Environmental Science Nano This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. PERSPECTIVE View Article Online View Journal | View Issue Probing the immune responses to nanoparticles across environmental species. A perspective of the Open Access Article. Published on 14 October 2020. Downloaded on 11/22/2022 11:54:47 AM. Cite this: Environ. Sci.: Nano, 2020, 7, 3216 EU Horizon 2020 project PANDORA Annalisa Pinsino, *a Neus G. Bastús,b Martí Busquets-Fité, c Laura Canesi,d Paola Cesaroni,e Damjana Drobne,f Albert Duschl, g Marie-Ann Ewart,h Ignasi Gispert,c Jutta Horejs-Hoeck,g Paola Italiani,ij Birgit Kemmerling,k Peter Kille,l Petra Procházková, m Victor F. Puntes,bno David J. Spurgeon, p Claus Svendsen,p Colin J. Wilde h and Diana Boraschi *ij Understanding how engineered nanomaterials affect immune responses of living organisms requires a strong collaborative effort between immunologists, toxicologists, ecologists, physiologists, inorganic chemists, nanomaterial scientists and experts in law and risk management. This perspective aims to provide a new viewpoint on the interaction between engineered nanomaterials and the immune defensive systems across living species, gained within the EU Horizon 2020 project PANDORA. We consider the effects of nanoparticle exposure on immune functions in plants, marine and terrestrial invertebrates and their relation to the current state of knowledge for vertebrates (in particular humans). These studies can shed light on the broader perspective of defensive and homeostatic mechanisms (immunity, inflammation, stress Received 14th July 2020, responses, microbiota, stem cell differentiation) suggesting ways to: i) perform a comparative analysis of Accepted 12th October 2020 the nanoparticle impact on immunity across model organisms; ii) inspire best practices in experimental methodologies for nanosafety/nanotoxicity studies; iii) regroup and harmonise fragmented research DOI: 10.1039/d0en00732c activities; iv) improve knowledge transfer strategies and nano-security; v) propose innovative tools and rsc.li/es-nano realistic solutions, thereby helping in identifying future research needs and tackling their challenges. Environmental significance This perspective is based on the experience gained within a collaborative work that compared the immune reaction to NPs in plants, invertebrates and vertebrates. Each species uses a shared number of core defensive mechanisms upon exposure to NPs (recognition, uptake, gene regulation, production of defensive molecules), and then adapts them and adds others to its own specific requirements. In general, an immune reaction to NPs is not detrimental for the organism and resolves rapidly with NP elimination, while in few cases it can produce damage or have beneficial effects. Each species has its own peculiarities, but the commonality of their basic defensive mechanisms becomes a valuable tool for NP-related environmental health risk assessment and management. a i Institute for Biomedical Research and Innovation, National Research Council, Institute of Biochemistry and Cell Biology, National Research Council, Napoli Palermo 90146, Italy. E-mail: annalisa.pinsino@irib.cnr.it 80131, Italy. E-mail: diana.boraschi@ibbc.cnr.it b j Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST, Stazione Zoologica Anton Dohrn, Napoli 80121, Italy Campus UAB, Bellaterra, Barcelona, Spain k Center for Plant Molecular Biology – ZMBP, Eberhard-Karls University Tübingen, c Applied Nanoparticles S.L., Barcelona, Spain Tübingen 72076, Germany d l Department of Earth Environment and Life Sciences, University of Genoa, Genoa School of Biosciences, Cardiff University, Cardiff CF10 3AX, UK m 16126, Italy Institute of Microbiology of the Czech Academy of Sciences, Prague 142 20, Czech e ALTA S.r.l.u, Siena, Italy Republic f n Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona 08010, 1000, Slovenia Spain g o Department of Biosciences, Paris-Lodron University Salzburg, Salzburg 5020, Austria Vall d Hebron Institut de Recerca (VHIR), Barcelona 08035, Spain h p AvantiCell Science Ltd, Ayr KA6 5HW, UK UK Centre for Ecology and Hydrology, Wallingford OX10 8BB, UK 3216 | Environ. Sci.: Nano, 2020, 7, 3216–3232 This journal is © The Royal Society of Chemistry 2020 View Article Online Environmental Science: Nano Perspective 1. Nanoparticles: health, immunity physiological conditions, again implying hyperreactivity.4 and environment While such reactions may be at the basis of inflammatory and other pathologies, on the other hand the NP capability to Assessing the safety of nanoparticles (NPs) has been activate, regulate or modulate immune responses is of great This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. identified as a priority, because of the potential challenges interest, because of potential developments into treatment that this kind of agent could pose to the environmental and strategies for numerous disorders, such as autoimmune and human health. Over the past decades, there has been a inflammatory diseases, and cancer.5 Thus, assessing the significant increase in the number and production volumes impact of NPs on the immune system not only is an issue of of nano-enabled products coming to market. As the diversity common immuno-nanosafety (reactive mechanisms shared of NP production and use has increased, the need to identify, among all organisms), but it can also provide an improved Open Access Article. Published on 14 October 2020. Downloaded on 11/22/2022 11:54:47 AM. measure and manage the associated hazards and risk has understanding of the impact of NP exposure on the nano- been recognised. Despite significant progress, many gaps still immune interaction that can be exploited for targeted remain in our knowledge of how NPs interact with immunomodulation, disease management and crop organisms. Gaps in understanding include details of the protection. mechanistic pathways of NP toxicity at the cellular level (e.g., By studying and comparing humans, plants and interactions with biological macromolecules, effects of signal invertebrates, we can learn much about the general and specific transduction mechanisms, cellular stress and cell function), mechanisms of the NP effects on immunity. The interactions of at the level of tissues/organs (e.g., blood vessels, immune NPs with cells and molecules of the innate immune system cells), individual organisms and population/community (e.g., represents the best ground for understanding the common resource allocation species sensitivity, ecosystem process mechanisms of immune defence. Innate immunity includes impacts). Such information would significantly improve the natural barriers at the organism–environment interface that basis for NP risk assessment and management. limit internal exposure to pathogens, and an array of different One of the major shortcomings in our understanding of cellular, molecular and chemical effector and regulatory players the effects of NPs relates to the impact of NP exposure on the participating to the defensive responses. The full operational immune system. The conserved nature of many immune capacity of innate immunity is a major determinant in the mechanisms suggests that comparative studies on the survival and fitness of all organisms. Therefore, the immune responses of organisms covering different phyla and immunosafety of engineered NPs is a key element of hierarchical levels of organization can together provide a environmental nanosafety. The fact that living species have been knowledge base for understanding the mechanisms and consistently exposed to potential pathogens and other threats consequences of NP exposure on human and environmental through their extended evolutionary history has directed the health. Key topics for comparative nano-immunosafety evolution of innate immune protective signalling pathways and studies include: i) the identification of species differences mechanisms towards diversification (mainly dependent on and/or species similarities in the immunological mechanisms species and environmental characteristics) of specialized triggered by NPs; ii) the extrapolation of predictive markers immune-related molecules, which mediate cellular responses.6 of risk vs. safety by a cross-species comparison of innate Ecological immunology attempts to explain variability in immune defence capacity; iii) the design of predictive in vitro immune defence, and, in turn, shows that the optimal immune assays, in the practical and vigilant application of the 3R response changes with environmental changes, establishing an principle, to measure the nano-immunological risk to the evolutionary ecology framework around immunity.7 Since the environment and human health, for industrial and appropriate functioning of the immune system is important for environmental nanosafety testing application. maintaining individual health, an agent that compromises its The full functioning of innate immunity is of central function can have implications for survival, reproduction and, importance for the survival and fitness of organisms, because hence, population