The longer the worse : a combined proteomic and targeted study of the long-term versus short-term effects of silver nanoparticles on macrophages †

2032 | Environ. Sci.: Nano, 2020, 7, 2032–2046 This journal is © The Roya a Chemistry and Biology of Metals, Univ. Grenoble Alpes, CNRS UMR5249, CEA, IRIG-DIESE-CBM, F-38054 Grenoble, France. E-mail: thierry.rabilloud@cnrs.fr b Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg, CNRS, IPHC UMR 7178, 67000 Strasbourg, France c Université Grenoble-Alpes, CEA, Nanosafety Platform, Medical Biology Laboratory (LBM), 17 rue des Martyrs, F-38054 Grenoble, France Univ. Grenoble-Alpes, CEA, CNRS UMR 5819, INAC-SyMMES, Chimie Interface Biologie pour l'Environnement, la Santé et la Toxicologie (CIBEST), F-38054 Grenoble, France † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c9en01329f ‡ Deceased Nov. 21st, 2018. This paper is dedicated to her memory. Environmental significance


Introduction
Silver nanoparticles are more and more frequently used as biocides in many consumer products and in healthcare products such as wound dressing, or as a surface biocide in catheters and prostheses. 1 The biocidal activity is mainly due to liberation of toxic silver ions, the particles themselves showing weak intrinsic toxicity. 2 While well tolerated, products containing silver nanoparticles are not without effects. For example, a localized argyria has been demonstrated to occur when using silver coated healthcare products. 3,4 It can be seen as a repeated exposure of proximal tissue to silver doses that are higher than the occupational doses, as the latter do not induce any argyria. 5 In parallel with the local argyria, repeated exposure to moderate dose of silver nanoparticles has been shown to alter physiological parameters, while being non lethal. [6][7][8][9][10] In the frame of occupational and foodderived exposures, in vitro long term studies of the effects of silver nanoparticles on cells have concentrated on the barrier cell types, i.e. epithelial cells. [11][12][13][14][15] For all exposure routes, including liberation in the bloodstream, 16 which is a frequent route for healthcare products, resident macrophages represent the common scavenger cell type that internalizes the particular material. In the case of silver nanoparticles, reported effects of a chronic exposure to silver nanoparticles appear to be generally deleterious and often show altered inflammatory responses, 6,9,10 although the internalization of silver nanoparticles by macrophages can help them in killing otherwise resistant bacteria, and thus be helpful. 17 The deleterious effects of silver nanoparticles on macrophages have been previously reported in several papers (e.g. [18][19][20]. However, these studies use a single dose of silver nanoparticles at relatively high concentrations to investigate the cellular responses.
As this exposure scenario is quite different from the in vivo situations, e.g. in case of use of silver nanoparticlescontaining healthcare products, we therefore decided to investigate in more details the responses of macrophage to a repeated exposure to silver nanoparticles, and to compare the results to those obtained with a single high dose, which is the standard practice in nanotoxicology. To this purpose, we used a proteomic approach, which has given interesting insights for the study of cellular responses to acute exposures to several nanoparticles, [21][22][23][24][25][26][27] including silver nanoparticles. [28][29][30]

Material and methods
All experiments were carried out on at least three independent biological replicates. Unless specified otherwise, all chemicals were purchased from Sigma-Aldrich and were at least 99% pure.

Nanoparticles
PVP-coated silver nanoparticles (Ag-NP) were purchased from Sigma, directly as a concentrated suspension (catalog number #758329). The zeta potential, size distribution and agglomeration state of Ag-NP suspensions were characterized by dynamic light scattering on a ZetaSizer nanoZS (Malvern Instrument). TEM images of Ag-NPs were obtained on a JEOL 1200EX TEM operating at 80 kV (Grenoble Institut des Neurosciences, Grenoble, France).

