Caspase isoforms in inflammasome activation
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Abstract
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.