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Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
Published on: May 22, 2014
Staphylococcus aureus alpha-hemolysin activates the NLRP3-inflammasome in human and mouse monocytic cells
Robin R Craven1, Xi Gao, Irving C Allen
1Department of Medicine-Division of Infectious Diseases, University of North Carolina, Chapel Hill, North Carolina, United States of America.
Abstract:
Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal alpha-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus alpha-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood. Using highly purified recombinant alpha-hemolysin, we now demonstrate that alpha-hemolysin activates the Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3)-inflammasome, a host inflammatory signaling complex involved in responses to pathogens and endogenous danger signals. Non-cytolytic mutant alpha-hemolysin molecules fail to elicit NLRP3-inflammasome signaling, demonstrating that the responses are not due to non-specific activation of this innate immune signaling system by bacterially derived proteins. In monocyte-derived cells from humans and mice, inflammasome assembly in response to alpha-hemolysin results in activation of the cysteine proteinase, caspase-1. We also show that inflammasome activation by alpha-hemolysin works in conjunction with signaling by other CA-MRSA-derived Pathogen Associated Molecular Patterns (PAMPs) to induce secretion of pro-inflammatory cytokines IL-1beta and IL-18. Additionally, alpha-hemolysin induces cell death in these cells through an NLRP3-dependent program of cellular necrosis, resulting in the release of endogenous pro-inflammatory molecules, like the chromatin-associated protein, High-mobility group box 1 (HMGB1). These studies link the activity of a major S. aureus virulence factor to a specific host signaling pathway. The cellular events linked to inflammasome activity have clear relevance to the disease processes associated with CA-MRSA including tissue necrosis and inflammation.
Insights
Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) alpha-hemolysin activates the NLRP3-inflammasome, a key inflammatory pathway. This leads to cell death and cytokine release, contributing to severe infections.
Area of Science:
- Immunology
- Microbiology
- Molecular Biology
Background:
- Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) causes severe necrotizing infections.
- Staphylococcal alpha-hemolysin is a critical virulence factor in CA-MRSA infections.
- The precise mechanisms by which alpha-hemolysin induces inflammation and cell death were not fully understood.
Purpose of the Study:
- To elucidate the molecular mechanisms by which staphylococcal alpha-hemolysin triggers host inflammatory responses.
- To investigate the role of the NLRP3-inflammasome in mediating alpha-hemolysin-induced cellular events.
- To link alpha-hemolysin activity to specific host signaling pathways relevant to CA-MRSA pathogenesis.
Main Methods:
- Utilized highly purified recombinant alpha-hemolysin and non-cytolytic mutants.
- Employed monocyte-derived cells from human and mouse origins.
- Assessed inflammasome assembly, caspase-1 activation, cytokine secretion (IL-1beta, IL-18), and cell death pathways.
Main Results:
- Alpha-hemolysin directly activates the Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3)-inflammasome.
- Inflammasome activation by alpha-hemolysin requires its cytolytic function and leads to caspase-1 activation.
- Alpha-hemolysin promotes pro-inflammatory cytokine secretion and induces NLRP3-dependent necrosis, releasing HMGB1.
Conclusions:
- Staphylococcal alpha-hemolysin is a direct activator of the NLRP3-inflammasome pathway.
- This activation is crucial for inducing inflammatory signaling, cell death, and cytokine release in CA-MRSA infections.
- The findings provide a molecular link between a key virulence factor and host inflammatory responses, offering insights into CA-MRSA pathogenesis.
