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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

Analysis of inflammasome-related and inflammasome-complex proteins for GP63-induced manipulation.(A) In silico GP63 cleavage sites analysis of indicated components of the NLRP3 inflammasome complex was done using the ScanProsite platform. (B) PMA-differentiated THP-1 cells or LPS-primed BMDMs were infected with Leishmania mexicana (Lmex), L. major GP63 wild type (Lmj WT), L. major GP63-/- (Lmj KO) or L. major GP63 rescue (Lmj Res) for 2 hrs. Cells were washed, cell extracts were collected and subjected to Western blot analysis with the indicated antibodies. (F) denotes the generation of GP63-dependent cleavage fragments. (C) Summary of potential cleavage sites in murine and human inflammasome and inflammasome-related proteins. Data shown is representative of three independent experiments.
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pntd.0003868.g005: Analysis of inflammasome-related and inflammasome-complex proteins for GP63-induced manipulation.(A) In silico GP63 cleavage sites analysis of indicated components of the NLRP3 inflammasome complex was done using the ScanProsite platform. (B) PMA-differentiated THP-1 cells or LPS-primed BMDMs were infected with Leishmania mexicana (Lmex), L. major GP63 wild type (Lmj WT), L. major GP63-/- (Lmj KO) or L. major GP63 rescue (Lmj Res) for 2 hrs. Cells were washed, cell extracts were collected and subjected to Western blot analysis with the indicated antibodies. (F) denotes the generation of GP63-dependent cleavage fragments. (C) Summary of potential cleavage sites in murine and human inflammasome and inflammasome-related proteins. Data shown is representative of three independent experiments.

Mentions: Our previous data showed that Leishmania infections of macrophages prevent the maturation of pro-IL-1β to mature IL-1β upon stimulation possibly intervening with a host protective effect. Thus far, the impairment of IL-1β after infection was attributed to a suppression of PKC-dependent signaling and the loss of ROS production. As the enclosed data supports that GP63 is closely associated with these events, we wanted to examine if Leishmania parasites and GP63 may also interfere with inflammasome activation through proteolytic cleavage of inflammasome components. We and others demonstrated that GP63 can cleave targets containing the following amino acid-motives: polar/hydrophobic/basic/basic amino acids (P1- P’1-P’2-P’3) [43,46]. A first indication for GP63-dependent cleavage of inflammasome components was obtained by experiments using BMDMs. There, we observed that after infection with Leishmania we were able to observe cleavage of pro-IL-1β (Fig 1C). An in silico sequence analysis for putative GP63 cleavage sites revealed the possibility of additional GP63 cleavage sites in the sequences of inflammasome complex and associated proteins. Thus, the sequences of human and murine NLRP3; pro-IL1β and TXNIP—a protein that has been suggested to possibly be involved in ROS-mediated inflammasome activation [40]–contain putative cleavage sites for GP63 (Fig 5A and 5C). As GP63 facilitated cleavage is not necessarily restricted to the proposed cleavage motif and to confirm our in silico findings we performed Western blotting analysis of infected THP1 and LPS-primed BMDM cells. In accordance with the in silico data, GP63 seemed to be able to directly interfere with the inflammasome complex. Thus, we observed cleavage of NLRP3 after infection with Leishmania (Fig 5). This process was GP63-dependent as illustrated by the results for L. major wt, GP63KO and GP63 rescue parasites. Although, the in silico analysis did not suggest a GP63-mediated cleavage of ASC or Caspase-1 we choose to analyze both as they are an integral part of the inflammasome complex. Neither pro-caspase-1 nor ASC showed any cleavage after infection. In addition, as anticipated cleavage and/or cleavage fragments were detected for pro-IL-1β and TXNIP in lysates of cells infected with L. mexicana, L. major wt or L. major GP63 rescue expressing GP63, but not in cells infected with L. major GP63 KO. Taken together our data suggests that Leishmania is able to impair inflammasome activation through different GP63-dependent alterations of proteins and signaling pathways.


