The internal dose makes the poison: higher internalization of polystyrene particles induce increased perturbation of macrophages.

Plastics are emerging pollutants of great concern. Macroplastics released in the environment degrade into microplastics and nanoplastics. Because of their small size, these micro and nano plastic particles can enter the food chain and contaminate humans with still unknown biological effects. Plastics being particulate pollutants, they are handled in the human body by scavenger cells such as macrophages, which are important players in the innate immune system. Using polystyrene as a model of micro and nanoplastics, with size ranging from under 100 nm to 6 microns, we have showed that although non-toxic, polystyrene nano and microbeads alter the normal functioning of macrophages in a size and dose-dependent manner. Alterations in the oxidative stress, lysosomal and mitochondrial functions were detected, as well as changes in the expression of various surface markers involved in the immune response such as CD11a/b, CD18, CD86, PD-L1, or CD204. For each beads size tested, the alterations were more pronounced for the cell subpopulation that had internalized the highest number of beads. Across beads sizes, the alterations were more pronounced for beads in the supra-micron range than for beads in the sub-micron range. Overall, this means that internalization of high doses of polystyrene favors the emergence of subpopulations of macrophages with an altered phenotype, which may not only be less efficient in their functions but also alter the fine balance of the innate immune system.

HMGB1 cleavage by complement C1s and its potent anti-inflammatory product.

Complement C1s association with the pathogenesis of several diseases cannot be simply explained only by considering its main role in activating the classical complement pathway. This suggests that non-canonical functions are to be deciphered for this protease. Here the focus is on C1s cleavage of HMGB1 as an auxiliary target. HMGB1 is a chromatin non-histone nuclear protein, which exerts in fact multiple functions depending on its location and its post-translational modifications. In the extracellular compartment, HMGB1 can amplify immune and inflammatory responses to danger associated molecular patterns, in health and disease. Among possible regulatory mechanisms, proteolytic processing could be highly relevant for HMGB1 functional modulation. The unique properties of HMGB1 cleavage by C1s are analyzed in details. For example, C1s cannot cleave the HMGB1 A-box fragment, which has been described in the literature as an inhibitor/antagonist of HMGB1. By mass spectrometry, C1s cleavage was experimentally identified to occur after lysine on position 65, 128 and 172 in HMGB1. Compared to previously identified C1s cleavage sites, the ones identified here are uncommon, and their analysis suggests that local conformational changes are required before cleavage at certain positions. This is in line with the observation that HMGB1 cleavage by C1s is far slower when compared to human neutrophil elastase. Recombinant expression of cleavage fragments and site-directed mutagenesis were used to confirm these results and to explore how the output of C1s cleavage on HMGB1 is finely modulated by the molecular environment. Furthermore, knowing the antagonist effect of the isolated recombinant A-box subdomain in several pathophysiological contexts, we wondered if C1s cleavage could generate natural antagonist fragments. As a functional readout, IL-6 secretion following moderate LPS activation of RAW264.7 macrophage was investigated, using LPS alone or in complex with HMGB1 or some recombinant fragments. This study revealed that a N-terminal fragment released by C1s cleavage bears stronger antagonist properties as compared to the A-box, which was not expected. We discuss how this fragment could provide a potent brake for the inflammatory process, opening the way to dampen inflammation.

Immediate and Sustained Effects of Cobalt and Zinc-Containing Pigments on Macrophages.

