Caspase isoforms in inflammasome activation

Inflammasomes are macromolecular signalling platforms composed of a receptor (for example NLRP3 or NLRC4), an adaptor (ASC) and effectors (caspases) that are thought to play critical roles in the host defence against microbial infections. Activation of inflammasomes leads to the processing of the pro-inflammatory cytokines pro-IL-1β and pro- IL-18 to their active form and cleavage of gasdermin D to induce pyroptosis. Caspase-1 is the main inflammatory caspase responsible for cytokine maturation and induction of cell death, while human caspase-4, -5 and the mouse orthologue caspase-11 play an essential role in cytosolic bacterial LPS recognition. The putative importance of inflammasomes suggest that their constituents should be conserved across different animal species, but there are major differences, particularly in the caspase repertoire, in both invertebrates and vertebrates. The dog (Canis lupus familiaris), for example, has a pseudogene for NLRC4 and has a unique caspase-1/4/11 hybrid gene (CASP1-4/5/11) comprising the caspase-1 caspase recruitment domain (CARD) and the catalytic domain of caspase-4/5/11. Dogs produce bioactive IL-1β despite the apparent lack of the catalytically active domain of caspase-1, but how this occurs and whether inflammasome activation of CASP1-4/5/11 is required remains to be resolved. In this study I characterised inflammasome function in dog macrophages (DH82 cell line) and mouse macrophages where a caspase 1/4/11 fusion protein that is functional equivalent to CASP1-4/5/11 (DogMo) had been generated by CRISPR/Cas9 gene editing. I used imaging (ASC and active caspase speck formation), cell death analysis and IL-β production as readouts before and after editing of key genes in inflammasome formation using CRISPR-Cas9 gene editing approaches. The NLRP3 inflammasome is functional in dog macrophages, but the rate of inflammasome formation is lower compared to wild-type murine macrophages. This suggests that dog macrophages may be adapted to be tolerant to NLRP3 inflammasome activation only undergoing lytic cell death and IL-1β maturation when exposed to high concentrations of NLRP3 inflammasome activating ligands. DogMo cells retained full NLRP3 functionality. Cytosolic LPS-induced non-canonical inflammasome activation induced IL-1β maturation and secretion without concomitant cell death induction in dog macrophages. No ASC speck formation or activated caspase speck was seen suggesting there are alternative pathways for cytosolic LPS recognition and the consequential IL-1β maturation. DogMo cells did not induce non-canonical inflammasome formation. The NLRC4 inflammasome should not function in the dog and indeed DH82 cells infected with wild-type S. Typhimurium showed no ASC speck formation or active caspase recruitment and delayed cell death (compared to their wild-type mouse counterpart), but cleaved IL-1β was present in the supernatant. The processed IL-1β was smaller than the p17 fragment normally associated with cleavage by caspase-1. DogMo cells, which express NLRC4, did not process IL-1β as expected due to the lack of the catalytic domain of caspase-1 but still induced cell death albeit at a reduced level to wild-type mouse macrophages. S.Typhimurium infection of CRISPR/Cas9 gene edited DH82 cells to remove CASP1-4/5/11showed that cell death was independent of this caspase. Collectively, these data indicate that indog cells processing of pro-IL-1β and cell death occurs by inflammasome-independent pathways.CRISPR/Cas9 gene editing of DH82 cells to remove the necroptotic cell death initiator RIP kinase1 and apoptotic cell death initiator caspase-8 genes identified an essential role for RIPK1 in pro-IL-1β priming, and potentially, for caspase-8 in IL-1β maturation. The characterisation of caspase functionality in dog and DogMo cells show marked differences despite their expression of a similar protein. The deficit in inflammasome functionality in the dog could point to an evolutionary adaptation to tolerate microorganisms associated with their lifestyle as an opportunistic scavenger to protect them against severe enteric infections from gut bacterial pathogens.Medical Research Council (MRC

Loss of the chromatin modifier Kdm2aa causes BrafV600E-independent spontaneous melanoma in zebrafish.

KDM2A is a histone demethylase associated with transcriptional silencing, however very little is known about its in vivo role in development and disease. Here we demonstrate that loss of the orthologue kdm2aa in zebrafish causes widespread transcriptional disruption and leads to spontaneous melanomas at a high frequency. Fish homozygous for two independent premature stop codon alleles show reduced growth and survival, a strong male sex bias, and homozygous females exhibit a progressive oogenesis defect. kdm2aa mutant fish also develop melanomas from early adulthood onwards which are independent from mutations in braf and other common oncogenes and tumour suppressors as revealed by deep whole exome sequencing. In addition to effects on translation and DNA replication gene expression, high-replicate RNA-seq in morphologically normal individuals demonstrates a stable regulatory response of epigenetic modifiers and the specific de-repression of a group of zinc finger genes residing in constitutive heterochromatin. Together our data reveal a complex role for Kdm2aa in regulating normal mRNA levels and carcinogenesis. These findings establish kdm2aa mutants as the first single gene knockout model of melanoma biology

A high-resolution mRNA expression time course of embryonic development in zebrafish.

