Titanium dioxide nanoparticles enhance macrophage activation by LPS through a TLR4-dependent intracellular pathway

Although causing moderate cytotoxicity and inflammatory effects, TiO2 nanoparticles ( NPs) are considered relatively safe materials. However, it is known that TiO2 NPs bind bioactive environmental contaminants, such as bacterial lipopolysaccharide ( LPS, endotoxin), and it is possible that this interaction leads to increased biological activity. In this report we have investigated the pro- inflammatory responses of Raw264.7 murine macrophages exposed to two preparations of TiO2 NPs, co- administered with LPS. The simultaneous exposure to NPs and LPS produced marked increases in Nos2 mRNA, Nos2 protein and medium nitrite concentration ( an indicator of NO production) well beyond the levels observed with LPS or TiO2 NP alone. TiO2 NPs also synergized LPS effects on Ptgs2 expression and cytokine secretion. The cytoskeletal drug cytochalasin B lowered the amount of NPs internalized by the cells and suppressed the synergy between TiO2 NPs and LPS in NO production and cytokine secretion. Pre- treatment with the TLR4 inhibitors polymyxin B and CLI- 095 eliminated the synergy that was also partially hampered by the inhibition of p38 MAPK, but not of ERK1/ 2. Moreover, p38 phosphorylation was synergistically enhanced by the combined treatment at 6 h of incubation. It is concluded that TiO2 NPs enhance macrophage activation by LPS via a TLR4- dependent mechanism that involves p38 and is mainly triggered from an intracellular site. These findings suggest that the simultaneous exposure to LPS and TiO2 NPs may exacerbate the inflammatory response in vivo.