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PKC/ROS-Mediated NLRP3 Inflammasome Activation Is Attenuated by Leishmania Zinc-Metalloprotease during Infection.

Shio MT, Christian JG, Jung JY, Chang KP, Olivier M - PLoS Negl Trop Dis (2015)

Bottom Line: Using PMA-differentiated THP-1 cells, we demonstrate that Leishmania infection effectively inhibits macrophage IL-1β production upon stimulation.Leishmania-dependent suppression of IL-1β secretion is accompanied by an inhibition of reactive oxygen species (ROS) production that has previously been shown to be associated with NLRP3 inflammasome activation.Collectively for the first time, we herein provide evidence that the protozoan parasite Leishmania, through its surface metalloprotease GP63, can significantly inhibit NLRP3 inflammasome function and IL-1β production.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada; Department of Microbiology, Immunology and Parasitology, Universidade Federal de São Paulo, São Paulo, Brazil.

ABSTRACT
Parasites of the Leishmania genus infect and survive within macrophages by inhibiting several microbicidal molecules, such as nitric oxide and pro-inflammatory cytokines. In this context, various species of Leishmania have been reported to inhibit or reduce the production of IL-1β both in vitro and in vivo. However, the mechanism whereby Leishmania parasites are able to affect IL-1β production and secretion by macrophages is still not fully understood. Dependent on the stimulus at hand, the maturation of IL-1β is facilitated by different inflammasome complexes. The NLRP3 inflammasome has been shown to be of pivotal importance in the detection of danger molecules such as inorganic crystals like asbestos, silica and malarial hemozoin, (HZ) as well as infectious agents. In the present work, we investigated whether Leishmania parasites modulate NLRP3 inflammasome activation. Using PMA-differentiated THP-1 cells, we demonstrate that Leishmania infection effectively inhibits macrophage IL-1β production upon stimulation. In this context, the expression and activity of the metalloprotease GP63 - a critical virulence factor expressed by all infectious Leishmania species - is a prerequisite for a Leishmania-mediated reduction of IL-1β secretion. Accordingly, L. mexicana, purified GP63 and GP63-containing exosomes, caused the inhibition of macrophage IL-1β production. Leishmania-dependent suppression of IL-1β secretion is accompanied by an inhibition of reactive oxygen species (ROS) production that has previously been shown to be associated with NLRP3 inflammasome activation. The observed loss of ROS production was due to an impaired PKC-mediated protein phosphorylation. Furthermore, ROS-independent inflammasome activation was inhibited, possibly due to an observed GP63-dependent cleavage of inflammasome and inflammasome-related proteins. Collectively for the first time, we herein provide evidence that the protozoan parasite Leishmania, through its surface metalloprotease GP63, can significantly inhibit NLRP3 inflammasome function and IL-1β production.

No MeSH data available.


Related in: MedlinePlus

ROS-independent IL-1β generation is dampened after infection with Leishmania.(A) LPS-primed BMDMs were infected with Leishmania mexicana (Lmex), for 2 hrs. Cells were washed three times and incubated in serum free MEM alpha medium for 16 hrs in the presence of linezolid (100 μg/ml) or HZ (200 μg/ml) as indicated. Supernatants were collected and subjected to IL-1β specific ELISA analysis. Data shows mean values with SEM of 3 independent experiments. Differences were considered significant for p < 0.05(*). (B) PMA-differentiated THP-1 cells were infected with Leishmania mexicana (Lmex). Cells were washed three times and incubated in serum free MEM alpha medium for 16 hrs in the presence of linezolid (100 μg/ml) as indicated. Afterwards, cell extracts were collected and subjected to Western blot analysis with the indicated antibodies. Data shown is representative of three independent experiments.
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pntd.0003868.g006: ROS-independent IL-1β generation is dampened after infection with Leishmania.(A) LPS-primed BMDMs were infected with Leishmania mexicana (Lmex), for 2 hrs. Cells were washed three times and incubated in serum free MEM alpha medium for 16 hrs in the presence of linezolid (100 μg/ml) or HZ (200 μg/ml) as indicated. Supernatants were collected and subjected to IL-1β specific ELISA analysis. Data shows mean values with SEM of 3 independent experiments. Differences were considered significant for p < 0.05(*). (B) PMA-differentiated THP-1 cells were infected with Leishmania mexicana (Lmex). Cells were washed three times and incubated in serum free MEM alpha medium for 16 hrs in the presence of linezolid (100 μg/ml) as indicated. Afterwards, cell extracts were collected and subjected to Western blot analysis with the indicated antibodies. Data shown is representative of three independent experiments.