fitness. Here we discuss the potential of NPs of the critical role of immunity in preserving tissue and to act as agents affecting the immune status in different cellular homeostasis. The particulate nature of NPs dictates a environmentally relevant species, with a focus on the emerging preferential interaction with those cells in the immune experimental models launched by the EU Commission H2020 system that are deputed to recognition and elimination of project PANDORA (probing safety of nano-objects by defining foreign particulate matters.1 NPs can interact with the immune responses of environmental organisms).8 immune system in a number of ways, which in the majority of cases end in a defensive reaction that resolves with the elimination of the particles and the re-establishment of 2. Nanosafety and nanotoxicity: homeostasis.2,3 In some instances, however, reaction to NPs considerations from an may trigger an anomalous immune response. This can be immunological perspective evident as a quantitatively exaggerated or insufficient response (immune hyperreactivity or immunodeficient Living organisms have developed highly efficient defence response) and also as a response sustained over extended biological barriers to block foreign species/substances and timescales, beyond those taking place under normal prevent damage to the organism's tissues. In the context of This journal is © The Royal Society of Chemistry 2020 Environ. Sci.: Nano, 2020, 7, 3216–3232 | 3217 View Article Online Perspective Environmental Science: Nano understanding the biological effects of NPs, an important physiological and biochemical barriers vary widely from one consideration is the extent by which particles may enter the species to another, all organisms are organized into similar organism, distribute across tissues, pass into cells and levels of anatomical structures (sub-cellular entity, cell, interact with organelles, DNA and other macromolecules. The tissue, organ, apparatus, organism). In the Fig. 1 are shown This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. external body surfaces (e.g., skin, lungs and intestine) are the anatomical maps of some invertebrates, in which the where NPs first interact with a living organism. Their active location of immune cells is shown, relative to the main defensive barrier functions determine the extent of NP–tissue barriers. interaction and internal distribution. Plants and Changes in cell/tissue/organ functions following NP invertebrates possess additional types of physical/biological exposure may indirectly affect immune functions. A relevant barriers (e.g., exoskeleton, peritrophic membrane) that can example is the effect of biocidal NPs on the structure and/or Open Access Article. Published on 14 October 2020. Downloaded on 11/22/2022 11:54:47 AM. further prevent the NP entry.9 These barriers largely rely on function of the gut microbiota.15 The resident microbiota cells working as a basic functional unit (e.g., epithelium), plays a central role in the stimulation, training, and function which prevent NPs to go deeper into tissues.10 If poor tissue of the host immune system. In healthy physiological permeability is a significant barrier to particle entry, NPs may conditions, the immune system and microbiota cooperate in still enter tissues in a number of ways.11 The physico- inducing protective responses to foreign substances and in chemical properties of NPs (e.g., size, surface charge and maintaining regulatory pathways.16 All organisms initiate shape), their bio-modification during translocation in the immune reactions to maintain metabolic homeostasis and body, and the physiological and pathological conditions of self-integrity after suffering from stress, injury or invaders. the animal, all influence the NP capability of interact with They require mechanisms to recognize damage, to the barriers. On the other hand, there is no available discriminate between the molecular signatures of invading evidence that the effects on barriers depend on the NP pathogens (“non-self” molecules) and cellular constituents concentration. As an example of the complexity of NP–barrier that generally pose no health risk (“self” molecules), and to interaction, NP translocation through the intestinal barrier is discriminate “self” from “damaged self”.17 An interesting a multistep process that involves NP diffusion through the question is whether it could be possible to predict innate mucus layer, contact with enterocytes and/or M cells, uptake defence responses against NPs based on how the immune via endocytosis, and translocation to the subepithelial tissue system manages pathogens. NPs may (or may not) set the via transcytosis.12 Some invertebrates (e.g., mussels, immune system in motion, communicate with cells, earthworms, arthropods) have fine food sorting systems, and receptors and proteins, trigger signalling cascades, and food particles are taken and digested intracellularly by cells generate unpredictable immune responses (activation or in the gut lumen.13,14 NPs may follow the same route as the suppression) as well as other harmful outcomes,18 depending food particles. In some coelomates (e.g., mussels, on their physico-chemical characteristics, especially their echinoderms) free circulating immune cells are present in all surface properties.19 In the case of nanomedical applications, coelomic spaces, including the perivisceral coelomic cavities it may be attractive to design NPs able to engage with the and the open water-vascular system, and in the major organs immune system in a specific manner, e.g., and tissues. In the open circulatory system, the NPs are likely immunostimulation or immunosuppression, depending on to be entrapped by phagocytic cells and to be moved quickly the intended use. This has encouraged the development of through the body fluids. Thus, some types of barriers may NP and cell-based hybrid systems that cannot be detected/ likely fail to prevent the NP entry. Although the anatomical, eliminated by the immune system, making them highly Fig. 1 Anatomical maps of some invertebrates, showing the location of immune cells relative to organs and barriers. A) Mussel; B) echinoderm; C) arthropod. 3218 | Environ. Sci.: Nano, 2020, 7, 3216–3232 This journal is © The Royal Society of Chemistry 2020 View Article Online Environmental Science: Nano Perspective biocompatible and physiologically integrable within tissues while in some cases NPs can aggregate, forming irreversible in humans.11 For example, nanoporous silicon NPs coated larger clusters. Heteroaggregation/agglomeration, in which with cellular membranes purified from leukocytes are able to NPs are admixed with other environmental molecules/ elude the immune system, avoiding opsonization and particles, is likely the most common form in which NPs are This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. phagocytosis, and to transmigrate across the endothelial present in the environment and come in contact with living barrier to enter a tissue.20 organisms. Such complexes can also associate with other Notably, some NPs may selectively accumulate in organs environmental biological and non-biological contaminants, with high adsorptive, filtering and detoxification capacity such as bacterial toxins (e.g. Gram-negative endotoxin) and (e.g., spleen, liver, kidney, intestine and lung), engulfed by chemicals.23 Since these complexes are the real-life forms in the mononuclear phagocytic system (MPS) that includes both which NPs come in contact with living organisms, the Open Access Article. Published on 14 October 2020. Downloaded on 11/22/2022 11:54:47 AM. resident macrophages and circulating blood monocytes.21 future ecotoxicological assays should consider using Phagocytic cells are responsible of several important complex environmental matrices to adequately reflect the processes, including clearance (engulfment/elimination of exposure conditions in real life. Although nude particles putative threats), biodistribution (selective localisation in can rapidly aggregate/agglomerate in a fluid, coated organs and tissues) and balance between tolerance and particles acquire chemical functions of the coat that modify toxicity (e.g., between production of reactive oxygen species the particle behaviour, for example by reducing the –ROS– and antioxidant response). Each organ can count on formation of aggregates/agglomerates. Within biological the availability of an exclusive equipment of cells that fluids (blood, haemolymph, coelomic fluid, mucus, etc.), coordinate and perform local innate immune reactions. The which are rich in biomacromolecules (e.g., lipids, sugars, cells responsible of innate immune activities include both nucleic acids, and particularly proteins), NPs readily adsorb “professional” immune cells (such as mononuclear and on their surface various organic molecules that form a polymorphonuclear phagocytes, mast cells and innate complex biomolecular corona and lead to an increase in the lymphoid cells) and non-professional defence cells, such as NP hydrodynamic range.24 Large