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Immune System
Abstract
This chapter deals with adverse effects of engineered nanomaterials on the immune system. Giving special attention to the immune system in the context of nanosafety is well justified; the immune system is not only enriched at the portals of entry of pathogenic microorganisms, but also nanoparticles, into the body. Moreover, the immune system has evolved to recognize foreign (nonself) entities and to determine whether or not they pose a threat to the organism. We will discuss effects on both the innate and adaptive arms of the immune system. It is worth noting that an inflammatory reaction is only a sign of a defense reaction and does not as such indicate whether the reaction is a detrimental one. We also discuss immune activation and immune suppression by nanomaterials and the implications of such effects for the biomedical exploitation of nanomaterials.
... Second, knowing how the immune system recognises and eliminates NPs will help with designing nanomedical products (e.g., drug delivery systems) not only capable of modulating the immune responses, but also of escaping immune surveillance thereby exerting more effectively their therapeutic potential. Thus, in the current safe-by-design approach to nanotechnological and nanomedical products, assessing how NPs interact with the immune defences is a crucial issue in determining NP safety versus hazard for human as well as for environmental health [1][2][3][4]. ...
... In these functions, innate immune factors (danger signals, inflammatory cytokines) are part of the extra-and intracellular environment that determines the outcome of exposure to an antigen. The resulting adaptive immune response towards nanoparticles has been discussed in detail elsewhere [4]. ...
The innate immune system provides the first line of defence against foreign microbes and particulate materials. Engineered nanoparticles can interact with the immune system in many different ways. Nanoparticles may thus elicit inflammation with engagement of neutrophils, macrophages and other effector cells; however, it is important to distinguish between acute and chronic inflammation in order to identify the potential hazards of nanoparticles for human health. Nanoparticles may also interact with and become internalised by dendritic cells, key antigen-presenting cells of the immune system, where a better understanding of these processes could pave the way for improved vaccination strategies. Nanoparticle characteristics such as size, shape and deformability also influence nanoparticle uptake by a plethora of immune cells and subsequent immune responses. Furthermore, the corona of adsorbed biomolecules on nanoparticle surfaces should not be neglected. Complement activation represents a special case of regulated and dynamic corona formation on nanoparticles with important implications in clearance and safety. Additionally, the inadvertent binding of bacterial lipopolysaccharide to nanoparticles is important to consider as this may skew the outcome and interpretation of immunotoxicological studies. Here, we discuss nanoparticle interactions with different cell types and soluble mediators belonging to the innate immune system.
... The key elements in the identification of NM-induced immunotoxicity are duration and deregulation of the inflammatory response. Therefore, reliable testing requires relevant in vitro and in vivo models, that can distinguish between normal and pathological responses (Boraschi et al., 2017). Due to their small size, NMs may escape the particleclearing defensive mechanisms (e.g.phagocytosis), and so many of them do not trigger a direct inflammatory response (Kunzmann et al., 2011). ...
... Another factor contributing to autoimmunity is the defective clearance of apoptotic cells by scavenger phagocytes (Mu~ noz et al., 2010). Activation of inflammasomes can occur via several mechanisms (rupture of lysosomes, direct recognition of NMs by toll-like receptors) (Boraschi et al., 2017). Neutrophils or polymorphonuclear granulocytes play a key role in NM-induced inflammation (Bhattacharya et al., 2013). ...
The unique properties of nanomaterials (NMs) are beneficial in numerous industrial and medical applications. However, they could also induce unintended effects. Thus, a proper strategy for toxicity testing is essential in human hazard and risk assessment. Toxicity can be tested in vivo and in vitro; in compliance with the 3Rs, alternative strategies for in vitro testing should be further developed for NMs. Robust, standardized methods are of great importance in nanotoxicology, with comprehensive material characterization and uptake as an integral part of the testing strategy. Oxidative stress has been shown to be an underlying mechanism of possible toxicity of NMs, causing both immunotoxicity and genotoxicity. For testing NMs in vitro, a battery of tests should be performed on cells of human origin, either cell lines or primary cells, in conditions as close as possible to an in vivo situation. Novel toxicity pathways particularly epigenetic modification should be assessed along with conventional toxicity testing methods. However, to initiate epigenetic toxicity screens for NM exposure, there is a need to better understand their adverse effects on the epigenome, to identify robust and reproducible causal links between exposure, epigenetic changes and adverse phenotypic endpoints and to develop improved assays to monitor epigenetic toxicity.
... The immune system comprises of the innate and acquired immune system. The major components of the innate immune system are the professional phagocytic cells such as macrophages, along with neutrophils, mast cells, eosinophils and basophils (Farrera and Fadeel, 2015;Boraschi et al., 2017). Natural killer (NK) cells also belong to the innate immune system and are specialized in warding of cancerous cells. ...
The Institute of Environmental Medicine (IMM), a department at Karolinska Institutet in Sweden, has published a report on nanotoxicology. The report is a collaboration between scientists from several different units at IMM, and the work has been coordinated by professor Bengt Fadeel, head of the unit of molecular toxicology and chair of SweNanoSafe´s Expert Panel.
The report entitled Nanotoxicology – State-of-the-Art and Future Research Needs provides an overview of current knowledge within the area of nanotoxicology, with a particular emphasis on the hazard assessment of nanomaterials for human health. The effects on other species in the natural environment are not discussed. In addition, knowledge gaps and future research needs in nanosafety are identified.
... Many studies showed that NPs may have adverse effects on health, mainly due to their small size, large surface area per mass and highly reactive properties (Bast us et al. 2008;Ge et al. 2012;Li et al. 2012;Wang et al. 2013). Among the different NP effects in biological systems, activation of immune responses is considered a central element for assessing health risks of NPs (Boraschi and Duschl 2012;). ...
Nanoparticles (NPs) are easily contaminated by bacterial endotoxin (lipopolysaccharide [LPS]). The presence of LPS can be responsible for many immune/inflammatory effects attributed to NPs. In this study, we examined the effects of LPS adsorption on the NP surface on the formation of a biocorona in biological fluids and on the subsequent inflammation-inducing activity of NPs. Different gold (Au) NPs with sizes ranging from 10 to 80 nm and with different surface functionalization (sodium citrate, lipoic acid, and branched polyethyleneimine (BPEI), or polyethylene glycol (PEG)) were exposed to E. coli LPS under different conditions. The binding capacity of LPS to the surface of AuNPs was dose- and time-dependent. LPS attached to sodium citrate and lipoic acid coatings, but did not adhere to BPEI- or PEG-coated NPs. By computational simulation, the binding of LPS to AuNPs seems to follow the Langmuir absorption isotherm. The presence of LPS on AuNP surface interfered and caused a decrease in the formation of the expected biomolecular corona upon incubation in human plasma. LPS-coated AuNPs, but not the LPS-free NPs, induced significant inflammatory responses in vitro. Notably, while free LPS did also induce an anti-inflammatory response, LPS bound to NPs appeared unable to do so. In conclusion, the unintentional adsorption of LPS onto the NP surface can affect the biocorona formation and the inflammatory properties of NPs. Thus, for an accurate interpretation of NP interactions with cells, it is extremely important to be able to distinguish the intrinsic NP biological effects from those caused by biologically active contaminants such as endotoxin.
