Lipopolysaccharide Adsorbed to the Bio-Corona of TiO2 Nanoparticles Powerfully Activates Selected Pro-inflammatory Transduction Pathways.

Massimiliano G Bianchi,Anna L Costa,Manfredi Allegri,Enrico Bergamaschi,Simona Ortelli,Magda Blosi,Martina Chiu,Ovidio Bussolati

doi:10.3389/fimmu.2017.00866

Abstract

It is known that the adsorption of bioactive molecules provides engineered nanoparticles (NPs) with novel biological activities. However, the biological effects of the adsorbed molecules may also be modified by the interaction with NP. Bacterial lipopolysaccharide (LPS), a powerful pro-inflammatory compound, is a common environmental contaminant and is present in several body compartments such as the gut. We recently observed that the co-incubation of LPS with TiO2 NPs markedly potentiates its pro-inflammatory effects on murine macrophages, suggesting that, when included in a NP bio-corona, LPS activity is enhanced. To distinguish the effects of adsorbed LPS from those of the free endotoxin, a pellet fraction, denominated P25/LPS, was isolated by centrifugation from a mixture of P25 TiO2 NP (128 µg/ml) and LPS (10 ng/ml) in the presence of fetal bovine serum. Western blot analysis of the pellet eluate indicated that the P25/LPS fraction contained, besides proteins, also LPS, pointing to the presence of LPS-doped NP. The effects of adsorbed or free LPS were then compared in Raw264.7 murine macrophages. RT-PCR was used to evaluate the induction of cytokine genes, whereas active, phosphorylated isoforms of proteins involved in signaling pathways were assessed with western blot. At a nominal LPS concentration of 40 pg/ml, P25/LPS induced the expression of both NF-κB and IRF3-dependent cytokines at levels comparable with those observed with free LPS (10 ng/ml), although with different time courses. Moreover, compared to free LPS, P25/LPS caused a more sustained phosphorylation of p38 MAPK and a more prolonged induction of STAT1-dependent genes. Cytochalasin B partially inhibited the induction of Tnfa by P25/LPS, but not by free LPS, and suppressed the induction of IRF3-dependent genes by either P25/LPS or free LPS. These data suggest that, when included in the bio-corona of TiO2 NP, LPS exhibits enhanced and time-shifted pro-inflammatory effects. Thus, in assessing the hazard of NP in real life, the enhanced effects of adsorbed bioactive molecules should be taken into account.

Highlights

When introduced in organic fluids, engineered nanoparticles (NP), due to their high ratio surface/ volume, adsorb proteins, lipids, and other bioactive molecules present in the medium, forming a corona that is of fundamental relevance for the interactions with cells and tissues [1,2,3]
LPS adsorption to NP has been considered one of the possible factors that interfere with cell-based immunological tests employing NP [19], because either contamination may be overlooked or adsorption may modulate the effects of LPS on innate immune cells [19]. Investigating this latter possibility, we have recently demonstrated that the co-exposure of murine macrophages to TiO2 NP and LPS in protein-rich medium powerfully synergizes the pro-inflammatory effects of the endotoxin [20]
On the basis of those results, we proposed that TiO2 NPs adsorb LPS and enhance macrophage activation by the endotoxin via a toll-like receptor 4 (TLR4)-dependent mechanism that is mainly triggered from an intracellular site [20]

Summary

Introduction

When introduced in organic fluids, engineered nanoparticles (NP), due to their high ratio surface/ volume, adsorb proteins, lipids, and other bioactive molecules present in the medium, forming a corona that is of fundamental relevance for the interactions with cells and tissues [1,2,3]. LPS-Doped Nanoparticles Enhance Endotoxin Effects to change in media or organic fluids of different composition [4]. The interaction with the nanomaterial may change the conformation and/or the bioavailability of the adsorbed molecules [5], leading to enhancement or inhibition of their effects. The formation and biological effects of protein corona have been extensively studied, other, non-protein bioactive molecules are expected to be adsorbed by nanomaterials. Signal transduction starts at the plasma membrane and later involves an endosomal compartment after the internalization of the complex LPS–TLR4 [10,11,12]

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Results

Conclusion