Cell culture
The mouse macrophage cell line RAW264.7 was obtained from the European Cell Culture Collection (Salisbury, UK). The cells were cultured in RPMI 1640 medium + 10% fetal bovine serum (FBS). For routine culture, cells were seeded on non-adherent flasks (e.g. suspension culture flasks from Greiner) at 200 000 cells per ml and harvested 48 hours later, at 100 0000 cells per ml. Cell viability was measured by a dye exclusion assay, either with eosin (1 mg ml −1 ) under the microscope 31 or with propidium iodide (1 μg ml −1 ) in a flow cytometry mode. 32 For determination of the useful dose, cells were seeded at 800 000 cells per ml. They were exposed to nanoparticles on the following day and harvested after a further 24 hours in culture.
For treatment with nanoparticles, cells were seeded at 800 000 cells per ml on classical, adherent cell culture flasks and left for 24 hours at 37°C for cell adhesion. The medium volume was adjusted to keep a constant medium height for all the culture supports used (2 ml for 6-well plates, 5 ml for 25 cm 2 flasks, 15 ml for 75 cm 2 flasks and 35 ml for 175 cm 2 flasks). The cells were grown to confluence for 48 hours. From then on, the culture medium was changed every 48 hours. For the acute exposure, the cells were kept post-confluence for 2 days. They were then treated with silver nanoparticles for 24 hours. After this period of time, the cell culture medium was removed and the cells were collected by scraping in PBS, then rinsed twice in PBS and processed. In the chronic exposure scheme, the treatment started just after confluence was reached. The cells were treated daily with 1 μg ml −1 silver nanoparticles for 20 days, with a culture medium change every two days. The cells were then collected as described above. Thus, four conditions were eventually analyzed: Acute control: cells let at confluence for 8 days Acute treated: cells let at confluence for 7 days, then treated with silver nanoparticles (20 μg ml −1 ) for 1 day Chronic control: cells let at confluence for 20 days Chronic treated: cells treated at confluence for 20 days (1 μg ml −1 per day silver nanoparticles)

Phagocytosis and particle internalization assay
The phagocytic activity was measured using fluorescent latex beads (1 μm diameter, green labelled, catalog number L4655 from Sigma), as described previously. 33 After a 2 h 30 treatment with the fluorescent beads in culture medium at 37°C, the cells were harvested in PBS with propidium iodide (1 μg mL −1 ). Viability and phagocytic activity were measured simultaneously by flow cytometry on a FacsCalibur instrument (Beckton Dickinson). The dead cells (propidium positive) were excluded from the analysis.

Mitochondrial transmembrane potential measurement
The mitochondrial transmembrane potential was assessed by rhodamine 123 uptake, 27 using a low rhodamine concentration (80 nM) to avoid intramitochondrial fluorescence quenching that would result in a poor estimation of the mitochondrial potential. 34

Enzyme assays
The enzymes were assayed according to published procedures (see below).
The cell extracts for enzyme assays were prepared as described previously. 27 The dehydrogenases or dehydrogenases-coupled activities were assayed at 500 nm using the phenazine methosulfate/iodonitrotetrazolium coupled assay. 35 The enzyme assay buffer contained 25 mM Hepes NaOH pH 7.5, 5 mM magnesium acetate, 100 mM potassium nitrate and 1% Triton X-100. It also contained 30 μM phenazine methosulfate, 200 μM iodonitrotetrazolium chloride, 250 μM of the adequate cofactor (NAD or NADP) and 1-5 mM of the organic substrate, which was used to start the reaction. Triose phosphate isomerase was assayed with dihydroxyacetone phosphate and a glyceraldehyde dehydrogenase-coupled assay. 36 Glutathione reductase was assayed through NADPH consumption. 37

Reduced glutathione assay
Classical glutathione assays are difficult to apply to macrophages, as the oxidizing activities present in their phagolysosomes oxidize the cytosolic reduced glutathione when the cells are homogeneized. To alleviate this problem, we used a spectrophotometric assay utilizing the cellular glutathione S-transferases and the chlorodinitrobenzene substrate. 38,39 After their exposure to silver in 6 well plates, cells were finally treated for 30 minutes with 25 μM chlorodinitrobenzene in the culture medium, resulting in the conjugation of glutathione to the substrate and consumption of the free reduced glutathione. 40,41 The culture medium was removed and the cell layer rinsed twice in Hepes NaOH buffer (10 mM pH 7.5) containing 2 mM magnesium acetate and 0.25 M sucrose. The cell layer was then lysed on a rocking table for 15 minutes in 800 μl of Hepes NaOH buffer (10 mM pH 7.5) containing 2 mM magnesium acetate and 1 mg ml −1 tetradecyldimethylammonio propane sulfonate (SB [3][4][5][6][7][8][9][10][11][12][13][14]. The suspension was collected and centrifuged at 15 000g for 15 minutes to pellet particulate material. The concentration of glutathione was determined by reading the absorbance at 340 nm, using an extinction coefficient of 9600 M −1 cm −1 . 38 To normalize the results, the protein concentration of the extracts was determined by a modified dye-binding assay, 42 and the final results were expressed in mM glutathione/mg protein.

