Cytokine profiling of primary human macrophages exposed to endotoxin-free graphene oxide: Size-independent NLRP3 inflammasome activation

Graphene-based materials including graphene oxide (GO) are envisioned for a variety of biomedical applications. However, there are conflicting results concerning the biocompatibility of these materials. Here we asked whether GO with small (GO-S) or large (GO-L) lateral dimensions triggered cytotoxicity and/or cytokine responses in primary human monocyte-derived macrophages. We found that GO sheets produced under sterile conditions by a modified Hummers’ method were taken up by macrophages without signs of cytotoxicity. Then, multiplex arrays were used for profiling of pro- and anti-inflammatory responses. Notably, GO suppressed the lipopolysaccharide (LPS)-triggered induction of several chemokines and cytokines, including the anti-inflammatory cytokine, IL-10. No production of pro-inflammatory TNF-α was observed. However, GO elicited caspase-dependent IL-1β expression, a hallmark of inflammasome activation, in LPS-primed macrophages. Furthermore, GO-triggered IL-1β production required NADPH oxidase-generated reactive oxygen species and cellular uptake of GO and was accompanied by cathepsin B release and potassium efflux. Using THP-1 knockdown cells, a role for the inflammasome sensor, NLRP3, the adaptor protein, ASC, and caspase-1 for GO-induced IL-1β secretion was demonstrated. Finally, our studies showed that inflammasome activation was independent of the lateral dimensions of the GO sheets. These studies have provided novel insights regarding the immunomodulatory properties of endotoxin-free GO.