Volume-activated chloride channels contribute to lipopolysaccharide plus nigericin-induced pyroptosis in bone...

The representative morphological features of pyroptosis are excessive cell swelling and subsequent membrane rupture. However, the mechanism underlying the cell's inherent inability to regulate volume during the progression of pyroptosis is poorly understood. In the current study, we found that both volume-activated chloride currents (I_{cl, vol}) and the regulatory volume decrease (RVD) were markedly decreased in bone marrow-derived macrophages (BMDMs) undergoing pyroptosis induced by lipopolysaccharides (LPS) and nigericin. The inhibition of I_{Cl, vol} and RVD by the chloride channel blockers, tamoxifen or DCPIB, led to the emergence of pyroptosis-like phenotypes such as activated-caspase-1, pyroptotic-body-like bubbles, and a fried-egg-like appearance. Moreover, the expression of the volume-activated chloride channel (VRAC) constituent protein Leucine-Rich Repeat-Containing 8A (LRRC8A) was significantly down-regulated in pyroptotic BMDMs treated with LPS and nigericin. The silencing of LRRC8A expression by small interfering RNA (si)-LRRC8A transfection not only reduced I_{Cl, vol} and RVD, but also caused BMDMs to show pyroptosis-like manifestations such as activated-caspase-1, membrane bubbles, and have a fried-egg-like appearance. These results reveal a new mechanism for the loss of volume regulation in the process of pyroptotic cell swelling and strongly suggest that a functional deficiency of VRAC/LRRC8A plays a key role in this disorder.