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Apoptosis-associated speck-like protein (ASC) controls Legionella pneumophila infection in human monocytes.

ABSTRACT: The ability of Legionella pneumophila to cause pneumonia is determined by its capability to evade the immune system and grow within human monocytes and their derived macrophages. Human monocytes efficiently activate caspase-1 in response to Salmonella but not to L. pneumophila. The molecular mechanism for the lack of inflammasome activation during L. pneumophila infection is unknown. Evaluation of the expression of several inflammasome components in human monocytes during L. pneumophila infection revealed that the expression of the apoptosis-associated speck-like protein (ASC) and the NOD-like receptor NLRC4 are significantly down-regulated in human monocytes. Exogenous expression of ASC maintained the protein level constant during L. pneumophila infection and conveyed caspase-1 activation and restricted the growth of the pathogen. Further depletion of ASC with siRNA was accompanied with improved NF-?B activation and enhanced L. pneumophila growth. Therefore, our data demonstrate that L. pneumophila manipulates ASC levels to evade inflammasome activation and grow in human monocytes. By targeting ASC, L. pneumophila modulates the inflammasome, the apoptosome, and NF-?B pathway simultaneously.

SUBMITTER: Abdelaziz DH

PROVIDER: S-EPMC3030324 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Apoptosis-associated speck-like protein (ASC) controls Legionella pneumophila infection in human monocytes.

Abdelaziz Dalia H DH Gavrilin Mikhail A MA Akhter Anwari A Caution Kyle K Kotrange Sheetal S Khweek Arwa Abu AA Abdulrahman Basant A BA Grandhi Jaykumar J Hassan Zeinab A ZA Marsh Clay C Wewers Mark D MD Amer Amal O AO

The Journal of biological chemistry 20101119 5

The ability of Legionella pneumophila to cause pneumonia is determined by its capability to evade the immune system and grow within human monocytes and their derived macrophages. Human monocytes efficiently activate caspase-1 in response to Salmonella but not to L. pneumophila. The molecular mechanism for the lack of inflammasome activation during L. pneumophila infection is unknown. Evaluation of the expression of several inflammasome components in human monocytes during L. pneumophila infectio ...[more]

PMID: 21097506

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Project description:UnlabelledNucleotide-binding domain, leucine-rich repeat containing proteins (NLRs) activate caspase-1 in response to a variety of bacterium-derived signals in macrophages. NLR-mediated activation of caspase-1 by Legionella pneumophila occurs through both an NLRC4/NAIP5-dependent pathway and a pathway requiring the adapter protein Asc. Both pathways are needed for maximal activation of caspase-1 and for the release of the cytokines interleukin-1β (IL-1β) and IL-18. Asc is not required for caspase-1-dependent pore formation and cell death induced upon infection of macrophages by L. pneumophila. Here, temporal and spatial localization of caspase-1-dependent processes was examined to better define the roles of Asc and NLRC4 during infection. Imaging studies revealed that caspase-1 localized to a single punctate structure in infected cells containing Asc but not in cells lacking this adapter. Both endogenous Asc and ectopically produced NLRC4 tagged with green fluorescent protein (GFP) were found to localize to caspase-1 puncta following L. pneumophila infection, suggesting that NLRC4 and Asc coordinate signaling through this complex during caspase-1 activation. Formation of caspase-1-containing puncta correlated with caspase-1 processing, suggesting a role for the Asc/NLRC4/caspase-1 complex in caspase-1 cleavage. In cells deficient for Asc, NLRC4 did not assemble into discrete puncta, and pyroptosis occurred at an accelerated rate. These data indicate that Asc mediates integration of NLR components into caspase-1 processing platforms and that recruitment of NLR components into an Asc complex can dampen pyroptotic responses. Thus, a negative feedback role of complexes containing Asc may be important for regulating caspase-1-mediated responses during microbial infection.ImportanceCaspase-1 is a protease activated during infection that is central to the regulation of several innate immune pathways. Studies examining the macromolecular complexes containing this protein, known as inflammasomes, have provided insight into the regulation of this protease. This work demonstrates that the intracellular bacterium Legionella pneumophila induces formation of complexes containing caspase-1 by multiple mechanisms and illustrates that an adapter molecule called Asc integrates signals from multiple independent upstream caspase-1 activators in order to assemble a spatially distinct complex in the macrophage. There were caspase-1-associated activities such as cytokine processing and secretion that were controlled by Asc. Importantly, this work uncovered a new role for Asc in dampening a caspase-1-dependent cell death pathway called pyroptosis. These findings suggest that Asc plays a central role in controlling a distinct subset of caspase-1-dependent activities by both assembling complexes that are important for cytokine processing and suppressing processes that mediate pyroptosis.

Project description:Inflammasomes are multiprotein complexes of the innate immune response that recognize a diverse range of intracellular sensors of infection or cell damage and recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into an inflammasome signaling complex. The recruitment, polymerization and cross-linking of ASC is upstream of caspase-1 activation and interleukin-1β release. Here we provide evidence that IC 100, a humanized IgG4κ monoclonal antibody against ASC, is internalized into the cell and localizes with endosomes, while another part is recycled and redistributed out of the cell. IC 100 binds intracellular ASC and blocks interleukin-1β release in a human whole blood cell inflammasome assay. In vitro studies demonstrate that IC 100 interferes with ASC polymerization and assembly of ASC specks. In vivo bioluminescence imaging showed that IC 100 has broad tissue distribution, crosses the blood brain barrier, and readily penetrates the brain and spinal cord parenchyma. Confocal microscopy of fluorescent-labeled IC 100 revealed that IC 100 is rapidly taken up by macrophages via a mechanism utilizing the Fc region of IC 100. Coimmunoprecipitation experiments and confocal immunohistochemistry showed that IC 100 binds to ASC and to the atypical antibody receptor Tripartite motif-containing protein-21 (TRIM21). In A549 WT and TRIM21 KO cells treated with either IC 100 or IgG4κ isotype control, the levels of intracellular IC 100 were higher than in the IgG4κ-treated controls at 2 hours, 1 day and 3 days after administration, indicating that IC 100 escapes degradation by the proteasome. Lastly, electron microscopy studies demonstrate that IC 100 binds to ASC filaments and alters the architecture of ASC filaments. Thus, IC 100 readily penetrates a variety of cell types, and it binds to intracellular ASC, but it is not degraded by the TRIM21 antibody-dependent intracellular neutralization pathway.

Dataset Information

Apoptosis-associated speck-like protein (ASC) controls Legionella pneumophila infection in human monocytes.

Publications

Apoptosis-associated speck-like protein (ASC) controls Legionella pneumophila infection in human monocytes.

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