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Rapid pathogen-induced apoptosis: a mechanism used by dendritic cells to limit intracellular replication of Legionella pneumophila.

Nogueira CV, Lindsten T, Jamieson AM, Case CL, Shin S, Thompson CB, Roy CR - PLoS Pathog. (2009)

Bottom Line: There are several examples where DCs have been shown to be more efficient at restricting the intracellular replication of pathogens compared to macrophages, a property that could prevent DCs from enhancing pathogen dissemination.Eliminating the pro-apoptotic proteins Bax and Bak or overproducing the anti-apoptotic protein Bcl-2 were both found to restore L. pneumophila replication in DCs.Thus, DCs have a microbial response pathway that rapidly activates apoptosis to limit pathogen replication.

View Article: PubMed Central - PubMed

Affiliation: Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT, USA.

ABSTRACT
Dendritic cells (DCs) are specialized phagocytes that internalize exogenous antigens and microbes at peripheral sites, and then migrate to lymphatic organs to display foreign peptides to naïve T cells. There are several examples where DCs have been shown to be more efficient at restricting the intracellular replication of pathogens compared to macrophages, a property that could prevent DCs from enhancing pathogen dissemination. To understand DC responses to pathogens, we investigated the mechanisms by which mouse DCs are able to restrict replication of the intracellular pathogen Legionella pneumophila. We show that both DCs and macrophages have the ability to interfere with L. pneumophila replication through a cell death pathway mediated by caspase-1 and Naip5. L. pneumophila that avoided Naip5-dependent responses, however, showed robust replication in macrophages but remained unable to replicate in DCs. Apoptotic cell death mediated by caspase-3 was found to occur much earlier in DCs following infection by L. pneumophila compared to macrophages infected similarly. Eliminating the pro-apoptotic proteins Bax and Bak or overproducing the anti-apoptotic protein Bcl-2 were both found to restore L. pneumophila replication in DCs. Thus, DCs have a microbial response pathway that rapidly activates apoptosis to limit pathogen replication.

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Bax and Bak are required for L. pneumophila growth restriction in DCs.(A) The graph shows percentage of B6 (open bars) and Bax−/−Bak−/− DCs (closed bars) infected with L. pneumophila WT, ΔdotA or ΔflaA that were TUNEL positive at 6 h post infection. Data represent the mean±SD of 300 cells counted per coverslip in triplicate. ** p<0.01. (B) B6, Bak−/− and Bax−/−Bak−/− were infected with either L. pneumophila WT (white bars) or ΔflaA (black bars) for 36 h. Intracellular replication was determined by dividing L. pneumophila CFUs recovered at 36 h by the CFUs recovered at 1 h post infection. Data for each time point are the average of values obtained from three independent wells. ** p<0.01. (C) Fluorescence micrographs of B6, Bak−/− and Bax−/−Bak−/− DCs that were infected with L. pneumophila ΔflaA and fixed at either 2 h or 10 h post infection. DCs were stained with an antibody specific for MHC II (red), DAPI (blue) and an anti-L. pneumophila antibody (green). On the right are graphical representations of the percentage of B6, Bak−/− and Bax−/−Bak−/− DCs infected at 2 h and the percentage of infected B6; Bak−/− and Bax−/−Bak−/− DCs with vacuoles containing replicating bacteria at 10 h post infection. Data represent the mean±SD of 500 cells counted per coverslip in triplicate. All cells had a dominant Lgn1 allele producing a functional Naip5 protein. R.V. = vacuoles containing replicating bacteria. N.D. = not detectable. ** p<0.01. Bar = 10 µm.
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ppat-1000478-g005: Bax and Bak are required for L. pneumophila growth restriction in DCs.(A) The graph shows percentage of B6 (open bars) and Bax−/−Bak−/− DCs (closed bars) infected with L. pneumophila WT, ΔdotA or ΔflaA that were TUNEL positive at 6 h post infection. Data represent the mean±SD of 300 cells counted per coverslip in triplicate. ** p<0.01. (B) B6, Bak−/− and Bax−/−Bak−/− were infected with either L. pneumophila WT (white bars) or ΔflaA (black bars) for 36 h. Intracellular replication was determined by dividing L. pneumophila CFUs recovered at 36 h by the CFUs recovered at 1 h post infection. Data for each time point are the average of values obtained from three independent wells. ** p<0.01. (C) Fluorescence micrographs of B6, Bak−/− and Bax−/−Bak−/− DCs that were infected with L. pneumophila ΔflaA and fixed at either 2 h or 10 h post infection. DCs were stained with an antibody specific for MHC II (red), DAPI (blue) and an anti-L. pneumophila antibody (green). On the right are graphical representations of the percentage of B6, Bak−/− and Bax−/−Bak−/− DCs infected at 2 h and the percentage of infected B6; Bak−/− and Bax−/−Bak−/− DCs with vacuoles containing replicating bacteria at 10 h post infection. Data represent the mean±SD of 500 cells counted per coverslip in triplicate. All cells had a dominant Lgn1 allele producing a functional Naip5 protein. R.V. = vacuoles containing replicating bacteria. N.D. = not detectable. ** p<0.01. Bar = 10 µm.

