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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

Leishmania parasites inhibit IL-1β production induced by HZ in GP63-dependent manner.PMA-differentiated THP-1 cells (1x106 cells/mL) were pre-infected with Leishmania parasites (A) or incubated (B) with culture medium (LCM) from 7 days old parasite cultures of either Leishmania mexicana (Lmex), L. major GP63 wild type (Lmj WT), L. major GP63-/- (Lmj KO) or L. major GP63 rescue (Lmj Res). Afterwards, cells were washed and stimulated with 200 μg/mL of HZ. (C) Bone-marrow derived macrophages (1.5x106 cells/mL) were pre-infected for 2 hrs with Leishmania mexicana (Lmex). Following infection, cells were washed and stimulated with 200 μg/mL of HZ. 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 two independent experiments. (D) Western blot of Leishmania culture medium (LCM) for GP63. Data shown is representative of two independent experiments. (E) PMA-differentiated THP-1 cells (1x106 cells/mL) were incubated with different concentrations of LCM from 7 days old parasite cultures of L. mexicana. Afterwards, cells were washed and stimulated with either 200 μg/mL of HZ, 200 μg/mL of silica or 200 μg/mL of asbestos for 6 hrs. Supernatants and cell extracts were collected and subjected to Western blot analysis with the indicated antibodies.
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pntd.0003868.g001: Leishmania parasites inhibit IL-1β production induced by HZ in GP63-dependent manner.PMA-differentiated THP-1 cells (1x106 cells/mL) were pre-infected with Leishmania parasites (A) or incubated (B) with culture medium (LCM) from 7 days old parasite cultures of either Leishmania mexicana (Lmex), L. major GP63 wild type (Lmj WT), L. major GP63-/- (Lmj KO) or L. major GP63 rescue (Lmj Res). Afterwards, cells were washed and stimulated with 200 μg/mL of HZ. (C) Bone-marrow derived macrophages (1.5x106 cells/mL) were pre-infected for 2 hrs with Leishmania mexicana (Lmex). Following infection, cells were washed and stimulated with 200 μg/mL of HZ. 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 two independent experiments. (D) Western blot of Leishmania culture medium (LCM) for GP63. Data shown is representative of two independent experiments. (E) PMA-differentiated THP-1 cells (1x106 cells/mL) were incubated with different concentrations of LCM from 7 days old parasite cultures of L. mexicana. Afterwards, cells were washed and stimulated with either 200 μg/mL of HZ, 200 μg/mL of silica or 200 μg/mL of asbestos for 6 hrs. Supernatants and cell extracts were collected and subjected to Western blot analysis with the indicated antibodies.

Mentions: Upon activation macrophages can produce a large array of pro-inflammatory molecules including IL-1β, which is produced by inflammasome complexes. The NLRP3 inflammasome acts as an intracellular signaling platform which is able to sense a variety of exogenous signals like asbestos, silica [17] as well as the malarial pigment HZ [20,31] and DAMPs such as ATP [18] and MSU [21]. In initial dose-response experiments using synthetic HZ we confirmed the ability of HZ to induce IL-1β maturation in PMA-differentiated THP-1 cells (S1 Fig). The observed, dose-dependent IL-1β secretion was comparable to the results obtained in the case of silica or MSU treatment of cells. Infection of THP-1 cells with Leishmania did not induce IL-1β release as shown for L. mexicana (S2 Fig). Leishmania parasites are well known for their ability to block and inhibit various microbicidal functions of macrophages [32], therefore we sought to elucidate whether infections with Leishmania would inhibit IL-1β production by macrophages, which has been indicated to possibly serve a host protective role in murine models of infection [33,34]. As previously introduced the NLRP3 inflammasome activator HZ causes the IL-1β maturation and secretion in PMA-differentiated macrophages. Pre-infection with L. mexicana and L. major, revealed a parasite-dependent block of IL-1β maturation and release (Fig 1A). The impaired production of IL-1β was not restricted to a system of human cell culture but was also observable when parasite infection preceded inflammasome activation in murine BMDMs (Fig 1C). In those experiments due to the availability of antibodies the processing of caspase-1 into its active fragments p10 and p20 could be observed. Notably processing of caspase-1 was absent after infection of cells possibly due to the lack of inflammasome activation or complex formation. Identical experimental setups for infections as in Fig 1A using L. major GP63 KO and L. major GP63 rescue parasites support the hypothesis that Leishmania’s capability to inhibit IL-1β maturation and release was GP63-dependent (Fig 1A–1E). Thus, IL-1β secretion was not impaired in the absence of the protease (Fig 1A). This finding was further supported by experiments using pretreatment of THP-1 cells with Leishmania culture supernatant (LCM) instead of parasites (Fig 1B). The attenuated IL-1β secretion coincided with the presence of GP63 in the LCM. We were able to show that LCM of L. mexicana and L. major GP63 wild type parasite cultures contained GP63 (Fig 1D). To further evaluate the impact of secreted leishmanial factors like GP63 on IL-1β production the supernatant of L. mexicana cultures was concentrated and used in titration experiment on PMA-differentiated THP-1 cells (Fig 1E). IL-1β maturation and release were stimulated by a variety of known NLRP3 inflammasome inducers, namely HZ, silica and asbestos. In all cases we observed an inhibition of IL-1β secretion in a dose-dependent manner by the L. mexicana culture supernatant. Leishmania GP63 can be found either intracellular in the protozoan endoplasmic reticulum, membrane bound via a GPI anchor or secreted without the GPI anchor. During infection GPI anchored, membrane bound GP63 (GPI-GP63) can also be cleaved and released into the supernatant [2,35]. Therefore, the similarities of our results using either Leishmania infections or Leishmania culture supernatant are most likely to be attributed to the presence and activity of 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)