Silicon-based materials and their oxides are widely used in drug delivery, dietary supplements, implants and dental fillers. Silica nanoparticles (SiNPs) interact with immunocompetent cells and induce immunotoxicity. However, the toxic effects of SiNPs on the immune system have been inadequately reviewed. The toxicity of SiNPs to the immune system depends on their physicochemical properties and the cell type. Assessments of immunotoxicity include determining cell dysfunctions, cytotoxicity and genotoxicity. This review focuses on the immunotoxicity of SiNPs and investigates the underlying mechanisms. The main mechanisms were proinflammatory responses, oxidative stress and autophagy. Considering the toxicity of SiNPs, surface and shape modifications may mitigate the toxic effects of SiNPs, providing a new way to produce these nanomaterials with less toxic impaction.
Carbon-based nanomaterials including carbon nanotubes (CNTs) have been shown to trigger inflammation. However, how these materials are 'sensed' by immune cells is not known. Here we compared the effects of two carbon-based nanomaterials, single-walled CNTs (SWCNTs) and graphene oxide (GO), on primary human monocyte-derived macrophages. Genome-wide transcriptomics assessment was performed at sub-cytotoxic doses. Pathway analysis of the microarray data revealed pronounced effects on chemokine-encoding genes in macrophages exposed to SWCNTs, but not in response to GO, and these results were validated by multiplex array-based cytokine and chemokine profiling. Conditioned medium from SWCNT-exposed cells acted as a chemoattractant for dendritic cells. Chemokine secretion was reduced upon inhibition of NF-κB, as predicted by upstream regulator analysis of the transcriptomics data, and Toll-like receptors (TLRs) and their adaptor molecule, MyD88 were shown to be important for CCL5 secretion. Moreover, a specific role for TLR2/4 was confirmed by using reporter cell lines. Computational studies to elucidate how SWCNTs may interact with TLR4 in the absence of a protein corona suggested that binding is guided mainly by hydrophobic interactions. Taken together, these results imply that CNTs may be 'sensed' as pathogens by immune cells.
Nanomaterials may be contaminated with bacterial endotoxin during production and handling, which may confound toxicological testing of these materials, not least when assessing for immunotoxicity. In the present study, we evaluated the conventional Limulus amebocyte lysate (LAL) assay for endotoxin detection in graphene based material (GBM) samples, including graphene oxide (GO) and few-layered graphene (FLG). Our results showed that some GO samples interfered with various formats of the LAL assay. To overcome this problem, we developed a TNF-α expression test (TET) using primary human monocyte-derived macrophages incubated in the presence or absence of the endotoxin inhibitor, polymyxin B sulfate, and found that this assay, performed with non-cytotoxic doses of the GBM samples, enabled unequivocal detection of endotoxin with a sensitivity that is comparable to the LAL assay. FLG also triggered TNF-α production in the presence of the LPS inhibitor, pointing to an intrinsic pro-inflammatory effect. Finally, we present guidelines for the preparation of endotoxin-free GO, validated by using the TET.
Background
Carbonaceous nanoparticles (CNP) represent a major constituent of urban particulate air pollution, and inhalation of high CNP levels has been described to trigger a pro-inflammatory response of the lung. While several studies identified specific particle characteristics driving respiratory toxicity of low-solubility and low-toxicity particles such as CNP, the major lung cell type, which initiates and drives that response, remains still uncertain. Since alveolar macrophages (AM) are known to effectively phagocytose inhaled particles and play a crucial role for the initiation of pulmonary inflammation caused by invading microbes, we aimed to determine their role for sterile stimuli such as CNP by profiling the primary alveolar cell compartments of the lung. We exposed C57BL/6 mice to 20 μg CNP by intratracheal instillation and comprehensively investigated the expression of the underlying mediators during a time span of 3 to 72 h in three different lung cell populations: CD45- (negative) structural cells, CD45+ (positive) leukocytes, and by BAL recovered cells.
Results
Bronchoalveolar lavage (BAL) analysis revealed an acute inflammatory response characterized by the most prominent culmination of neutrophil granulocytes from 12 to 24 h after instillation, which declined to basal levels by day 7. As early as 3 h after CNP exposure 50 % of the AM revealed particle laden. BAL concentrations and lung gene expression profiles of TNFα, and the neutrophil chemoattractants CXCL1,-2 and-5 preceded the neutrophil recruitment and showed highest levels after 12 h of CNP exposure, pointing to a significant activation of the inflammation-evoking lung cells at this point of time. AM, isolated from lungs 3 to 12 h after CNP instillation, however, did not show a pro-inflammatory signature. On the contrary, gene expression analysis of different lung cell populations isolated 12 h after CNP instillation revealed CD45-, mainly representing alveolar epithelial type II (ATII) cells as major producer of inflammatory CXCL cytokines. Particularly by CD45- cells expressed Cxcl5 proved to be the most abundant chemokine, being 12 h after CNP exposure 24 (±11) fold induced.
Conclusion
Our data suggests that AM are noninvolved in the initiation of the inflammatory response. ATII cells, which induced highest CXCL levels early on, might in contrast be the driver of acute neutrophilic inflammation upon pulmonary CNP exposure.
Electronic supplementary material
The online version of this article (doi:10.1186/s12989-016-0144-6) contains supplementary material, which is available to authorized users.
The study of toxicity and potential risks of engineered nanoparticles is of particular importance in nanomedicine. Endotoxin, a common contaminant of bacterial origin, has biological effects that can mask the true biological effects of nanoparticles, if its presence is overlooked. In this review, we report the features of nanoparticle contamination by endotoxin, and the different biological effects of endotoxin-contaminated nanoparticles. We will describe different methods for endotoxin detection applied to nanoparticles, and discuss their pros and cons. Eventually, we describe various methods for eliminating endotoxin contamination in nanoparticles. Although there is no universal technique for efficiently removing endotoxin from nanoparticles, specific solutions can be found case by case, which can allow us to perform nanosafety studies in biologically relevant conditions.
Engineered nanomaterials (ENMs) interact with different biomolecules as soon as they are in contact, resulting in the formation of a biomolecule ‘corona’. Hence, the ‘corona’ defines the biological identity of the ENMs and could affect the response of the immune system to ENM exposure. With up to 40 % of the world population suffering from type I allergy, a possible modulation of allergen effects by binding to ENMs is highly relevant with respect to work place and consumer safety. Therefore, the aim of this present study was to gain an insight into the interactions of gold nanoparticles with different seasonally and perennially occurring outdoor and indoor allergens.
Gold nanoparticles (AuNPs) were conjugated with the major allergens of birch pollen (Bet v 1), timothy grass pollen (Phl p 5) and house dust mite (Der p 1). The AuNP-allergen conjugates were characterized by means of TEM negative staining, dynamic light scattering (DLS), z-potential measurements and hyperspectral imaging. Furthermore, 3D models were constructed, based on the characterization data, to visualize the interaction between the allergens and the AuNPs surface. Differences in the activation of human basophil cells derived from birch/grass pollen- and house dust mite-allergic patients in response to free allergen and AuNP-allergen conjugates were determined using the basophil activation assay (BAT). Potential allergen corona replacement during BAT was controlled for using Western blotting. The protease activity of AuNP-Der p 1 conjugates compared to free Der p 1 was assessed, by an enzymatic activity assay and a cellular assay pertaining to lung type II alveolar epithelial cell tight junction integrity.
