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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 mast cells.Primary mouse BMMCs were left untreated (NT) or infected with P. aeruginosa strain 8821 at an MOI of 1∶100. Lysates were collected at the time points indicated and subjected to Western blot analysis for LC3 and actin loading control (A). The fold change in the ratio of LC3-I to LC3-II normalized to actin was determined by scanning densitometry (B) (n = 3 ± SEM, *p<0.05). HMC-1 5C6 human mast cell line stably expressing LC3-GFP reporter were left untreated (NT) or infected with P. aeruginosa at an MOI of 1∶100 for 18 hours (Ps.a) before being fixed and examined by laser scanning confocal microscopy (C). HMC-1 5C6 cells stably expressing LC3-GFP were left untreated (NT) or infected with P. aeruginosa strain 8821 at an MOI of 1∶100. At the indicated time points cells were fixed for the study by confocal microscopy, and the percentage of cells displaying 5 or more GFP puncta was assessed (D). (n = 3± SEM, *p<0.05, **p<0.01). Lysates were also prepared from HMC-1 5C6 cells stably expressing LC3-GFP and Western blot analysis for free GFP and actin loading control was performed (E).
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pone-0072263-g001: P. aeruginosa induces autophagy in mast cells.Primary mouse BMMCs were left untreated (NT) or infected with P. aeruginosa strain 8821 at an MOI of 1∶100. Lysates were collected at the time points indicated and subjected to Western blot analysis for LC3 and actin loading control (A). The fold change in the ratio of LC3-I to LC3-II normalized to actin was determined by scanning densitometry (B) (n = 3 ± SEM, *p<0.05). HMC-1 5C6 human mast cell line stably expressing LC3-GFP reporter were left untreated (NT) or infected with P. aeruginosa at an MOI of 1∶100 for 18 hours (Ps.a) before being fixed and examined by laser scanning confocal microscopy (C). HMC-1 5C6 cells stably expressing LC3-GFP were left untreated (NT) or infected with P. aeruginosa strain 8821 at an MOI of 1∶100. At the indicated time points cells were fixed for the study by confocal microscopy, and the percentage of cells displaying 5 or more GFP puncta was assessed (D). (n = 3± SEM, *p<0.05, **p<0.01). Lysates were also prepared from HMC-1 5C6 cells stably expressing LC3-GFP and Western blot analysis for free GFP and actin loading control was performed (E).

Mentions: Mast cells are important sentinel cells of the immune system, playing a critical role in sensing invading pathogens and coordinating the appropriate immune response against P. aeruginosa[5], [7]. Due to the high density of the cells along the airways, and their phagocyte capacity, mast cells also represent the first line of defense against pathogens within the respiratory tract. Our lab has extensively studied the roles of mast cells during host defense against P. aeruginosa. However the role of autophagy in mast cells in the context of host defense, as well as the biological role of autophagy during P. aeruginosa infections remains unknown. In order to examine the role of autophagy in mast cells during P. aeruginosa infection, bone marrow derived mast cells (BMMCs) were cultured from C57/BL6 mice. These cells were then infected with P. aeruginosa strain 8821 at an MOI of 1∶100. Whole cell lysates were prepared at various time points as indicated, and subjected to western blot analysis for microtubule associated protein light chain 3 (LC3) and actin loading control. Upon the induction of autophagy the cytoplasmic form of LC3 (LC3-I) becomes cleaved and conjugated to phophotidylethanolamine (PE) through a ubiquitin like conjugation pathway. This PE conjugated form of the protein (LC3-II) becomes and remains associated with autophagosomal membrane throughout the maturation cycle of the vesicle. The conversion of cytosolic LC3-I to autophagosome associated LC3-II is diagnostic of autophagy and can be tracked by Western blot analysis [46]. In untreated cells, the PE conjugated LC3-II form of the protein predominated within the cells, consistent with a previously describe role for LC3 in mast cell granule formation [47]. However while the cytoplasmic LC3-I levels remained unchanged upon stimulation with P. aeruginosa, the levels of LC3-II accumulated well above basal levels, peaking around 18 hours post infection (hpi), indicating an induction of autophagy (Figure 1A and 1B).