Pigments are among the oldest nanoparticulate products known to mankind, and their use in tattoos is also very old. Nowadays, 25% of American people aged 18 to 50 are tattooed, which poses the question of the delayed effects of tattoos. In this article, we investigated three cobalt [Pigment Violet 14 (purple color)] or cobalt alloy pigments [Pigment Blue 28 (blue color), Pigment Green 14 (green color)], and one zinc pigment [Pigment White 4 (white color)] which constitute a wide range of colors found in tattoos. These pigments contain microparticles and a significant proportion of submicroparticles or nanoparticles (in either aggregate or free form). Because of the key role of macrophages in the scavenging of particulate materials, we tested the effects of cobalt- and zinc-based pigments on the J774A.1 macrophage cell line. In order to detect delayed effects, we compared two exposure schemes: acute exposure for 24 hours and an exposure for 24 hours followed by a 3-day post-exposure recovery period. The conjunction of these two schemes allowed for the investigation of the delayed or sustained effects of pigments. All pigments induced functional effects on macrophages, most of which were pigment-dependent. For example, Pigment Green 19, Pigment Blue 28, and Pigment White 4 showed a delayed alteration of the phagocytic capacity of cells. Moreover, all the pigments tested induced a slight but significant increase in tumor necrosis factor secretion. This effect, however, was transitory. Conversely, only Pigment Blue 28 induced both a short and sustained increase in interleukin 6 secretion. Results showed that in response to bacterial stimuli (LPS), the secretion of tumor necrosis factor and interleukin 6 declined after exposure to pigments followed by a recovery period. For chemoattractant cytokines (MCP-1 or MIP-1α), delayed effects were observed with a secretion decreased in presence of Pigment Blue 28 and Pigment violet 14, both with or without LPS stimuli. The pigments also induced persisting changes in some important macrophage membrane markers such as CD11b, an integrin contributing to cell adhesion and immunological tolerance. In conclusion, the pigments induced functional disorders in macrophages, which, in some cases, persist long after exposure, even at non-toxic doses.

Repeated Exposure of Macrophages to Synthetic Amorphous Silica Induces Adaptive Proteome Changes and a Moderate Cell Activation.

Synthetic amorphous silica (SAS) is a nanomaterial used in a wide variety of applications, including the use as a food additive. Two types of SAS are commonly employed as a powder additive, precipitated silica and fumed silica. Numerous studies have investigated the effects of synthetic amorphous silica on mammalian cells. However, most of them have used an exposure scheme based on a single dose of SAS. In this study, we have used instead a repeated 10-day exposure scheme in an effort to better simulate the occupational exposure encountered in daily life by consumers and workers. As a biological model, we have used the murine macrophage cell line J774A.1, as macrophages are very important innate immune cells in the response to particulate materials. In order to obtain a better appraisal of the macrophage responses to this repeated exposure to SAS, we have used proteomics as a wide-scale approach. Furthermore, some of the biological pathways detected as modulated by the exposure to SAS by the proteomic experiments have been validated through targeted experiments. Overall, proteomics showed that precipitated SAS induced a more important macrophage response than fumed SAS at equal dose. Nevertheless, validation experiments showed that most of the responses detected by proteomics are indeed adaptive, as the cellular homeostasis appeared to be maintained at the end of the exposure. For example, the intracellular glutathione levels or the mitochondrial transmembrane potential at the end of the 10 days exposure were similar for SAS-exposed cells and for unexposed cells. Similarly, no gross lysosomal damage was observed after repeated exposure to SAS. Nevertheless, important functions of macrophages such as phagocytosis, TNFα, and interleukin-6 secretion were up-modulated after exposure, as was the expression of important membrane proteins such as the scavenger receptors, MHC-II, or the MAC-1 receptor. These results suggest that repeated exposure to low doses of SAS slightly modulates the immune functions of macrophages, which may alter the homeostasis of the immune system.

About the Transient Effects of Synthetic Amorphous Silica: An In Vitro Study on Macrophages.

Silica (either crystalline or amorphous) is widely used for different applications and its toxicological assessment depends on its characteristics and intended use. As sustained inflammation induced by crystalline silica is at the root of silicosis, investigating the inflammatory effects induced by amorphous silicas and their persistence is needed. For the development of new grades of synthetic amorphous silicas, it is also desirable to be able to understand better the factors underlying potential adverse effects. Therefore, we used an optimized in vitro macrophage system to investigate the effects of amorphous silicas, and their persistence. By using different amorphous silicas, we demonstrated that the main driver for the adverse effects is a low size of the overall particle/agglomerate; the second driver being a low size of the primary particle. We also demonstrated that the effects were transient. By using silicon dosage in cells, we showed that the transient effects are coupled with a decrease of intracellular silicon levels over time after exposure. To further investigate this phenomenon, a mild enzymatic cell lysis allowed us to show that amorphous silicas are degraded in macrophages over time, explaining the decrease in silicon content and thus the transiency of the effects of amorphous silicas on macrophages.

Proteomic analysis of Rhodospirillum rubrum after carbon monoxide exposure reveals an important effect on metallic cofactor biosynthesis.