The formation of a stable corona was found for all three allergens used. Our data suggest, that depending on the allergen, different effects are observed after binding to ENMs, including enhanced allergic responses against Der p 1 and also, for some patients, against Bet v 1. Moreover elevated protease activity of AuNP-Der p 1 conjugates compared to free Der p 1 was found.
In summary, this study presents that conjugation of allergens to ENMs can modulate the human allergic response, and that protease activity can be increased.
Graphical Abstract
Cross-linking of IgE receptors and degranulation of human basophils due to epitope alignment of nanoparticle-coated allergens.
This study evaluated the in vitro effects of 62.5 µg/mL silica nanoparticles (SiO2 NPs) on MRC-5 human lung fibroblast cells for 24, 48 and 72 h. The nanoparticles’ morphology, composition, and structure were investigated using high resolution transmission electron microscopy, selected area electron diffraction and X-ray diffraction. Our study showed a decreased cell viability and the induction of cellular oxidative stress as evidenced by an increased level of reactive oxygen species (ROS), carbonyl groups, and advanced oxidation protein products after 24, 48, and 72 h, as well as a decreased concentration of glutathione (GSH) and protein sulfhydryl groups. The protein expression of Hsp27, Hsp60, and Hsp90 decreased at all time intervals, while the level of protein Hsp70 remained unchanged during the exposure. Similarly, the expression of p53, MDM2 and Bcl-2 was significantly decreased for all time intervals, while the expression of Bax, a marker for apoptosis, was insignificantly downregulated. These results correlated with the increase of pro-caspase 3 expression. The role of autophagy in cellular response to SiO2 NPs was demonstrated by a fluorescence-labeled method and by an increased level of LC3-II/LC3-I ratio. Taken together, our data suggested that SiO2 NPs induced ROS-mediated autophagy in MRC-5 cells as a possible mechanism of cell survival.
Unlabelled:
Carbon-based nanomaterials including carbon nanotubes, graphene oxide, fullerenes and nanodiamonds are potential candidates for various applications in medicine such as drug delivery and imaging. However, the successful translation of nanomaterials for biomedical applications is predicated on a detailed understanding of the biological interactions of these materials. Indeed, the potential impact of the so-called bio-corona of proteins, lipids, and other biomolecules on the fate of nanomaterials in the body should not be ignored. Enzymatic degradation of carbon-based nanomaterials by immune-competent cells serves as a special case of bio-corona interactions with important implications for the medical use of such nanomaterials. In the present review, we highlight emerging biomedical applications of carbon-based nanomaterials. We also discuss recent studies on nanomaterial 'coronation' and how this impacts on biodistribution and targeting along with studies on the enzymatic degradation of carbon-based nanomaterials, and the role of surface modification of nanomaterials for these biological interactions.
From the clinical editor:
Advances in technology have produced many carbon-based nanomaterials. These are increasingly being investigated for the use in diagnostics and therapeutics. Nonetheless, there remains a knowledge gap in terms of the understanding of the biological interactions of these materials. In this paper, the authors provided a comprehensive review on the recent biomedical applications and the interactions of various carbon-based nanomaterials.
In the last years, nanotechnologies have raised great interest because of the potential applications of engineered nanoparticles in nanomedicine (i.e., in vaccination, in diagnostic imaging procedures, and as therapeutic drug delivery systems). The use of nanoparticles in medicine has brought about the issue of their interaction with the immune system for two main reasons: first, understanding how long nanomedicines could persist in the organism and exert their beneficial effects before being recognized and eliminated by our defensive systems; second, understanding how the immune responses can be modulated by nanoparticles in order to obtain optimal effects. This issue is crucial in vaccine formulations based on the use of nanoparticles, which can operate both as a delivery system to enhance antigen processing and as an immunostimulatory adjuvant to induce and amplify protective immunity, in part because of their ability to activate the inflammasome and induce the maturation of interleukin 1β. Nanoparticles can be excellent adjuvants due to their biocompatibility and their physicochemical properties (e.g., size, shape, and surface charge), which can be tailored to obtain different immunological effects. This review provides an overview of recent strategies for the use of nanoparticles as promising/attractive adjuvants for novel prophylactic and therapeutic vaccines. The use of nanovaccines, with their practically infinite possibilities of specific design, could open the way to precision vaccinology, i.e., vaccine formulations tailored on the individual immune reactivity status.
Recent findings in the field of immune memory have demonstrated that B and T cell mediated immunity following infections are enhanced by the so-called
trained immunity
. This effect has been most extensively investigated for the tuberculosis vaccine strain Bacillus Calmette-Guérin (BCG). Epidemiological studies suggest that this vaccine is associated with a substantial reduction in overall child mortality that cannot be solely explained by prevention of the target disease but that it seems to rely on inducing resistance to other infections. Upon infection or vaccination, monocytes/macrophages can be functionally reprogrammed so as to display an enhanced defensive response against unrelated infections. Epigenetic modifications seem to play a key role in the induction of this “innate memory.” These findings are revolutionising our knowledge of the immune system, introducing the concept of memory also for mammalian innate immunity. Thus, vaccines are likely to nonspecifically affect the overall immunological status of individuals in a clinically relevant manner. As a consequence, future vaccine strategies ought to take into account the contribution of innate memory through appropriate design of formulations and administration scheduling.
The adsorption of proteins and their layering onto nanoparticle surfaces has been called the ‘protein corona’. This dynamic process of protein adsorption has been extensively studied following in vitro incubation of many different nanoparticles with plasma proteins. However, the formation of protein corona under dynamic, in vivo conditions remains largely unexplored. Extrapolation of in vitro formed protein coronas to predict the fate and possible toxicological burden from nanoparticles in vivo is of great interest. However complete lack of such direct comparisons for clinically used nanoparticles makes the study of in vitro and in vivo formed protein coronas of great importance. Our aim was to study the in vivo protein corona formed onto intravenously injected, clinically used liposomes, based on the composition of the PEGylated liposomal formulation that constitutes the anticancer agent Doxil®. The formation of in vivo protein corona was determined after the recovery of the liposomes from the blood circulation of CD-1 mice 10 minutes post-injection. In comparison, in vitro protein corona was formed by the incubation of liposomes in CD-1 mouse plasma. In vivo and in vitro formed protein coronas were compared in terms of morphology, composition and cellular internalisation. The protein coronas on bare (non-PEGylated) and monoclonal antibody (IgG) targeted liposomes of the same lipid composition were also comparatively investigated. A network of linear fibrillary structures constituted the in vitro formed protein corona, whereas the in vivo corona had a different morphology but did not appear to coat the liposome surface entirely. Even though the total amount of protein attached on circulating liposomes correlated with that observed from in vitro incubations, the variety of molecular species in the in vivo corona were considerably wider. Both in vitro and in vivo formed protein coronas were found to significantly reduce receptor binding and cellular internalisation of antibody-conjugated liposomes, however the in vivo corona formation did not lead to complete ablation of their targeting capability.
