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Acute exposure to silica nanoparticles enhances mortality and increases lung permeability in a mouse model of Pseudomonas aeruginosa pneumonia.

Delaval M, Boland S, Solhonne B, Nicola MA, Mornet S, Baeza-Squiban A, Sallenave JM, Garcia-Verdugo I - Part Fibre Toxicol (2015)

Bottom Line: Furthermore, internalisation of SiO2 nanoparticles by primary alveolar macrophages did not reduce the capacity of the cells to clear Pseudomonas aeruginosa.In our murine model, SiO2 nanoparticle pre-exposure preferentially enhanced Pseudomonas aeruginosa-induced lung permeability (the latter assessed by the measurement of alveolar albumin and IgM concentrations) rather than contributing to Pseudomonas aeruginosa-induced lung inflammation (as measured by leukocyte recruitment and cytokine concentration in the alveolar compartment).The deleterious effects of SiO2 nanoparticle exposure during Pseudomonas aeruginosa-induced pneumonia are related to alterations of the alveolar-capillary barrier rather than to modulation of the inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: Univ Paris Diderot. Sorbone Paris Cité. Unit of Functional and Adaptive Biology (BFA) UMR 8251, CNRS, Laboratory of Molecular and Cellular Responses to Xenobiotics, 5 rue Thomas Mann, 75013, Paris, France. mathilde.delaval@gmail.com.

ABSTRACT

Background: The lung epithelium constitutes the first barrier against invading pathogens and also a major surface potentially exposed to nanoparticles. In order to ensure and preserve lung epithelial barrier function, the alveolar compartment possesses local defence mechanisms that are able to control bacterial infection. For instance, alveolar macrophages are professional phagocytic cells that engulf bacteria and environmental contaminants (including nanoparticles) and secrete pro-inflammatory cytokines to effectively eliminate the invading bacteria/contaminants. The consequences of nanoparticle exposure in the context of lung infection have not been studied in detail. Previous reports have shown that sequential lung exposure to nanoparticles and bacteria may impair bacterial clearance resulting in increased lung bacterial loads, associated with a reduction in the phagocytic capacity of alveolar macrophages.

Results: Here we have studied the consequences of SiO2 nanoparticle exposure on Pseudomonas aeruginosa clearance, Pseudomonas aeruginosa-induced inflammation and lung injury in a mouse model of acute pneumonia. We observed that pre-exposure to SiO2 nanoparticles increased mice susceptibility to lethal pneumonia but did not modify lung clearance of a bioluminescent Pseudomonas aeruginosa strain. Furthermore, internalisation of SiO2 nanoparticles by primary alveolar macrophages did not reduce the capacity of the cells to clear Pseudomonas aeruginosa. In our murine model, SiO2 nanoparticle pre-exposure preferentially enhanced Pseudomonas aeruginosa-induced lung permeability (the latter assessed by the measurement of alveolar albumin and IgM concentrations) rather than contributing to Pseudomonas aeruginosa-induced lung inflammation (as measured by leukocyte recruitment and cytokine concentration in the alveolar compartment).

Conclusions: We show that pre-exposure to SiO2 nanoparticles increases mice susceptibility to lethal pneumonia but independently of macrophage phagocytic function. The deleterious effects of SiO2 nanoparticle exposure during Pseudomonas aeruginosa-induced pneumonia are related to alterations of the alveolar-capillary barrier rather than to modulation of the inflammatory responses.

No MeSH data available.