Some carboxydotrophs like Rhodospirillum rubrum are able to grow with CO as their sole source of energy using a Carbone monoxide dehydrogenase (CODH) and an Energy conserving hydrogenase (ECH) to perform anaerobically the so called water-gas shift reaction (WGSR) (CO + H2O → CO2 + H2). Several studies have focused at the biochemical and biophysical level on this enzymatic system and a few OMICS studies on CO metabolism. Knowing that CO is toxic in particular due to its binding to heme iron atoms, and is even considered as a potential antibacterial agent, we decided to use a proteomic approach in order to analyze R. rubrum adaptation in term of metabolism and management of the toxic effect. In particular, this study allowed highlighting a set of proteins likely implicated in ECH maturation, and important perturbations in term of cofactor biosynthesis, especially metallic cofactors. This shows that even this CO tolerant microorganism cannot avoid completely CO toxic effects associated with its interaction with metallic ions. SIGNIFICANCE: This proteomic study highlights the fact that even in a microorganism able to handle carbon monoxide and in some way detoxifying it via the intrinsic action of the carbon monoxide dehydrogenase (CODH), CO has important effects on metal homeostasis, metal cofactors and metalloproteins. These effects are direct or indirect via transcription regulation, and amplified by the high interdependency of cofactors biosynthesis.

Synthetic Amorphous Silica Nanoparticles Promote Human Dendritic Cell Maturation and CD4+ T-Lymphocyte Activation.

Innate immune cells such as dendritic cells (DCs) sense and engulf nanomaterials potentially leading to an adverse immune response. Indeed, as described for combustion-derived particles, nanomaterials could be sensed as danger signals, enabling DCs to undergo a maturation process, migrate to regional lymph nodes and activate naive T lymphocytes. Synthetic amorphous silica nanoparticles (SAS-NPs) are widely used as food additives, cosmetics, and construction materials. This work aimed to evaluate in vitro the effects of manufactured SAS-NPs, produced by thermal or wet routes, on human DCs functions and T-cell activation. Human monocyte-derived DCs (moDCs) were exposed for 16 h to 3 endotoxin-free test materials: fumed silica NPs from Sigma-Aldrich (no. S5505) or the JRC Nanomaterial Repository (NM-202) and colloidal LudoxTMA NPs. Cell viability, phenotypical changes, cytokines production, internalization, and allogeneic CD4+ T-cells proliferation were evaluated. Our results showed that all SAS-NPs significantly upregulated the surface expression of CD86 and CD83 activation markers. Secretions of pro-inflammatory cytokines (CXCL-8 and CXCL-12) were significantly enhanced in a dose-dependent manner in the moDCs culture supernatants by all SAS-NPs tested. In an allogeneic coculture, fumed silica-activated moDCs significantly increased T-lymphocyte proliferation at all T-cell: DC ratios compared with unloaded moDCs. Moreover, analysis of coculture supernatants regarding the production of T-cell-derived cytokines showed a significant increase of IL-9 and IL-17A and F, as well as an upregulation of IL-5, consistent with the pro-inflammatory phenotype of treated moDCs. Taken together, these results suggest that SAS-NPs could induce functional moDCs maturation and play a role in the immunization process against environmental antigens.

Impact of Physico-Chemical Properties of Cellulose Nanocrystal/Silver Nanoparticle Hybrid Suspensions on Their Biocidal and Toxicological Effects.

There is a demand for nanoparticles that are environmentally acceptable, but simultaneously efficient and low cost. We prepared silver nanoparticles (AgNPs) grafted on a native bio-based substrate (cellulose nanocrystals, CNCs) with high biocidal activity and no toxicological impact. AgNPs of 10 nm are nucleated on CNCs in aqueous suspension with content from 0.4 to 24.7 wt%. XANES experiments show that varying the NaBH4/AgNO3 molar ratio affects the AgNP oxidation state, while maintaining an fcc structure. AgNPs transition from 10 nm spherical NPs to 300 nm triangular-shaped AgNPrisms induced by H2O2 post-treatment. The 48 h biocidal activity of the hybrid tested on B. Subtilis is intensified with the increase of AgNP content irrespective of the Ag+/Ag0 ratio in AgNPs, while the AgNSphere-AgNPrism transition induces a significant reduction of biocidal activity. A very low minimum inhibitory concentration of 0.016 mg AgNP/mL is determined. A new long-term biocidal activity test (up to 168 h) proved efficiency favorable to the smaller AgNPs. Finally, it is shown that AgNPs have no impact on the phagocytic capacity of mammalian cells.