A thorough understanding of how proteins induce nanoparticle (NP) aggregation is crucial when designing in vitro and in vivo assays and interpreting experimental results. This knowledge is also crucial when developing nano-applications and formulation for drug delivery systems. In this study, we found that extraction of immunoglobulin G (IgG) from cow serum results in lower polystyrene NPs aggregation. Moreover, addition of isolated IgG or fibrinogen to fetal cow serum enhanced this aggregation, thus demonstrating that these factors are major drivers of NP aggregation in serum. Counter-intuitively, NP aggregation was inversely dependent on protein concentration; i.e., low protein concentrations induced large aggregates, whereas high protein concentrations induced small aggregates. Protein-induced NP aggregation and aggregate size were monitored by absorbance at 400 nm and dynamic light scattering, respectively. Here, we propose a mechanism behind the protein concentration dependent aggregation; this mechanism involves the effects of multiple protein interactions on the NP surface, surface area limitations, aggregation kinetics, and the influence of other serum proteins.
Liposome-protamine-DNA nanoparticles (LPD) are safe, effective, and non-toxic adjuvants that induce Th1-like immune responses. We hypothesized that encapsulation of allergens into liposomes could be an appropriate option for immunotherapy. The present study evaluated the immunotherapeutic potential of a recombinant hybrid molecule (rHM) encapsulated in LPD nanoparticles in a murine model of Chenopodium album allergy. BALB/c mice were sensitized with the allergen in alum, and the immunotherapy procedure was performed by subcutaneous injections of LPD-rHM, rHM, or empty LPD at weekly intervals. Sensitized mice developed a Th2-biased immune response characterized by strong specific IgG1 and IgE production, IL-4, and the transcription factor GATA3 in spleen cell cultures. Treatment with the LPD-rHM resulted in a reduction in IgE and a marked increase in IgG2a. The LPD-rHM induced allergen-specific responses with relatively high interferon-gamma production, as well as expression of the transcription factor T-bet in stimulated splenocytes. In addition, lymphoproliferative responses were higher in the LPD-rHM-treated mice than in the other groups. Removal of the nanoparticles from the rHM resulted in a decrease in the allergen's immunogenicity. These results indicate that the rHM complexed with LPD nanoparticles has a marked suppressive effect on the allergic response and caused a shift toward a Th1 pathway.
During last decades, changes have been observed in the frequency of different histological subtypes of lung cancer - one of the most common causes of morbidity and mortality - with a declining proportion of squamous cell carcinomas and an increasing proportion of adenocarcinomas, particularly in developed countries. This suggests the emergence of new etiological factors and mechanisms including those defining the lung microenvironment promoting tumor growth. Assuming that the lung is the main portal of entry for broadly used nanomaterials and their established pro-inflammatory propensities, we hypothesized that nanomaterials may contribute to changes facilitating tumor growth. Here we report that an acute exposure to single-walled carbon nanotubes (SWCNT) induces recruitment and accumulation of lung-associated myeloid-derived suppressor cells (MDSC) and MDSC-derived production of TGF-β resulting in up-regulated tumor burden in the lung. The production of TGF-β by MDSC requires their interaction with both SWCNT and tumor cells. We conclude that pulmonary exposure to SWCNT favors the formation of a niche that supports ingrowth of lung carcinoma in vivo via activation of TGF-β production by SWCNT-attracted and pre-sensitized MDSC.
Copyright © 2015, American Association for Cancer Research.
In the past decade, the increasing amount of nanoparticles (NPs) and nanomaterials used in multiple applications led the scientific community to investigate the potential toxicity of NPs. Many studies highlighted the cytotoxic effects of various NPs, including titanium dioxide, zinc oxide and silver nanoparticles (AgNPs). In a few studies, endoplasmic reticulum (ER) stress was found to be associated with NP cytotoxicity leading to apoptosis in different cell types. In this study, we report for the first time that silver nanoparticles of 15 nm (AgNP15), depending on the concentration, induced different signature ER stress markers in human THP-1 monocytes leading to a rapid ER stress response with degradation of the ATF-6 sensor. Also, AgNP15 induced pyroptosis and activation of the NLRP-3 inflammasome as demonstrated by the processing and increased activity of caspase-1, secretion of IL-1β and ASC pyroptosome formation. Transfection of THP-1 cells with siRNA targeting NLRP-3 decreased the AgNP15-induced IL-1β production. Absence of caspase-4 expression resulted in a significant reduction of pro-IL-1β. However, caspase-1 activity was significantly higher in caspase-4 deficient cells compared to WT cells. Inhibition of AgNP15-induced ATF-6 degradation with Site-2 protease inhibitors completely blocked the effect of AgNP15 on pyroptosis and secretion of IL-1β, indicating that ATF-6 is crucial for the induction of this type of cell death. We conclude that AgNP15 induce degradation of the ER stress sensor ATF-6, leading to activation of the NLRP-3 inflammasome regulated by caspase-4 in human monocytes.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c+ dendritic cells (DCs) with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002-2 ng/ml). We show that CD1c+ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated with high CD14 expression levels observed in CD1c+ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.
In contrast to short-lived neutrophils, macrophages display persistent presence in the lung of animals after pulmonary exposure to carbon nanotubes. While effective in clearance of bacterial pathogens and injured host cells, the ability of macrophages to "digest" carbonaceous nanoparticles has not been documented. Here, we used chemical, biochemical, cell and animal models and demonstrated oxidative biodegradation of oxidatively functionalized single walled carbon nanotubes via superoxide/NO*-> peroxynitrite driven oxidative pathways of activated macrophages facilitating clearance of nanoparticles from the lung.
Modulation of immune responses for therapeutic purposes is a particularly relevant area, given the central role of anomalous immunity in a wide variety of diseases, from the most typically immune-related syndromes (autoimmune diseases, allergy and asthma, immunodeficiencies) to those in which altered immunity and inflammation define the pathological outcomes (chronic infections, tumors, chronic inflammatory and degenerative diseases, metabolic disorders, etc.). This brief review will summarize some of the most promising perspectives of immunopharmacology, in particular in the area of immunosuppression, by considering the following aspects: molecular pathways: investigating the fine mechanisms of immune modulation, to define novel targets and new clinical applications, with particular emphasis to approaches for re-education of anomalous immunity, including induction of tolerance; personalized medicine: how pharmacogenomics can help in selecting the optimal treatment for each patient, focusing on human immunology for obtaining relevant information that animal models cannot provide; microbiome: considering immune responses as the result of the interaction between human cells and commensal bacteria, for a new perspective in the use of immunosuppressive treatments; natural products: providing a scientific basis to the huge body of pharmacological knowledge and products of traditional medicine for identifying new active molecules and new treatment concepts; nanomedicine: exploiting the intelligent design of engineered nanoparticles offered by nanotechnologies in tailoring shape-, size-, surface-controlled carriers for targeted delivery of drugs.
