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Front Immunol
2019 Jan 01;10:2261. doi: 10.3389/fimmu.2019.02261.
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Sea Urchin Extracellular Proteins Design a Complex Protein Corona on Titanium Dioxide Nanoparticle Surface Influencing Immune Cell Behavior.
Alijagic A
,
Benada O
,
Kofroňová O
,
Cigna D
,
Pinsino A
.
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Extensive exploitation of titanium dioxide nanoparticles (TiO2NPs) augments rapid release into the marine environment. When in contact with the body fluids of marine invertebrates, TiO2NPs undergo a transformation and adhere various organic molecules that shape a complex protein corona prior to contacting cells and tissues. To elucidate the potential extracellular signals that may be involved in the particle recognition by immune cells of the sea urchin Paracentrotus lividus, we investigated the behavior of TiO2NPs in contact with extracellular proteins in vitro. Our findings indicate that TiO2NPs are able to interact with sea urchin proteins in both cell-free and cell-conditioned media. The two-dimensional proteome analysis of the protein corona bound to TiO2NP revealed that negatively charged proteins bound preferentially to the particles. The main constituents shaping the sea urchin cell-conditioned TiO2NP protein corona were proteins involved in cellular adhesion (Pl-toposome, Pl-galectin-8, Pl-nectin) and cytoskeletal organization (actin and tubulin). Immune cells (phagocytes) aggregated TiO2NPs on the outer cell surface and within well-organized vesicles without eliciting harmful effects on the biological activities of the cells. Cells showed an active metabolism, no oxidative stress or caspase activation. These results provide a new level of understanding of the extracellular proteins involved in the immune-TiO2NP recognition and interaction in vitro, confirming that primary immune cell cultures from P. lividus can be an optional model for swift and efficient immune-toxicological investigations.
Figure 1. The interaction between Sea urchin secretome and TiO2NPs, extracellular signaling and protein corona identification. (A) One-dimensional profiling of the formed non-cell-conditioned protein corona at 1, 10, and 100 μg mL−1 TiO2NPs after 24 h of incubation. (B) Two-dimensional profiling of the TiO2NP protein corona after 24 h of incubation in the non-cell-conditioned medium (Cell free CF plus CCM). (C) One-dimensional profiling of the protein corona formed in situ (in vitro) at 3 and 24 h. (D) Representative immunoblotting results show limited influence of the TiO2NPs on sea urchin extracellular signaling and reveal the main constituents shaping the cell-conditioned protein corona (Pl-toposome, Pl-galectin-8, Pl-nectin, actin, tubulin). *, see text.
Figure 2. Scanning and transmission electron microscopy of the in situ (in vitro) interaction between TiO2NPs and sea urchin immune cells. (A,C) Control cells not exposed to TiO2NPs. (B,D) Immune cells exposed to 1 μg mL−1 TiO2NPs for 24 h. These figures illustrate interactions and selective binding of TiO2NPs on the immune cell (phagocyte) surface. Black arrowheads indicate small aggregate. (E,F) Sea urchin phagocyte after exposure to 1 μg mL−1 TiO2NPs for 24 h shows internalized TiO2NPs within a vesicular structure (black arrowhead) that is localized in the proximity with lysosomes (L). N, nucleus, M, mitochondria.
Figure 3. Impacts of TiO2NPs on the viability, toxicity, reactive oxygen species production and caspase activity on sea urchin immune cells. (A) Real-time viability assay over 72 h shows that only the highest dose of TiO2NPs (100 μg/mL) induces decrease in cell viability. (B) Cell toxicity shows an increase only in response to the highest TiO2NPs dose. (C) A Luciferase-based ROS assay does not indicate any significant increase in the ROS levels in response to TiO2NPs. Positive control (+ control): Hydrogen peroxide activates a strong increase in ROS production (24 h, 4 mM). (D) Caspase 3/7 activity shows that TiO2NPs (at all doses) does not induce an increase in the activity of these apoptotic enzymes. The positive controls (+ control) are cells exposed for 24 h to Iron oxide nanoparticles (100 μg/mL). Assays involved five biological replicates except for Caspase 3/7 assay, which had three replicates. Data are reported as the mean ± SE; stars (*) indicate significant differences among groups (*p < 0.05; ****p < 0.0001). RLU, Relative Luminescence Units; RFU, Relative Fluorescence Units. Negative control (- control) was medium plus particles without cells.
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