We have produced an mRNA expression time course of zebrafish development across 18 time points from 1 cell to 5 days post-fertilisation sampling individual and pools of embryos. Using poly(A) pulldown stranded RNA-seq and a 3' end transcript counting method we characterise temporal expression profiles of 23,642 genes. We identify temporal and functional transcript co-variance that associates 5024 unnamed genes with distinct developmental time points. Specifically, a class of over 100 previously uncharacterised zinc finger domain containing genes, located on the long arm of chromosome 4, is expressed in a sharp peak during zygotic genome activation. In addition, the data reveal new genes and transcripts, differential use of exons and previously unidentified 3' ends across development, new primary microRNAs and temporal divergence of gene paralogues generated in the teleost genome duplication. To make this dataset a useful baseline reference, the data can be browsed and downloaded at Expression Atlas and Ensembl

The age of heterozygous telomerase mutant parents influences the adult phenotype of their offspring irrespective of genotype in zebrafish

Background: Mutations in proteins involved in telomere maintenance lead to a range of human diseases, including dyskeratosis congenita, idiopathic pulmonary fibrosis and cancer. Telomerase functions to add telomeric repeats back onto the ends of chromosomes, however non-canonical roles of components of telomerase have recently been suggested. Methods: Here we use a zebrafish telomerase mutant which harbours a nonsense mutation in tert to investigate the adult phenotypes of fish derived from heterozygous parents of different ages. Furthermore we use whole genome sequencing data to estimate average telomere lengths. Results: We show that homozygous offspring from older heterozygotes exhibit signs of body wasting at a younger age than those of younger parents, and that offspring of older heterozygous parents weigh less irrespective of genotype. We also demonstrate that tert homozygous mutant fish have a male sex bias, and that clutches from older parents also have a male sex bias in the heterozygous and wild-type populations. Telomere length analysis reveals that the telomeres of younger heterozygous parents are shorter than those of older heterozygous parents. Conclusions: These data indicate that the phenotypes observed in offspring from older parents cannot be explained by telomere length. Instead we propose that Tert functions outside of telomere length maintenance in an age-dependent manner to influence the adult phenotypes of the next generation

The age of heterozygous telomerase mutant parents influences the adult phenotype of their offspring irrespective of genotype in zebrafish [version 2; referees: 2 approved]

Background: Mutations in proteins involved in telomere maintenance lead to a range of human diseases, including dyskeratosis congenita, idiopathic pulmonary fibrosis and cancer. Telomerase functions to add telomeric repeats back onto the ends of chromosomes, however non-canonical roles of components of telomerase have recently been suggested. Methods: Here we use a zebrafish telomerase mutant which harbours a nonsense mutation in tert to investigate the adult phenotypes of fish derived from heterozygous parents of different ages. Furthermore we use whole genome sequencing data to estimate average telomere lengths. Results: We show that homozygous offspring from older heterozygotes exhibit signs of body wasting at a younger age than those of younger parents, and that offspring of older heterozygous parents weigh less irrespective of genotype. We also demonstrate that tert homozygous mutant fish have a male sex bias, and that clutches from older parents also have a male sex bias in the heterozygous and wild-type populations. Telomere length analysis reveals that the telomeres of younger heterozygous parents are shorter than those of older heterozygous parents. Conclusions: These data indicate that the phenotypes observed in offspring from older parents cannot be explained by telomere length. Instead we propose that Tert functions outside of telomere length maintenance in an age-dependent manner to influence the adult phenotypes of the next generation

Raw data for sex ratios of offspring from different aged tert heterozygous parents.

Raw data for sex ratios of offspring from different aged tert<i>^sa6541/+</i> heterozygous parents.<br>Numbers of male and female fish of each genotype are given for 7 lines. Parental age at time of conception is shown.<br><br

Raw data for sperm collection.

Sperm extraction was attempted on tert<i>^{sa6541/sa6541}</i> males and their wild-type siblings from four lines. The number of males tested and the number that produced sperm are shown.<br><br

Raw data for weights of adult fish.

Weights of 8 month old homozygous, heterozygous and wild-type tert<i>^sa6541</i> adult males from parents aged 4 or 16 months at time of conception and which were raised at the same time in the nursery are provided. Weights of 6 month old wild-type fish raised at the same time from parents aged 5 or 30 months at time of conception are shown.<br><br

Raw data of sex ratios

Sex ratio (as percentage Male) of zebrafish lines compared to the age in days of the mother and father

Raw data of Fertilisation Rates

Data on fertilisation and phenotype rates of clutches laid by wild-type and heterozygous tert<i>^sa6541</i> intercrosses