Limits...
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: 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.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

Leishmania parasites and purified GP63 inhibit generation of ROS induced by inflammasome activator.(A and B) PMA-differentiated THP-1 cells (0.1x106 cells/100 μL) were stimulated with indicated concentration of HZ or silica (A) or pre-infected/incubated with either Leishmania or purified GP63 (pGP63) for 2 hrs, and then stimulated with 200 μg/mL of HZ or 100 μg/mL silica (B). After indicated time of incubation (A) or 1.5 hrs (B), ROS production was measured by fluorescence using DCFA. Differences were considered significant for p < 0.05. Data shows mean values with SEM of 3 independent experiments. (#) denotes significant changes between uninfected and HZ treated samples. (*) denotes significant changes of ROS production due to Leishmania infection or pGP63. Both Leishmania and purified GP63 inhibit ROS production induced by inflammasome activators.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482689&req=5

pntd.0003868.g003: Leishmania parasites and purified GP63 inhibit generation of ROS induced by inflammasome activator.(A and B) PMA-differentiated THP-1 cells (0.1x106 cells/100 μL) were stimulated with indicated concentration of HZ or silica (A) or pre-infected/incubated with either Leishmania or purified GP63 (pGP63) for 2 hrs, and then stimulated with 200 μg/mL of HZ or 100 μg/mL silica (B). After indicated time of incubation (A) or 1.5 hrs (B), ROS production was measured by fluorescence using DCFA. Differences were considered significant for p < 0.05. Data shows mean values with SEM of 3 independent experiments. (#) denotes significant changes between uninfected and HZ treated samples. (*) denotes significant changes of ROS production due to Leishmania infection or pGP63. Both Leishmania and purified GP63 inhibit ROS production induced by inflammasome activators.

Mentions: Activation of the NLRP3 inflammasome due to DAMPs is often associated with ROS production and ROS-induced or -dependent signaling [38,39]. In this context the molecular basis of ROS generation has been under debate for some time and recent hypothesis include damage to mitochondria as a possible ROS-source and propose thioredoxin-interacting protein TXNIP may act as a ROS-sensor [40]. Leishmania has been shown to interfere with the generation of ROS and other microbicidal molecules [15] and has been described to be involved in the inflammasome activation [15]. Using known danger molecules like HZ and silica we determined, that both crystalline agents readily induce the generation of ROS in THP1 cells (Fig 3A). Therefore, we hypothesized that a Leishmania-dependent decrease of ROS-species or an impaired ROS production could be the basis of the diminished IL-1β maturation/release previously observed. Consequently, infection of THP-1 cells with L. mexicana led to an abrogated ROS production even after HZ or silica stimulation, supporting our hypothesis (Fig 3B). As our previous results suggested the possibility of a GP63-mediated inflammasome suppression, we included purified L. mexicana GP63 (pGP63) in our experimental setup. Pretreatment of THP cells with pGP63 from L. mexicana supernatant was also sufficient to reduce ROS levels to a similar extend as Leishmania infections (Fig 3B).


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)

Leishmania parasites and purified GP63 inhibit generation of ROS induced by inflammasome activator.(A and B) PMA-differentiated THP-1 cells (0.1x106 cells/100 μL) were stimulated with indicated concentration of HZ or silica (A) or pre-infected/incubated with either Leishmania or purified GP63 (pGP63) for 2 hrs, and then stimulated with 200 μg/mL of HZ or 100 μg/mL silica (B). After indicated time of incubation (A) or 1.5 hrs (B), ROS production was measured by fluorescence using DCFA. Differences were considered significant for p < 0.05. Data shows mean values with SEM of 3 independent experiments. (#) denotes significant changes between uninfected and HZ treated samples. (*) denotes significant changes of ROS production due to Leishmania infection or pGP63. Both Leishmania and purified GP63 inhibit ROS production induced by inflammasome activators.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0003868.g003: Leishmania parasites and purified GP63 inhibit generation of ROS induced by inflammasome activator.(A and B) PMA-differentiated THP-1 cells (0.1x106 cells/100 μL) were stimulated with indicated concentration of HZ or silica (A) or pre-infected/incubated with either Leishmania or purified GP63 (pGP63) for 2 hrs, and then stimulated with 200 μg/mL of HZ or 100 μg/mL silica (B). After indicated time of incubation (A) or 1.5 hrs (B), ROS production was measured by fluorescence using DCFA. Differences were considered significant for p < 0.05. Data shows mean values with SEM of 3 independent experiments. (#) denotes significant changes between uninfected and HZ treated samples. (*) denotes significant changes of ROS production due to Leishmania infection or pGP63. Both Leishmania and purified GP63 inhibit ROS production induced by inflammasome activators.
Mentions: Activation of the NLRP3 inflammasome due to DAMPs is often associated with ROS production and ROS-induced or -dependent signaling [38,39]. In this context the molecular basis of ROS generation has been under debate for some time and recent hypothesis include damage to mitochondria as a possible ROS-source and propose thioredoxin-interacting protein TXNIP may act as a ROS-sensor [40]. Leishmania has been shown to interfere with the generation of ROS and other microbicidal molecules [15] and has been described to be involved in the inflammasome activation [15]. Using known danger molecules like HZ and silica we determined, that both crystalline agents readily induce the generation of ROS in THP1 cells (Fig 3A). Therefore, we hypothesized that a Leishmania-dependent decrease of ROS-species or an impaired ROS production could be the basis of the diminished IL-1β maturation/release previously observed. Consequently, infection of THP-1 cells with L. mexicana led to an abrogated ROS production even after HZ or silica stimulation, supporting our hypothesis (Fig 3B). As our previous results suggested the possibility of a GP63-mediated inflammasome suppression, we included purified L. mexicana GP63 (pGP63) in our experimental setup. Pretreatment of THP cells with pGP63 from L. mexicana supernatant was also sufficient to reduce ROS levels to a similar extend as Leishmania infections (Fig 3B).

Bottom Line: 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.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