Biological effects of cobalt-chromium nanoparticles and ions on dural fibroblasts and dural epithelial cells.

The introduction of metal-on-metal total disc replacements motivated studies to evaluate the effects of cobalt-chromium (CoCr) nanoparticles on cells of the dura mater. Porcine fibroblasts and epithelial cells isolated from the dura mater were cultured with clinically-relevant CoCr nanoparticles and the ions, generated by the particles over 24 h, at doses up to 121 μm(3)per cell. Cell viability and production of proinflammatory cytokines was assessed over 4 days. The capacity of the particles to induce oxidative stress in the cells was evaluated at 24 h. The CoCr particles and their ions significantly reduced the viability of the dural epithelial cells in a dose-dependent manner but not the fibroblasts. Both cell types secreted IL-8 in response to particle exposure at doses of 60.5 μm(3) (epithelial cells) and 121 μm(3) (fibroblasts, epithelial cells) per cell. No significant release of IL-6 was observed in both cell types at any dose. Reactive oxygen species were induced in both cell types at 50 μm(3) per cell after 24 h exposure. The data suggested novel differences in the resistance of the dural epithelial cells and fibroblasts to CoCr nanoparticle/ion toxicity and demonstrated the inflammatory potential of the particles. The data contributes to a greater understanding of the potential biological consequences of the use of metal-on-metal total disc prostheses

Intracellular Host Defense in Microbial Infections

Bacterial and viral infections represent major health and economic burdens on the global population. In this study, we unravel the molecular mechanisms governing bacterial sepsis and picornavirus infections. Specifically, we delineate the structural determinants of the sepsis endotoxin LPS and provide evidence that the LPS core sugars are necessary for its cytosolic access and engagement with the noncanonical inflammasome. Mice injected with E. coli lacking LPS inner core exhibited diminished activation of inflammasome-dependent cytokines while normally activating LPS-induced TLR4 signaling. We also establish galectin-8, a member of the galectin family of proteins, as a critical component in the host defense against picornavirus infection. Mice lacking galectin-8 succumbed faster to encephalomyocarditis virus (EMCV) infection while wild-type mice were more resistant to the virus. Mechanistically, cells lacking galectin-8 displayed reduced autophagic flux and increased viral burdens during picornavirus infection, implicating galectin-8 in the autophagy-mediated degradation of EMCV. Our findings provide mechanistic insights into the molecular mechanisms underlying bacterial and viral infections in an effort to contribute to the development of effective therapeutic strategies against microbial pathogens

Trace Levels of Staphylococcal Enterotoxin Bioactivity Are Concealed in a Mucosal Niche during Pulmonary Inflammation.

Pathogen and cellular by-products released during infection or trauma are critical for initiating mucosal inflammation. The localization of these factors, their bioactivity and natural countermeasures remain unclear. This concept was studied in mice undergoing pulmonary inflammation after Staphylococcal enterotoxin A (SEA) inhalation. Highly purified bronchoalveolar lavage fluid (BALF) fractions obtained by sequential chromatography were screened for bioactivity and subjected to mass spectrometry. The Inflammatory and inhibitory potentials of the identified proteins were measured using T cells assays. A potent pro-inflammatory factor was detected in BALF, and we hypothesized SEA could be recovered with its biological activity. Highly purified BALF fractions with bioactivity were subjected to mass spectrometry. SEA was the only identified protein with known inflammatory potential, and unexpectedly, it co-purified with immunosuppressive proteins. Among them was lactoferrin, which inhibited SEA and anti-CD3/-CD28 stimulation by promoting T cell death and reducing TNF synthesis. Higher doses of lactoferrin were required to inhibit effector compared to resting T cells. Inhibition relied on the continual presence of lactoferrin rather than a programming event. The data show a fraction of bioactive SEA resided in a mucosal niche within BALF even after the initiation of inflammation. These results may have clinical value in human diagnostic since traces levels of SEA can be detected using a sensitive bioassay, and may help pinpoint potential mediators of lung inflammation when molecular approaches fail

Lactoferrin inhibits IL-2 secretion.

<p>(<b>A</b>) Mouse splenocytes stimulated with SEA (10 ng/ml) (top panel) and soluble anti-CD3/-CD28 (each 1 μg/ml) (bottom panel) were co-incubated with increasing doses of BSA (negative control), lactoferrin and transferrin. Culture supernatants were assayed for IL-2 after 18 h and the bar graphs show IL-2 secretion. Data are representative of the average of 3 independent experiments with n = 3. The error bars indicate the standard error of the mean between 3 biological replicates. Statistical significance between treatments was evaluated by two-tailed Student’s <i>t</i> tests. * p<0.05, ** p<0.01, *** p<0.001. (<b>B</b>) Mouse splenocytes from either unimmunized mice (<i>Naïve</i>) or 3 day post immunization with 0.5 μg SEA and 10 μg LPS (<i>Effector</i>) were incubated with lactoferrin (white bars) or BSA (grey bars) (5 mg/ml) at 37°C with PMA (50 ng/ml) + Ionomycin (1 μg/ml). Culture supernatants were assayed for IL-2 after 18 h and the data are representative of the average of 3 independent experiments with n = 3. The error bars indicate the standard error of the mean between 3 biological replicates. Statistical significance between lactoferrin and BSA was evaluated by two-tailed Student’s <i>t</i> tests. (<b>C</b>) Cells were incubated with lactoferrin or BSA (5 mg/ml) for 3 h at 37°C, washed 4 times with tissue culture medium, and then stimulated with SEA (10 ng/ml) or PMA (50 ng/ml) + Ionomycin (1 μg/ml). Culture supernatants were assayed for IL-2 and IFN-γ after 18 h. (<b>D</b>) The four successive washes described above in <b>C</b> were analyzed by SDS-PAGE and immunoblotted with anti-lactoferrin as described in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141548#pone.0141548.g005" target="_blank">Fig 5a</a>. Representative of 1 out 3 experiments is shown. (<b>E</b>) IL-2 and IFN-γ secretion from the culture supernatants (as described above in <b>C</b>) are shown as bar graphs. Data representative of the average of 3 independent experiments are presented. The error bars indicate the standard error of the mean between triplicates.</p