NO production and cytokines production
The cells were grown to confluence in a 6 well plate and pretreated with nanoparticles as described above for flasks. For the final 18 hours of culture, half of the wells were treated with 100 ng ml −1 LPS (from Salmonella, purchased from Sigma), and arginine monohydrochloride was added to all the wells (5 mM final concentration) to give a high concentration of substrate for the nitric oxide synthase. After 18 hours of incubation, the cell culture medium was recovered, centrifuged at 10 000g for 10 minutes to remove cells and debris, and the nitrite concentration in the supernatants was read at 540 nm after addition of an equal volume of Griess reagent and incubation at room temperature for 30 minutes.
For cytokine production, a commercial kit (BD cytometric bead array, catalog number 552364 from BD Biosciences) was used.

Silver quantification
For measuring the Ag-NP uptake and dissolution in cells, 5 ml cell cultures in 25 cm 2 culture flasks were used, with the exposure scheme described above. At the end of the exposure period, the NP-containing medium was removed and the cell layer was gently rinsed twice with complete culture medium.
The cells were then harvested by scraping in buffer A (Hepes 50 mM pH 7.5, sorbitol 200 mM, magnesium acetate 2 mM), and collected by centrifugation (500 g, 5 min). After a further rinse in buffer A, the cells were lysed by incubation in 10-cell pellet volume of lysis buffer (Hepes 50 mM pH 7.5, magnesium acetate 2 mM, tetradecyldimethylammonio propane sulfonate (SB 3-14), 0.15% (w/v)) for 20 minutes on ice. Non sedimentable (soluble) silver is obtained by centrifugation of an aliquot of the extracts (15 000g, 30 minutes, 4°C) and collection of the upper half of the supernatant.
The extracts were mineralized by the addition of one volume of suprapure 65% HNO 3 and incubation on a rotating wheel at room temperature for 18 h.
Mineralized samples were then diluted in ultrapure grade HNO 3 (1% v/v) and analyzed on a Nexion 300X ICP-MS (Perkin Elmer, Waltham, MA) equipped with a concentric nebulizer and operated in standard mode. External calibration was performed using a certified ionic Ag ICP-MS standard and Yttrium was used as an internal standard.
The technic was validated based on linearity, repeatability, reproducibility, accuracy, inter-sample contamination criterions, and the analytical limit of detection determined as the sum of the mean and three standard deviations of 20 ultrapure water samples was 0.035 μg L −1 of Ag in injected sample. Multielemental quality controls were used to validate each run of analysis.
In some experiments, inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Shimadzu ICP 9000 with mini plasma torch in axial reading mode) was used to measure the metal content. Standard solutions of Ag for atomic absorption spectroscopy (Sigma Aldrich) were used for quantification (calibration curve between 1.9 to 1000 μg L −1 with 1% HNO 3 (Fluka)). Mineralized samples were diluted with pure water to a final concentration of 1% of HNO 3 prior to analysis.
To normalize the results, the protein concentration of the extracts was determined by a modified dye-binding assay. 42 Environmental Science: Nano Paper