A proteomic-informed view of the changes induced by loss of cellular adherence: The example of mouse macrophages.

Except cells circulating in the bloodstream, most cells in vertebrates are adherent. Studying the repercussions of adherence per se in cell physiology is thus very difficult to carry out, although it plays an important role in cancer biology, e.g. in the metastasis process. In order to study how adherence impacts major cell functions, we used a murine macrophage cell line. Opposite to the monocyte/macrophage system, where adherence is associated with the acquisition of differentiated functions, these cells can be grown in both adherent or suspension conditions without altering their differentiated functions (phagocytosis and inflammation signaling). We used a proteomic approach to cover a large panel of proteins potentially modified by the adherence status. Targeted experiments were carried out to validate the proteomic results, e.g. on metabolic enzymes, mitochondrial and cytoskeletal proteins. The mitochondrial activity was increased in non-adherent cells compared with adherent cells, without differences in glucose consumption. Concerning the cytoskeleton, a rearrangement of the actin organization (filopodia vs sub-cortical network) and of the microtubule network were observed between adherent and non-adherent cells. Taken together, these data show the mechanisms at play for the modification of the cytoskeleton and also modifications of the metabolic activity between adherent and non-adherent cells.

Silver Staining of 2D Electrophoresis Gels.

Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap equipment and chemicals. For its use in proteomics, two important additional features must be considered, compatibility with mass spectrometry and quantitative response. Both features are discussed in this chapter, and optimized silver staining protocols are proposed.

The Whereabouts of 2D Gels in Quantitative Proteomics.

Two-dimensional gel electrophoresis has been instrumental in the development of proteomics. Although it is no longer the exclusive scheme used for proteomics, its unique features make it a still highly valuable tool, especially when multiple quantitative comparisons of samples must be made, and even for large samples series. However, quantitative proteomics using two-dimensional gels is critically dependent on the performances of the protein detection methods used after the electrophoretic separations. This chapter therefore examines critically the various detection methods, (radioactivity, dyes, fluorescence, and silver) as well as the data analysis issues that must be taken into account when quantitative comparative analysis of two-dimensional gels is performed.

A proteomic view of cellular responses of macrophages to copper when added as ion or as copper-polyacrylate complex.

Copper is an essential metal for life, but is toxic at high concentrations. In mammalian cells, two copper transporters are known, CTR1 and CTR2. In order to gain insights on the possible influence of the import pathway on cellular responses to copper, two copper challenges were compared: one with copper ion, which is likely to use preferentially CTR1, and one with a copper-polyacrylate complex, which will be internalized via the endosomal pathway and is likely to use preferentially CTR2. A model system consisting in the J774A1 mouse macrophage system, with a strong endosomal/lysosomal pathway, was used. In order to gain wide insights into the cellular responses to copper, a proteomic approach was used. The proteomic results were validated by targeted experiments, and showed differential effects of the import mode on cellular physiology parameters. While the mitochondrial transmembrane potential was kept constant, a depletion in the free glutahione content was observed with copper (ion and polylacrylate complex). Both copper-polyacrylate and polyacrylate induced perturbations in the cytoskeleton and in phagocytosis. Inflammatory responses were also differently altered by copper ion and copper-polyacrylate. Copper-polyacrylate also perturbed several metabolic enzymes. Lastly, enzymes were used as a test set to assess the predictive value of proteomics. SIGNIFICANCE: Proteomic profiling provides an in depth analysis of the alterations induced on cells by copper under two different exposure modes to this metal, namely as the free ion or as a complex with polyacrylate. The cellular responses were substantially different between the two exposure modes, although some cellular effects are shared, such as the depletion in free glutathione. Targeted experiments were used to confirm the proteomic results. Some metabolic enzymes showed altered activities after exposure to the copper-polyacrylate complex. The basal inflammatory responses were different for copper ion and for the copper-polyacrylate complex, while the two forms of copper inhibited lipopolysaccharide-induced inflammatory responses.

A Low-Serum Culture System for Prolonged in Vitro Toxicology Experiments on a Macrophage System.