The success of nanoparticle-based cancer therapies ultimately depends on their ability to selectively and efficiently accumulate in regions of disease. Outfitting nanoparticles to actively target tumor-specific markers has improved specificity, yet it remains a challenge to amass adequate therapy in a selective manner. To help address this challenge, we have developed a mechanism of nanoparticle amplification based on stigmergic (environment-modifying) signalling, in which a "Signalling" population of gold nanorods induces localized unveiling of cryptic collagen epitopes, which are in turn targeted by "Responding" nanoparticles bearing gelatin-binding fibronectin fragments. We demonstrate that this two-particle system results in significantly increased, selective recruitment of responding particles. Such amplification strategies have the potential to overcome limitations associated with single-particle targeting by leveraging the capacity of nanoparticles to interact with their environment to create abundant new binding motifs.
Interaction of nanoparticles with proteins is the basis of nanoparticle bio-reactivity. This interaction gives rise to the formation of a dynamic nanoparticle-protein corona. The protein corona may influence cellular uptake, inflammation, accumulation, degradation and clearance of the nanoparticles. Furthermore, the nanoparticle surface can induce conformational changes in adsorbed protein molecules which may affect the overall bio-reactivity of the nanoparticle. In depth understanding of such interactions can be directed towards generating bio-compatible nanomaterials with controlled surface characteristics in a biological environment. The main aim of this review is to summarise current knowledge on factors that influence nanoparticle-protein interactions and their implications on cellular uptake.
Although the potential human health impacts from exposure to engineered nanoparticles (ENPs) are uncertain, past epidemiological studies have established correlations between exposure to ambient air pollution particulates and the incidence of pneumonia and lung infections. Using amorphous silica and superparamagnetic iron oxide (SPIO) as model high production volume ENPs, we examined how macrophage activation by bacterial lipopolysaccharide (LPS) or the lung pathogen Streptococcus pneumoniae is altered by ENP pre-treatment. Neither silica nor SPIO treatment elicited direct cytotoxic or pro-inflammatory effects in bone marrow-derived macrophages. However, pre-treatment of macrophages with SPIO caused extensive reprogramming of nearly 500 genes regulated in response to LPS challenge, hallmarked by exaggerated activation of oxidative stress response pathways and suppressed activation of both pro- and anti-inflammatory pathways. Silica pre-treatment altered regulation of only 67 genes, but there was strong correlation with gene sets affected by SPIO. Macrophages exposed to SPIO displayed a phenotype suggesting an impaired ability to transition from a M1 to M2-like activation state, characterized by suppressed IL-10 induction, enhanced TNF production, and diminished phagocytic activity toward S. pneumoniae. Studies in macrophages deficient in scavenger receptor A (SR-A) showed SR-A participates in cell uptake of both the ENPs and S. pneumonia, and co-regulates the anti-inflammatory IL-10 pathway. Thus, mechanisms for dysregulation of innate immunity exist by virtue that common receptor recognition pathways are used by some ENPs and pathogenic bacteria, although the extent of transcriptional reprogramming of macrophage function depends on the physicochemical properties of the ENP after internalization. Our results also illustrate that biological effects of ENPs may be indirectly manifested only after challenging normal cell function. Nanotoxicology screening strategies should therefore consider how exposure to these materials alters susceptibility to other environmental exposures.
Many people suffer from metal allergy, and the recently demonstrated presence of naturally occurring metal nanoparticles in our environment could present a new candidate for inducing metal allergy. Here, we show that mice pretreated with silver nanoparticles (nAg) and lipopolysaccharides, but not with the silver ions that are thought to cause allergies, developed allergic inflammation in response to the silver. nAg-induced acquired immune responses depended on CD4(+) T cells and elicited IL-17A-mediated inflammation, similar to that observed in human metal allergy. Nickel nanoparticles also caused sensitization in the mice, whereas gold and silica nanoparticles, which are minimally ionizable, did not. Quantitative analysis of the silver distribution suggested that small nAg (≤10 nm) transferred to the draining lymph node and released ions more readily than large nAg (>10 nm). These results suggest that metal nanoparticles served as ion carriers to enable metal sensitization. Our data demonstrate a potentially new trigger for metal allergy.
The current gold standard to reduce non-specific cellular uptake of drug delivery vehicles is by covalent attachment of poly(ethylene glycol) (PEG). It is thought that PEG can reduce protein adsorption and thereby confer a stealth effect. Here, we show that polystyrene nanocarriers that have been modified with PEG or poly(ethyl ethylene phosphate) (PEEP) and exposed to plasma proteins exhibit a low cellular uptake, whereas those not exposed to plasma proteins show high non-specific uptake. Mass spectrometric analysis revealed that exposed nanocarriers formed a protein corona that contains an abundance of clusterin proteins (also known as apolipoprotein J). When the polymer-modified nanocarriers were incubated with clusterin, non-specific cellular uptake could be reduced. Our results show that in addition to reducing protein adsorption, PEG, and now PEEPs, can affect the composition of the protein corona that forms around nanocarriers, and the presence of distinct proteins is necessary to prevent non-specific cellular uptake.
There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. The evidence suggests marginal uptake of C60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Computational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal deformation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. The surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis.
Neutrophils express pattern recognition receptors (PRRs) and regulate immune responses via PRR-dependent cytokine production. An emerging theme is that neutrophil PRRs often exhibit cell type-specific adaptations in their signalling pathways. This prompted us to examine inflammasome signalling by the PRR NLRP3 in murine neutrophils, in comparison to well-established NLRP3 signalling pathways in macrophages. Here, we demonstrate that while murine neutrophils can indeed signal via the NLRP3 inflammasome, neutrophil NLRP3 selectively responds to soluble agonists but not to the particulate/crystalline agonists that trigger NLRP3 activation in macrophages via phagolysosomal rupture. In keeping with this, alum did not trigger IL-1β production from human PMN, and the lysosomotropic peptide Leu-Leu-OMe stimulated only weak NLRP3-dependent IL-1β production from murine neutrophils, suggesting that lysosomal rupture is not a strong stimulus for NLRP3 activation in neutrophils. We validated our in vitro findings for poor neutrophil NLRP3 responses to particles in vivo, where we demonstrated that neutrophils do not significantly contribute to alum-induced IL-1β production in mice. In all, our studies highlight that myeloid cell identity and the nature of the danger signal can strongly influence signalling by a single PRR, thus shaping the nature of the resultant immune response. This article is protected by copyright. All rights reserved
Unlabelled:
When inhaled nanoparticles deposit in the lungs, they transit through respiratory tract lining fluid (RTLF) acquiring a biomolecular corona reflecting the interaction of the RTLF with the nanomaterial surface. Label-free snapshot proteomics was used to generate semi-quantitative profiles of corona proteins formed around silica (SiO2) and poly(vinyl) acetate (PVAc) nanoparticles in RTLF, the latter employed as an archetype drug delivery vehicle. The evolved PVAc corona was significantly enriched compared to that observed on SiO2 nanoparticles (698 vs. 429 proteins identified); however both coronas contained a substantial contribution from innate immunity proteins, including surfactant protein A, napsin A and complement (C1q and C3) proteins. Functional protein classification supports the hypothesis that corona formation in RTLF constitutes opsonisation, preparing particles for phagocytosis and clearance from the lungs. These data highlight how an understanding of the evolved corona is necessary for the design of inhaled nanomedicines with acceptable safety and tailored clearance profiles.