aggregates/agglomerates endothelial cells, epithelial cells, and fibroblasts.22 are transported in vesicles via different mechanisms of Experimental methodologies for nanosafety/nanotoxicity endocytosis/exocytosis (based on the size, shape and surface studies must allow us to capture the relevant details of a characteristics, and the cell internalization machinery).25 complex nano-immune interaction. While in vitro models For example, NPs of <200 nm can be internalized via facilitate the analysis of particle effects on isolated single clathrin-coated pits by non-phagocytic cells, whereas at cells and tissues, in vivo models allow for a clearer evaluation increasing size a shift towards caveolae-mediated of the NP effects in whole organs or in the entire organism. internalization become predominant.26 NPs can also enter While exposure time, route, and NP concentration help in cells by a passive uptake, which can or not disturb the determining the modes of interaction between NPs and the membrane depending the size of the NPs. Small NPs have a immune system (or any other system they come in contact higher probability to be internalized by passive uptake than with), there is a parameter that is of major importance in any large ones.25 kind of nano-bio interaction, i.e., the changes particles The bio-corona composition may vary among different experience when coming in contact with biological fluids. NPs and upon contact with different biological fluids that Mimicking such interactions in the laboratory is always contain different mixtures of biomolecules. The bio-corona challenging because of the complexity and multiplicity of evolves with time, with a progressive loss of molecules of interactions taking place in real life environments. For lower avidity (soft corona) and the establishment of a hard instance, the presence of natural organic matter (including corona of strongly interacting biomolecules. The structure toxins) may play a role in the NP partition, which is difficult of this macromolecular coating is most likely a key factor in to control but that should be considered, as they are part of a making the NPs “visible” to cells for uptake, contributing to natural environment. This is a critical point in the context of clearance from circulation and possibly reducing their ecotoxicological assays and how and to which purpose we interactions with other cells and tissues.27 On the other want to use them. Thus, given the advantages of the in vivo hand, functionalizing particles with some capping agents and vitro/ex vivo approaches in providing nanosafety (e.g., polyethylene glycol) reduces accumulation of proteins information, future studies should aim to improving the on the NP surface, making them functionally “invisible” to exposure conditions of the ecotoxicological assays in order to the immune system in humans.28,29 The majority of the bio- better mimicking complex environments and provide realistic corona studies have been performed in vitro, and have and valid nanosafety data. contributed to unravel the role of the bio-corona in the Aggregation/agglomeration influences the fate of the NPs immune recognition of NPs. Conversely, the in vivo in the environment, particularly in aqueous environments. approach is important for a better understanding of the NP colloidal stability is influenced by the solution ionic mechanisms of particle clearance vs. toxicity, which would strength, organic matter composition, pH and solvent be greatly improved by examining the differences in composition. In some conditions NPs can agglomerate, and clearance kinetics and type of toxic effect across living return in colloidal solution when the conditions change, species. This journal is © The Royal Society of Chemistry 2020 Environ. Sci.: Nano, 2020, 7, 3216–3232 | 3219 View Article Online Perspective Environmental Science: Nano Vertebrates and invertebrates display common immune attention, as alteration of immune memory can modify the mechanisms but they have also developed divergent immune capability of an organism to adapt to its environment. NPs tools for attaining challenge-specific protection. As an can modulate stimulus-induced memory in human example, whereas vertebrates use somatic rearrangement of monocytes, mostly by shifting the memory response towards This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. immune receptors and immunoglobulins, invertebrates tolerance.36 Innate memory in plants is known as systemic privilege alternative splicing of pattern-recognition genes.30 acquired resistance (SAR), which leads to the activation of a Whereas vertebrates have developed an array of different cell better protective response (e.g., increase in the levels of types as effectors of innate immune responses (e.g., pattern-recognition receptors, accumulation of dormant monocytes, macrophages, mast cells, neutrophils, basophils, signalling enzymes, alterations in chromatin state) not only eosinophils, NK cells, ILC, epithelial cells, fibroblasts), in the tissue re-exposed to the same or similar insult but also Open Access Article. Published on 14 October 2020. Downloaded on 11/22/2022 11:54:47 AM. invertebrates use a limited range of multifunctional cells. in an unexposed tissue.37 If found able to induce an Plants do not have specialised immune cells, with all cells epigenetic and metabolic reprogramming of immune being able of innate immune reactivity and with systemically responses, as they are able to promote root growth (B. spreading signals emanating from injured sites.31 Kemmerling, unpublished results), NPs would support the The innate immune system also exhibits adaptive traits, a design of new agriculture strategies able to reduce yield feature that has been termed “innate immune memory” (or losses, thereby allowing for reduction of conventional “trained immunity” in vertebrates).32 The innate immune pesticides. Invertebrates show a high degree of memory in memory is the ability of the immune system to record and their immune responses, including recall immunity to recall information of previously met pathogens to activate a infections, natural transplantation immunity, and individual/ faster and more efficient protective response upon re- trans-generational immunological priming.34 How NPs can exposure to the same or different pathogens/stressful affect the establishment of innate memory in invertebrates is agents.33 The phenomenon is well known in plants and however still largely unknown. The only available results invertebrates, which also show some degree of specificity,34,35 describing nanoplastics-induced innate memory and some and has been also observed in vertebrates (in particular underlying molecular and cellular mechanisms come from mouse and man), in which however the memory is as non- mussels.38 Upon a primary exposure to amino-modified specific as innate immunity itself.32 How NPs may induce/ nanopolystyrene, Mytilus haemocytes respond with changes alter innate memory is an open issue that requires special in some immunological parameters (e.g., lysozyme release in Fig. 2 Impact of NPs on the microbiota–immune axis in vivo: differences across environmental organisms. 3220 | Environ. Sci.: Nano, 2020, 7, 3216–3232 This journal is © The Royal Society of Chemistry 2020 View Article Online Environmental Science: Nano Perspective the haemolymph, lysosomal acidification, membrane destabilization), whereas a second exposure induces an immunological memory by shifting the immune parameters towards a tolerant condition (recovery). This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. An important issue that should be considered when examining the interaction between NPs and immunity is the role of microbiota. This is particularly important for NPs that are ingested (as in the case of most aquatic and terrestrial/ earth-dwelling invertebrates and vertebrates) but also for NPs to which organisms are exposed through the skin or Open Access Article. Published on 14 October 2020. Downloaded on 11/22/2022 11:54:47 AM. inhalation, because symbiotic and commensal microbiota populate the gut and all the barrier surfaces or environmental organisms and it is well known that microbiota profoundly influence and shape immunity at the barrier level.39 However, little is currently known on how the interaction between NPs and commensal microbes can Fig. 3 Overview of the experimental models for investigating the impact on immune responses, in particular in the long term interactions of nanomaterials with the immune system used within the EU Commission H2020 project PANDORA: from the plants to humans. (Fig. 2). Recent data show that TiO2NPs impact the immune system of mussels and induce a shift in the microbiota composition of the haemolymph40 upon exposure in vivo, scientists to select different model species for their studies. while CuONPs cause a shift in the earthworm gut microbiota As a consequence, our current knowledge of the immune despite the absence of an evident immune reaction.41 The functions of species used in ecotoxicology is faulty, and only available data