Related in: MedlinePlus

Silica nanoparticles are internalized by alveolar macrophages and colocalize with alveolar epithelial cells. C57Bl/6j mice were instilled with FITC-SiO2 NPs (5 mg/kg;100 μg/mice). A) 5 h later, mice were sacrificed and cells present in BALs were analysed by flow cytometry after labelling with specific antibodies directed against alveolar macrophages markers (CD11c, F4/80). Panel B) shows that 15 ± 2% (n = 4, mice) of the cells were FITC positive. Among them, over 95% were CD11c + and 85% CD11c + F4/80+ (panel C). The FACs analysis from one representative BAL from a total of 4 is shown. In D) BAL cells were cytospined and stained with anti-mouse CD45 antibody followed by Alexa 664 Fluor (A664) coupled secondary antibody (red) to confirm internalisation of FITC-SiO2 NPs (white arrows). E) Cryo-sections of lung tissues recovered from mice 24 h after instillation of FITC- SiO2 NPs (green) were stained with anti-proSPC antibody (a marker of alveolar type II epithelial cells) followed by A664 coupled secondary antibody (red). White arrows indicate co-localisation of NPs and alveolar epithelial cells. White scale bar = 100 μm or 20 μm (upper and lower panel, respectively). Nuclei were stained with DAPI (blue). Fluorescence of stained cells and tissues were analysed under structured illumination (ApoTome system). Superposed images from FITC, A664, DAPI and differential interferential contrast (D, lower panel) filters are shown. The ‘A’ within the figures indicates the alveolar spaces.
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Fig3: Silica nanoparticles are internalized by alveolar macrophages and colocalize with alveolar epithelial cells. C57Bl/6j mice were instilled with FITC-SiO2 NPs (5 mg/kg;100 μg/mice). A) 5 h later, mice were sacrificed and cells present in BALs were analysed by flow cytometry after labelling with specific antibodies directed against alveolar macrophages markers (CD11c, F4/80). Panel B) shows that 15 ± 2% (n = 4, mice) of the cells were FITC positive. Among them, over 95% were CD11c + and 85% CD11c + F4/80+ (panel C). The FACs analysis from one representative BAL from a total of 4 is shown. In D) BAL cells were cytospined and stained with anti-mouse CD45 antibody followed by Alexa 664 Fluor (A664) coupled secondary antibody (red) to confirm internalisation of FITC-SiO2 NPs (white arrows). E) Cryo-sections of lung tissues recovered from mice 24 h after instillation of FITC- SiO2 NPs (green) were stained with anti-proSPC antibody (a marker of alveolar type II epithelial cells) followed by A664 coupled secondary antibody (red). White arrows indicate co-localisation of NPs and alveolar epithelial cells. White scale bar = 100 μm or 20 μm (upper and lower panel, respectively). Nuclei were stained with DAPI (blue). Fluorescence of stained cells and tissues were analysed under structured illumination (ApoTome system). Superposed images from FITC, A664, DAPI and differential interferential contrast (D, lower panel) filters are shown. The ‘A’ within the figures indicates the alveolar spaces.

Mentions: To study the consequences of NP exposure on P.a lung infection, we set up a protocol where mice were first treated with SiO2 NPs (5 mg/kg) and 5 h later infected with the P.a strain PAK (Figure 1). For most of our experiments, we selected 5 h time after NP treatment as a time point for infection because at that time, we did not observed obvious signs of inflammation (including neutrophil accumulation) in the lungs of NP treated mice compared to controls (Figure 2). In addition, because neutrophil influx usually starts 6 hours post-LPS instillation [23,24], this indirectly also confirms that LPS is undetectable and if present, is biologically inactive in our NP preparations. However, in one parallel experiment, 24 h after NP instillation a slight inflammation was detected in the lungs characterized by increased levels of KC (CXCL-1, a neutrophil chemokine) and a slight neutrophil accumulation (less than 10% of total cells) (Figure 2). Higher doses of SiO2 NPs increased pro-inflammatory cytokines 24 h after treatment (data not shown). Because our goal was to study NP effects independently of its pro-inflammatory activity, we therefore selected a dose of 5 mg/Kg for the SiO2 NP treatments and chose 5 h post NP instillation as the time point when P.a was administered (see below). At that time point and in accordance with the quasi-exclusive presence of AM (over 90%) and the absence of neutrophils in BALs, FITC-SiO2 or unlabelled SiO2 NP treatment did not increase the basal levels of TNFα, KC, or IL-6 in BAL fluid (Figure 2). Interestingly, at 5 h post-NP instillation, over 15% of the cells present in the BAL (mostly AM) internalized FITC-SiO2 NPs (Figure 3B and D, white arrows). In accordance with the cytospin analysis (Figure 2) most of the cells that internalized SiO2 NPs presented an alveolar macrophage phenotype (CD11c+ F4/80+) (Figure 3C). Therefore, these data and the absence of inflammation after NP treatment alone allowed us to study the consequences of NP exposure on target cells without the confounding indirect effects of pro-inflammatory response associated with NPs treatment. Indeed, 24 h after FITC-SiO2 instillation, cryo-sections of lungs showed that FITC-SiO2 NPs were associated with lung epithelium and colocalized with alveolar type II epithelial cells (labelled with an anti-pro surfactant protein-C (SPC) antibody) (Figure 3E). Association of FITC-SiO2 with alveolar epithelial cells (AEC) could explain the increased levels of KC (a typically epithelial cell secreted chemokine) found in BALs 24 h post instillation (Figure 2).Figure 1


Acute exposure to silica nanoparticles enhances mortality and increases lung permeability in a mouse model of Pseudomonas aeruginosa pneumonia.

