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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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L. pneumophila sdhA mutants induce rapid cell death in macrophages and DCs by a pathway that does not require Bax and Bak.(A) Intracellular replication of L. pneumophila ΔflaA, ΔdotA and ΔflaA, sdhA::kan was measured in B6 (black bars) and Bax−/−Bak−/− BMMs (white bars) at 72 h after infection. The fold increase in intracellular replication was determined by dividing L. pneumophila CFUs recovered at 72 h by the CFUs recovered at 1 h post infection. (B) Intracellular replication of L. pneumophila ΔflaA, ΔdotA and ΔflaA, sdhA::kan in B6 DCs (black bars) and Bax−/−Bak−/− DCs (white bars) at 36 h after infection. The fold increase in intracellular replication was determined by dividing L. pneumophila CFUs recovered at 36 h by the L. pneumophila CFUs recovered at 1 h post infection. Data are the mean±SD from three independent wells. N.D. = not detectable. (C) The graph shows the percentage of B6 and Tg (bcl2) 535rm BMMs or (D) DCs infected with L. pneumophila ΔflaA, ΔdotA or ΔflaA, sdhA::kan that were TUNEL positive at 1 h (black bars), 4 h (gray bars) and 8 h (white bars) after infection. All cells had a dominant Lgn1 allele producing a functional Naip5 protein. Data are represented by the mean±SD of 300 cells counted per each coverslip in triplicate.
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ppat-1000478-g009: L. pneumophila sdhA mutants induce rapid cell death in macrophages and DCs by a pathway that does not require Bax and Bak.(A) Intracellular replication of L. pneumophila ΔflaA, ΔdotA and ΔflaA, sdhA::kan was measured in B6 (black bars) and Bax−/−Bak−/− BMMs (white bars) at 72 h after infection. The fold increase in intracellular replication was determined by dividing L. pneumophila CFUs recovered at 72 h by the CFUs recovered at 1 h post infection. (B) Intracellular replication of L. pneumophila ΔflaA, ΔdotA and ΔflaA, sdhA::kan in B6 DCs (black bars) and Bax−/−Bak−/− DCs (white bars) at 36 h after infection. The fold increase in intracellular replication was determined by dividing L. pneumophila CFUs recovered at 36 h by the L. pneumophila CFUs recovered at 1 h post infection. Data are the mean±SD from three independent wells. N.D. = not detectable. (C) The graph shows the percentage of B6 and Tg (bcl2) 535rm BMMs or (D) DCs infected with L. pneumophila ΔflaA, ΔdotA or ΔflaA, sdhA::kan that were TUNEL positive at 1 h (black bars), 4 h (gray bars) and 8 h (white bars) after infection. All cells had a dominant Lgn1 allele producing a functional Naip5 protein. Data are represented by the mean±SD of 300 cells counted per each coverslip in triplicate.

Mentions: Previous studies have shown that macrophages infected with a L. pneumophila mutant deficient in the effector protein SdhA undergo rapid cell death by an unknown pathway [57]. This observation suggests that one possible reason DCs die quickly after L. pneumophila infection is because a proposed anti-apoptotic activity mediated by the translocated SdhA protein might not be effective at preventing cell death in DCs. This would explain why the phenotype of DCs infected by L. pneumophila capable of translocating the SdhA protein appears to be similar to the phenotype of macrophages infected by an sdhA mutant. If this hypothesis is correct, then perturbing cell death pathways activated by Bax and Bak should restore replication of an sdhA mutant in macrophages, and the elimination of SdhA should not affect replication of L. pneumophila in DCs deficient in Bax and Bak signaling. To test this hypothesis we inactivated sdhA in the L. pneumophila ΔflaA strain to generate L. pneumophila ΔflaA, sdhA::kan. Elimination of Bax and Bak did not restore replication of L. pneumophila ΔflaA, sdhA::kan in macrophages (Figure 9A) and the L. pneumophila ΔflaA, sdhA::kan strain was unable to replicate in Bax−/−Bak−/− DCs (Figure 9B). After infection by L. pneumophila ΔflaA, sdhA::kan, cell death levels measured by TUNEL staining were similar in Tg(bcl2) 535rm macrophages and control B6 macrophages (Figure 9C). The L. pneumophila ΔflaA, sdhA::kan strain also induced cell death in DCs derived from Tg(bcl2) 535rm mice (Figure 9D). Thus, the L. pneumophila sdhA mutant phenotype was similar in both macrophages and DCs, which indicates that SdhA is necessary to prevent L. pneumophila from killing both macrophages and DCs by a pathway that does not require Bax and Bak function.


