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INFLAMMASOME LAB RESOURCES


Animations and teaching tools

Animated film - The Inflammasome signalling pathway

Associate Professor Kate Schroder explains the signal transduction pathway of inflammasomes - molecular machines at the heart of host defence, inflammation and disease.

© 2017 The University of Queensland.  This film may be reproduced and communicated to the public (in whole or in part) for non-commercial research, education or study purposes only, provided all copyright and attribution notices appear in the copied or communicated film.  Subject to exceptions mandated by law, this film many not otherwise be reproduced or communicated to the public without the written permission of The University of Queensland.

For commercial use of this film, please contact Kate Schroder (K.Schroder@uq.edu.au). 

 

Inflammasome lab published protocols

Assessment of inflammasome formation by flow cytometry.
Current Protocols in Immunology Aug1;114:14.40.1-14.40.29. Pubmed
Sester DP, Zamoshnikova A, Thygesen SJ, Vajjhala PR, Cridland SO, Schroder K, Stacey KJ. (2016).

Inflammasomes are large protein complexes formed in response to cellular stresses that are platforms for recruitment and activation of caspase 1. Central to most inflammasome functions is the adapter molecule ASC (apoptosis-associated speck-like protein containing a caspase-recruitment dåomain) that links the inflammasome initiator protein to the recruited caspases. ASC is normally diffuse within the cell but within minutes of inflammasome activation relocates to a dense speck in the cytosol. The dramatic redistribution of ASC can be monitored by flow cytometry using parameters of fluorescence peak height and width when immunostained or tagged with a fluorescent protein. This can be used to define cells with active inflammasomes within populations of primary macrophages and monocytes, allowing quantification of responses and flow-sorting of responding cells. Protein structural requirements for ASC speck formation and recruitment of caspases to ASC specks can be assessed by expressing components in HEK293 cells. This provides rapid quantification of responding cell number and correlation with the expression level of inflammasome components within single cells. © 2016 by John Wiley & Sons, Inc.

 

A novel flow cytometric method to assess inflammasome formation.
Journal of Immunology 194(1):455-462. Pubmed
Sester DP, Thygesen SJ, Sagulenko V, Vajjhala PR, Cridland JA, Vitak N, Chen KW, Osborne GW, Schroder K, Stacey KJ (2015).

Inflammasomes are large protein complexes induced by a wide range of microbial, stress, and environmental stimuli that function to induce cell death and inflammatory cytokine processing. Formation of an inflammasome involves dramatic relocalization of the inflammasome adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into a single speck. We have developed a flow cytometric assay for inflammasome formation, time of flight inflammasome evaluation, which detects the change in ASC distribution within the cell. The transit of ASC into the speck is detected by a decreased width or increased height of the pulse of emitted fluorescence. This assay can be used to quantify native inflammasome formation in subsets of mixed cell populations ex vivo. It can also provide a rapid and sensitive technique for investigating molecular interactions in inflammasome formation, by comparison of wild-type and mutant proteins in inflammasome reconstitution experiments.

 

Protocols we recommend

Measuring the inflammasome.
Methods in Molecular Biology 844:199-222. Pubmed
Gross O (2012).

Inflammasomes are multiprotein complexes whose activity has been implicated in physiological and pathological inflammation. The hallmarks of inflammasome activation are the secretion of the mature forms of Caspase-1 and IL-1β from cells of the innate immune system. This protocol covers the methods required to study inflammasome activation using mouse bone marrow-derived dendritic cells (BMDCs) as a model system. The protocol includes the generation and handling of BMDCs, the stimulation of BMDCs with established Nlrp3 inflammasome activators, and the measurement of activation by both ELISA and western blot. These methods can be useful for the study of potential inflammasome activators, and of the signaling pathways involved in inflammasome activation. General considerations are provided that may help in the design and optimization of modified methods for the study of other types of inflammasomes and in other cell types.

 

Gene expression in LPS-stimulated human and mouse macrophages

Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages.
Proceedings of the National Academy of Sciences U S A 109: E944-53. Pubmed
Schroder K, Irvine KM, Taylor MS, Bokil NJ, Le Cao KA, Masterman KA, Labzin LI, Semple CA, Kapetanovic R, Fairbairn L, Akalin A, Faulkner GJ, Baillie JK, Gongora M, Daub CO, Kawaji H, McLachlan GJ, Goldman N, Grimmond SM, Carninci P, Suzuki H, Hayashizaki Y, Lenhard B, Hume DA, Sweet MJ (2012).

Evolutionary change in gene expression is generally considered to be a major driver of phenotypic differences between species. We investigated innate immune diversification by analyzing interspecies differences in the transcriptional responses of primary human and mouse macrophages to the Toll-like receptor (TLR)-4 agonist lipopolysaccharide (LPS). By using a custom platform permitting cross-species interrogation coupled with deep sequencing of mRNA 5' ends, we identified extensive divergence in LPS-regulated orthologous gene expression between humans and mice (24% of orthologues were identified as "divergently regulated"). We further demonstrate concordant regulation of human-specific LPS target genes in primary pig macrophages. Divergently regulated orthologues were enriched for genes encoding cellular "inputs" such as cell surface receptors (e.g., TLR6, IL-7Rα) and functional "outputs" such as inflammatory cytokines/chemokines (e.g., CCL20, CXCL13). Conversely, intracellular signaling components linking inputs to outputs were typically concordantly regulated. Functional consequences of divergent gene regulation were confirmed by showing LPS pretreatment boosts subsequent TLR6 responses in mouse but not human macrophages, in keeping with mouse-specific TLR6 induction. Divergently regulated genes were associated with a large dynamic range of gene expression, and specific promoter architectural features (TATA box enrichment, CpG island depletion). Surprisingly, regulatory divergence was also associated with enhanced interspecies promoter conservation. Thus, the genes controlled by complex, highly conserved promoters that facilitate dynamic regulation are also the most susceptible to evolutionary change.
 

MacGate: Macrophage Comparitive Expression Gateway
MacGate is a publicly available resource focused on divergent gene regulation in human and mouse macrophages. The website displays expression data generated from a custom microarray, which we specifically collected and analysed to investigate divergent gene regulation in response to lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls that stimulates inflammatory responses via Toll-like receptor 4. These and other analyses are published in Schroder et al.,2012, PNAS USA. The expression of 3041 human and 2945 mouse genes was interrogated over a timecourse of LPS stimulation in human macrophages and two mouse macrophage populations respectively. The expression data pages for each gene interrogated on the array are arranged according to orthology clusters, and can be retrieved by searching for gene names or identifiers, or by simply browsing the database.