From the clinical editor:
Inhaled nanoparticles often acquire a layer of protein corona while they go through the respiratory tract. Here, the authors investigated the identity of these proteins. The proper identification would improve the understanding of the use of inhaled nanoparticles in future therapeutics.
The delivery of drugs, antigens, and imaging agents benefits from using nanotechnology-based carriers. The successful translation of nanoformulations to the clinic involves thorough assessment of their safety profiles, which, among other end-points, includes evaluation of immunotoxicity. The past decade of research focusing on nanoparticle interaction with the immune system has been fruitful in terms of understanding the basics of nanoparticle immunocompatibility, developing a bioanalytical infrastructure to screen for nanoparticle-mediated immune reactions, beginning to uncover the mechanisms of nanoparticle immunotoxicity, and utilizing current knowledge about the structure-activity relationship between nanoparticles' physicochemical properties and their effects on the immune system to guide safe drug delivery. In the present review, we focus on the most prominent pieces of the nanoparticle-immune system puzzle and discuss the achievements, disappointments, and lessons learned over the past 15years of research on the immunotoxicity of engineered nanomaterials.
Notwithstanding rapid advances of nanotechnology in diagnostic imaging and drug delivery, the engineered nanocarriers still exhibit substantial lack of hemocompatibility. Thus, when injected systemically, nanoparticles are avidly recognized by blood leukocytes and platelets, but the mechanisms of immune recognition are not well understood and strategies to mitigate these phenomena remain underexplored. Using superparamagnetic dextran iron oxide (SPIO) nanoworms (NWs) we demonstrate an efficient and predominantly complement-dependent uptake by mouse lymphocytes, neutrophils and monocytes from normal and tumor bearing mice in vitro. Following intravenous injection into wild type mice, blood leukocytes as well as platelets became magnetically labeled, while the labeling was decreased by 95% in complement C3-deficient mice. Using blood cells from healthy and cancer patient donors, we demonstrated that neutrophils, monocytes, lymphocytes and eosinophils took up SPIO NWs, and the uptake was prevented by EDTA (a general complement inhibitor) and by antiproperdin antibody (an inhibitor of the alternative pathway of the complement system). Cross-linking and hydrogelation of SPIO NWs surface by epichlorohydrin decreased C3 opsonization in mouse serum, and consequently reduced the uptake by mouse leukocytes by more than 70% in vivo. Remarkably, the cross-linked particles did not show a decrease in C3 opsonization in human serum, but showed a significant decrease (over 60%) of the uptake by human leukocytes. The residual uptake of cross-linked nanoparticles was completely blocked by EDTA. These findings demonstrate species differences in complement-mediated nanoparticle recognition and uptake by leukocytes, and further show that human hemocompatibility could be improved by inhibitors of complement alternative pathway and by nanoparticle surface coating. These results provide important insights into the mechanisms of hemocompatibility of nanomedicines.
The goal of this study was to investigate whether poly (lactic-co-glycolic) acid (PLGA) nanoparticles could enhance sublingual immunotherapy (SLIT) efficacy. BALB/c mice sensitized to rChe a 3 were treated sublingually either with soluble rChe a 3 (100μg/dose) or PLGA-encapsulated rChe a 3 (5, 25, or 50μg/dose). SLIT with PLGA-encapsulated rChe a 3 (equivalent to 25 and 50μg rChe a 3 per dose) led to significantly increased antigen-specific IgG2a, along with no effect on allergen-specific IgE and IgG1 antibody levels. In addition, interleukin 4 (IL-4) levels in restimulated splenocytes were significantly less, while interferon-γ (IFN-γ), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) levels, as well as Foxp3 expression, were significantly greater than in the control groups. Our findings suggest that PLGA nanoparticle-based vaccination may help rational development of sublingual immunotherapy through reduction of the needed allergen doses and also significantly enhanced systemic T regulatory (Treg) and T helper 1 (Th1) immune responses.
Despite numerous applications, the cellular-clearance mechanism of multi-walled carbon nanotubes (MWCNTs) has not been clearly established yet. Previous in vitro studies showed the ability of oxidative enzymes to induce nanotube degradation. Interestingly, these enzymes have the common capacity to produce reactive oxygen species (ROS). Here, we combined material and life science approaches for revealing an intra-cellular way taken by macrophages to degrade carbon nanotubes. We report the in situ monitoring of ROS-mediated-MWCNT degradation by liquid-cell transmission electron microscopy. Two degradation mechanisms induced by hydroxyl radicals were extracted from these unseen dynamic nanoscale investigations: a non-site-specific thinning process of the walls and a site-specific transversal drilling process on pre-existing defects of nanotubes. Remarkably, similar ROS-induced structural injuries were observed on MWCNTs after aging into macrophages from one to seven days. Beside unraveling oxidative transformations of MWCNT structure, we elucidated an important, albeit not exclusive, biological pathway for MWCNT degradation in macrophages, involving NOX2 complex activation, superoxide production and hydroxyl radical attack, which highlights the critical role of oxidative stress in cellular processing of MWCNTs.
The new generation vaccines are safe but poorly immunogenic, and thus they require the use of adjuvants, and adjuvants which can control the balance and induction level of cellular and humoral immunities are urgently required for the treatment and/or protection of infectious diseases and cancers. Although, there have no adjuvants which can achieve these requirements. In this study, amphiphilic poly(γ-glutamic acid) (γ-PGA) with various kinds of hydrophobic amino acid ethyl esters (AAE), was synthesized (γ-PGA-AAE) and prepared antigen-encapsulated nanoparticles (NPs). γ-PGA-graft-Leu (γ-PGA-Leu, Leu; leucine ethyl ester), γ-PGA-graft-Phe (γ-PGA-Phe, Phe; phenylalanine ethyl ester), and γ-PGA-graft-Trp (γ-PGA-Trp, Trp; tryptophan ethyl ester) formed monodispersed NPs that encapsulated ovalbumin (OVA). The type and the induction level of the antigen-specific cellular and humoral immunities could be controlled by the kinds of hydrophobic segments and vaccine formulation (encapsulation or mixture). When OVA was encapsulated into NPs, the cellular immunity was dominantly induced, while humoral immunity was dominant when OVA was mixed with NPs. These results are a first report that demonstrated the balance and induction level of cellular and humoral immunities could be controlled by modifying compositions of NPs and vaccine formulation. Our results suggest that γ-PGA-AAE NPs can provide safe and efficient nanoparticle-based vaccine adjuvants, and provide guidelines in the rational design of amphiphilic polymers as vaccine adjuvants which can control the balance of immune responses.
The immune system represents our primary defense system against foreign intrusion, including pathogens as well as particles. In order to understand the potential toxicity of engineered nanomaterials of ever increasing sophistication, it is necessary to understand the sophistication of the immune system with its multiple, specialized cell types and soluble mediators. Moreover, it is important to consider not only material-intrinsic properties of the pristine nanomaterial, but also the acquired, context-dependent 'identity' of a nanomaterial in a living system resulting from the adsorption of biomolecules on its surface. The immune system has evolved to recognize a vast array of microboes through so-called pattern recognition; we discuss in the present review whether engineered nanomaterials with or without a corona of biomolecules could also be sensed as 'pathogens' by immune-competent cells.