Proteomics
The 2D gel based proteomic experiments were essentially carried out as previously described, 26,27 on independent biological triplicates, except for the acutely exposed cells for which a quadruplicate was obtained. The isoelectric focusing dimension used a linear 4-8 pH gradient, and the second dimension a homogeneous 10% acrylamide gel spanning a molecular mass window of 14-200 kDa. Protein detection was carried out by silver staining. Two-dimensional gel images were analyzed by the Delta2D software, and the spot intensities are expressed as a percentage of the sum of all detected spots, thereby normalizing the intensities between gels. Statistical analysis was carried out by a heteroschedastic Student T-test (also known as Welch T test). For the general analysis a first pass filter of p < 0.25 was carried out. This allowed decreasing the noise brought by proteins that do not show a consistent variation in the biological phenomena under investigation. For the detailed analysis only proteins showing a p ≤ 0.05 in one T-test (acutely exposed cells vs. acute control or chronicallyexposed cells vs. chronic control) were selected.
Protein identification was carried out by mass spectrometry as described previously. 27 The MS/MS data were interpreted using a local Mascot server with MASCOT 2.5.1 algorithm (Matrix Science, London, UK) against UniProtKB/ SwissProt (version 2017_09, 555594 sequences). The research was carried out in all species. Spectra were searched with a mass tolerance of 15 ppm for MS and 0.05 Da for MS/MS data, allowing a maximum of one trypsin missed cleavage. Carbamidomethylation of cysteine residues and oxidation of methionine residues were specified as variable modifications. Protein identifications were validated with at least two peptides with Mascot ion score above 30.

Silver nanoparticles characterization and viability effects
First, the characteristics of the silver nanoparticles were verified and their effect on cell viability was assayed. From the results shown in Fig. 1, the silver nanoparticles showed a spherical shape with a diameter of 49.3 ± 12.6 nm. Their hydrodynamic diameter (Z-average) in water was 103 ± 2 nm (polydispersity index: 0.15 ± 0.04) (B). This value did not change significantly upon dilution of Ag-NP suspension in exposure medium, where it was 110 ± 2 nm (polydispersity index: 0.11 ± 0.01). After 24 h at 37°C, the size distribution of Ag-NPs in water and medium slightly decreased (Z-average: 98 ± 1 nm/Pdi: 0,08 ± 0,01 and Z-average: 94 ± 1 nm/Pdi: 0,23 ± 0,01 in water and medium, respectively). Their zeta potential, when diluted in water, was −11.4 ± 1,0 mV. From the viability curve, a dose of 20 μg ml −1 was selected for all the subsequent experiments, corresponding to the LD 20 . This dose ensures strong effects on cells while keeping cellular Fig. 1 Ag-NP physico-chemical characterization. AgNPs were imaged by TEM (panel A) and their size distribution was characterized by DLS (panel B). Cell viability (panel C) was measured by dye exclusion. The cells were seeded at 800 000 cells ml −1 in non-adherent culture flasks and exposed to silver nanoparticles for 24 hours before measurement of cell viability. As previous experiments had shown that the cellular differentiated features were kept for up to 20 days of confluence culture, the dose was split at 1 μg ml −1 per day silver nanoparticles for the chronic exposure scheme. Experiments were then conducted to determine the actual silver dose reaching the cells after a 24 hours exposure at the two concentrations used (1 and 20 μg ml −1 ). The results, shown in Table S1, † indicated that ca. 75% of the silver input was internalized by the cells, regardless of the input concentration.

Global analysis of the proteomic results
From the total protein abundances data obtained from the gel analysis software, a subset of putatively variable proteins (T-test p < 0.25 in either the acute exposure vs. control or the chronic exposure vs. control comparisons) was extracted. This subset was then tested using the PAST statistical suite. 43 As a first test, a hierarchical clustering was performed, and the results are shown in Fig. 2. This test clearly showed that the cells chronically exposed to silver stood apart from all the other groups, and that the second most important variable was the cell aging, as the chronic control group also stood apart from the two acute groups (control and treated).

Detailed analysis of the proteomic results
We extracted the intensity values for the spots that were significantly altered (T-test p ≤ 0.05) between one of the silver-treated groups and its corresponding control. We thus obtained two subsets, one for the spots changed in the chronic exposure scheme and one for the spots changed in the acute exposure scheme. The two subsets, accounting in total for 152 spots, were then merged into a single table of 85 variable and identified proteins ( Table 1). The corresponding 2D gel images are shown on Fig. 3 and Fig. S1 and S2. † The mass spectrometry identification parameters are detailed in Table S2 † and the mass spectrometry data are deposited in Pride under the identifier PXD016379. 44 In order to cope with the multiple testing issue, we used several approaches such as the Benjamini-Hochberg approach, 45 the sequential goodness of fit approach 46 and the sequential Fisher test approach. 47 All selected spots showed a very low q-value for the condition for which they were variable compared to the corresponding control (Table S3 †).
In order to further analyze the proteomic results, we applied a pathway analysis through the DAVID tool 48,49 (publicly available at https://david.ncifcrf.gov/), and the results of these analyses are shown in Tables S4 and S5. † Several important cellular pathways were highlighted by this analysis, such as antioxidant pathways (e.g. glutathione reductase, glutamate cysteine ligase, flavin reductase), mitochondrial proteins (e.g. ATP synthase, respiratory complexes I-III, pyruvate dehydrogenase, oxoglutarate dehydrogenase), lysosomal proteins (e.g. cathepsins B, D, S and Z), or central metabolism enzymes (e.g. sorbitol dehydrogenase, triose phosphate isomerase). We also used knowledge obtained for other nanoparticles using a proteomic screen, as detailed in the following sections.

