Cell Swelling and the NLRP3 Inflammasome

Multiple chemically and biologically diverse stimuli activate the NLRP3 inflammasome, but the precise mechanism for activation remains to be determined (Davis, Wen, & Ting, 2011). Cellular perturbation and homeostatic destabilisation also activate NLRP3. Consistent with this, in the September 2012 issue of Immunity, Compan et al., 2012, presented data suggesting that the NLRP3 inflammasome is activated by cell swelling (Compan et al., 2012). Cell-swelling followed by a restorative reduction in volume resulted in caspase-1 activation in murine bone-marrow-derived macrophages and macrophages from the teleost fish Sparus aurata (Gilthead Seabream) (Figure 2 in (Compan et al., 2012)). Murine cells lacking NLRP3 (or ASC, or caspase-1) did not process interleukin-1β in response to cell swelling leading to the conclusion that the NLRP3 inflammasome mediates the cellular response to cell swelling and that this is evolutionarily conserved from fish to mammals.

Various NLR-specific expansions have been identified in fish. Generally they cannot be identified as full orthologues of specific mammalian NLRs. However, a number show greatest similarity to the NACHT domain of NLRC3 (Huang et al., 2008; Laing, Purcell, Winton, & Hansen, 2008). To date there are, to our knowledge, no published reports of direct NLRP3 orthologues in fish.

We have consequently performed a detailed search of the currently available fish genome assemblies in ENSEMBL for evidence of NLRP3 orthologues. These encompassed: Takigugu rubripes (Fugu), Xiphophorus maculatus (Platyfish), Gasterosteus aculeatus (Stickleback), Gadus morhua (Cod), Latimeria chalumnae (Coelacanth), Tetraodon nigroviridis (Tetraodon), Oreochromis niloticus (Tilapia) and Danio Rerio (Zebrafish).

BLASTP searches using the amino acid sequence of human NLRP3 (NCBI Accession: NP_004886.3) identified a gene on Zebrafish chromosome 17 (ENSEMBL gene ID ENSDARG00000090946) which upon reciprocal BLASTP analysis against the non-redundant human protein database returned NLRP3 as the most significant hit. However, the domain organisation of the zebrafish protein differs to that of mammalian NLRP3; consisting of an NTPase domain, leucine-rich repeats and a C-terminal PRY-SPRY domain. Homology searches failed to identify a specific N-terminal effector domain. Hence it is questionable whether this protein can be described as a true NLRP3 orthologue. Importantly we were unable to identify any other putative NLRP3 orthologues in the other fish genomes using human NLRP3, or indeed the supposed zebrafish NLRP3, as search terms. Instead NLR expansions with greatest similarity to the NLRC3 NTPase were returned as hits. All the fish, except the tetraodon, possessed clear orthologues of ASC.

It is plausible that NLRP3-like functionality is provided by a fish specific NLR-expansion, such as NLR-B, the NACHT of which phylogentically clusters with the mammalian NLRPs (Laing et al., 2008). However, Sparus aurata macrophages do not respond to classical NLRP3 activators including ATP, nigericin, alum crystals, and monosodium urate crystals (Figure 2E in (Compan et al., 2012)) suggesting that this may not be the case. The absence of direct NLRP3 orthologues and the lack of classical NLRP3 activity in the teleost indicate that reconsideration of the evolutionary and mechanistic basis for the observed inflammatory response to cell swelling is needed.