Delaval M, Boland S, Solhonne B, Nicola MA, Mornet S, Baeza-Squiban A, Sallenave JM, Garcia-Verdugo I - Part Fibre Toxicol (2015)

Silica nanoparticles are internalized by alveolar macrophages and colocalize with alveolar epithelial cells. C57Bl/6j mice were instilled with FITC-SiO2 NPs (5 mg/kg;100 μg/mice). A) 5 h later, mice were sacrificed and cells present in BALs were analysed by flow cytometry after labelling with specific antibodies directed against alveolar macrophages markers (CD11c, F4/80). Panel B) shows that 15 ± 2% (n = 4, mice) of the cells were FITC positive. Among them, over 95% were CD11c + and 85% CD11c + F4/80+ (panel C). The FACs analysis from one representative BAL from a total of 4 is shown. In D) BAL cells were cytospined and stained with anti-mouse CD45 antibody followed by Alexa 664 Fluor (A664) coupled secondary antibody (red) to confirm internalisation of FITC-SiO2 NPs (white arrows). E) Cryo-sections of lung tissues recovered from mice 24 h after instillation of FITC- SiO2 NPs (green) were stained with anti-proSPC antibody (a marker of alveolar type II epithelial cells) followed by A664 coupled secondary antibody (red). White arrows indicate co-localisation of NPs and alveolar epithelial cells. White scale bar = 100 μm or 20 μm (upper and lower panel, respectively). Nuclei were stained with DAPI (blue). Fluorescence of stained cells and tissues were analysed under structured illumination (ApoTome system). Superposed images from FITC, A664, DAPI and differential interferential contrast (D, lower panel) filters are shown. The ‘A’ within the figures indicates the alveolar spaces.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
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Fig3: Silica nanoparticles are internalized by alveolar macrophages and colocalize with alveolar epithelial cells. C57Bl/6j mice were instilled with FITC-SiO2 NPs (5 mg/kg;100 μg/mice). A) 5 h later, mice were sacrificed and cells present in BALs were analysed by flow cytometry after labelling with specific antibodies directed against alveolar macrophages markers (CD11c, F4/80). Panel B) shows that 15 ± 2% (n = 4, mice) of the cells were FITC positive. Among them, over 95% were CD11c + and 85% CD11c + F4/80+ (panel C). The FACs analysis from one representative BAL from a total of 4 is shown. In D) BAL cells were cytospined and stained with anti-mouse CD45 antibody followed by Alexa 664 Fluor (A664) coupled secondary antibody (red) to confirm internalisation of FITC-SiO2 NPs (white arrows). E) Cryo-sections of lung tissues recovered from mice 24 h after instillation of FITC- SiO2 NPs (green) were stained with anti-proSPC antibody (a marker of alveolar type II epithelial cells) followed by A664 coupled secondary antibody (red). White arrows indicate co-localisation of NPs and alveolar epithelial cells. White scale bar = 100 μm or 20 μm (upper and lower panel, respectively). Nuclei were stained with DAPI (blue). Fluorescence of stained cells and tissues were analysed under structured illumination (ApoTome system). Superposed images from FITC, A664, DAPI and differential interferential contrast (D, lower panel) filters are shown. The ‘A’ within the figures indicates the alveolar spaces.
Mentions: To study the consequences of NP exposure on P.a lung infection, we set up a protocol where mice were first treated with SiO2 NPs (5 mg/kg) and 5 h later infected with the P.a strain PAK (Figure 1). For most of our experiments, we selected 5 h time after NP treatment as a time point for infection because at that time, we did not observed obvious signs of inflammation (including neutrophil accumulation) in the lungs of NP treated mice compared to controls (Figure 2). In addition, because neutrophil influx usually starts 6 hours post-LPS instillation [23,24], this indirectly also confirms that LPS is undetectable and if present, is biologically inactive in our NP preparations. However, in one parallel experiment, 24 h after NP instillation a slight inflammation was detected in the lungs characterized by increased levels of KC (CXCL-1, a neutrophil chemokine) and a slight neutrophil accumulation (less than 10% of total cells) (Figure 2). Higher doses of SiO2 NPs increased pro-inflammatory cytokines 24 h after treatment (data not shown). Because our goal was to study NP effects independently of its pro-inflammatory activity, we therefore selected a dose of 5 mg/Kg for the SiO2 NP treatments and chose 5 h post NP instillation as the time point when P.a was administered (see below). At that time point and in accordance with the quasi-exclusive presence of AM (over 90%) and the absence of neutrophils in BALs, FITC-SiO2 or unlabelled SiO2 NP treatment did not increase the basal levels of TNFα, KC, or IL-6 in BAL fluid (Figure 2). Interestingly, at 5 h post-NP instillation, over 15% of the cells present in the BAL (mostly AM) internalized FITC-SiO2 NPs (Figure 3B and D, white arrows). In accordance with the cytospin analysis (Figure 2) most of the cells that internalized SiO2 NPs presented an alveolar macrophage phenotype (CD11c+ F4/80+) (Figure 3C). Therefore, these data and the absence of inflammation after NP treatment alone allowed us to study the consequences of NP exposure on target cells without the confounding indirect effects of pro-inflammatory response associated with NPs treatment. Indeed, 24 h after FITC-SiO2 instillation, cryo-sections of lungs showed that FITC-SiO2 NPs were associated with lung epithelium and colocalized with alveolar type II epithelial cells (labelled with an anti-pro surfactant protein-C (SPC) antibody) (Figure 3E). Association of FITC-SiO2 with alveolar epithelial cells (AEC) could explain the increased levels of KC (a typically epithelial cell secreted chemokine) found in BALs 24 h post instillation (Figure 2).Figure 1