Copyright © 2015. Published by Elsevier B.V.
Abstract The increasing risk of incidental exposure to nanomaterials has led to mounting concerns regarding nanotoxicity. Zinc oxide nanoparticles (ZnO NPs) are produced in large quantities and have come under scrutiny due to their capacity to cause cytotoxicity in vitro and potential to cause harm in vivo. Recent evidence has indicated that ZnO NPs promote autophagy in cells; however, the signaling pathways and the role of ion release inducing toxicity remain unclear. In this study, we report that ZnO NPs are immunotoxic to primary and immortalized immune cells. Importantly, such immunotoxicity is observed in mice in vivo, since death of splenocytes is seen after intranasal exposure to ZnO NPs. We determined that ZnO NPs release free Zn(2+) that can be taken up by immune cells, resulting in cell death. Inhibiting free Zn(2+) ions in solution with EDTA or their uptake with CaCl2 abrogates ZnO NP-induced cell death. ZnO NP-mediated immune cell death was associated with increased levels of intracellular reactive oxygen species (ROS). ZnO NP death was not due to apoptosis, necroptosis or pyroptosis. Exposure of immune cells to ZnO NPs resulted in autophagic death and increased levels of LC3A, an essential component of autophagic vacuoles. Accordingly, ZnO NP-mediated upregulation of LC3A and induction of immune cell death were inhibited by blocking autophagy and ROS production. We conclude that release of Zn(2+) from ZnO NPs triggers the production of excessive intracellular ROS, resulting in autophagic death of immune cells. Our findings suggest that exposure to ZnO NPs has the potential to impact host immunity.
Neutrophils are critical for antifungal defense, but the mechanisms that clear hyphae and other pathogens that are too large to be phagocytosed remain unknown. We found that neutrophils sensed microbe size and selectively released neutrophil extracellular traps (NETs) in response to large pathogens, such as Candida albicans hyphae and extracellular aggregates of Mycobacterium bovis, but not in response to small yeast or single bacteria. NETs were fundamental in countering large pathogens in vivo. Phagocytosis via dectin-1 acted as a sensor of microbe size and prevented NET release by downregulating the translocation of neutrophil elastase (NE) to the nucleus. Dectin-1 deficiency led to aberrant NET release and NET-mediated tissue damage during infection. Size-tailored neutrophil responses cleared large microbes and minimized pathology when microbes were small enough to be phagocytosed.
Respiratory syncytial virus (RSV) is one of the most common causes of viral deaths in infants worldwide, yet no effective vaccines are available. Here, we report an osmotically-active polysaccharide-based polysorbitol transporter (PST) prepared from sorbitol diacrylate and low molecular weight polyethylenimine (PEI) showing a potent, yet safe, adjuvant activity and acting as an effective delivery tool for RSV glycoprotein (RGp) antigen. PST showed no toxicity in vitro and in vivo, unlike PEI and the well-known experimental mucosal adjuvant cholera toxin (CT). PST formed nano-sized complexes with RGp by simple mixing, without affecting antigenic stability. The complexes exhibited negative surface charges that made them highly efficient in the selective activation of phagocytic cells and enhancement of phagocytic uptake. This resulted in an improved cytokine production and in the significant augmentation of RGp-specific antibody production, which persisted for over 200 days. Interestingly, PST/RGp enhanced phagocytic uptake owing to the osmotic property of PST and its negative zeta potential, suggesting that PST could selectively stimulate phagocytic cells thereby facilitating long-lived antigen-specific immune response, which was presumably further enhanced by the polysaccharide properties of PST.
Abstract Endotoxin is often used to activate NF-κB in vitro when assessing NLRP3 inflammasome activation by various exogenous particles including nanoparticles. However, the endogenous source of this signal 1 is unknown. High-mobility group box 1 (HMGB1) is known to play a critical role in acute lung injury, however the potential contribution of the alarmin HMGB1 to NLRP3 Inflammasome activation has not been determined in response to nanoparticles in vivo. In this study, the ability of multi-walled carbon nanotubes (MWCNT) to cause release of HMGB1 in vitro and in vivo, as well as the potential of HMGB1 to function as signal 1 in vitro and in vivo, was determined. HMGB1 activity in vivo was assessed by administration of HMGB1 neutralization antibodies following MWCNT exposure. Caspase-1(-/-) mice were utilized to elucidate the dependence of HMGB1 secretion on NLRP3 inflammasome activity. MWCNT exposure increased extracellular HMGB1 levels in primary alveolar macrophages from C57Bl/6 mice and C10 mouse epithelial cell culture supernatants, and in C57Bl/6 mouse lung lavage fluid. MWCNT-induced HMGB1 secretion was dependent upon caspase-1. HMGB1 increased MWCNT-induced IL-1β release from macrophages in vitro, and neutralization of extracellular HMGB1 reduced MWCNT-induced IL-1β secretion in vivo. HMGB1 neutralization was accompanied with overall decreased inflammation. In summary, this study suggests extracellular HMGB1 participates in NLRP3 inflammasome activity and regulates IL-1β associated sterile inflammation induced by MWCNT.
Neutrophils extrude neutrophil extracellular traps (NETs) consisting of a network of chromatin decorated
with antimicrobial proteins to enable non-phagocytic killing of microorganisms. Here, utilizing a model
of ex vivo activated human neutrophils, we present evidence of entrapment and degradation of
carboxylated single-walled carbon nanotubes (SWCNTs) in NETs. The degradation of SWCNTs was
catalyzed by myeloperoxidase (MPO) present in purified NETs and the reaction was facilitated by the
addition of H2O2 and NaBr. These results show that SWCNTs can undergo acellular, MPO-mediated
biodegradation and imply that the immune system may deploy similar strategies to rid the body of
offending microorganisms and engineered nanomaterials.
While plasma proteins can influence the physicochemical properties of nanoparticles, the adsorption of protein to the surface of nanomaterials can also alter the structure and function of the protein. Here, we show that plasma proteins form a hard corona around synthetic layered silicate nanoparticles (LSN) and that one of the principle proteins is serum albumin. The protein corona was required for recognition of the nanoparticles by scavenger receptors, a major scavenger receptor family associated with the mononuclear phagocyte system (MPS). Albumin alone could direct nanoparticle uptake by human macrophages, which involved Class A but not Class B scavenger receptors. Upon binding to LSN, albumin unfolded to reveal a cryptic epitope that could also be exposed by heat denaturation. This work provides an understanding of how albumin, and possibly other proteins, can promote nanomaterial recognition by the MPS without albumin requiring chemical modification for scavenger receptor recognition. These findings also demonstrate an additional function for albumin in vivo.