Mentions: Bax and Bak play a central role in regulating apoptosis. When activated by members of the BH3-only protein family, Bax and Bak create a channel in the membrane of mitochondria that releases cytochrome c. This results in activation of the apoptosome and the subsequent activation of effector caspases, such as caspase-3 [48]–[51]. DCs derived from C57BL/6 (B6) and from mice deficient in Bak (Bak−/−) or both Bax and Bak (Bax−/−Bak−/−) were analyzed to determine if Bax and Bak have a role in cell death induced by L. pneumophila. TUNEL analysis demonstrated that WT L. pneumophila induced equivalent levels of cell death in DCs derived from B6 and Bax−/−Bak−/− mice (Figure 5A), suggesting that the Naip5-dependent pathway of cell death remained functional in DCs. A L. pneumophila strain containing an in-frame deletion of the flaA gene encoding flagellin was used to bypass Naip5-mediated cell death [38]–[40]. A dramatic reduction in cell death was observed for Bax−/−Bak−/− DCs infected with L. pneumophila ΔflaA (Figure 5A). Measurements of caspase-3/7 activity following infection of DCs confirmed that Bax and Bak were required for induction of apoptosis by L. pneumophila ΔflaA (Table 1). Thus, L. pneumophila independently induces DC cell death by a Bax/Bak-dependent pathway and a Naip5-dependent pathway.


Rapid pathogen-induced apoptosis: a mechanism used by dendritic cells to limit intracellular replication of Legionella pneumophila.

Nogueira CV, Lindsten T, Jamieson AM, Case CL, Shin S, Thompson CB, Roy CR - PLoS Pathog. (2009)

Bax and Bak are required for L. pneumophila growth restriction in DCs.(A) The graph shows percentage of B6 (open bars) and Bax−/−Bak−/− DCs (closed bars) infected with L. pneumophila WT, ΔdotA or ΔflaA that were TUNEL positive at 6 h post infection. Data represent the mean±SD of 300 cells counted per coverslip in triplicate. ** p<0.01. (B) B6, Bak−/− and Bax−/−Bak−/− were infected with either L. pneumophila WT (white bars) or ΔflaA (black bars) for 36 h. Intracellular replication was determined by dividing L. pneumophila CFUs recovered at 36 h by the CFUs recovered at 1 h post infection. Data for each time point are the average of values obtained from three independent wells. ** p<0.01. (C) Fluorescence micrographs of B6, Bak−/− and Bax−/−Bak−/− DCs that were infected with L. pneumophila ΔflaA and fixed at either 2 h or 10 h post infection. DCs were stained with an antibody specific for MHC II (red), DAPI (blue) and an anti-L. pneumophila antibody (green). On the right are graphical representations of the percentage of B6, Bak−/− and Bax−/−Bak−/− DCs infected at 2 h and the percentage of infected B6; Bak−/− and Bax−/−Bak−/− DCs with vacuoles containing replicating bacteria at 10 h post infection. Data represent the mean±SD of 500 cells counted per coverslip in triplicate. All cells had a dominant Lgn1 allele producing a functional Naip5 protein. R.V. = vacuoles containing replicating bacteria. N.D. = not detectable. ** p<0.01. Bar = 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2689937&req=5

