Sara J. Thygesen

AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC.

Inflammasomes are protein complexes assembled upon recognition of infection or cell damage signals, and serve as platforms for clustering and activation of procaspase-1. Oligomerisation of initiating proteins such as AIM2 (absent in melanoma-2) and NLRP3 (NOD-like receptor family, pyrin domain-containing-3) recruits procaspase-1 via the inflammasome adapter molecule ASC (apoptosis-associated speck-like protein containing a CARD). Active caspase-1 is responsible for rapid lytic cell death termed pyroptosis. Here we show that AIM2 and NLRP3 inflammasomes activate caspase-8 and -1, leading to both apoptotic and pyroptotic cell death. The AIM2 inflammasome is activated by cytosolic DNA. The balance between pyroptosis and apoptosis depended upon the amount of DNA, with apoptosis seen at lower transfected DNA concentrations. Pyroptosis had a higher threshold for activation, and dominated at high DNA concentrations because it happens more rapidly. Gene knockdown showed caspase-8 to be the apical caspase in the AIM2- and NLRP3-dependent apoptotic pathways, with little or no requirement for caspase-9. Procaspase-8 localised to ASC inflammasome ‘specks’ in cells, and bound directly to the pyrin domain of ASC. Thus caspase-8 is an integral part of the inflammasome, and this extends the relevance of the inflammasome to cell types that do not express caspase-1.

Molecular Mechanism for p202-Mediated Specific Inhibition of AIM2 Inflammasome Activation

Mouse p202 containing two hemopoietic expression, interferon inducibility, nuclear localization (HIN) domains antagonizes AIM2 inflammasome signaling and potentially modifies lupus susceptibility. We found that only HIN1 of p202 binds double-stranded DNA (dsDNA), while HIN2 forms a homotetramer. Crystal structures of HIN1 revealed that dsDNA is bound on face opposite the site used in AIM2 and IFI16. The structure of HIN2 revealed a dimer of dimers, the face analogous to the HIN1 dsDNA binding site being a dimerization interface. Electron microscopy imaging showed that HIN1 is flexibly linked to HIN2 in p202, and tetramerization provided enhanced avidity for dsDNA. Surprisingly, HIN2 of p202 interacts with the AIM HIN domain. We propose that this results in a spatial separation of the AIM2 pyrin domains, and indeed p202 prevented the dsDNA-dependent clustering of apoptosis-associated speck-like protein containing caspase recruitment domain (ASC) and AIM2 inflammasome activation. We hypothesize that while p202 was evolutionarily selected to limit AIM2-mediated inflammation in some mouse strains, the same mechanism contributes to increased interferon production and lupus susceptibility.

A Novel Flow Cytometric Method To Assess Inflammasome Formation

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.

Deficient NLRP3 and AIM2 Inflammasome Function in Autoimmune NZB Mice

Inflammasomes are protein complexes that promote caspase activation, resulting in processing of IL-1β and cell death, in response to infection and cellular stresses. Inflammasomes have been anticipated to contribute to autoimmunity. The New Zealand Black (NZB) mouse develops anti-erythrocyte Abs and is a model of autoimmune hemolytic anemia. These mice also develop anti-nuclear Abs typical of lupus. In this article, we show that NZB macrophages have deficient inflammasome responses to a DNA virus and fungal infection. Absent in melanoma 2 (AIM2) inflammasome responses are compromised in NZB by high expression of the AIM 2 antagonist protein p202, and consequently NZB cells had low IL-1β output in response to both transfected DNA and mouse CMV infection. Surprisingly, we also found that a second inflammasome system, mediated by the NLR family, pyrin domain containing 3 (NLRP3) initiating protein, was completely lacking in NZB cells. This was due to a point mutation in an intron of the Nlrp3 gene in NZB mice, which generates a novel splice acceptor site. This leads to incorporation of a pseudoexon with a premature stop codon. The lack of full-length NLRP3 protein results in NZB being effectively null for Nlrp3, with no production of bioactive IL-1β in response to NLRP3 stimuli, including infection with Candida albicans. Thus, this autoimmune strain harbors two inflammasome deficiencies, mediated through quite distinct mechanisms. We hypothesize that the inflammasome deficiencies in NZB alter the interaction of the host with both microflora and pathogens, promoting prolonged production of cytokines that contribute to development of autoantibodies.

Correcting the NLRP3 inflammasome deficiency in macrophages from autoimmune NZB mice with exon skipping antisense oligonucleotides

Inflammasomes are molecular complexes activated by infection and cellular stress, leading to caspase‐1 activation and subsequent interleukin‐1β (IL‐1β) processing and cell death. The autoimmune NZB mouse strain does not express NLRP3, a key inflammasome initiator mediating responses to a wide variety of stimuli including endogenous danger signals, environmental irritants and a range of bacterial, fungal and viral pathogens. We have previously identified an intronic point mutation in the Nlrp3 gene from NZB mice that generates a splice acceptor site. This leads to inclusion of a pseudoexon that introduces an early termination codon and is proposed to be the cause of NLRP3 inflammasome deficiency in NZB cells. Here we have used exon skipping antisense oligonucleotides (AONs) to prevent aberrant splicing of Nlrp3 in NZB macrophages, and this restored both NLRP3 protein expression and NLRP3 inflammasome activity. Thus, the single point mutation leading to aberrant splicing is the sole cause of NLRP3 inflammasome deficiency in NZB macrophages. The NZB mouse provides a model for addressing a splicing defect in macrophages and could be used to further investigate AON design and delivery of AONs to macrophages in vivo.

Assessment of Inflammasome Formation by Flow Cytometry

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 domain) 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.

Compromised NLRP3 and AIM2 inflammasome function in autoimmune NZB/W F1 mouse macrophages

Inflammasomes are protein complexes activated by infection and cellular stress that promote caspase‐1 activation and subsequent inflammatory cytokine processing and cell death. It has been anticipated that inflammasome activity contributes to autoimmunity. However, we previously showed that macrophages from autoimmune New Zealand Black (NZB) mice lack NLRP3 inflammasome function, and their absent in melanoma 2 (AIM2) inflammasome responses are compromised by high expression of the AIM2 antagonist protein p202. Here we found that the point mutation leading to lack of NLRP3 expression occurred early in the NZB strain establishment, as it is shared with the related obese strain New Zealand Obese, but not with the unrelated New Zealand White (NZW) strain. The first cross progeny of NZB and NZW mice develop more severe lupus nephritis than the NZB strain. We have compared AIM2 and NLRP3 inflammasome function in macrophages from NZB, NZW, and NZB/W F1 mice. The NZW parental strain showed strong inflammasome function, whereas the NZB/W F1 have haploinsufficient expression of NLRP3 and show reduced NLRP3 and AIM2 inflammasome responses, particularly at low stimulus strength. It remains to be established whether the low inflammasome function could contribute to loss of tolerance and the onset of autoimmunity in NZB and NZB/W F1. However, with amplifying inflammatory stimuli through the course of disease, the NLRP3 response in the NZB/W F1 may be sufficient to contribute to kidney damage at later stages of disease.

UNIT 14.40 Assessment of Inflammasome Formation by Flow Cytometry

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 domain) 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.

Assessment of Inflammasome Formation by Flow Cytometry: Assessment of Inflammasome Formation

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 domain) 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.