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)

Analysis of inflammasome-related and inflammasome-complex proteins for GP63-induced manipulation.(A) In silico GP63 cleavage sites analysis of indicated components of the NLRP3 inflammasome complex was done using the ScanProsite platform. (B) PMA-differentiated THP-1 cells or LPS-primed BMDMs were infected with Leishmania mexicana (Lmex), L. major GP63 wild type (Lmj WT), L. major GP63-/- (Lmj KO) or L. major GP63 rescue (Lmj Res) for 2 hrs. Cells were washed, cell extracts were collected and subjected to Western blot analysis with the indicated antibodies. (F) denotes the generation of GP63-dependent cleavage fragments. (C) Summary of potential cleavage sites in murine and human inflammasome and inflammasome-related proteins. 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.g005: Analysis of inflammasome-related and inflammasome-complex proteins for GP63-induced manipulation.(A) In silico GP63 cleavage sites analysis of indicated components of the NLRP3 inflammasome complex was done using the ScanProsite platform. (B) PMA-differentiated THP-1 cells or LPS-primed BMDMs were infected with Leishmania mexicana (Lmex), L. major GP63 wild type (Lmj WT), L. major GP63-/- (Lmj KO) or L. major GP63 rescue (Lmj Res) for 2 hrs. Cells were washed, cell extracts were collected and subjected to Western blot analysis with the indicated antibodies. (F) denotes the generation of GP63-dependent cleavage fragments. (C) Summary of potential cleavage sites in murine and human inflammasome and inflammasome-related proteins. Data shown is representative of three independent experiments.
Mentions: Our previous data showed that Leishmania infections of macrophages prevent the maturation of pro-IL-1β to mature IL-1β upon stimulation possibly intervening with a host protective effect. Thus far, the impairment of IL-1β after infection was attributed to a suppression of PKC-dependent signaling and the loss of ROS production. As the enclosed data supports that GP63 is closely associated with these events, we wanted to examine if Leishmania parasites and GP63 may also interfere with inflammasome activation through proteolytic cleavage of inflammasome components. We and others demonstrated that GP63 can cleave targets containing the following amino acid-motives: polar/hydrophobic/basic/basic amino acids (P1- P’1-P’2-P’3) [43,46]. A first indication for GP63-dependent cleavage of inflammasome components was obtained by experiments using BMDMs. There, we observed that after infection with Leishmania we were able to observe cleavage of pro-IL-1β (Fig 1C). An in silico sequence analysis for putative GP63 cleavage sites revealed the possibility of additional GP63 cleavage sites in the sequences of inflammasome complex and associated proteins. Thus, the sequences of human and murine NLRP3; pro-IL1β and TXNIP—a protein that has been suggested to possibly be involved in ROS-mediated inflammasome activation [40]–contain putative cleavage sites for GP63 (Fig 5A and 5C). As GP63 facilitated cleavage is not necessarily restricted to the proposed cleavage motif and to confirm our in silico findings we performed Western blotting analysis of infected THP1 and LPS-primed BMDM cells. In accordance with the in silico data, GP63 seemed to be able to directly interfere with the inflammasome complex. Thus, we observed cleavage of NLRP3 after infection with Leishmania (Fig 5). This process was GP63-dependent as illustrated by the results for L. major wt, GP63KO and GP63 rescue parasites. Although, the in silico analysis did not suggest a GP63-mediated cleavage of ASC or Caspase-1 we choose to analyze both as they are an integral part of the inflammasome complex. Neither pro-caspase-1 nor ASC showed any cleavage after infection. In addition, as anticipated cleavage and/or cleavage fragments were detected for pro-IL-1β and TXNIP in lysates of cells infected with L. mexicana, L. major wt or L. major GP63 rescue expressing GP63, but not in cells infected with L. major GP63 KO. Taken together our data suggests that Leishmania is able to impair inflammasome activation through different GP63-dependent alterations of proteins and signaling pathways.

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