Reduction of NLRP3 inflammasome-mediated inflammation in a novel inducible NEK7 knockout mouse model

Abstract ID 90520 Poster Board 403 NEK7, which belongs to the NIMA-related kinase family, has been reported to play a key role in the formation of the NLRP3 inflammasome by directly interacting with the leucine-rich repeat (LRR) domain of NLRP3 and licensing its activation. The formation of the inflammasome complex results in autoproteolytic cleavage of procaspase-1 into its active form, and subsequent caspase-1-mediated cleavage of pro-IL-1β and pro-IL-18 to produce mature cytokines. Once released into the extracellular space, these inflammatory effectors can further propagate inflammation. Global NEK7 knockout (KO) mice are embryonic lethal. We therefore created a novel tamoxifen inducible NEK7 conditional KO mouse model (NEK7 cKO) to evaluate the effects of NEK7 knockdown on NLRP3 inflammasome activation in vivo. Mice were injected with tamoxifen (2mg/day, for 5 days IP) and bone marrow cells were isolated from Nek7 cKOs after 2 wk, differentiated into macrophages (BMDMs), and stimulated with LPS and ATP. In addition, Nek7 cKOs were injected with either LPS/ATP (IP) or Monosodium Urate (MSU, IP) crystals to determine the effects of NEK7 reduction in vivo in these acute peritonitis models. Lastly, Nek7 cKOs were evaluated in an MSU-induced gouty arthritis model. NEK7 gene expression was significantly downregulated in heart, liver, kidney, and spleen of NEK7 cKOs. Protein levels were also significantly reduced in these tissues. In BMDMs, NEK7 protein levels were reduced by >90%. After LPS priming followed by ATP stimulation, significant inhibition of IL-1β secretion was observed in BMDMs from Nek7 cKOs compared to wildtype (WT) controls. Furthermore, the N-terminal Gasdermin D fragment (a cleavage product of caspase-1 downstream of NLRP3 activation) was not detected after LPS/ATP treatment. In addition, we observed a significant decrease in phosphorylation of pSTAT3 (705) and pSTAT1 confirming the attenuation of interferon-gamma and IL-6 signaling pathways after NEK7 reduction. Injection of LPS and ATP caused a significant increase in plasma IL-1β and IL-18 levels in WT mice. These levels were significantly reduced by ∼70% and ∼755%, respectively, in NEK7 cKOs. A mouse cytokine array demonstrated a significant reduction in at least 29 proinflammatory cytokines in plasma from NEK7 cKOs after LPS/ATP. Additionally, we investigated the effects of reducing NEK7 levels in spleen as it contains immune precursor cells. We observed a decrease in expression of inflammatory genes, including IL 1β, MCP 1, IL 10, IL 1 α, IL 6 TNF α, and IFN-gamma, in this tissue after LPS/ATP. Injection of MSU caused a significant increase in proinflammatory markers MPO and IL-1β in peritoneal lavage fluid in WT control mice. These levels were significantly reduced in NEK7 cKOs. In the MSU-induced gouty arthritis model, NEK7 cKOs exhibited a lower paw diameter (53%), reduced paw volume (51%), and reduced clinical score (54%) compared to WT controls. NLRP3 inflammasome activation was attenuated after acute knockdown of NEK7 in vivo and confirms that NEK7 is a key mediator of NLRP3 inflammasome activation in mice. This novel Nek7 cKO mouse strain will allow for further elucidation of NEK7-dependent effects on inflammation in vivo. The authors acknowledge support from the MRL Postdoctoral Research Program