on impact of NPs on the human likewise we have an incomplete knowledge of the microbiota–immune axis come from in vitro studies, which ecotoxicological aspects in species whose immune functions underlines an essential a lack of information on the realistic are well known. impact of the NPs ingested via food in humans.39 Whether In this section, we report and discuss recent studies chronic dietary exposure to NPs may be viewed as a risk aiming at integrating nanotoxicological evaluations with factor that facilitates disease development remains an open assessment of immune-related effects in plant, human and issue. invertebrate animal models, in vivo and in vitro (Fig. 3). We In the next section, we will provide an overview of the will examine the similarities and differences in the defensive environmentally relevant organisms and approaches used, responses of each organism upon exposure to NPs. within the framework of the EU PANDORA project, to study Comparison will contribute to increasing our knowledge of nano-immune interactions and identify their common vs. the commonalities in the immune reaction to NPs, and allow divergent mechanisms. us to identify pathways and biomarkers of nano-risk that are shared across species. Notably, several features must be considered in order to correctly evaluate the NP interaction 3. Nanotoxicological evaluations: with the immune defences in the selected experimental in vivo and in vitro models for models: experimental conditions (in vivo, in vitro/ex vivo); studying nano-immune interactions environmental factors; basal immunological state; immunological memory. The 3R principle of reducing, refining, and replacing animal experimentation42 has led to new approaches to in vivo experimental studies, with an increasing use of invertebrate 3.1 Plants animal models. Studying the interactions between NPs and Plants are in constant contact with the rising number of NPs the immune system across animal phyla allows us to identify released into the environment. Given the importance of both conserved mechanisms and species-specific defence plants in determining ecosystem functions such as stability solutions. Plants differ from animals because they are and resistance,43,44 studying plant-NP interactions is an autotrophs, but nevertheless show conserved/converging important element to assess the risk of NPs for the defensive mechanisms that could greatly help in clarifying environment. the mechanisms of defence and repair in animal species. Plants have a well understood immune system able to Some invertebrate organisms, by virtue of being easy to detect and react to danger, and are easily accessible to handle, breed and maintain, are becoming popular genetic and biochemical analyses and therefore provide a experimental models, in particular in ecotoxicological valuable tool for NP-related risk assessment.45 Plant research and in comparative immunology. However, the immunity is an exceptional example of sensing capability different demands of the two disciplines have brought based on networks of immune receptors that act as sensors This journal is © The Royal Society of Chemistry 2020 Environ. Sci.: Nano, 2020, 7, 3216–3232 | 3221 View Article Online Perspective Environmental Science: Nano of invading pathogens.46 Receptors evolve rapidly to keep up self” substances, including anatomical and chemical with the pathogens' variability, and interact with each other protective barriers (e.g., skin, mucopolysaccharides), cellular creating systemic signals emanating from infection sites that responses (phagocytosis, nodule formation and induce an effective immune response. The task of mounting encapsulation, blood coagulation and wound repair) and This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. an immune reaction is not dependent on specialized humoral responses (antibacterial proteins, lysozyme-like immune cells, but each plant cell is able to react against proteins, proteases, cytolytic proteins, agglutinins, invaders. The plant immune system is organised in three haemolysins, opsonins, lectin-like and pattern recognition layers, the pathogen-associated molecular patterns (PAMP)- molecules).57 Direct contact between microorganisms and the triggered immunity (PTI) (preliminary defence), the effector earthworm immune components occurs at the mucosal triggered immunity (ETI) (secondary defence), and the surfaces of two body cavities, the coelomic cavity and the Open Access Article. Published on 14 October 2020. Downloaded on 11/22/2022 11:54:47 AM. exosome-mediated cross-kingdom RNA interference (CKRI) digestive tract (gut). Both cavities are in permanent contact system (additional defence).47 Resistance against a non- with the soil microorganisms. Different defensive strategies adapted pathogen involves the rapid production of cell wall are in place in these two cavities. The coelomic cavity appositions (physical barriers), and inducible-specialized contains free circulating cells (effector immunocytes called metabolites at the infection sites via PTI and ETI coelomocytes) and indigenous microflora (bacteria, signalling.48,49 protozoans and fungi) that work together to prevent The growth and metabolic functions in response to NPs colonization and invasion by pathogens or other vary among plants.50 Reports reveal contradictory effects of dangers.58–60 Coelomocytes produce a broad spectrum of NPs depending on the experimental conditions (field or lab antimicrobial molecules and release them into the coelomic studies), medium of plant growth, duration of exposure and fluid to control the levels of the indigenous microflora.61 On species.51 For example, TiO2NPs improve light absorption the other hand, the gut is in permanent contact with the efficiency and promote the activity of Rubisco activase, indigenous microflora and other exogenous bacteria that are accelerating growth in spinach;52 whereas they reduce the ingested with the soil particles and that may have beneficial photosynthetic rate thereby decelerating growth in Ulmus effects on the integrity and health of the gut epithelium.62 elongata.53 Arabidopsis thaliana leaf cells exposed to large Notably, the human gut microbiota is known to play a key particles of single-walled carbon nanotubes form role in protecting against pathogens and in preserving endocytosis-like structures in the plasma membrane, whereas immune and metabolic homeostasis.63 the same particles fail to penetrate the cell walls in other The mechanisms of interaction of NPs interact with plants.54 Notably, the interaction of plant cells with NPs leads different components of the earthworm immune system are to the modulation of plant gene expression and related still not well defined. There are data showing that AgNPs, biological pathways, which in turn influence plant growth both in vivo and in vitro on Eisenia fetida coelomocytes, can and development.55 For example, exposure of A. thaliana to transiently affect the expression of a number of immune- gold NPs induces the expression of a few key regulatory related genes, such as those involved in oxidative stress (CAT, molecules and microRNAs (miRs) thereby stimulating seed SOD), energy metabolism (aspartate aminotransferase, germination, growth, and antioxidant metabolic activity.56 A. glucose-6-phosphate isomerise, phosphoglucomutase) and thaliana is a small model plant with a short life cycle, which immune response (lysozyme, MyD88, PKC, MEKK1).64,65 The allows for easy manipulation and study. The observations intracellular signalling pathway involved in the AgNP- made in this species for studying NP uptake and metabolism induced modulation of earthworm immunity apparently encourage more relevant and significant research in this involve the myeloid differentiation factor 88 (MyD88) field, due to commonalities between Arabidopsis and edible (encoding a central adaptor protein of toll-like receptors), plants. Well controlled and field studies will allow us to get and the mitogen activated protein kinase (MAPK) cascades.65 an overview about the potential of plants to recognize and This upstream signalling cascade induces activation of react to NPs, keeping in mind that NPs bear also the nuclear factor (NF)-κB, resulting in transcription of genes potential to improve plant yield and health and reduce crop encoding inflammatory cytokines in humans.66 Cytokine loss by specific, localized and efficient application of nano- production is necessary for defence, but it can also induce to fertilizers, pesticides or immunostimulating agents.51 tissue damage by inducing the generation of ROS, enzymes and other toxic defensive molecules.67 These findings underline the presence of conserved innate immune 3.2 Protostomes activation mechanisms and pathways between earthworm Earthworms. Earthworms (annelid subgroup) are the most and human immunity. In agreement, the 1H NMR-based abundant invertebrates present in soils worldwide. The metabolomic analysis of Eisenia fetida earthworms exposed to permeable skin and the digestive tract place earthworms in TiO2NPs in soil showed significant changes