Enzymatic activities
For some of the modulated proteins, we first checked whether their enzyme activities (Table 2), which is the relevant parameter in terms of cell physiology, followed or not the proteomic results. For triose phosphate isomerase, sorbitol dehydrogenase and flavin reductase the enzymatic activity followed the trend indicated by proteomics, i.e. a decrease for triose phosphate isomerase and an increase for sorbitol dehydrogenase and flavin reductase. The results were different for glutathione reductase, for which the enzymatic activity was constant, while proteomics indicated an increase in the protein amount upon cell exposure to silver in both modes. In this case, however, we showed that the enzymatic activity is inhibited by silver ion. Thus, the decrease in the relative enzymatic activity observed upon silver treatment (compared to the protein amount) may be explained by the inhibition of the enzyme by the silver ion present in the cells in this case (as shown by the ICP-MS results).

Mitochondrial potential
The keyword mitochondrion appeared in pathway analyses. Correspondingly, several mitochondrial proteins appeared modulated upon exposure of macrophages to silver nanoparticles, among which a few subunits of the respiratory complexes, some components of the mitochondrial matrix involved in electron transfer (electron transfer flavoprotein) and some proteins involved in the general energy metabolism (pyruvate dehydrogenase, oxoglutarate dehydrogenase). As this may suggest perturbations in the mitochondrial functions, we investigated the mitochondrial transmembrane potential. The results, shown on Fig. 4, indicated that nearly all viable cells accumulated rhodamine 123 and thus had a strong mitochondrial transmembrane potential. Nonetheless, the amount of accumulated rhodamine 123 was lower in cells  View Article Online chronically exposed to silver nanoparticles compared to the corresponding control, and this effect, albeit of low magnitude, was statistically significant. In cells acutely exposed to silver, no significant change in the mitochondrial membrane potential was observed. In addition, the proportion of cells with a high mitochondrial potential is also a good indicator of viability. The results shown in Fig. 4 indicated that both treatment schemes with silver nanoparticles induced a decrease in cell viability (−15% for the acute exposure, −21% for the chronic exposure) that is in line with the LD 20 .

Glutathione level
Beside glutathione reductase, which has been studied in section 3.4, one of the proteins increased after exposure to silver nanoparticles is glutamate cysteine ligase subunit M, i.e. the regulatory subunit of the enzyme involved in the first step of glutathione biosynthesis, which is the limiting step of the pathway. 50 In a previous work on copper nanoparticles, 33 a change in the amount of this protein correlated with a change in the level of free reduced glutathione. Thus, this induction could be a response of cells to decreased intracellular glutathione levels due to exposure to Ag-NPs. Moreover, we previously reported that the levels of free glutathione was reduced in primary macrophages exposed to silver nanoparticles, 51 and that intracellular dissolution of silver nanoparticles led to the formation of silver-glutathione complexes. 52 In the present exposure conditions, a statistically significant reduction ( p ≤ 0.05, Mann-Whitney U test) of the intracellular free, reduced glutathione content was measured: These values are of the same order than those previously described using a different glutathione assay. 53 Both exposure schemes decreased the intracellular reduced glutathione concentration. Here again, the chronic exposure resulted in a stronger effect than the acute exposure. Thus, the increase in the glutamate cysteine ligase subunit M amount can be interpreted as a cellular mechanism activated to compensate for the decrease of free glutathione when it reaches a critical level.

