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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: 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

Purified GP63 and leishmanial exosomes containing GP63 inhibit IL-1β maturation induced by inflammasome activators.Silver staining of purified GP63 (A). PMA-differentiated THP-1 cells (1x106 cells/mL) were pre-treated (2 hrs) with or without the indicated concentration of purified GP63 (pGP63) (B) or either leishmanial exosome (exo) or secretome (sec) (C) for 2 hrs and stimulated with 200 μg/mL of the malarial pigment—HZ, or MSU as specified. After 6 hrs of incubation, supernatant and cell extracts were collected and subjected to Western blot analysis with the indicated antibodies. Data shown is representative of three independent experiments. Materials containing GP63 leads to inhibition of inflammasome activation.
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pntd.0003868.g002: Purified GP63 and leishmanial exosomes containing GP63 inhibit IL-1β maturation induced by inflammasome activators.Silver staining of purified GP63 (A). PMA-differentiated THP-1 cells (1x106 cells/mL) were pre-treated (2 hrs) with or without the indicated concentration of purified GP63 (pGP63) (B) or either leishmanial exosome (exo) or secretome (sec) (C) for 2 hrs and stimulated with 200 μg/mL of the malarial pigment—HZ, or MSU as specified. After 6 hrs of incubation, supernatant and cell extracts were collected and subjected to Western blot analysis with the indicated antibodies. Data shown is representative of three independent experiments. Materials containing GP63 leads to inhibition of inflammasome activation.

Mentions: In recent years, several studies have described the observation that proteins are secreted as exosecretome by Leishmania parasites upon 37°C temperature shock [28] or as exosomes during the culture of parasites [36,37]. Both Leishmania secretome and exosomes have been shown to contain GP63. Thus, we evaluated whether leishmanial secretome or exosome preparations would inhibit IL-1β maturation and secretion. As expected the results were in accordance with the data acquired using parasite infections and culture supernatant treatment of cells, with both secretome and exosomes inhibiting IL-1β production induced by either HZ or MSU (Fig 2C). The importance of the metalloprotease GP63 was clearly demonstrated through experiments using purified GPI-GP63 (Fig 2A and 2B) from L. mexicana stationary phase cultures to pretreat THP-1 cells. This resulted in an attenuation of the HZ-induced IL-1β maturation and its release (Fig 2B) identical to infection experiments previously shown. Collectively, these results provide convincing evidence that Leishmania GP63 is the causative factor for an impaired IL-1β production by the NLRP3 inflammasome complex, which was observed after infections with Leishmania parasites prior to cell stimulation.


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)

Purified GP63 and leishmanial exosomes containing GP63 inhibit IL-1β maturation induced by inflammasome activators.Silver staining of purified GP63 (A). PMA-differentiated THP-1 cells (1x106 cells/mL) were pre-treated (2 hrs) with or without the indicated concentration of purified GP63 (pGP63) (B) or either leishmanial exosome (exo) or secretome (sec) (C) for 2 hrs and stimulated with 200 μg/mL of the malarial pigment—HZ, or MSU as specified. After 6 hrs of incubation, supernatant and cell extracts were collected and subjected to Western blot analysis with the indicated antibodies. Data shown is representative of three independent experiments. Materials containing GP63 leads to inhibition of inflammasome activation.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0003868.g002: Purified GP63 and leishmanial exosomes containing GP63 inhibit IL-1β maturation induced by inflammasome activators.Silver staining of purified GP63 (A). PMA-differentiated THP-1 cells (1x106 cells/mL) were pre-treated (2 hrs) with or without the indicated concentration of purified GP63 (pGP63) (B) or either leishmanial exosome (exo) or secretome (sec) (C) for 2 hrs and stimulated with 200 μg/mL of the malarial pigment—HZ, or MSU as specified. After 6 hrs of incubation, supernatant and cell extracts were collected and subjected to Western blot analysis with the indicated antibodies. Data shown is representative of three independent experiments. Materials containing GP63 leads to inhibition of inflammasome activation.
Mentions: In recent years, several studies have described the observation that proteins are secreted as exosecretome by Leishmania parasites upon 37°C temperature shock [28] or as exosomes during the culture of parasites [36,37]. Both Leishmania secretome and exosomes have been shown to contain GP63. Thus, we evaluated whether leishmanial secretome or exosome preparations would inhibit IL-1β maturation and secretion. As expected the results were in accordance with the data acquired using parasite infections and culture supernatant treatment of cells, with both secretome and exosomes inhibiting IL-1β production induced by either HZ or MSU (Fig 2C). The importance of the metalloprotease GP63 was clearly demonstrated through experiments using purified GPI-GP63 (Fig 2A and 2B) from L. mexicana stationary phase cultures to pretreat THP-1 cells. This resulted in an attenuation of the HZ-induced IL-1β maturation and its release (Fig 2B) identical to infection experiments previously shown. Collectively, these results provide convincing evidence that Leishmania GP63 is the causative factor for an impaired IL-1β production by the NLRP3 inflammasome complex, which was observed after infections with Leishmania parasites prior to cell stimulation.

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