 2B). Highly vesicularized double membrane bound vesicles characteristic of autophagosomes were observed in both untreated (NT) and P. aeruginosa (Ps.a) treated cells. The percentage of cytosol contained within autophagosomes, as well as the number of autophagosomes per cross-section was significantly increased in P. aeruginosa treated cells (Figure 2C and 2D). Furthermore, P. aeruginosa bacteria were repeatedly seen inside the cell contained within double membrane bound vesicles (insert), although these observations were infrequent. Together these results demonstrate that autophagy is induced by P. aeruginosa in primary human and mouse mast cells, and that the bacteria can become incorporated into autophagosomes in vitro.


Autophagy enhances bacterial clearance during P. aeruginosa lung infection.

Junkins RD, Shen A, Rosen K, McCormick C, Lin TJ - PLoS ONE (2013)

P. aeruginosa induces autophagy in primary human and mouse mast cells, and becomes incorporated into autophagosomes.Primary human cord blood derived mast cells (CBMCs) and mouse bone marrow-derived mast cells (BMMCs) were left untreated (NT) or infected with P. aeruginosa strain 8821 at an MOI of 1∶100 for 8 hours. Cells were then fixed and processed for transmission electron microscopic study. Representative images of untreated (A) and P. aeruginosa treated (B) CBMCs are shown. P. aeruginosa could clearly be seen inside double membrane bound vesicles inside the mast cells (insert). The percentage of cytosol encompassed within autophagosomes (C) and the number of autophagosomes per cross section (D) in CBMCs and BMMCs was calculated by area analysis of cross sections containing a portion of the cell nucleus (n = 21± SEM, *p<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756076&req=5

pone-0072263-g002: P. aeruginosa induces autophagy in primary human and mouse mast cells, and becomes incorporated into autophagosomes.Primary human cord blood derived mast cells (CBMCs) and mouse bone marrow-derived mast cells (BMMCs) were left untreated (NT) or infected with P. aeruginosa strain 8821 at an MOI of 1∶100 for 8 hours. Cells were then fixed and processed for transmission electron microscopic study. Representative images of untreated (A) and P. aeruginosa treated (B) CBMCs are shown. P. aeruginosa could clearly be seen inside double membrane bound vesicles inside the mast cells (insert). The percentage of cytosol encompassed within autophagosomes (C) and the number of autophagosomes per cross section (D) in CBMCs and BMMCs was calculated by area analysis of cross sections containing a portion of the cell nucleus (n = 21± SEM, *p<0.05).
Mentions: We next set out to examine the ultrastructural characteristics of autophagosomes in primary human and murine mast cells. To address this question we infected primary human cord blood derived mast cells (CBMCs) and primary mouse BMMCs with P. aeruginosa strain 8821 at an MOI of 1∶100. Eight hours later cells were fixed and processed for TEM viewing (Figure 2A and 2B). Highly vesicularized double membrane bound vesicles characteristic of autophagosomes were observed in both untreated (NT) and P. aeruginosa (Ps.a) treated cells. The percentage of cytosol contained within autophagosomes, as well as the number of autophagosomes per cross-section was significantly increased in P. aeruginosa treated cells (Figure 2C and 2D). Furthermore, P. aeruginosa bacteria were repeatedly seen inside the cell contained within double membrane bound vesicles (insert), although these observations were infrequent. Together these results demonstrate that autophagy is induced by P. aeruginosa in primary human and mouse mast cells, and that the bacteria can become incorporated into autophagosomes in vitro.

Bottom Line: We further demonstrate that inhibition of autophagy through pharmacological means or protein knockdown inhibits clearance of intracellular P. aeruginosa in vitro, while pharmacologic induction of autophagy significantly increased bacterial clearance.Finally we find that pharmacological manipulation of autophagy in vivo effectively regulates bacterial clearance of P. aeruginosa from the lung.Together our results demonstrate that autophagy is required for an effective immune response against P. aeruginosa infection in vivo, and suggest that pharmacological interventions targeting the autophagy pathway could have considerable therapeutic potential in the treatment of P. aeruginosa lung infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada ; Department of Pediatrics, IWK Health Centre, Halifax, Nova Scotia, Canada ; Beatrice Hunter Cancer Research Institute, Halifax, Nova Scotia, Canada.

ABSTRACT
Pseudomonas aeruginosa is an opportunistic bacterial pathogen which is the leading cause of morbidity and mortality among cystic fibrosis patients. Although P. aeruginosa is primarily considered an extacellular pathogen, recent reports have demonstrated that throughout the course of infection the bacterium acquires the ability to enter and reside within host cells. Normally intracellular pathogens are cleared through a process called autophagy which sequesters and degrades portions of the cytosol, including invading bacteria. However the role of autophagy in host defense against P. aeruginosa in vivo remains unknown. Understanding the role of autophagy during P. aeruginosa infection is of particular importance as mutations leading to cystic fibrosis have recently been shown to cause a blockade in the autophagy pathway, which could increase susceptibility to infection. Here we demonstrate that P. aeruginosa induces autophagy in mast cells, which have been recognized as sentinels in the host defense against bacterial infection. We further demonstrate that inhibition of autophagy through pharmacological means or protein knockdown inhibits clearance of intracellular P. aeruginosa in vitro, while pharmacologic induction of autophagy significantly increased bacterial clearance. Finally we find that pharmacological manipulation of autophagy in vivo effectively regulates bacterial clearance of P. aeruginosa from the lung. Together our results demonstrate that autophagy is required for an effective immune response against P. aeruginosa infection in vivo, and suggest that pharmacological interventions targeting the autophagy pathway could have considerable therapeutic potential in the treatment of P. aeruginosa lung infection.

Show MeSH
Related in: MedlinePlus