in the metabolic constant contact with the soil particles, which may contain profile consistent with oxidative stress as the main an unknown number of pathogens and pollutants. To face mechanism of immune-related inflammation/toxicity.68 Upon this challenge, earthworms developed a variety of defence exposure to another type of particle, CuO2NPs, in soil, the mechanisms efficiently recognizing and responding to “non- earthworm Metaphire posthuma showed an immune 3222 | Environ. Sci.: Nano, 2020, 7, 3216–3232 This journal is © The Royal Society of Chemistry 2020 View Article Online Environmental Science: Nano Perspective metabolic impairment in term of nitric oxide generation, microscale particles.80,81 Mussels are an excellent model for superoxide dismutase, phenoloxidase, catalase and alkaline understanding the interaction between NPs and innate phosphatase activity.69 immunity, because their biochemical and physiological Since earthworms ingest soil and their associate responses to a variety of environmental stressors and This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. microbiota, the NP effects on the soil microbial pathogens are well known,79,82 they are easy to handle communities70–72 will affect the composition of the experimentally, including the non-invasive sampling of microbiota in the earthworm gut. This is particularly immune cells (haemocytes) and blood (haemolymph). important, in terms of effects on immune functions, because However, long-term cultures of haemocytes and in vitro the interaction between immunity and microbiota at the gut studies are still not feasible. Upon in vivo exposure to level defines the defensive capacity of an organism (from environmentally relevant concentrations of different NPs, it Open Access Article. Published on 14 October 2020. Downloaded on 11/22/2022 11:54:47 AM. earthworms to human beings), and any variation of is important to note that mussels show little particle microbiota can affect immune competence. Unfortunately, accumulation and toxicity, but that nevertheless immune little is known on how NPs might interact with earthworms' functions can be affected.82 bacterial communities and even less on how this might affect The interaction of NPs with the mussel biological fluids, in their immunity. Earthworms capture NPs from contaminated particular the haemolymph, allowed for the identification of soil using mucus glycoproteins/glycans of gut/microbial specific proteins forming a stable biocorona on the NP surface, origin.73 Exposing the oligochaete Enchytraeus crypticus to 50– and to assess the features of the haemocyte interaction with 100 nm plastic particles caused a significant shift in the gut the biocorona-coated NPs. Depending on the NPs and its bio- microbiota, including a significant decrease in the relative corona, this interaction can lead to distinct responses abundance of the families Rhizobiaceae, Xanthobacteraceae (recognition, internalization, activation/inhibition of immune and Isosphaeraceae,74 but how these changes may affect responses), and therefore results in distinct immunity is unknown. The earthworm Eisenia fetida can immunomodulatory pathways.83 These findings underlie the survive in soil containing high ZnONP concentrations, and strong similarities with the features of the nano-immune accumulate zinc ions in the body. ZnONPs enhance the interactions in mammalian cells, suggesting that this model activity of cellulolytic bacteria (Bacillus and Pseudomonas sp.) could be suitable for investigating the mechanistic basis and in the gut, contributing to bioconversion (digestion) of the biological outcomes of such interactions in conditions that lignocellulosic waste.75 The metagenomic analysis of Eisenia reflect those in both mammals and environmental organisms. fetida earthworms exposed to ZnNPs in soil shows a decrease Notably, recent data showed that the immunomodulatory in biodiversity of the gut microbiota, mainly due to a effects of different types of NPs are linked with shifts in the significant decrease in the relative abundance of the phylum microbiota composition of the haemolymph, underlying a Firmicutes.76 Reduction in the abundance of these bacteria relationship between innate immunity and host microbiota in correlates with inflammatory/autoimmune diseases in mussels, as it occurs in mammals.40 mammals.77 Although there is no strong evidence that this Terrestrial isopods. Terrestrial isopods (arthropod type of correlation occurs in earthworms, we can speculate subgroup) comprise a group of crustaceans that have that NPs impacting the gut microbiota can consequently acquired a terrestrial lifestyle. They are keystone species in affect the earthworm immunity upon in vivo exposure. This is terrestrial ecosystems, contributing to the decomposition of based on the consideration that amoebocytes, the most active plant detritus and regulating the microbial food mesh.84 The immune cells in the earthworm, are involved in a broad terrestrial isopod Porcellio scaber is increasingly used as range of defensive functions in the gut78 reflecting the model organism to evaluate the toxicity of chemicals and the abundance of immune cells in the gut of vertebrates. bioaccumulation of pollutants in soil, including NPs. The However, recent data show that in vivo exposure to CuONPs main cellular effectors of P. scaber immune system are the can cause significant changes in the gut microbiota and haemocytes, free moving sentinel cells capable of microbiome of earthworms, but it does not affect the phagocytosing, encapsulating and sequestering pathogens. As expression of immune-related genes (e.g., coelomic cytolytic mammalian macrophages, haemocytes are also involved in factor, lysenin/fetidin, lysozyme) and resistance/susceptibility carbohydrate metabolism and in transporting and storing to a bacterial infection.41 Future studies of gene expression amino acids and proteins. Chronic exposure to NPs in food based on a greater time resolution of immune responses may (AgNPs, CeO2NPs) did not result in immune activation, aid the understanding of whether and how NPs could impact although it caused changes in proteins, lipids, nucleic acids on the dialogue between gut commensals and immune cells and carbohydrates at the tissue level.85,86 It is likely that a in the earthworms. direct contact between NPs and haemocytes cannot take Mussels. Mussels (mollusc subgroup) edible bivalves place, because the mineralised cuticle that surrounds most of widespread in estuarine and coastal area, have long been the gut epithelium is believed to reduce translocation and used as a model to evaluate the biological impact of uptake of NPs from the gut into the haemocoel. This is an contaminants.79 As suspension-feeding invertebrates, they important notion, which must be considered also in human are particularly prone to NP exposure, due to their extremely exposure: the physical/mechanical barriers at the body well-developed systems for the uptake of nano- and surfaces block the vast majority of external particles from This journal is © The Royal Society of Chemistry 2020 Environ. Sci.: Nano, 2020, 7, 3216–3232 | 3223 View Article Online Perspective Environmental Science: Nano entering the body. On the other hand (and this is again true particle surface in vitro.93 The protein-coated TiO2NPs are also in mammals), NPs may affect P. scaber immunity internalised more efficiently, transiently down-regulating the indirectly, for example by impacting the gut microbiota.87 expression of genes encoding for proteins involved in immune response (NF-κB, FGFR2, JUN, p38MAPK), and This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. increasing a few antioxidant metabolic pathways (pentose 3.3 Deuterostomes phosphate, cysteine–methionine, glycine–serine).2 Conversely, Echinoderms. Echinoderms (ambulacraria subgroup) studies on sea urchin phagocytes exposed to PVP-AuNPs represent the most developed marine invertebrates and the in vitro underline the transient involvement of the TLR4/ERK bridge leading to the primitive chordates (urochordates and signalling pathway based on increased protein levels.94 The cephalochordates). Echinoderms (crinoids, ophiuroids, notion that these NPs do not cause a pathological immune Open Access Article. Published on 14 October 2020. Downloaded on 11/22/2022 11:54:47 AM. asteroids, holothuroids, echinoids) share several common response and associated irreversible damage in sea urchins characteristics, such as a penta-radial symmetry and a highly may prompt us to consider them immunologically safe also specialized water-based vascular system open to the outside. for other environmental species and human beings, given the This last characteristic explains why these animals are commonality of involved defensive mechanisms. particularly sensitive to rapid water chemistry variations (e.g., Thus, the combined use of in vivo and in vitro ion content) and why they have developed a complex, prompt investigations, its proximity to mammalian reactivity as well and sophisticated immune system.74 Environmental pressure