Immunotoxicology sensu lato comprises not only toxicity toward immune cells, but also biological reactions from immune cells exposed to toxicants, reactions that may have deleterious effects at the organismal level. Within this wide frame, a specific case of interest is represented by the response of macrophages to particulate materials, with the epitome examples of asbestos and crystalline silica. For such toxicants that are both persistent and often encountered in an occupational setting, i.e. at low but repeated doses, there is a need for in vitro systems that can take into account these two parameters. Currently, most in vitro systems are used in an acute exposure mode, i.e., with a single dose and a readout made shortly if not immediately after exposure. We describe here how adequate changes of the culture methods applied to the murine macrophage cell line J774A.1 enable longer periods of culture (several days), which represents a first opportunity to address the persistence and dose-rate issues. To respond to this, the protocol uses a reduction in the concentration of the animal serum used for cell culture, as well as a switch from fetal to adult serum, which is less rich in proliferation factors. By doing so, we have considerably reduced cell proliferation, which is a problem with cell lines when they are supposed to represent slowly-dividing cells such as resident macrophages. We also succeeded in maintaining the differentiated functions of macrophages, such as phagocytosis or inflammatory responses, over the whole culture period. Furthermore, the presence of serum, even at low concentrations, provides excellent cell viability and keeps the presence of a protein corona on particulate materials, a feature that is known to strongly modulate their effects on cells and is lost in serum-free culture. Besides data showing the impact of these conditions on macrophages cell line cultures, illustrative examples are shown on silica- and cobalt-based pigments.

Hydroxyl groups on cellulose nanocrystal surfaces form nucleation points for silver nanoparticles of varying shapes and sizes.

In this study, we investigate the interactions between the cellulose surface and Ag nanoparticles (AgNPs) for the purpose of manufacturing hybrid nanomaterials using bacterial cellulose nanocrystals (BCNs) as a model substrate. We focus on the role of the BCN surface chemistry on the AgNP nucleation obtained by chemical reduction of Ag+ ions. Homogeneous hybrid suspensions of BCN/AgNP are produced, regardless of whether the BCNs are quasi-neutral, negatively (TBCNs) or positively charged (ABCNs). The characterization of BCN/AgNP hybrids identifies the -OH surface groups as nucleation points for AgNPs, of about 20 nm revealing that surface charges only improve the accessibility to OH groups. X-ray Absorption technics (XANES and EXAFS) revealed a high metallic Ag0 content ranging from 88% to 97%. Moreover, the grafting of hydrophobic molecules on a BCN surface (HBCNs) does not prevent AgNP nucleation, illustrating the versatility of our method and the possibility to obtain bifunctional NPs. A H2O2 redox post-treatment on the hybrid induces an increase in AgNPs size, up to 90 nm as well as a shape variation (i.e., triangular). In contrast, H2O2 induces no size/shape variation for aggregated hybrids, emphasizing that the accessibility to -OH groups ensures the nucleation of bigger Ag nano-objects.

ZnO and TiO2 nanoparticles alter the ability of Bacillus subtilis to fight against a stress.

Due to the physicochemical properties of nanoparticles, the use of nanomaterials increases over time in industrial and medical processes. We herein report the negative impact of nanoparticles, using solid growth conditions mimicking a biofilm, on the ability of Bacillus subtilis to fight against a stress. Bacteria have been exposed to sublethal doses of nanoparticles corresponding to conditions that bacteria may meet in their natural biotopes, the upper layer of soil or the gut microbiome. The analysis of the proteomic data obtained by shotgun mass spectrometry have shown that several metabolic pathways are affected in response to nanoparticles, n-ZnO or n-TiO2, or zinc salt: the methyglyoxal and thiol metabolisms, the oxidative stress and the stringent responses. Nanoparticles being embedded in the agar medium, these impacts are the consequence of a physiological adaptation rather than a physical cell injury. Overall, these results show that nanoparticles, by altering bacterial physiology and especially the ability to resist to a stress, may have profound influences on a "good bacteria", Bacillus subtilis, in its natural biotope and moreover, on the global equilibrium of this biotope.

How Reversible Are the Effects of Fumed Silica on Macrophages? A Proteomics-Informed View.

