16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Review www.small-journal.com Addressing Nanomaterial Immunosafety by Evaluating Innate Immunity across Living Species Diana Boraschi,* Andi Alijagic, Manon Auguste, Francesco Barbero, Eleonora Ferrari, Szabolcs Hernadi, Craig Mayall, Sara Michelini, Natividad I. Navarro Pacheco, Alessandra Prinelli, Elmer Swart, Benjamin J. Swartzwelter, Neus G. Bastús, Laura Canesi, Damjana Drobne, Albert Duschl, Marie-Ann Ewart, Jutta Horejs-Hoeck, Paola Italiani, Birgit Kemmerling, Peter Kille, Petra Prochazkova, Victor F. Puntes, David J. Spurgeon, Claus Svendsen, Colin J. Wilde, and Annalisa Pinsino* 1. Introduction The interaction of a living organism with external foreign agents is a cen- tral issue for its survival and adaptation to the environment. Nanosafety Interaction of living organisms with their surrounding environment seeks to main- should be considered within this perspective, and it should be examined tain a dynamic equilibrium that allows that how different organisms interact with engineered nanomaterials (NM) the organism’s survival and development. by either mounting a defensive response or by physiologically adapting to When confronted with unknown envi- them. Herein, the interaction of NM with one of the major biological sys- ronmental agents that may be potential tems deputed to recognition of and response to foreign challenges, i.e., the threats (e.g., a pathogenic or damaging agent), organisms may behave in two ways. immune system, is specifically addressed. The main focus is innate immunity, Either they react to the external challenge the only type of immunity in plants, invertebrates, and lower vertebrates, with a defensive response, or they change and that coexists with adaptive immunity in higher vertebrates. Because of their physiological status as an adaptive their presence in the majority of eukaryotic living organisms, innate immune response to their new conditions. This responses can be viewed in a comparative context. In the majority of cases, concept of dynamic equilibrium between the interaction of NM with living organisms results in innate immune reac- defensive reaction and adaptive behavior is also relevant to the interpretation of tions that eliminate the possible danger with mechanisms that do not lead nanotoxicological data. In this context, to damage. While in some cases such interaction may lead to pathological exposure of organisms to nanomaterials consequences, in some other cases beneficial effects can be identified. (NM) and the resulting response can be Prof. D. Boraschi, B. J. Swartzwelter, Dr. P. Italiani F. Barbero, Dr. N. G. Bastús, Prof. V. F. Puntes Institute of Biochemistry and Cell Biology Institut Català de Nanosciència i Nanotecnologia (ICN2) National Research Council Bellaterra, Barcelona 08193, Spain Napoli 80131, Italy E. Ferrari, Dr. B. Kemmerling E-mail: diana.boraschi@ibbc.cnr.it Center for Plant Molecular Biology - ZMBP A. Alijagic, Dr. A. Pinsino Eberhard-Karls University Tübingen Institute for Biomedical Research and Innovation Tübingen 72076, Germany National Research Council S. Hernadi, Prof. P. Kille Palermo 90146, Italy School of Biosciences E-mail: annalisa.pinsino@irib.cnr.it Cardiff University M. Auguste, Prof. L. Canesi Cardiff CF10 3AX, UK Department of Earth C. Mayall, Prof. D. Drobne Environment and Life Sciences Department of Biology University of Genova Biotechnical Faculty Genova 16126, Italy University of Liubljana The ORCID identification number(s) for the author(s) of this article Ljubljana 1000, Slovenia can be found under https://doi.org/10.1002/smll.202000598. S. Michelini, Prof. A. Duschl, Prof. J. Horejs-Hoeck Department of Biosciences © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Paris-Lodron University Salzburg Weinheim. This is an open access article under the terms of the Creative Salzburg 5020, Austria Commons Attribution License, which permits use, distribution and repro- N. I. Navarro Pacheco, Dr. P. Prochazkova duction in any medium, provided the original work is properly cited. Institute of Microbiology of the Czech Academy of Sciences The copyright line for this article was changed on 26 May 2020 after original Prague 142 20, Czech Republic online publication. A. Prinelli, Dr. M.-A. Ewart, Dr. C. J. Wilde AvantiCell Science Ltd DOI: 10.1002/smll.202000598 Ayr KA6 5HW, UK Small 2020, 16, 2000598 2000598 (1 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com interpreted as a response to a foreign dangerous agent. On this basis, rethinking nano-immunosafety would imply a more Diana Boraschi is an comprehensive, holistic organism-based evaluation of the inter- immunologist with over action of engineered NM with the immune system, as one 40 years of experience of the major biological systems that living organisms use for in innate immunity and probing the external environment, sensing potential threats inflammation, with a focus and mounting defensive or adaptive responses. on inflammation-related What does immunosafety mean in a nanoecotoxicological pathologies and strategies context? If an organism meets a NM, recognizes it as an alien for modulating anomalous substance (potential threat) and mounts a successful immune immune reactions. Since reaction (e.g., that eliminates it), does this represents in itself several years, she has been an adverse response? In our opinion it does not, because a suc- studying the effects of cessful immune reaction is a physiological health-preserving engineered nanomaterials defensive response to foreign agents and not alone a sign of on innate immunity in