Lactoferrin significantly inhibits early intracellular TNF synthesis in naïve over that made by effector T cells.

<p>(<b>A</b>) Splenocytes from naïve mice were incubated with lactoferrin or BSA (5 mg/ml), in the presence of BFA and stimulated with or without PMA + Ionomycin for 4 h at 37°C. Cells were stained for CD4 and TCR Vβ3, fixed, permeabilized and stained for intracellular TNF. Representative dot plots show the gating strategy. The percentage of SEA-specific T cells: CD4⁺Vβ3⁺, is indicated in top right quadrant (left panels) and the percentage of those cells producing TNF is indicated as histograms in the right panels. (<b>B</b>) The mean percentage +/- standard error of the mean of CD4⁺Vβ3⁺ cells producing TNF from 3 independent biological replicates was analyzed as described in <b>A</b> is shown. Hence, naïve and effector splenocytes were obtained as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141548#pone.0141548.g006" target="_blank">Fig 6</a>. One set of cells were directly incubated with lactoferrin or BSA in the presence of BFA and PMA + Ionomycin (<i>No wash</i>, left panels) and stained as described in <b>A</b>. Another set of cells (<i>Washed</i>, right panels) was first incubated for 3 h with lactoferrin or BSA, washed 4 times as described in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141548#pone.0141548.g006" target="_blank">Fig 6c</a> and then stimulated with or without PMA + Ionomycin for 4 h at 37°C. Data are combined from 3 experiments with n = 3 and displayed as mean +/- standard error of the mean. Statistical significance between BSA and lactoferrin was evaluated by two-tailed Student’s <i>t</i> tests. * p<0.05, ** p<0.01, *** p<0.001.</p

SEA induces classical pro-inflammatory factors in BALF.

<p>BALF obtained 2–88 h after i.n. SEA inhalation was tested for the presence of IL-6, TNF and SAA by ELISA. Plots show cytokine secretion, each symbol represents one mouse. BALF were collected from 10 experiments. The error bars indicate the standard error of the mean between the mice in one group. Statistical significance between the groups was evaluated by two-tailed Student’s <i>t</i> tests.</p

Overnight incubation with lactoferrin affects cell viability.

<p>Cell were treated with proteins at 5 mg/ml, except Exp. #4* (10 mg/ml), stimulated for 18 h as described in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141548#pone.0141548.g006" target="_blank">Fig 6a and 6b</a>. Viability of cells was then assayed by flow cytometry using Mitoflow as a marker of mitochondrial integrity, the percentage of cell alive is reported in the table.</p><p>Overnight incubation with lactoferrin affects cell viability.</p

3 h incubation with lactoferrin does not affect cell viability.

<p>Cell were treated with proteins (5 mg/ml), washed as described as in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141548#pone.0141548.g006" target="_blank">Fig 6c</a>, and stimulated for 18 h as described in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141548#pone.0141548.g006" target="_blank">Fig 6c and 6e</a>. Viability of cells was then assessed by flow cytometry using Mitoflow as a measurement of mitochondrial integrity, the percentage of cells alive is reported in the table.</p><p>3 h incubation with lactoferrin does not affect cell viability.</p

Lactoferrin co-migrates with SEA.

<p>(<b>A</b>) BALF-SEA was resolved by 4–15% gradient SDS-PAGE under reducing and denaturing conditions, transferred to nitrocellulose membrane and probed with anti-lactoferrin and anti-SEA antibodies as described in the Materials and Methods. Purified Lactoferrin and SEA (30 and 10 ng) were loaded in parallel to serve as positive controls. Specificity of the antibody was assessed using isotype control. The data are representative of 2 (lactoferrin) and 3 (SEA) independent experiments. (<b>B</b>) 16 h BALF-SEA was directly fractionated by cation exchange chromatography. Fractions were tested in a bioassay and immunoblotted for lactoferrin (inset). Chromatogram of protein absorption at 280nM (top panel) and bar graphs of IL-2 secretion (bottom panel) are shown. Representative of 1 out 3 experiments is shown. (<b>C</b>) BALF from naïve mice, and BALF from mice having received a time course of i.n. SEA and BSS were immunoblotted with anti-lactoferrin antibodies and 3 experiments are shown.</p