Black Carbon Induces Complement Activation via NLRP3 Inflammasome in Human Corneal Epithelial Cells

Abstract

ABSTRACT Purpose To investigate the effect of black carbon (BC) particles on complement activation in human corneal epithelial cells (HCECs) and determine whether this effect can be attenuated by inhibiting the NLPR3 inflammasome pathway. Materials and Methods HCECs were treated with fresh BC (FBC) or ozone-oxidized BC (OBC) particles at a concentration of 200 μg/ml for 72 hours. Complement activation was observed by detecting C5b-9 protein level in cell culture supernatant using ELISA. HCECs were transfected with duplexes of siRNA targeting NLRP3 (NLRP3-siRNA) at 0.1 pmol/µl for 24 hours to inhibit the NLPR3 inflammasome pathway. RT-qPCR was performed to examine the efficacy of NLRP3-siRNA for inhibition; a random siRNA duplex was used for control siRNA. Results Both FBC exposure and OBC exposure for 72 hours significantly increased the C5b-9 protein level compared to negative control cells (all P < .05). However, the difference in C5b-9 level after FBC exposure and OBC exposure was not statistically significant (P > .05). NLRP3-siRNA transfection reduced C5b-9 protein levels in FBC-treated and OBC-treated HCECs compared to control (lowered by 27% in the FBC-treated group and by 23% in the OBC-treated group, all P < .05). Conclusions BC particles, including FBC and OBC, triggered complement activation, increasing the protein level of C5b-9 in cultured HCECs. siRNA targeting NLRP3 to inhibit NLRP3 generation reduced C5b-9 protein level in HCECs treated with FBC or OBC particles, indicating that BC induces complement activation potentially through the NLRP3 inflammasome in HCECs.