Synthetic amorphous silica is one of the most used nanomaterials, and numerous toxicological studies have studied its effects. Most of these studies have used an acute exposure mode to investigate the effects immediately after exposure. However, this exposure modality does not allow the investigation of the persistence of the effects, which is a crucial aspect of silica toxicology, as exemplified by crystalline silica. In this paper, we extended the investigations by studying not only the responses immediately after exposure but also after a 72 h post-exposure recovery phase. We used a pyrolytic silica as the test nanomaterial, as this variant of synthetic amorphous silica has been shown to induce a more persistent inflammation in vivo than precipitated silica. To investigate macrophage responses to pyrolytic silica, we used a combination of proteomics and targeted experiments, which allowed us to show that most of the cellular functions that were altered immediately after exposure to pyrolytic silica at a subtoxic dose, such as energy metabolism and cell morphology, returned to normal at the end of the recovery period. However, some alterations, such as the inflammatory responses and some aldehyde detoxification proteins, were persistent. At the proteomic level, other alterations, such as proteins implicated in the endosomal/lysosomal pathway, were also persistent but resulted in normal function, thus suggesting cellular adaptation.

How Do the Different Proteomic Strategies Cope with the Complexity of Biological Regulations in a Multi-Omic World? Critical Appraisal and Suggestions for Improvements.

In this second decade of the 21st century, we are lucky enough to have different types of proteomic analyses at our disposal. Furthermore, other functional omics such as transcriptomics have also undergone major developments, resulting in mature tools. However, choice equals questions, and the major question is how each proteomic strategy is fit for which purpose. The aim of this opinion paper is to reposition the various proteomic strategies in the frame of what is known in terms of biological regulations in order to shed light on the power, limitations, and paths for improvement for the different proteomic setups. This should help biologists to select the best-suited proteomic strategy for their purposes in order not to be driven by raw availability or fashion arguments but rather by the best fitness for purpose. In particular, knowing the limitations of the different proteomic strategies helps in interpreting the results correctly and in devising the validation experiments that should be made downstream of the proteomic analyses.

What Room for Two-Dimensional Gel-Based Proteomics in a Shotgun Proteomics World?

Two-dimensional gel electrophoresis was instrumental in the birth of proteomics in the late 1980s. However, it is now often considered as an outdated technique for proteomics-a thing of the past. Although this opinion may be true for some biological questions, e.g., when analysis depth is of critical importance, for many others, two-dimensional gel electrophoresis-based proteomics still has a lot to offer. This is because of its robustness, its ability to separate proteoforms, and its easy interface with many powerful biochemistry techniques (including western blotting). This paper reviews where and why two-dimensional gel electrophoresis-based proteomics can still be profitably used. It emerges that, rather than being a thing of the past, two-dimensional gel electrophoresis-based proteomics is still highly valuable for many studies. Thus, its use cannot be dismissed on simple fashion arguments and, as usual, in science, the tree is to be judged by the fruit.

Toxicity to RAW264.7 Macrophages of Silica Nanoparticles and the E551 Food Additive, in Combination with Genotoxic Agents.

Synthetic amorphous silica (SAS) is used in a plethora of applications and included in many daily products to which humans are exposed via inhalation, ingestion, or skin contact. This poses the question of their potential toxicity, particularly towards macrophages, which show specific sensitivity to this material. SAS represents an ideal candidate for the adsorption of environmental contaminants due to its large surface area and could consequently modulate their toxicity. In this study, we assessed the toxicity towards macrophages and intestinal epithelial cells of three SAS particles, either isolated SiO2 nanoparticles (LS30) or SiO2 particles composed of agglomerated-aggregates of fused primary particles, either food-grade (E551) or non-food-grade (Fumed silica). These particles were applied to cells either alone or in combination with genotoxic co-contaminants, i.e., benzo[a]pyrene (B[a]P) and methane methylsulfonate (MMS). We show that macrophages are much more sensitive to these toxic agents than a non-differenciated co-culture of Caco-2 and HT29-MTX cells, used here as a model of intestinal epithelium. Co-exposure to SiO2 and MMS causes DNA damage in a synergistic way, which is not explained by the modulation of DNA repair protein mRNA expression. Together, this suggests that SiO2 particles could adsorb genotoxic agents on their surface and, consequently, increase their DNA damaging potential.