as the similar features with invertebrate immune responses, favoured phenotypic plasticity in echinoderms, conferring an make the sea urchin a valuable model for the study of the individual ability to generate multiple phenotypic states and nano-immune interactions across evolution. activities (e.g., metabolism).88 The survey of the sea urchin Representative images of the invertebrate models genome has disclosed strong similarities between sea urchin described in this section and their immune cells are shown and human innate immune-related genes, and also provides in the Fig. 4. clues of alternative adaptive or anticipatory immune functions Humans. The study of nano-immune interactions in humans that are shared with humans.89 For these reasons, the sea (Homo sapiens, chordate subgroup) has two main directions, the urchin can be considered an excellent proxy for human model NP immunosafety, when NPs are present as environmental for investigating the nano-immune interaction in vivo90–91 and, contaminants or when used as nanomedicines, and their more recently, also in vitro. The establishment of long-term sea potential use for medical modulation of immune responses. urchin phagocyte cultures that maintain the physiological Most human in vitro models for assessing NP immune effects functions of fresh cells will allow for a deeper understanding of use human cancer cell lines or immortalized cell lines, which the mechanisms of immune activation by NPs.92 have the advantage of easy handling and high reproducibility. Current studies of the interaction of NPs with the immune However, as these cells lines mostly derive from cancer cells, system of the sea urchin Paracentrotus lividus show that, as in they have physiological and metabolic features that are humans, a number of sea urchin extracellular proteins significantly different from those of normal primary immune (glycoproteins, cytoskeletal proteins) form a biocorona on the cells, and therefore they may not be predictive for human Fig. 4 From the earthworm to the sea urchin: invertebrate models for the in vivo and in vitro studies of nano-immune interactions. A) The earthworm Eisenia andrei. B) SEM image of a free circulating E. andrei coelomocyte. C) The mussel Mytilus galloprovincialis. D) SEM image of a free circulating M. galloprovincialis haemocyte. E) The terrestrial isopod Porcellio scaber. F) SEM image of a P. scaber macrophage resident in the hepatopancreas; G) A violet adult Paracentrotus lividus collected from the Mediterranean Sea. H) SEM image of P. lividus phagocytes cultured for 24 h. 3224 | Environ. Sci.: Nano, 2020, 7, 3216–3232 This journal is © The Royal Society of Chemistry 2020 View Article Online Environmental Science: Nano Perspective responses.95 Thus, the use of primary immune cells from the NPs effect in vivo. Also, it warrants a personalised evaluation healthy tissue sources may provide much more reliable and of the NP effects on immunity, as each individual subject may representative responses on the nano-immune interactions.96 In react differently to an NP challenge. particular, leucocytes from peripheral blood from healthy In light of the notion that an unbalanced immune response This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. donors are readily available and include all the major types of underlies many diseases including autoimmunity, inflammatory professional immune cells, including the main innate cell types, diseases and inflammation-related cancers, the potential effects i.e., the mononuclear phagocytes (monocytes, dendritic cells), of NPs on the human immune responses requires extensive the polymorphonuclear phagocytes (neutrophils, eosinophils, attention. To obtain a realistic overview on the effects of NPs on basophils) and innate lymphoid cells. Thus, among the huge human immune responses, it is necessary to examine both the number of in vitro studies on human nano-immune direct effects of NPs on innate immune effector cells (capacity to Open Access Article. Published on 14 October 2020. Downloaded on 11/22/2022 11:54:47 AM. interactions, those performed on human primary immune cells induce an innate/inflammatory reaction that does not resolve are to be considered the most reliable/realistic. and become chronic) and their interference with an ongoing An efficient and predictive method that can be applied for defensive innate/inflammatory response (capacity to exacerbate assessing the general impact of NPs on human blood is the or inhibit a defensive response). In the latter context, the effects whole blood assay,97,98 in which an aliquot of anti-coagulated of NPs on the development of innate memory could be of great blood is challenged with NPs. This assay encompasses NP interest. NPs could be appropriately designed for modulating coating with serum proteins and interaction with all types of innate memory to treat two opposing conditions, i.e., diseases circulating cells, without however allowing for the identification where an innate/trained memory should be inhibited of specific cell types affected by NPs. Since phagocytes are the (autoimmune, inflammatory and cardiovascular diseases) and first defence cells that confront particulate matter, we can those in which memory responses should be potentiated (cancer, consider as the best models of innate immunity those based on sepsis, immunosuppression). Recent work describes that AuNPs monocytes isolated from blood of healthy donors (MAT, could interfere with the induction of BCG-induced memory by monocytes activation test), and on macrophages and dendritic shifting the human memory monocytes towards a tolerant cells differentiated in vitro from fresh monocytes.99 These phenotype.36 Another recent study shows that pristine graphene models allow us to observe not only the direct activation of can directly prime mouse macrophages towards a potentiated immune cells by NPs but also the capacity of NPs to interfere secondary response to TLR-activating stimuli, by increasing the with the physiological course of an inflammatory response4 or production of inflammatory cytokines and decreasing the with the induction of innate memory.36 The in vitro systems production of regulatory factors.101 The effect of NPs on innate based on human primary cells present the main disadvantage of memory is a new field with great therapeutic potential. A all in vitro systems, i.e., they do not reflect the complexity of an representative in vitro model for assessing the capacity of NPs to in vivo system, and, in addition, they suffer of donor-to-donor induce innate immune memory is shown in the Fig. 5. variations. Such variations are however sufficiently limited in the case of innate immune responses, with a general 4. Exploiting similarities and homogeneity of qualitative reaction and variability evident only in quantitative terms.100 Conversely, in the case of innate differences: improving models and memory responses the variability is much higher, as also tools for basic and translational observed in adaptive immune responses, as memory depends studies on nano-immune interactions on the individual history of exposure. This notion underlines the need of using human primary cells in immunoassays for A number of aspects determine the outcome and the possible assessing nano-risk, since they are more predictive evaluation of risk to environmental and human health posed by exposure Fig. 5 In vitro model for assessing the NP effect on innate memory induction in human monocytes and monocyte-derived macrophages. Cells are primed with NPs or with stimuli that induce memory (Bacille Calmette–Guérin, BCG, that induces potentiation of subsequent responses, and lipopolysaccharide, LPS, that induces tolerance) in the absence or presence of NPs for 24 h. Supernatants are collected, and cells rested for 6 days until reaching again a quiescent state. On day 7, the cells are challenged with a strong inflammatory stimulus (e.g., LPS), and their innate response measured in terms of production of inflammatory and anti-inflammatory cytokines. This journal is © The Royal Society of Chemistry 2020 Environ. Sci.: Nano, 2020, 7, 3216–3232 | 3225 View Article Online Perspective Environmental Science: Nano to engineered NPs, which include the inherent nature of the model gives us the opportunity to unravel a piece of the story. particle, the level of environmental exposure, the route of the We believe that the new frontier in our understanding of exposure, and the susceptibility of the living species and of nano-immune interactions also revolves around the the single individuals. As our studies focus on immunity, and differences. Studies on resistance and tolerance to This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. more specifically on innate immunity, we have examined the anthropogenic challenges (including engineered NPs and interaction of NPs with the innate immune system of nano-/microplastics) will most likely contribute to identifying different living species to assess similarities and differences. not only some key species-specific or common protective The