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Updated: Feb 7, 2026
Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
Published on: May 22, 2014
Membrane vesicles from Pseudomonas aeruginosa activate the noncanonical inflammasome through caspase-5 in human
Natalie J Bitto1, Paul J Baker2,3, Jennifer K Dowling1
1Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, 3168, Australia.
Abstract:
Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria both in vivo and in vitro. These lipid-bound structures carry a range of immunogenic components derived from the parent cell, which are transported into host target cells and activate the innate immune system. Recent advances in the field have shed light on some of the multifaceted roles of OMVs in host-pathogen interactions. In this study, we investigated the ability of OMVs from two clinically important pathogens, Pseudomonas aeruginosa and Helicobacter pylori, to activate canonical and noncanonical inflammasomes. P. aeruginosa OMVs induced inflammasome activation in mouse macrophages, as evidenced by "speck" formation, as well as the cleavage and secretion of interleukin-1β and caspase-1. These responses were independent of AIM2 and NLRC4 canonical inflammasomes, but dependent on the noncanonical caspase-11 pathway. Moreover, P. aeruginosa OMVs alone were able to activate the inflammasome in a TLR-dependent manner, without requiring an exogenous priming signal. In contrast, H. pylori OMVs were not able to induce inflammasome activation in macrophages. Using CRISPR/Cas9 knockout THP-1 cells lacking the human caspase-11 homologs, caspase-4 and -5,we demonstrated that caspase-5 but not caspase-4 is required for inflammasome activation by P. aeruginosa OMVs in human monocytes. In contrast, free P. aeruginosa lipopolysaccharide (LPS) transfected into cells induced inflammasome responses via caspase-4. This suggests that caspase-4 and caspase-5 differentially recognize LPS depending on its physical form or route of delivery into the cell. These findings have relevance to Gram-negative infections in humans and the use of OMVs as novel vaccines.
Insights
Outer membrane vesicles (OMVs) from Pseudomonas aeruginosa activate inflammasomes via the caspase-11 pathway in mouse macrophages, but not in human cells, revealing differential caspase roles. Helicobacter pylori OMVs did not activate inflammasomes.
Area of Science:
- Bacteriology
- Immunology
- Cell Biology
Background:
- Outer membrane vesicles (OMVs) are released by Gram-negative bacteria and play roles in host-pathogen interactions.
- OMVs carry bacterial components that can activate the host innate immune system.
- Inflammasomes are key components of the innate immune system, regulating inflammation and cell death.
Purpose of the Study:
- To investigate the ability of OMVs from Pseudomonas aeruginosa and Helicobacter pylori to activate canonical and noncanonical inflammasomes.
- To elucidate the specific inflammasome pathways and caspases involved in OMVs-mediated immune responses.
- To compare the inflammasome-activating potential of OMVs with free lipopolysaccharide (LPS).
Main Methods:
- Incubation of mouse macrophages and human THP-1 cells with OMVs from P. aeruginosa and H. pylori.
- Assessment of inflammasome activation through speck formation, IL-1β cleavage, and caspase-1 activity.
- Utilized CRISPR/Cas9 gene editing to generate knockout cell lines lacking caspase-4 and caspase-5.
- Stimulation of cells with free P. aeruginosa LPS to compare immune responses.
Main Results:
- P. aeruginosa OMVs induced inflammasome activation in mouse macrophages, dependent on the noncanonical caspase-11 pathway, not AIM2 or NLRC4.
- H. pylori OMVs did not induce inflammasome activation in macrophages.
- In human monocytes, P. aeruginosa OMVs required caspase-5, but not caspase-4, for inflammasome activation.
- Free P. aeruginosa LPS activated inflammasomes via caspase-4, indicating differential recognition based on LPS form and delivery.
Conclusions:
- P. aeruginosa OMVs activate inflammasomes through a TLR-dependent, noncanonical pathway in mouse macrophages.
- Caspase-11 in mice and caspase-5 in humans are crucial for OMV-induced inflammasome activation.
- Differential recognition of LPS by caspase-4 and caspase-5 highlights the importance of LPS presentation.
- These findings have implications for understanding Gram-negative infections and developing OMV-based vaccines.
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