A recombinant hybrid composed of the two major allergens of the Parietaria pollen Par j 1 and Par j 2 has been generated by DNA recombinant technology (PjED). This hybrid was produced in E.coli at high levels of purity. Then, the engineered derivative has been combined with a synthetic polyaminoacidic derivative having a poly(hydroxyethyl)aspartamide (PHEA) backbone and bearing both butyryl groups (C4) and succinyl (S) moieties in the side chain (PHEA-C4-S). The allergen-copolymer nanoaggregate was characterized by means of DLS, Zeta Potential, electrophoretic mobility and Atom Force Microscopy analysis displaying the formation of a stable complex. Its safety has been proved in vitro on a murine cell line, human erythrocytes and basophils. Moreover, the formation of the complex did not alter the ability of the allergens to cross-link surface bound specific IgE demonstrating that the combination of an engineered hybrid with a copolymer did not interfere with its biological activity suggesting its employment as potential vaccine against Parietaria-induced allergies.
In biological fluids, proteins bind to the surface of nanoparticles to form a coating known as the protein corona, which can critically affect the interaction of the nanoparticles with living systems. As physiological systems are highly dynamic, it is important to obtain a time-resolved knowledge of protein-corona formation, development and biological relevancy. Here we show that label-free snapshot proteomics can be used to obtain quantitative time-resolved profiles of human plasma coronas formed on silica and polystyrene nanoparticles of various size and surface functionalization. Complex time- and nanoparticle-specific coronas, which comprise almost 300 different proteins, were found to form rapidly (<0.5 minutes) and, over time, to change significantly in terms of the amount of bound protein, but not in composition. Rapid corona formation is found to affect haemolysis, thrombocyte activation, nanoparticle uptake and endothelial cell death at an early exposure time.
Protein adsorption influences greatly the performance of materials used in biotechnology and biomedicine. The binding of fibrinogen (Fg) to nanoparticles (NPs) can result in protein unfolding and exposure of cryptic epitopes that subsequently interact with cell surface receptors. The response and its degree are dependent on the size, charge, and concentration of the NPs. In this study the binding kinetics of human Fg to negatively charged 11-mercaptoundecanoic acid-functionalized gold nanoparticles (AuNPs-COOH) ranging from 5.6 to 64.5 nm were examined. The larger NPs bound Fg with a larger number of proteins per square unit and a higher dissociation rate (Kd'), but with decreased affinity. By contrast, the 5.6 nm AuNPs-COOH behaved in a cooperative manner for Fg adsorption. In the presence of excess Fg, only the 64.5 nm AuNPs-COOH showed severe aggregation, whose degree was alleviated in a dilute Fg solution. The Fg is adsorbed through a side-on configuration and both side-on and end-on configurations on the smaller (5.6 and 14.2 nm) and 31.5 nm AuNPs-COOH, respectively. It also retains the native conformation. By contrast, on the 64.5 nm AuNPs-COOH the Fg adopts the end-on configuration and loses most of the secondary structure.
The NLRP3 inflammasome is an important component of the innate immune system. However, its mechanism of activation remains largely unknown. We show that NLRP3 activators including bacterial pore-forming toxins, nigericin, ATP, and particulate matter caused mitochondrial perturbation or the opening of a large membrane pore, but this was not required for NLRP3 activation. Furthermore, reactive oxygen species generation or a change in cell volume was not necessary for NLRP3 activation. Instead, the only common activity induced by all NLRP3 agonists was the permeation of the cell membrane to K(+) and Na(+). Notably, reduction of the intracellular K(+) concentration was sufficient to activate NLRP3, whereas an increase in intracellular Na(+) modulated but was not strictly required for inflammasome activation. These results provide a unifying model for the activation of the NLRP3 inflammasome in which a drop in cytosolic K(+) is the common step that is necessary and sufficient for caspase-1 activation.
Carbon nanotubes have gained tremendous interest in a wide range of applications due to their unique physical, chemical, and electronic properties. Needless to say, close attention to the potential toxicity of carbon nanotubes is of paramount importance. Numerous studies have linked exposure of carbon nanotubes to the induction of inflammation, a complex protective response to harmful stimuli including pathogens, damaged or dying cells, and other irritants. However, inflammation is a double-edged sword as chronic inflammation can lead to destruction of tissues thus compromising the homeostasis of the organism. Here, we provide an overview of the process of inflammation, the key cells and the soluble mediators involved, and discuss research on carbon nanotubes and inflammation, including recent studies on the activation of the so-called inflammasome complex in macrophages resulting in secretion of pro-inflammatory cytokines. Moreover, recent work has shown that inflammatory cells i.e. neutrophils and eosinophils are capable of enzymatic degradation of carbon nanotubes, with mitigation of the pro-inflammatory and pro-fibrotic effects of nanotubes thus underscoring that inflammation is both good and bad.
The currently applied immunotherapy of type I allergy with aluminium hydroxide (alum) as adjuvant elicits -among other side effects- an initial IgE-boost. In contrast, CpG-oligodeoxynucleotides (ODNs) drive the immune response towards Th1. The biodegradable material protamine can spontaneously form nanoparticles together with such ODNs. Our aim was to investigate the immune response induced by protamine-based nanoparticles (proticles) with CpG-ODN as an allergen delivery system. Proticles complexed with Ara h 2 extracted from raw peanuts as model allergen were injected subcutaneously into naïve BALB/c mice. Ara h 2-specific antibodies were analyzed by ELISA and rat basophilic leukemia (RBL) cell assay. Cytokine levels were investigated in supernatants of stimulated splenocytes. The in vivo distribution after subcutaneous injection was examined via fluorescence imaging. BMDCs were stimulated with proticles and expression of stimulation and maturation markers as well as cytokines in supernatants were investigated. A favorable increase of Ara h 2-specific IgG2a antibodies was found after immunization with proticles-Ara h 2, whereas Ara h 2-specific IgE was not detectable. Accordingly, the ratio of IL-5/IFN-gamma was low in this group. Granuloma formation was completely absent at injection sites of proticles. The distribution of Ara h 2 after subcutaneous injection was markedly decelerated when complexed to proticles. Stimulation of BMDCs with proticles-Ara h 2 caused upregulation of CD11c and CD80 as well as an increased IL-6 production. Our data suggest that biodegradable protamine-based nanoparticles with CpG-ODN counteract the Th2-dominated immune response induced by an allergen and therefore are suitable as novel carrier system for immunotherapy of allergy.
Eosinophil peroxidase (EPO) is one of the major oxidant-producing enzymes during inflammatory states in the human lung. The degradation of single-walled carbon nanotubes (SWCNTs) upon incubation with human EPO and H2 O2 is reported. Biodegradation of SWCNTs is higher in the presence of NaBr, but neither EPO alone nor H2 O2 alone caused the degradation of nanotubes. Molecular modeling reveals two binding sites for SWCNTs on EPO, one located at the proximal side (same side as the catalytic site) and the other on the distal side of EPO. The oxidized groups on SWCNTs in both cases are stabilized by electrostatic interactions with positively charged residues. Biodegradation of SWCNTs can also be executed in an ex vivo culture system using primary murine eosinophils stimulated to undergo degranulation. Biodegradation is proven by a range of methods including transmission electron microscopy, UV-visible-NIR spectroscopy, Raman spectroscopy, and confocal Raman imaging. Thus, human EPO (in vitro) and ex vivo activ