Phagocytosis
Phagocytosis is one of the most important functions of the macrophages. The proteomic analysis highlighted important changes in the levels of several cathepsins, which may indicate alterations in the phagolysosomal pathway.

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Moreover, actin binding appeared as a modulated pathway, and is important for phagocytosis. Thus, the phagocytic activity of macrophages exposed to silver nanoparticles was assessed. The proportion of phagocytic cells did not change appreciably upon treatment with silver nanoparticles in the acute exposure mode. Conversely, chronic exposure to silver nanoparticles significantly decreased their phagocytic capacity (Fig. 5A).  Table 1.

NO and cytokine production
Production of nitric oxide and of pro-inflammatory cytokines such as IL-6 and TNF upon stimulation is one of the specialized functions of macrophages. In addition, the proteomic screen highlighted proteins that may be related, directly or indirectly, with these processes. One interesting example is represented by sepiapterin reductase, which catalyses the production of tetrahydrobiopterin, an obligatory cofactor of NO synthase. Other examples are represented by potential regulators of transduction of the pro-inflammatory signal in the nucleus (e.g. COMM domain-containing protein 4 and interferon-induced 35 kDa protein) or more upstream in the cytosol (e.g. toll-interacting protein), or proteins known to regulate inflammation by indirect mechanisms (e.g. latexin). For some of these proteins, such as sepiapterin reductase and interferon-induced 35 kDa protein, changes observed at the proteomic level have been correlated to changes in the nitric oxide and cytokines production. 26,27 It was thus legitimate to evaluate the inflammatory status in cells exposed to silver nanoparticles. To this purpose IL6, TNF and NO production were measured after treatment of macrophages with silver nanoparticles under two schemes: i) treatment with nanoparticles only, or ii) treatment with nanoparticles and lipopolysaccharide (LPS) for the last 18 hours of exposure. The first scheme investigated the intrinsic pro-inflammatory action of the nanoparticles, while the second one investigated the interference of nanoparticles with a standard pro-inflammatory response induced by a bacterial stimulus. Silver nanoparticles did not show an intrinsic proinflammatory effect, as detected from NO production (Fig. 5B, grey bars). The situation was very different for the combined nanoparticles-LPS treatment ( Fig.  5B-D, black bars). In this case, the response to acute exposure did not differ significantly from that of the control cells (exposed to LPS only). Oppositely, the NO production was significantly lower in chronically-exposed cells compared to control cells. This trend was also observed for interleukin-6 production (where a slight but statistically significant was however observed upon acute exposure, Fig. 5C), while TNFalpha production was not changed significantly by exposure to silver in either mode. It is worth noting, however, that TNF-alpha production is sensitive to cell aging (Fig. 5D), with a 50% reduced production under LPS stimulation after 20 days of culture.
Finally, to better understand why chronically-exposed cells were more affected than acutely-exposed cells, we performed silver accumulation measurements, both for total silver and ionic silver. To this purpose, we exposed cells chronically to 1

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μg ml −1 per day for 5, 10, 15 and 20 days. In parallel, cells were exposed to the same cumulated dose (i.e. 5, 10, 15 and 20 μg ml −1 ) for 24 hours. After exposure, the total and soluble silver contents were determined for each point. The results, shown in Fig. 6, indicate that total accumulated silver was lower for chronically-exposed cells than for acutely exposed cells. For soluble silver, the concentrations were similar and relatively constant for chronically-exposed cells and for Fig. 6 Silver cellular incorporation. The incorporation of total (panel A) and soluble (panel B) silver was measured on tetraplicate samples. The results were normalized to the protein content and are thus expressed in ng silver per mg cellular protein. View Article Online acutely exposed cells for up to 15 days, and were then lower for chronically-exposed cells at 20 μg ml −1 cumulated dose.