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)

L. pneumophila sdhA mutants induce rapid cell death in macrophages and DCs by a pathway that does not require Bax and Bak.(A) Intracellular replication of L. pneumophila ΔflaA, ΔdotA and ΔflaA, sdhA::kan was measured in B6 (black bars) and Bax−/−Bak−/− BMMs (white bars) at 72 h after infection. The fold increase in intracellular replication was determined by dividing L. pneumophila CFUs recovered at 72 h by the CFUs recovered at 1 h post infection. (B) Intracellular replication of L. pneumophila ΔflaA, ΔdotA and ΔflaA, sdhA::kan in B6 DCs (black bars) and Bax−/−Bak−/− DCs (white bars) at 36 h after infection. The fold increase in intracellular replication was determined by dividing L. pneumophila CFUs recovered at 36 h by the L. pneumophila CFUs recovered at 1 h post infection. Data are the mean±SD from three independent wells. N.D. = not detectable. (C) The graph shows the percentage of B6 and Tg (bcl2) 535rm BMMs or (D) DCs infected with L. pneumophila ΔflaA, ΔdotA or ΔflaA, sdhA::kan that were TUNEL positive at 1 h (black bars), 4 h (gray bars) and 8 h (white bars) after infection. All cells had a dominant Lgn1 allele producing a functional Naip5 protein. Data are represented by the mean±SD of 300 cells counted per each coverslip in triplicate.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2689937&req=5

ppat-1000478-g009: L. pneumophila sdhA mutants induce rapid cell death in macrophages and DCs by a pathway that does not require Bax and Bak.(A) Intracellular replication of L. pneumophila ΔflaA, ΔdotA and ΔflaA, sdhA::kan was measured in B6 (black bars) and Bax−/−Bak−/− BMMs (white bars) at 72 h after infection. The fold increase in intracellular replication was determined by dividing L. pneumophila CFUs recovered at 72 h by the CFUs recovered at 1 h post infection. (B) Intracellular replication of L. pneumophila ΔflaA, ΔdotA and ΔflaA, sdhA::kan in B6 DCs (black bars) and Bax−/−Bak−/− DCs (white bars) at 36 h after infection. The fold increase in intracellular replication was determined by dividing L. pneumophila CFUs recovered at 36 h by the L. pneumophila CFUs recovered at 1 h post infection. Data are the mean±SD from three independent wells. N.D. = not detectable. (C) The graph shows the percentage of B6 and Tg (bcl2) 535rm BMMs or (D) DCs infected with L. pneumophila ΔflaA, ΔdotA or ΔflaA, sdhA::kan that were TUNEL positive at 1 h (black bars), 4 h (gray bars) and 8 h (white bars) after infection. All cells had a dominant Lgn1 allele producing a functional Naip5 protein. Data are represented by the mean±SD of 300 cells counted per each coverslip in triplicate.
Mentions: Previous studies have shown that macrophages infected with a L. pneumophila mutant deficient in the effector protein SdhA undergo rapid cell death by an unknown pathway [57]. This observation suggests that one possible reason DCs die quickly after L. pneumophila infection is because a proposed anti-apoptotic activity mediated by the translocated SdhA protein might not be effective at preventing cell death in DCs. This would explain why the phenotype of DCs infected by L. pneumophila capable of translocating the SdhA protein appears to be similar to the phenotype of macrophages infected by an sdhA mutant. If this hypothesis is correct, then perturbing cell death pathways activated by Bax and Bak should restore replication of an sdhA mutant in macrophages, and the elimination of SdhA should not affect replication of L. pneumophila in DCs deficient in Bax and Bak signaling. To test this hypothesis we inactivated sdhA in the L. pneumophila ΔflaA strain to generate L. pneumophila ΔflaA, sdhA::kan. Elimination of Bax and Bak did not restore replication of L. pneumophila ΔflaA, sdhA::kan in macrophages (Figure 9A) and the L. pneumophila ΔflaA, sdhA::kan strain was unable to replicate in Bax−/−Bak−/− DCs (Figure 9B). After infection by L. pneumophila ΔflaA, sdhA::kan, cell death levels measured by TUNEL staining were similar in Tg(bcl2) 535rm macrophages and control B6 macrophages (Figure 9C). The L. pneumophila ΔflaA, sdhA::kan strain also induced cell death in DCs derived from Tg(bcl2) 535rm mice (Figure 9D). Thus, the L. pneumophila sdhA mutant phenotype was similar in both macrophages and DCs, which indicates that SdhA is necessary to prevent L. pneumophila from killing both macrophages and DCs by a pathway that does not require Bax and Bak function.

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