Bottom Line: Furthermore, internalisation of SiO2 nanoparticles by primary alveolar macrophages did not reduce the capacity of the cells to clear Pseudomonas aeruginosa.In our murine model, SiO2 nanoparticle pre-exposure preferentially enhanced Pseudomonas aeruginosa-induced lung permeability (the latter assessed by the measurement of alveolar albumin and IgM concentrations) rather than contributing to Pseudomonas aeruginosa-induced lung inflammation (as measured by leukocyte recruitment and cytokine concentration in the alveolar compartment).The deleterious effects of SiO2 nanoparticle exposure during Pseudomonas aeruginosa-induced pneumonia are related to alterations of the alveolar-capillary barrier rather than to modulation of the inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: Univ Paris Diderot. Sorbone Paris Cité. Unit of Functional and Adaptive Biology (BFA) UMR 8251, CNRS, Laboratory of Molecular and Cellular Responses to Xenobiotics, 5 rue Thomas Mann, 75013, Paris, France. mathilde.delaval@gmail.com.

ABSTRACT

Background: The lung epithelium constitutes the first barrier against invading pathogens and also a major surface potentially exposed to nanoparticles. In order to ensure and preserve lung epithelial barrier function, the alveolar compartment possesses local defence mechanisms that are able to control bacterial infection. For instance, alveolar macrophages are professional phagocytic cells that engulf bacteria and environmental contaminants (including nanoparticles) and secrete pro-inflammatory cytokines to effectively eliminate the invading bacteria/contaminants. The consequences of nanoparticle exposure in the context of lung infection have not been studied in detail. Previous reports have shown that sequential lung exposure to nanoparticles and bacteria may impair bacterial clearance resulting in increased lung bacterial loads, associated with a reduction in the phagocytic capacity of alveolar macrophages.

Results: Here we have studied the consequences of SiO2 nanoparticle exposure on Pseudomonas aeruginosa clearance, Pseudomonas aeruginosa-induced inflammation and lung injury in a mouse model of acute pneumonia. We observed that pre-exposure to SiO2 nanoparticles increased mice susceptibility to lethal pneumonia but did not modify lung clearance of a bioluminescent Pseudomonas aeruginosa strain. Furthermore, internalisation of SiO2 nanoparticles by primary alveolar macrophages did not reduce the capacity of the cells to clear Pseudomonas aeruginosa. In our murine model, SiO2 nanoparticle pre-exposure preferentially enhanced Pseudomonas aeruginosa-induced lung permeability (the latter assessed by the measurement of alveolar albumin and IgM concentrations) rather than contributing to Pseudomonas aeruginosa-induced lung inflammation (as measured by leukocyte recruitment and cytokine concentration in the alveolar compartment).

Conclusions: We show that pre-exposure to SiO2 nanoparticles increases mice susceptibility to lethal pneumonia but independently of macrophage phagocytic function. The deleterious effects of SiO2 nanoparticle exposure during Pseudomonas aeruginosa-induced pneumonia are related to alterations of the alveolar-capillary barrier rather than to modulation of the inflammatory responses.

No MeSH data available.


Related in: MedlinePlus