Genome-wide CRISPR/Cas9 knockout screen for novel regulators of mechanical stress on NLRP3 inflammasome activation

Immune cells sense the microenvironment to fine-tune their inflammatory responses. Patients with cryopyrin associated periodic syndrome (CAPS), caused by mutations in the NLRP3 gene, present auto-inflammation and its manifestation is largely dependent on environmental cues. However, the underlying mechanisms are poorly understood. Here, we uncover that KCNN4, a calcium-activated potassium channel, links PIEZO-mediated mechanotransduction to NLRP3 inflammasome activation. Yoda1, a PIEZO1 agonist, lowers the threshold for NLRP3 inflammasome activation. PIEZO-mediated sensing of stiffness and shear stress increases NLRP3-dependent inflammation. Myeloid-specific deletion of PIEZO1/2 protects mice from gouty arthritis. Activation of PIEZO1 triggers calcium influx, which activates KCNN4 to evoke potassium efflux promoting NLRP3 inflammasome activation. Activation of PIEZO signaling is sufficient to activate the inflammasome in cells expressing CAPS-causing NLRP3 mutants via KCNN4. Finally, pharmacologic inhibition of KCNN4 alleviates auto-inflammation in CAPS patient cells and in CAPS-mimicking mice. Thus, PIEZO-dependent mechanical inputs augment inflammation in NLRP3-dependent diseases including CAPS. This is a pooled genome-wide CRISPR/Cas9 knockout screen for novel regulators downstream mechanical stress to promote NLRP3 inflammasome activation. THP-1 cells stably expressing Cas9 protein were infected with the pooled Brunello lentivirus library. After puromycin selection for 3 days, cells were further expanded. To ensure a representation of minimal 500 cells per sgRNA, at least 4.0×107 cells were used in each group and primed with LPS in suspension for 3 hours and treated with vehicle (control group) or R837 plus Yoda1 (treated group). When pyroptotic cell death reached ~85% in the treated group, R837 and Yoda1 were washed out and the survivals were expanded. When cell number reached 4.0 × 107 cells, these cells were repeatedly treated with R837 plus Yoda1 to reach reached ~85% of pyroptotic cell death. R837 and Yoda1 were washed out and the survivals were further expanded to reach 4.0 × 107 cells. Genomic DNA from both control- and treated groups was isolated using the phenol/chloroform extraction method. Fragments containing sgRNA were amplified from genomic DNA by PCR and subjected to deep sequencing. We sequenced control-treated cells and survivals of R837 plus Yoda1-treated cells for each of the two replicates and performed MAGeCK analysis to identify the top hits.