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 mast cells.Primary mouse BMMCs were left untreated (NT) or infected with P. aeruginosa strain 8821 at an MOI of 1∶100. Lysates were collected at the time points indicated and subjected to Western blot analysis for LC3 and actin loading control (A). The fold change in the ratio of LC3-I to LC3-II normalized to actin was determined by scanning densitometry (B) (n = 3 ± SEM, *p<0.05). HMC-1 5C6 human mast cell line stably expressing LC3-GFP reporter were left untreated (NT) or infected with P. aeruginosa at an MOI of 1∶100 for 18 hours (Ps.a) before being fixed and examined by laser scanning confocal microscopy (C). HMC-1 5C6 cells stably expressing LC3-GFP were left untreated (NT) or infected with P. aeruginosa strain 8821 at an MOI of 1∶100. At the indicated time points cells were fixed for the study by confocal microscopy, and the percentage of cells displaying 5 or more GFP puncta was assessed (D). (n = 3± SEM, *p<0.05, **p<0.01). Lysates were also prepared from HMC-1 5C6 cells stably expressing LC3-GFP and Western blot analysis for free GFP and actin loading control was performed (E).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756076&req=5

pone-0072263-g001: P. aeruginosa induces autophagy in mast cells.Primary mouse BMMCs were left untreated (NT) or infected with P. aeruginosa strain 8821 at an MOI of 1∶100. Lysates were collected at the time points indicated and subjected to Western blot analysis for LC3 and actin loading control (A). The fold change in the ratio of LC3-I to LC3-II normalized to actin was determined by scanning densitometry (B) (n = 3 ± SEM, *p<0.05). HMC-1 5C6 human mast cell line stably expressing LC3-GFP reporter were left untreated (NT) or infected with P. aeruginosa at an MOI of 1∶100 for 18 hours (Ps.a) before being fixed and examined by laser scanning confocal microscopy (C). HMC-1 5C6 cells stably expressing LC3-GFP were left untreated (NT) or infected with P. aeruginosa strain 8821 at an MOI of 1∶100. At the indicated time points cells were fixed for the study by confocal microscopy, and the percentage of cells displaying 5 or more GFP puncta was assessed (D). (n = 3± SEM, *p<0.05, **p<0.01). Lysates were also prepared from HMC-1 5C6 cells stably expressing LC3-GFP and Western blot analysis for free GFP and actin loading control was performed (E).
Mentions: Mast cells are important sentinel cells of the immune system, playing a critical role in sensing invading pathogens and coordinating the appropriate immune response against P. aeruginosa[5], [7]. Due to the high density of the cells along the airways, and their phagocyte capacity, mast cells also represent the first line of defense against pathogens within the respiratory tract. Our lab has extensively studied the roles of mast cells during host defense against P. aeruginosa. However the role of autophagy in mast cells in the context of host defense, as well as the biological role of autophagy during P. aeruginosa infections remains unknown. In order to examine the role of autophagy in mast cells during P. aeruginosa infection, bone marrow derived mast cells (BMMCs) were cultured from C57/BL6 mice. These cells were then infected with P. aeruginosa strain 8821 at an MOI of 1∶100. Whole cell lysates were prepared at various time points as indicated, and subjected to western blot analysis for microtubule associated protein light chain 3 (LC3) and actin loading control. Upon the induction of autophagy the cytoplasmic form of LC3 (LC3-I) becomes cleaved and conjugated to phophotidylethanolamine (PE) through a ubiquitin like conjugation pathway. This PE conjugated form of the protein (LC3-II) becomes and remains associated with autophagosomal membrane throughout the maturation cycle of the vesicle. The conversion of cytosolic LC3-I to autophagosome associated LC3-II is diagnostic of autophagy and can be tracked by Western blot analysis [46]. In untreated cells, the PE conjugated LC3-II form of the protein predominated within the cells, consistent with a previously describe role for LC3 in mast cell granule formation [47]. However while the cytoplasmic LC3-I levels remained unchanged upon stimulation with P. aeruginosa, the levels of LC3-II accumulated well above basal levels, peaking around 18 hours post infection (hpi), indicating an induction of autophagy (Figure 1A and 1B).

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