Leishmania parasites inhibit IL-1β production induced by HZ in GP63-dependent manner.PMA-differentiated THP-1 cells (1x106 cells/mL) were pre-infected with Leishmania parasites (A) or incubated (B) with culture medium (LCM) from 7 days old parasite cultures of either Leishmania mexicana (Lmex), L. major GP63 wild type (Lmj WT), L. major GP63-/- (Lmj KO) or L. major GP63 rescue (Lmj Res). Afterwards, cells were washed and stimulated with 200 μg/mL of HZ. (C) Bone-marrow derived macrophages (1.5x106 cells/mL) were pre-infected for 2 hrs with Leishmania mexicana (Lmex). Following infection, cells were washed and stimulated with 200 μg/mL of HZ. 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 two independent experiments. (D) Western blot of Leishmania culture medium (LCM) for GP63. Data shown is representative of two independent experiments. (E) PMA-differentiated THP-1 cells (1x106 cells/mL) were incubated with different concentrations of LCM from 7 days old parasite cultures of L. mexicana. Afterwards, cells were washed and stimulated with either 200 μg/mL of HZ, 200 μg/mL of silica or 200 μg/mL of asbestos for 6 hrs. Supernatants and cell extracts were collected and subjected to Western blot analysis with the indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0003868.g001: Leishmania parasites inhibit IL-1β production induced by HZ in GP63-dependent manner.PMA-differentiated THP-1 cells (1x106 cells/mL) were pre-infected with Leishmania parasites (A) or incubated (B) with culture medium (LCM) from 7 days old parasite cultures of either Leishmania mexicana (Lmex), L. major GP63 wild type (Lmj WT), L. major GP63-/- (Lmj KO) or L. major GP63 rescue (Lmj Res). Afterwards, cells were washed and stimulated with 200 μg/mL of HZ. (C) Bone-marrow derived macrophages (1.5x106 cells/mL) were pre-infected for 2 hrs with Leishmania mexicana (Lmex). Following infection, cells were washed and stimulated with 200 μg/mL of HZ. 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 two independent experiments. (D) Western blot of Leishmania culture medium (LCM) for GP63. Data shown is representative of two independent experiments. (E) PMA-differentiated THP-1 cells (1x106 cells/mL) were incubated with different concentrations of LCM from 7 days old parasite cultures of L. mexicana. Afterwards, cells were washed and stimulated with either 200 μg/mL of HZ, 200 μg/mL of silica or 200 μg/mL of asbestos for 6 hrs. Supernatants and cell extracts were collected and subjected to Western blot analysis with the indicated antibodies.
Mentions: Upon activation macrophages can produce a large array of pro-inflammatory molecules including IL-1β, which is produced by inflammasome complexes. The NLRP3 inflammasome acts as an intracellular signaling platform which is able to sense a variety of exogenous signals like asbestos, silica [17] as well as the malarial pigment HZ [20,31] and DAMPs such as ATP [18] and MSU [21]. In initial dose-response experiments using synthetic HZ we confirmed the ability of HZ to induce IL-1β maturation in PMA-differentiated THP-1 cells (S1 Fig). The observed, dose-dependent IL-1β secretion was comparable to the results obtained in the case of silica or MSU treatment of cells. Infection of THP-1 cells with Leishmania did not induce IL-1β release as shown for L. mexicana (S2 Fig). Leishmania parasites are well known for their ability to block and inhibit various microbicidal functions of macrophages [32], therefore we sought to elucidate whether infections with Leishmania would inhibit IL-1β production by macrophages, which has been indicated to possibly serve a host protective role in murine models of infection [33,34]. As previously introduced the NLRP3 inflammasome activator HZ causes the IL-1β maturation and secretion in PMA-differentiated macrophages. Pre-infection with L. mexicana and L. major, revealed a parasite-dependent block of IL-1β maturation and release (Fig 1A). The impaired production of IL-1β was not restricted to a system of human cell culture but was also observable when parasite infection preceded inflammasome activation in murine BMDMs (Fig 1C). In those experiments due to the availability of antibodies the processing of caspase-1 into its active fragments p10 and p20 could be observed. Notably processing of caspase-1 was absent after infection of cells possibly due to the lack of inflammasome activation or complex formation. Identical experimental setups for infections as in Fig 1A using L. major GP63 KO and L. major GP63 rescue parasites support the hypothesis that Leishmania’s capability to inhibit IL-1β maturation and release was GP63-dependent (Fig 1A–1E). Thus, IL-1β secretion was not impaired in the absence of the protease (Fig 1A). This finding was further supported by experiments using pretreatment of THP-1 cells with Leishmania culture supernatant (LCM) instead of parasites (Fig 1B). The attenuated IL-1β secretion coincided with the presence of GP63 in the LCM. We were able to show that LCM of L. mexicana and L. major GP63 wild type parasite cultures contained GP63 (Fig 1D). To further evaluate the impact of secreted leishmanial factors like GP63 on IL-1β production the supernatant of L. mexicana cultures was concentrated and used in titration experiment on PMA-differentiated THP-1 cells (Fig 1E). IL-1β maturation and release were stimulated by a variety of known NLRP3 inflammasome inducers, namely HZ, silica and asbestos. In all cases we observed an inhibition of IL-1β secretion in a dose-dependent manner by the L. mexicana culture supernatant. Leishmania GP63 can be found either intracellular in the protozoan endoplasmic reticulum, membrane bound via a GPI anchor or secreted without the GPI anchor. During infection GPI anchored, membrane bound GP63 (GPI-GP63) can also be cleaved and released into the supernatant [2,35]. Therefore, the similarities of our results using either Leishmania infections or Leishmania culture supernatant are most likely to be attributed to the presence and activity of 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