1146 Nuclear translocation of PKCα is important in neutrophil NETosis and UVB induced-skin inflammation

Abstract

Neutrophils are the most abundant leukocytes and the first to be recruited to the site of photodamage after ultraviolet B (UVB) exposure. However, little is known about the role of neutrophils in UVB-induced skin inflammation. Neutrophils release neutrophil extracellular traps (NETs) during NETosis, which are important in autoimmune inflammation, including lupus. Studies have shown the importance of actin cytoskeleton regulator Rho kinase (ROCK) in PMA-induced neutrophil NETosis, and we have found the NETotic neutrophils in UVB-induced skin inflammation. However it not clear how ROCK is involved in neutrophil NETosis and UVB-induced skin inflammation. To explore the effects of ROCK, we studied the effects of ROCK inhibition on neutrophil NETosis in vitro and on neutrophil NETosis in the UVB-induced skin inflammation in mice. Following PMA stimulation, cytosolic PKCα is gradually translocated to the nuclear membrane. Particularly the phosphorylated PKCα (phosphor-PKCα at ser657) is accumulated at the site of nuclear membrane rupture in the NETotic neutrophils in our confocal analysis. Inhibition of actin, myosin, MLCK (myosin light chain kinase) of actomyosin networks with cytochalasin D, Blebbistatin, or ML7 can attenuate both nuclear translocation of PKCα and neutrophil NETosis. Similarly, dual ROCK1/2 inhibitor HA1077 attenuated nuclear translocation of PKCα and neutrophil NETosis. Most importantly, application of HA1077 intraperitoneally (i.p.) can also attenuate neutrophil NETosis and skin inflammation in UVB-induced skin inflammation. Our preliminary studies therefore elucidates a novel mechanism that ROCK-mediated nuclear translocation and phosphorylation of PKCα regulates neutrophil NET formation, and demonstrates the protective role of ROCK inhibition in neutrophil NETosis in vitro and in vivo. These findings may provide insights into a novel therapeutic target for treatment of UVB-induced skin inflammation.