Discussion
Although completely irreplaceable on the level of relevance, animal experimentation in toxicology poses important challenges in terms of speed, cost and also ethical issues, as exemplified by studies on the long term effects of ammunition. 54 Furthermore, new regulations such as the REACH regulation in EU dramatically increase the amount of toxicological data to be produced. There is thus a general trend to decrease as much as possible the use of animal experimentation, following the 3R (refine, reduce, replace) guidelines. This however requires mandatorily to increase the relevance of data obtained on in vitro models and thus to refine these models and their use, with encouraging recent results regarding silver nanomaterials. [11][12][13][14][15]55 In in vitro nanotoxicological studies, the cell type, exposure scheme and nanoparticles used are critical features. Regarding silver nanoparticles, we tried to use an industrial product to maximise the relevance of our study. As such, PVPcoated silver nanoparticles have been used as antibacterial agents, 56 in conductive inks, [57][58][59] in electromechanics 60 or in sensors. 61 and thus appear a relevant choice.
Regarding the exposure schemes, most in vitro nanotoxicology studies use a single exposure to relatively high doses of materials (see for example ref. [62][63][64]. This strategy mimics an accidental exposure to a high dose, but not a repeated exposure to low doses, which is more realistic in mimicking occupational scenarios. Very few in vitro studies using a repeated exposure scheme have been reported in the literature. Interestingly, these studies report different outcomes than those observed for a single acute dose. Moreover, chronic exposure leads sometimes to stronger cell response compared to acute exposure, but the opposite also occurs. 11,65,66 Thus, cell responses to a repeated exposure scenario are still not well understood, and more data need to be generated.
Until now, macrophages have not been used in repeated exposure scenarios, despite the fact that they are the primary scavenger cells for all particulate material in the body and are relevant for many exposure routes. 16,67 To fill this gap of knowledge, we report here the cellular responses of macrophages to either acute or repeated exposure to silver nanoparticles, using an in vitro model. Primary macrophages cannot be maintained in vitro for a long period of time, which limits their use to a few days of exposure. 51 In the present study, the response of a macrophage cell line to silver nanoparticles was analysed, in such a way that cells were exposed at confluence, thereby limiting the effect of proliferation on cellular responses. This system is different from those used previously in other cell types, which are regularly split during the exposure period. [12][13][14][15] This difference is however in line with the in vivo physiology, as resident macrophages have a long life span 68 and self-renew slowly, 69 a feature that is mimicked by our confluent culture system.
Besides the culture scheme, the exposure dose used is also an important factor. We have used a dose (1 ppm per day) that is much higher than the dose previously used on keratinocytes (0.4 ppb), 11 but it is similar to doses used in recent papers on bronchial 15 and intestinal 14 cells. The latter doses have been calculated based on exposure data, so we are confident that the dose that we used should not be irrelevant as compared to real in vivo doses. Furthermore, the existence of localized argyria under some clinical circumstances 34 calls for the possibility of relatively high doses released under a chronic exposure scheme.
In addition to the chronic exposure scheme, we added a control experiment with the same cumulative dose delivered acutely, i.e. over 24 hours. This control experiment has been omitted for some studies on epithelial cells, [13][14][15] but included in other studies. 11,12,65,70 As these previous studies on epithelial cells have shown that short-and long-term responses are very different, such a control may have been seen as superfluous. However, macrophages are highly phagocytic cells that store the material that they cannot degrade (e.g. mineral material) as exemplified in. 67 Thus, it can be argued that a single exposure to the total dose may recapitulate what is observed upon repeated exposure, and this is why we included this control experiment. As cell aging has been shown to be an important parameter, 11 the acute exposure control could be performed on aged cells or on fresh cells. In line with a scheme validated by previous publications, 11,12 we decided to perform the control on fresh cells, as it allows the investigation of the molecular effects of cell aging, especially when using "omic" approaches (e.g. in ref. 11). More importantly, this scheme also allows the comparison of the data obtained in the acute exposure scheme with those present in the nanotoxicology literature, where fresh cells are always used.
Using this in vitro system, we showed that a repeated low dose exposure produced a stronger impact on cells compared to a single dose exposure at the same theoretical cumulative dose. Targeted experiments confirmed that a repeated exposure produces stronger effects on cellular metabolism and on specialized macrophages functions (e.g. phagocytosis and cytokine production) than a single high dose exposure. This suggests that chronic exposure to silver nanoparticles may produce stronger disturbances in the function of the innate immune system than acute exposure. Moreover, the set of proteins with modified intracellular content differs in the repeated exposure scheme compared to the acute one. In response to the chronic exposure scheme, the highlighted proteins belong essentially to antioxidant responses (e.g. glutathione reductase, glutamate cysteine ligase, flavin reductase), cytoskeletal proteins (e.g. plastin, cofilin, actin capping protein) energy metabolism (e.g. ATP synthase, respiratory complexes I-III, pyruvate dehydrogenase, oxoglutarate dehydrogenase, triose phosphate isomerase) and phagolysosomal proteins (e.g. cathepsins B, D, S and Z).
This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.