Mentions: A controversial question of inflammasome activation is the dependency of ROS for the activation and assembly of the complex. Recent data associates mitochondrial damage with the activation of the NLRP3 inflammasome. In this context cardiolipin seems to work as a DAMP, able to induce inflammasome complex formation and ultimately IL-1β. The previous data presented different ways how leishmanial GP63 can suppress inflammasome activity. This included the cleavage of NLRP3. Therefore, we wanted to know whether these processes might also have a direct effect on IL-1β production. Thus, we observed the IL-1β generation in a ROS-independent experimental setup using the antibiotic linezolid [19]. Linezolid stimulation of either THP-1 or LPS-primed BMDM cells resulted in the maturation of IL-1β as previously published (Fig 6A and 6B). When cells were infected prior to linezolid stimulation, IL-1β release appeared reduced for both murine and human cells. In accordance to previously shown data Leishmania infection alone did only lead to a minimal production of mature IL-1β (Fig 6A). Taken together this finding may indicate, that the infection of cells with Leishmania can abrogate both ROS-dependent and -independent inflammasome activation, possibly through different mechanisms as both ROS-inducing signaling events are blocked and inflammasome components are cleaved due to the leishmanial protease GP63.


PKC/ROS-Mediated NLRP3 Inflammasome Activation Is Attenuated by Leishmania Zinc-Metalloprotease during Infection.

Shio MT, Christian JG, Jung JY, Chang KP, Olivier M - PLoS Negl Trop Dis (2015)

ROS-independent IL-1β generation is dampened after infection with Leishmania.(A) LPS-primed BMDMs were infected with Leishmania mexicana (Lmex), for 2 hrs. Cells were washed three times and incubated in serum free MEM alpha medium for 16 hrs in the presence of linezolid (100 μg/ml) or HZ (200 μg/ml) as indicated. Supernatants were collected and subjected to IL-1β specific ELISA analysis. Data shows mean values with SEM of 3 independent experiments. Differences were considered significant for p < 0.05(*). (B) PMA-differentiated THP-1 cells were infected with Leishmania mexicana (Lmex). Cells were washed three times and incubated in serum free MEM alpha medium for 16 hrs in the presence of linezolid (100 μg/ml) as indicated. Afterwards, cell extracts were collected and subjected to Western blot analysis with the indicated antibodies. Data shown is representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482689&req=5