ppat-1000478-g005: Bax and Bak are required for L. pneumophila growth restriction in DCs.(A) The graph shows percentage of B6 (open bars) and Bax−/−Bak−/− DCs (closed bars) infected with L. pneumophila WT, ΔdotA or ΔflaA that were TUNEL positive at 6 h post infection. Data represent the mean±SD of 300 cells counted per coverslip in triplicate. ** p<0.01. (B) B6, Bak−/− and Bax−/−Bak−/− were infected with either L. pneumophila WT (white bars) or ΔflaA (black bars) for 36 h. Intracellular replication was determined by dividing L. pneumophila CFUs recovered at 36 h by the CFUs recovered at 1 h post infection. Data for each time point are the average of values obtained from three independent wells. ** p<0.01. (C) Fluorescence micrographs of B6, Bak−/− and Bax−/−Bak−/− DCs that were infected with L. pneumophila ΔflaA and fixed at either 2 h or 10 h post infection. DCs were stained with an antibody specific for MHC II (red), DAPI (blue) and an anti-L. pneumophila antibody (green). On the right are graphical representations of the percentage of B6, Bak−/− and Bax−/−Bak−/− DCs infected at 2 h and the percentage of infected B6; Bak−/− and Bax−/−Bak−/− DCs with vacuoles containing replicating bacteria at 10 h post infection. Data represent the mean±SD of 500 cells counted per coverslip in triplicate. All cells had a dominant Lgn1 allele producing a functional Naip5 protein. R.V. = vacuoles containing replicating bacteria. N.D. = not detectable. ** p<0.01. Bar = 10 µm.
Mentions: Bax and Bak play a central role in regulating apoptosis. When activated by members of the BH3-only protein family, Bax and Bak create a channel in the membrane of mitochondria that releases cytochrome c. This results in activation of the apoptosome and the subsequent activation of effector caspases, such as caspase-3 [48]–[51]. DCs derived from C57BL/6 (B6) and from mice deficient in Bak (Bak−/−) or both Bax and Bak (Bax−/−Bak−/−) were analyzed to determine if Bax and Bak have a role in cell death induced by L. pneumophila. TUNEL analysis demonstrated that WT L. pneumophila induced equivalent levels of cell death in DCs derived from B6 and Bax−/−Bak−/− mice (Figure 5A), suggesting that the Naip5-dependent pathway of cell death remained functional in DCs. A L. pneumophila strain containing an in-frame deletion of the flaA gene encoding flagellin was used to bypass Naip5-mediated cell death [38]–[40]. A dramatic reduction in cell death was observed for Bax−/−Bak−/− DCs infected with L. pneumophila ΔflaA (Figure 5A). Measurements of caspase-3/7 activity following infection of DCs confirmed that Bax and Bak were required for induction of apoptosis by L. pneumophila ΔflaA (Table 1). Thus, L. pneumophila independently induces DC cell death by a Bax/Bak-dependent pathway and a Naip5-dependent pathway.

Bottom Line: There are several examples where DCs have been shown to be more efficient at restricting the intracellular replication of pathogens compared to macrophages, a property that could prevent DCs from enhancing pathogen dissemination.Eliminating the pro-apoptotic proteins Bax and Bak or overproducing the anti-apoptotic protein Bcl-2 were both found to restore L. pneumophila replication in DCs.Thus, DCs have a microbial response pathway that rapidly activates apoptosis to limit pathogen replication.

View Article: PubMed Central - PubMed

Affiliation: Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT, USA.

ABSTRACT
Dendritic cells (DCs) are specialized phagocytes that internalize exogenous antigens and microbes at peripheral sites, and then migrate to lymphatic organs to display foreign peptides to naïve T cells. There are several examples where DCs have been shown to be more efficient at restricting the intracellular replication of pathogens compared to macrophages, a property that could prevent DCs from enhancing pathogen dissemination. To understand DC responses to pathogens, we investigated the mechanisms by which mouse DCs are able to restrict replication of the intracellular pathogen Legionella pneumophila. We show that both DCs and macrophages have the ability to interfere with L. pneumophila replication through a cell death pathway mediated by caspase-1 and Naip5. L. pneumophila that avoided Naip5-dependent responses, however, showed robust replication in macrophages but remained unable to replicate in DCs. Apoptotic cell death mediated by caspase-3 was found to occur much earlier in DCs following infection by L. pneumophila compared to macrophages infected similarly. Eliminating the pro-apoptotic proteins Bax and Bak or overproducing the anti-apoptotic protein Bcl-2 were both found to restore L. pneumophila replication in DCs. Thus, DCs have a microbial response pathway that rapidly activates apoptosis to limit pathogen replication.

Show MeSH
Related in: MedlinePlus