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Quite interestingly, these proteins did not respond to the acute exposure scheme. This suggests that the innate immune response may be affected differently by chronic and acute exposures to silver nanoparticles. Therefore, effort should be put in generating data from chronic exposure scenarios, which are more realistic. The next step was then to correlate the intensity of the impact on cells with the amount of internalized silver. There is ample evidence in the literature that particulate silver is very weakly toxic, 2,20 and that the toxicity of silver nanoparticles is mediated by the intracellular release of silver ions. 2,20,71,72 We thus measured total and soluble silver levels for both exposure schemes.
Measurements of total silver levels showed that they were lower for the repeated exposure scheme. This could be due to cell renewal over time, a decrease in silver uptake upon cell aging or a progressive effect of silver internalization on the phagocytic process itself, as supported by the lower phagocytic capacity observed for chronically-exposed cells.
It could be argued, however, that repeated exposure could result in a higher dissolution of silver because of longer dissolution time, thus explaining the stronger effects of a repeated exposure. However, we showed that dissolved silver intracellular content was constant for nontoxic doses. This suggests a sustained release of silver from cells, as documented in primary macrophages, 51 probably via a complexation to glutathione. 52 This hypothesis is further supported by the decrease in the free glutathione levels that we observed in repeatedly-exposed cells, a mechanism that may contribute to the deleterious effects of silver. 73 When toxicity begins to appear, i.e. at 20 μg ml −1 cumulated dose, the soluble silver content diverged between chronicallyexposed cells and acutely-exposed cells. In acutely-exposed cells, the soluble silver level increased, suggesting that the systems controlling intracellular silver ion content became overwhelmed, leading to toxicity. In chronically-exposed cells, the soluble silver level was close to the levels observed for earlier time points, suggesting a different toxicity mechanism, based on progressive attrition of cellular functions at this low but sustained silver ion concentration.

Conclusions
Thus, the overall and major conclusion of this work is that even with a lower intracellular release of silver ions, chronic exposure to silver nanoparticles, if sustained for enough time (e.g. in comparison with a four-day exposure 51 ) induces a progressive "wearing-down" of macrophage functions, which was not observed for an acute exposure to the same cumulative dose. This phenomenon may be part of the picture explaining the silver chronic toxicity observed in several in vivo models. [6][7][8][9][10] Indeed, body barriers are the combination of an epithelium that forms a physical barrier and of scavenging cells that either remove particulate material that has crossed the epithelial barrier (e.g. in skin or intestine), or keep the epithelial surface clean for exchanges (e.g. in lung alveolae). Moreover, it is now known that both cell types (epithelial and innate immune cells) can trigger an immune response. Thus, our study on macrophages complements previous studies on epithelial cells, [13][14][15] and shows that very different cell types share a deleterious response to chronic exposure to silver nanoparticles, even at sub-toxic doses. Put in the broader context of long term silver toxicity, the in vitro studies using repeated exposure to low doses shall be seen as a first step to bridge the current gap existing between classical in vitro toxicology using single exposures and results obtained with chronic exposures on animals.

Authors contributions
Experimental contributions BD and CAG performed the mitochondrial potential, phagocytosis, nitric oxide and cytokine production experiments, and the cell cultures required for these experiments. VCF performed the 2D gel experiments. HD and SC performed the mass spectrometry identifications of the proteins, while TR, JB and CC performed the computerized analysis of the proteomic experiments. CMD, JP and MD performed the ICP measurements. MC performed the nanoparticles characterization. TR provided the cell cultures needed for proteomics, and performed the glutathione assay experiments and the enzyme assays.

Redactional contributions
TR and MC drafted the initial manuscript, for which all the coauthors in charge of some experiments drafted the section corresponding to their experiments. The manuscript was critically reviewed, amended and approved by all co-authors.

Conflicts of interest
The authors report no conflicts of interest.