pntd.0003868.g006: ROS-independent IL-1β generation is dampened after infection with Leishmania.(A) LPS-primed BMDMs were infected with Leishmania mexicana (Lmex), for 2 hrs. Cells were washed three times and incubated in serum free MEM alpha medium for 16 hrs in the presence of linezolid (100 μg/ml) or HZ (200 μg/ml) as indicated. Supernatants were collected and subjected to IL-1β specific ELISA analysis. Data shows mean values with SEM of 3 independent experiments. Differences were considered significant for p < 0.05(*). (B) PMA-differentiated THP-1 cells were infected with Leishmania mexicana (Lmex). Cells were washed three times and incubated in serum free MEM alpha medium for 16 hrs in the presence of linezolid (100 μg/ml) as indicated. Afterwards, cell extracts were collected and subjected to Western blot analysis with the indicated antibodies. Data shown is representative of three independent experiments.
Mentions: A controversial question of inflammasome activation is the dependency of ROS for the activation and assembly of the complex. Recent data associates mitochondrial damage with the activation of the NLRP3 inflammasome. In this context cardiolipin seems to work as a DAMP, able to induce inflammasome complex formation and ultimately IL-1β. The previous data presented different ways how leishmanial GP63 can suppress inflammasome activity. This included the cleavage of NLRP3. Therefore, we wanted to know whether these processes might also have a direct effect on IL-1β production. Thus, we observed the IL-1β generation in a ROS-independent experimental setup using the antibiotic linezolid [19]. Linezolid stimulation of either THP-1 or LPS-primed BMDM cells resulted in the maturation of IL-1β as previously published (Fig 6A and 6B). When cells were infected prior to linezolid stimulation, IL-1β release appeared reduced for both murine and human cells. In accordance to previously shown data Leishmania infection alone did only lead to a minimal production of mature IL-1β (Fig 6A). Taken together this finding may indicate, that the infection of cells with Leishmania can abrogate both ROS-dependent and -independent inflammasome activation, possibly through different mechanisms as both ROS-inducing signaling events are blocked and inflammasome components are cleaved due to the leishmanial protease GP63.

Bottom Line: Using PMA-differentiated THP-1 cells, we demonstrate that Leishmania infection effectively inhibits macrophage IL-1β production upon stimulation.Leishmania-dependent suppression of IL-1β secretion is accompanied by an inhibition of reactive oxygen species (ROS) production that has previously been shown to be associated with NLRP3 inflammasome activation.Collectively for the first time, we herein provide evidence that the protozoan parasite Leishmania, through its surface metalloprotease GP63, can significantly inhibit NLRP3 inflammasome function and IL-1β production.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada; Department of Microbiology, Immunology and Parasitology, Universidade Federal de São Paulo, São Paulo, Brazil.

ABSTRACT
Parasites of the Leishmania genus infect and survive within macrophages by inhibiting several microbicidal molecules, such as nitric oxide and pro-inflammatory cytokines. In this context, various species of Leishmania have been reported to inhibit or reduce the production of IL-1β both in vitro and in vivo. However, the mechanism whereby Leishmania parasites are able to affect IL-1β production and secretion by macrophages is still not fully understood. Dependent on the stimulus at hand, the maturation of IL-1β is facilitated by different inflammasome complexes. The NLRP3 inflammasome has been shown to be of pivotal importance in the detection of danger molecules such as inorganic crystals like asbestos, silica and malarial hemozoin, (HZ) as well as infectious agents. In the present work, we investigated whether Leishmania parasites modulate NLRP3 inflammasome activation. Using PMA-differentiated THP-1 cells, we demonstrate that Leishmania infection effectively inhibits macrophage IL-1β production upon stimulation. In this context, the expression and activity of the metalloprotease GP63 - a critical virulence factor expressed by all infectious Leishmania species - is a prerequisite for a Leishmania-mediated reduction of IL-1β secretion. Accordingly, L. mexicana, purified GP63 and GP63-containing exosomes, caused the inhibition of macrophage IL-1β production. Leishmania-dependent suppression of IL-1β secretion is accompanied by an inhibition of reactive oxygen species (ROS) production that has previously been shown to be associated with NLRP3 inflammasome activation. The observed loss of ROS production was due to an impaired PKC-mediated protein phosphorylation. Furthermore, ROS-independent inflammasome activation was inhibited, possibly due to an observed GP63-dependent cleavage of inflammasome and inflammasome-related proteins. Collectively for the first time, we herein provide evidence that the protozoan parasite Leishmania, through its surface metalloprotease GP63, can significantly inhibit NLRP3 inflammasome function and IL-1β production.

No MeSH data available.


Related in: MedlinePlus