Simon O. Cridland

The Inflammasome Adaptor ASC Induces Procaspase-8 Death Effector Domain Filaments

Background: ASC mediates inflammasome assembly, recruiting procaspase-1 and procaspase-8 to initiate inflammation and cell death. Results: ASC pyrin domain (PYD) surfaces that mediate filament assembly bind procaspase-8 death effector domains (DEDs) and induce filaments. Conclusion: Procaspase-8 DED filaments are initiated from ASC PYD filaments. Significance: The data give insights into cross-talk between apoptotic and inflammatory pathways and procapase-8 activation. Inflammasomes mediate inflammatory and cell death responses to pathogens and cellular stress signals via activation of procaspases-1 and -8. During inflammasome assembly, activated receptors of the NLR or PYHIN family recruit the adaptor protein ASC and initiate polymerization of its pyrin domain (PYD) into filaments. We show that ASC filaments in turn nucleate procaspase-8 death effector domain (DED) filaments in vitro and in vivo. Interaction between ASC PYD and procaspase-8 tandem DEDs optimally required both DEDs and represents an unusual heterotypic interaction between domains of the death fold superfamily. Analysis of ASC PYD mutants showed that interaction surfaces that mediate procaspase-8 interaction overlap with those required for ASC self-association and interaction with the PYDs of inflammasome initiators. Our data indicate that multiple types of death fold domain filaments form at inflammasomes and that PYD/DED and homotypic PYD interaction modes are similar. Interestingly, we observed condensation of procaspase-8 filaments containing the catalytic domain, suggesting that procaspase-8 interactions within and/or between filaments may be involved in caspase-8 activation. Procaspase-8 filaments may also be relevant to apoptosis induced by death receptors.

A recessive screen for genes regulating hematopoietic stem cells

Identification of genes that regulate the development, self-renewal, and differentiation of stem cells is of vital importance for understanding normal organogenesis and cancer; such knowledge also underpins regenerative medicine. Here we demonstrate that chemical mutagenesis of mice combined with advances in hematopoietic stem cell reagents and genome resources can efficiently recover recessive mutations and identify genes essential for generation and proliferation of definitive hematopoietic stem cells and/or their progeny. We used high-throughput fluorescence-activated cell sorter to analyze 9 subsets of blood stem cells, progenitor cells, circulating red cells, and platelets in more than 1300 mouse embryos at embryonic day (E) 14.5. From 45 pedigrees, we recovered 6 strains with defects in definitive hematopoiesis. We demonstrate rapid identification of a novel mutation in the c-Myb transcription factor that results in thrombocythemia and myelofibrosis as proof of principal of the utility of our fluorescence-activated cell sorter-based screen. Such phenotype-driven approaches will provide new knowledge of the genes, protein interactions, and regulatory networks that underpin stem cell biology.

Genomic organisation and regulation of murine alpha haemoglobin stabilising protein by erythroid Kruppel-like factor

Alpha haemoglobin stabilising protein (AHSP) binds free α‐globin chains and plays an important role in the protection of red cells, particularly during β‐thalassaemia. Murine ASHP was discovered as a GATA‐1 target gene and human AHSP is directly regulated by GATA‐1. More recently, AHSP was rediscovered as a highly erythroid Kruppel‐like factor (EKLF) ‐dependent transcript. We have determined the organisation of the murine AHSP gene and compared it to orthologs. There are two CACC box elements in the proximal promoter. The proximal element is absolutely conserved, but does not bind EKLF as it is not a canonical binding site. In rodents, the distal element contains a 3 bp insertion that disrupts the typical EKLF binding consensus region. Nevertheless, EKLF binds this atypical site by gel mobility shift assay, specifically occupies the AHSP promoter in vivo in a chromatin immunoprecipitation assay, and transactivates AHSP through this CACC site in promoter–reporter assays. Our results suggest EKLF can occupy CACC elements in vivo that are not predictable from the consensus binding site inferred from structural studies. We also propose that absence of AHSP in EKLF‐null red cells exacerbates the toxicity of free α‐globin chains, which exist because of the defect in β‐globin gene activation.

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.

Indian hedgehog supports definitive erythropoiesis.

Indian hedgehog (Ihh) has been reported to stimulate haematopoiesis ex vivo. In this study we studied the consequences of loss of function of Ihh for murine haematopoietic development. Ihh has no essential role in primitive erythropoiesis, but it is required in a non cell autonomous fashion for definitive erythropoieisis. Many components of the hedgehog signaling pathway are present in the fetal liver, with Ihh and Gli1 being most highly expressed in the stroma and Ptc1 being most highly expressed in haematopoietic stem and progenitor cells. Ihh knockout HSC and progenitor cell populations are produced in normal numbers in vivo and respond normally to haematopoietic cytokines in vitro, but terminal erythroid differentiation is defective leading to fatal anemia in mid gestation in many Ihh knockout embryos. These loss-of-function studies are consistent with previous gain-of-function studies which show Ihh can induce blood from ectoderm or expand HSCs in stroma-dependent culture.

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.

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.