first general observation is that each species uses a molecules, but also the mechanisms at the basis of the number of similar defensive mechanisms, adapting them to individual innate memory profile, i.e., the pattern of its own specific requirements (due to environmental epigenetic reprogramming that defines the individual Open Access Article. Published on 14 October 2020. Downloaded on 11/22/2022 11:54:47 AM. conditions, range of microbial species within the capacity to optimally react to a challenge and that is shaped environment, lifestyle, etc.). Whether this is indeed by the previous exposure to other challenges. While in short- adaptation of common mechanisms/tools or parallel lived animals this immune memory lasts for a lifetime and is evolution is still a matter of debate, but we see that physical/ usually shared by the entire community (living in the same mechanical barriers are common and very efficient environment and exposed to the same challenges), the innate mechanisms of defence, that toll-like receptors are present in memory profiles in human beings are expected to be highly all living species, and are used to recognition of invaders and variable and therefore warrant to a personalised approach in initiators of a defensive reaction, and that animals use the nano-risk assessment. phagocytic cells in the gut as specialised innate immune Which are the models and tools that we can use to assess effectors. Phagocytosis and ROS production, for direct nano-immune interactions, which information will they pathogen elimination, and kinase-dependent signalling for provide, and how can we use them optimally to answer to upregulating expression of effector proteins are also common different questions? to many animal species. The number of known innate We have focused on protostomes, including the immune receptors and pathways of many species are rapidly earthworm Eisenia, the isopod P. scaber, the blue mussel increasing and provides a rich resource to improving our Mytilus, and the plant A. thaliana for in vivo studies, while understanding of the mechanisms of innate immunity. The deuterostomes (including human models and the sea urchin species-specific range of the relevant innate receptors is P. lividus) have been used directly in vitro/ex vivo, as obvious, as their number and their ligand selectivity can discussed previously. differ significantly among species. The question is if the In plants, uptake and response to NPs is dependent on differences are a problem or, on the contrary, they can be the plant species and the capacity of NPs to cross the considered precious tools for a deeper understanding of mechanical barriers. While in some cases NPs can induce human immunity and for knowledge exploitation towards stress responses, it is notable that in several instances the targeted immunomodulation for medical purposes. We tend presence of NPs showed positive effects on growth and yield. to favour the latter, based on the experience gained within a This opens novel opportunities in the use of NPs in collaborative work of comparative nano-immune interaction increasing the fitness of plants of agronomic interest. assessment across living species.17 Non-human models have Whether such effect includes an increased resistance to a great value in unravelling the signalling pathways used in pests, in terms of induction of immune memory, is currently innate immune responses, in identifying conserved under investigation. mechanisms/molecules of innate immunity, and in From earthworms and isopods, we learned the major evaluating the in vivo impact of exposure to NPs. For importance of gut and resident microbiota in determining instance, the use on invertebrate models reveals that the the immune response to foreign particles (including NPs). A large majority of NPs either do not cross the body barriers, or disturbed host-microbiota interaction, in particular in the are rapidly excreted, while only in some infrequent gut, can impact on the immune system, increasing conditions they are retained within the body. Another susceptibility to infections. NPs may have significant effects important notion is that the interaction between NPs and in altering the microbiota balance, indirectly affecting the immune cells occurs mainly in the gut (from invertebrates to host immune defensive competence. Thus, some protostomes human beings), that resident microbiota influences immune become a practical choice to study the effect of ingested NPs responses, and that phagocytic cells are the main/only on the gut microbiota interaction with the host immunity immune cells involved in the first interaction with the and its ability to overcome infections, a study that cannot be foreign particles. Each organism has its own array of few or easily performed in human beings. many phagocytic cell types, which may preferentially interact Mytilus shows, in response to exposure to high NP with one or another foreign agent, but the basic mechanism concentrations, a stress response that is very similar to is common: recognition, uptake, degranulation, gene that observed in humans, with activation of phagocytes regulation with the production of defensive molecules. As and release of ROS. This warrants the use of Mytilus as discussed previously, each model has its own advantages and easy in vivo model for evaluating the phagocyte response disadvantages, specificities and commonalities, thus each to NPs. 3226 | Environ. Sci.: Nano, 2020, 7, 3216–3232 This journal is © The Royal Society of Chemistry 2020 View Article Online Environmental Science: Nano Perspective Table 1 NP interaction with innate immunity: similarities and differences across environmental organisms Organism Parameter Plant Earthworm Mussel Terrestrial isopod Sea urchin Human This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Exposure route Soil Soil Seawater Soil Seawater Skin, mucosal cells in gastrointestinal and respiratory tract Contact level Root cells Gut and skin epithelial Gills, gut Microbiota. No Coelomic fluid, Gut and skin and cells, coelomocytes, epithelial cells, direct contact with free immune mucosal epithelial and mucus, microbiota haemolymph, gut epithelial cells cells, gut immune cells microbiota, and haemocytes epithelial cells Open Access Article. Published on 14 October 2020. Downloaded on 11/22/2022 11:54:47 AM. haemocytes Biocorona ND Lysenins Complement ND Depends on NP Depends on NP surface component C1q, surface characteristics and haemolymph characteristics route of exposure components (several (several proteins) proteins) Uptake by Endocytosis-like Endocytosis Endocytosis No uptake Endocytosis Endocytosis immune cells Immune-related Regulatory molecules, PRRs, MyD88, MAPKs, PRRs, MAPKs, Changes in PRRs, MAPKs, PRRs, MAPKs, NF-κB, effects/activation miRs, PRRs, MAPKs, NF-κB, coelomic cytolytic NF-κB, lysozyme, protein, lipid, NFκB, energy metabolism, energy metabolism, factors, lysozyme, energy energy nucleic acid and cytokines, cytokines, chemokines, upregulation Rubisco metabolism, metabolism, carbohydrate growth factors, complement system, activase antimicrobial peptides antimicrobial levels. No immune energy growth factors, etc. peptides response metabolism Oxidative stress ROS production, ROS production, ROS production, ND Antioxidant ROS production, and apoptosis antioxidant antioxidant metabolism, antioxidant metabolism antioxidant metabolism, Rubisco apoptotic enzymes metabolism, metabolism, apoptotic activase, apoptotic apoptotic enzymes enzymes enzymes Immune ND ND Compensatory ND ND Innate immune memory memory recall responses to maintain immune homeostasis The comparison of the sea urchin and human genomes transfusion by-products, distributed in multiwell plates, allows for discriminating between conserved immune genes either used immediately after isolation or differentiated into and new human-specific genes. The possibility of exploiting macrophages or dendritic cells, frozen with a novel protocol long-term in vitro cultures of sea urchin immune cells makes in analysis-ready format and, in the case of poorly-adherent it possible to compare in vivo effects of NPs with the cells such as dendritic cells, anchoring them on bio printable evaluation of cell-specific molecular mechanisms, and to scaffolds in NP-receptive 3D culture arrangement. Upon focus on conserved molecules across evolution. Table 1 thawing the different types of cells were able to react to NPs reports the main features and mechanisms involved in the in a fashion that is qualitatively and quantitatively nano-immune interaction across the selected species. superimposable to that of cells that did not experience Based on the above consideration, we can state that the freezing–thawing. The reproducibility of immune cell realistic evaluation of nanosafety risk demands a ‘toolbox’ of response, or more often the absence of response to NP cell-based models able to reproduce with fidelity the challenge, translated to user-friendly cryopreserved cell functions of barrier tissues and of the immune system. This models offers a robust platform