Charged Vesicles Potently Induce NLRP3 Inflammasome Activation

Extensive muscle inflammation is frequently observed in muscular dystrophy patients, particularly in those with dysferlin deficiency. Dysferlin has been shown to play multiple functions in skeletal muscle including the plasma membrane repair. However, the mechanisms underlying the extensive muscle inflammation in dysferlin-deficient muscular dystrophy are largely unknown. Our previous study revealed that genetic ablation of the complement system ameliorates the pathology in a mouse model of dysferlinopathy, indicating that the innate immune activation may be involved in the pathogenesis. However, it is unclear how the innate immune system contributes to the disease pathology in dysferlinopathy. Recently, we found that vesicles composed of charged lipids can potently activate the NLRP3 inflammasome, resulting in the maturation and release of pro-inflammatory cytokine interleukin-1β. Furthermore, mitochondrial reactive oxygen species-dependent calcium influx via the TRPM2 channel plays an essential role in activation of the NLRP3 inflammasome. Interestingly, NLRP3 inflammasome pathway has been shown to be up regulated in dysferlin-deficient muscle. To further investigate the regulatory signaling pathways involved in NLRP3 inflammasome activation, we established a cell screening system using THP-1 cells. NLRP3 inflammasome activation induces pyroptosis, a form of programmed cell death associated with antimicrobial responses during inflammation. LPS was used to stimulate PMA-differentiated THP1 cells, Nigericin was used to induce NLRP3 activation and over 80% cell death. To validate the system, we created NLRP3-deficient THP-1 cells using CRISPR technology. The NLPR3-deficient THP-1 cells showed significantly decreased cell death when compared to control cells. We will use this system to identify novel regulators of NLRP3 inflammasome.