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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
Dynamin inhibition interferes with inflammasome activation and cytokine gene expression in Streptococcus
S Latvala1, S M Mäkelä, M Miettinen
1Virology Unit, Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare, Helsinki, Finland.
Abstract:
In the present study, we have analysed the ability of Streptococcus pyogenes [Group A streptococcus (GAS)] to activate the NACHT-domain-, leucine-rich repeat- and PYD-containing protein 3 (NALP3) inflammasome complex in human monocyte-derived macrophages and the molecules and signalling pathways involved in GAS-induced inflammatory responses. We focused upon analysing the impact of dynamin-dependent endocytosis and the role of major streptococcal virulence factors streptolysin O (SLO) and streptolysin S (SLS) in the immune responses induced by GAS. These virulence factors are involved in immune evasion by forming pores in host cell membranes, and aid the bacteria to escape from the endosome-lysosome pathway. We analysed cytokine gene expression in human primary macrophages after stimulation with live or inactivated wild-type GAS as well as with live SLO and SLS defective bacteria. Interleukin (IL)-1β, IL-10, tumour necrosis factor (TNF)-α and chemokine (C-X-C motif) ligand (CXCL)-10 cytokines were produced after bacterial stimulation in a dose-dependent manner and no differences in cytokine levels were seen between live, inactivated or mutant bacteria. These data suggest that streptolysins or other secreted bacterial products are not required for the inflammatory responses induced by GAS. Our data indicate that inhibition of dynamin-dependent endocytosis in macrophages attenuates the induction of IL-1β, TNF-α, interferon (IFN)-β and CXCL-10 mRNAs. We also observed that pro-IL-1β protein was expressed and efficiently cleaved into mature-IL-1β via inflammasome activation after bacterial stimulation. Furthermore, we demonstrate that multiple signalling pathways are involved in GAS-stimulated inflammatory responses in human macrophages.
Insights
Streptococcus pyogenes activates the NALP3 inflammasome in macrophages, leading to cytokine release. Bacterial virulence factors like streptolysins are not required for this response, but dynamin-dependent endocytosis is crucial.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Streptococcus pyogenes (Group A Streptococcus, GAS) is a pathogen that can trigger inflammatory responses.
- The NACHT-domain-, leucine-rich repeat- and PYD-containing protein 3 (NALP3) inflammasome plays a key role in innate immunity.
- GAS virulence factors, such as streptolysin O (SLO) and streptolysin S (SLS), are known to mediate immune evasion.
Purpose of the Study:
- To investigate GAS activation of the NALP3 inflammasome in human macrophages.
- To identify molecules and signaling pathways involved in GAS-induced inflammation.
- To determine the role of dynamin-dependent endocytosis and streptolysins in these responses.
Main Methods:
- Human primary macrophages were stimulated with live, inactivated, or mutant GAS (SLO/SLS-defective).
- Cytokine gene expression (IL-1β, IL-10, TNF-α, CXCL-10) and protein levels were analyzed.
- The impact of inhibiting dynamin-dependent endocytosis on inflammatory mediator induction was assessed.
Main Results:
- GAS stimulation induced dose-dependent production of IL-1β, IL-10, TNF-α, and CXCL-10.
- No significant differences in cytokine levels were observed between live, inactivated, or mutant GAS, suggesting streptolysins are not required.
- Inhibition of dynamin-dependent endocytosis attenuated the induction of IL-1β, TNF-α, IFN-β, and CXCL-10 mRNAs.
- GAS induced pro-IL-1β expression and subsequent cleavage into mature IL-1β via inflammasome activation.
Conclusions:
- GAS activates the NALP3 inflammasome in macrophages, leading to the production of key inflammatory cytokines.
- Dynamin-dependent endocytosis is essential for the induction of inflammatory responses by GAS.
- Bacterial streptolysins (SLO and SLS) are not necessary for GAS-induced inflammatory cytokine production in this context.