against which effects of is true for both environmental and human health risk. An materials of uncharacterized or non-specific nano- important issue in this context is the accessibility of tests composition can be tested with confidence. and assays, which would allow their use across several The next-generation in vitro human models, including co- institutions, companies, agencies and private labs, without culture with multiple cell types, 3D spheroids, 3D tissue the need of specialised instrumentation and personnel. sections, microfluidic organ-on-a-chip, air–liquid interface Within the PANDORA consortium, we have contributed to systems, are showing great capacity in helping to bridge the this ‘toolbox’ through adaptation of human immune cell gap between in vitro and in vivo exposure/effect. Notably, 3D models to be readily-available and user-friendly, whilst organoids are self-organizing, multi-cellular aggregates that retaining the analytical reproducibility and predictive value closely resemble function and architecture of human obtainable only with primary cells (that retain the functions tissues.102 Furthermore, microfluidic technologies can model of their tissue of origin under test conditions in culture). To fluidic properties in organ-like tissues, making it possible to this end, human blood monocytes were extracted from blood study small fluidic volumes in motion.103,104 Despite the This journal is © The Royal Society of Chemistry 2020 Environ. Sci.: Nano, 2020, 7, 3216–3232 | 3227 View Article Online Perspective Environmental Science: Nano availability of outstanding organoid models representing the safety, sustainability, inter- and trans-disciplinarity, critical lungs, gastrointestinal tract and many other organs, one of creativity and elegance. the greatest challenges for the coming years will be to further improve organoid-like tissues to include the interaction of This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. tissues/organoids with the immune cells that in real life 6. Conclusions: what's next? reside in the organ and with those that enter it during an By evaluating the impact of engineered NPs on the immune inflammatory reaction. reactivity of many living organisms across evolution, from plants to human beings, we can draw some conclusions that 5. Inclusive and sustainable research can help us in choosing the direction of future research. First Open Access Article. Published on 14 October 2020. Downloaded on 11/22/2022 11:54:47 AM. and innovation: the future is that immune responses across evolution are not too different apart for some specialized mechanisms due to perspectives of research on nano- anatomical peculiarities (e.g., plant cells that do not move). immune interactions As a consequence, reaction to NPs turned out to be pretty similar in different species, and mainly based on immediate In the study of nano-immune interactions, which is elimination, no recognition, or recognition/reaction that specifically relating to nanosafety for both environmental rapidly resolves. Only in few cases a detrimental reaction and human health, an aspect that should be duly could be observed, depending on the dose and chemical considered is the so-called responsible research and composition of the NPs, while in other cases it was possible innovation (RRI). RRI has gained increasing importance in to observe beneficial effects. EU research policy (with its inclusion in Horizon 2020) and Based on these findings, what should we do in the future? academia. RRI considers scientific investigations in a larger Abandoning nanosafety research, only because in general context by including the environmental and societal NPs do not seem to be detrimental, is not a good idea perspective. RRI is a policy concept experiencing a dynamic because there are too many special situations that could development, in which sustainability, social justice/ increase risk. Just to make an example, the immune system inclusion, open innovation, open science and open in the of elderly or chronically ill individuals is weakened, and world are the overarching goals. These are the backbone of therefore we should expect that agents that are innocuous in the Horizon 2020 RRI strategy and that will inform the healthy people may cause significant damage. Another case: upcoming European Green Deal. The RRI approach even when exposure to NPs or other individual agents does consolidates a new instrumental model (transdisciplinary, not cause a damaging effect, co-exposure to several agents at network-governed, externally validated, problem-solving, the same time may synergize and provoke unexpected impact-driven).105 detrimental reactions. The RRI strategy (“Science with and for Society”) is the Thus, we may foresee that future research of nano- approach that the PANDORA partners have tried to use to immune interactions may explore these directions: increase the researchers' capacity to think and act towards a • Development and refinement of in vivo/in vitro models more responsible, acceptable, and ethical science. The EU- that can realistically predict the nanorisk associated to acute funded project PANDORA encompassed five Academic and chronic exposure to NPs. Goals will be having models Institutions, four Research Centres, and two small and that discriminate between a normal immune response and a medium-sized enterprises (SMEs) distributed within seven pre-pathological reaction, models that predict risk for many European countries (Italy, Austria, Czech Republic, Germany, different environmental species and human beings, models Slovenia, United Kingdom, Spain), all with proven experience that predict realistic risk upon exposure to combined in higher education and training. Eleven PhD students have challenges, models that predict risk in frail organisms. been involved in an overarching training programme • Exploiting the potential of NPs in modulating some including training-by-research, joint courses of technical, immune functions (such as innate memory) for designing scientific and transferrable skills, completed with an intense new immunotherapeutic or immunopreventive approaches inter-sectorial networking exchange plan. PANDORA merged for human beings and crop production in agriculture. scientists, NP producers and experts in law and risk Building on the environmental impact of nanosafety management into a broader approach to environmental research and its impact on society for reshaping all these nanosafety. Within this complex framework, the younger studies along the principles of RRI, contributing at the same scientists have been encouraged to critically reflect106 on the time in building a new generation of researchers that research process within, around and outside the laboratory, privilege rigor, cooperation and sustainability. and to aim at collaboration and quality by privileging care and sustainability in their research planning and conduction.107 Such attitude/behaviour was particularly Authors' contributions suited to the goals of their studies, dealing with environmental nanosafety, and helped them in progressing AP and DB conceived and wrote the manuscript. NGB, MBF, towards the fundamental dimensions of RRI: courage, rigor, LC, PC, DD, AD, MAE, IG, JHH, PI, BK, PK, PP, VFP, DJS, CS, 3228 | Environ. Sci.: Nano, 2020, 7, 3216–3232 This journal is © The Royal Society of Chemistry 2020 View Article Online Environmental Science: Nano Perspective CJW contributed to writing, critical reviewing and editing the 11 S. Barua and S. Mitragotri, Challenges associated with manuscript. penetration of nanoparticles across cell and tissue barriers: a review of current status and future prospects, Nano Conflicts of interest Today, 2014, 9, 223. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. 12 I. L. Bergin and F. A. Witzmann, Nanoparticle toxicity by There are no conflits to declare. the gastrointestinal route: evidence and knowledge gaps, Int. J. Biomed. Nanosci. Nanotechnol., 2013, 3. Acknowledgements 13 M. van der Zande, A. J. Kokalj, D. J. Spurgeon, S. Loureiro, This work is based on the EU Commission H2020 project P. V. Silva, Z. Khodaparast, D. Drobne, N. J. Clark, N. W. van den Brink, M. Baccaro, C. A. M. van Gestel, H. Open Access Article. Published on 14 October 2020. Downloaded on 11/22/2022 11:54:47 AM. PANDORA (GA 671881) that has supported the studies of all authors. The authors wish to acknowledge the EM Facility of Bouwmeesteraf and R. D. Handy, The gut barrier and the CAS (Prague, Czech Republic; supported by the project fate of engineered nanomaterials: a view from comparative LO1509 of the Ministry of Education, Youth and Sports) for physiology, Environ. Sci.: Nano, 2020, 7, 1874. providing the electron microscopy images. A special thanks to 14 L. C. Smith, V. Arizza, M. A. B. Hudgell, G. Barone, A. G. Giuliana Donini (EU Commission, Bruxelles, Belgium), who Bodnar, K. M. Buckley, V. Cunsolo, N. Dheilly, N. Franchi, has expertly supported this project throughout all stages of its S. D. Fugmann, R. Furukawa, J. 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