The intracellular C5 system is critical to DAMP sensing and cellular responses in human monocytes

Abstract

Abstract Cholesterol crystal (CC)-induced inflammasome activation is a key contributor to atherosclerosis. Phagocytosis of CC causes lysosomal damage, assembly of the NLRP3 inflammasome, activation of caspase-1 and release of mature IL-1β. We have demonstrated that complement activation by CC in serum leads to C5a-C5aR1 driven CR3 up-regulation, increased uptake and IL1B gene expression in monocytes. Intracellular complement activation now emerges as critical regulator of cellular responses for induction of Th1 cells. In this study we investigated if CC-induced NLRP3 activation and IL-1β release in monocytes involves intracellular C5 activation. We observed that resting monocytes contained stores of C5 and C5a that increased upon CC uptake. Reduction of intracellular C5 by siRNA treatment abrogated IL-1β secretion to the same extent as NLRP3 inhibition. We confirmed the importance of the intracellular C5a-C5aR1 axis using monocytes from a serum C5-deficient patient, which produced intracellular C5 and detectable C5a and mounted equally strong IL-1β response when compared to healthy donor monocytes. Monocytes expressed also active Factors B and D and contained C3 and C3 activation fragments suggesting that an intracellular C5 convertase could mediate intracellular C5 activation. This notion is supported by the finding that knockdown of Factor B and usage of a cell-permeable convertase inhibitor both reduced CC-induced IL-1β. These results indicate that intracellular C3/C5 convertase-driven C5 activation is required for DAMP sensing, such as for CC, and suggest a critical role of intracellular complement in sterile inflammation and related disease states – and, importantly, that this novel axis can be pharmacologically targeted.