humans and across evolution, to toxicity. Thus, not only are the agents to which the organisms describe efficacy and failures of innate defensive mecha- adapt (e.g., by actively preventing immune reaction) handled nisms. She is currently senior associated researcher at the in a controlled way, but also those that are recognized as for- Italian National Research Council. eign may be directed to the appropriate cellular elimination system by an immune defensive response. A very important Annalisa Pinsino is a biologist issue that we have to consider is that the real efficacy of a with 20 years of experience defensive immune reaction cannot be evaluated at the level of in molecular and cellular single cell types, as is the case in many in vitro toxicity assays. studies, with a focus on cell In fact, during a successful defensive immune reaction, which regulation, signaling, and implies inflammation, many immune cells involved in the differentiation mechanisms reaction may actually die, and some damage to the tissue may influencing development, life occur. However, the overall final outcome is the elimination of maintenance, and immune the threat, repair of the tissue damage and re-establishment of response. Her current tissue integrity and function. Thus, the death of some cell types research focuses on under- (such as polymorphonuclear phagocytes or monocytes) may not standing of the biosafety of be alone a sign of immunotoxicity but of a normal homeostatic nanoparticles in an emergent immune reaction. In this view, we should consider with critical proxy to human nonmammalian model for in vitro basic attention the results obtained with single cell types in in vitro and translational immunology: the sea urchin Paracentrotus toxicological assays, as these may lead to incorrect assumption lividus. She is currently a researcher at the Italian National of systemic impacts when in an in vivo exposure, while such Research Council, leading the group of Invertebrate responses may actually be part of the normal immunological Immunology. response to NM. Although immune reactions may be part of a benign response to NM exposure, there are ways by which the presence of NM such as a chronic inflammatory response with associated tissue may affect immune responses in a fashion that could cause damage? Based on such considerations, we can therefore con- problems to the organism health. For example, could NM alter sider NM as immunologically safe when they do not impair the normal defensive immune reaction to a threat, could the immune responses, and do not cause pathological immune NM-induced metabolic or epigenetic changes in immune cells reactions. Suitable assays, designed in a way that allows us to affect their capacity to mount an effective defensive response capture the relevant details of the nano-immune interaction, are to a real danger, for instance an infection? Or could foreign required for adequately addressing these questions. or endogenous agents, when associated with NM, undergo Nano-immunosafety, as in all toxicological approaches for structural changes thereby triggering an anomalous immune any type of particulate and non-particulate agents, also depends reaction, for instance become allergenic or triggering an auto- on the nature of the exposure. NM concentration makes a immune reaction? Also, could NM, alone or associated with difference, and the same NM can fail to induce an adverse other agents, trigger a potentially pathological immune reaction, immune response at a low dose, while triggering a strong reac- tion at higher concentrations. Although this concept may sound E. Swart, Prof. D. J. Spurgeon, Dr. C. Svendsen obvious in toxicological studies, it is nonetheless important to UK Centre for Ecology and Hydrology bear this in mind when assessing the putative toxicity of NM. Wallingford OX10 8BB, UK Also, the immune system of each organism can react/adapt dif- Prof. V. F. Puntes ferently to the same NM at the same concentration. Thus, NM Institució Catalana de Recerca i Estudis Avançats (ICREA) that elicit no adverse response at a specific concentration for Barcelona 08010, Spain immune cells (such as macrophages or dendritic cells (DC)) or Prof. V. F. Puntes Vall d Hebron for a given organism, may result in an adverse effect for other Institut de Recerca (VHIR) cells (such as epithelial cells) or other organisms at the same Barcelona 08035, Spain exposure level. This also is an obvious concept but it is worth Small 2020, 16, 2000598 2000598 (2 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com reiterating in the context of a potential adverse effect. Finally, secretion of endogenous danger signals, the so-called phyto- we should consider that the ability of the immune system to cytokines, that are able to alert adjacent cells.[2] The transport cope with NM strongly depends on the health conditions of route for water and nutrients, through xylem and phloem, is the organism, and that immature, aging or damaged/diseased used by agents such as small molecules, RNA, or peptides to organisms usually have impaired immune reactivity and may achieve systemic activation of plant responses. NM can be also be less able to adequately cope with potential threats or, in gen- transported within the plant,[3] and therefore all plant tissues eral, with foreign agents. Thus, we should be aware that it is could potentially sense them. The extent by which NM can impossible to define the immune response to a NM in a simple interfere with or be actively recognized by plants depends on universal context, as its immunosafety will depend on the type the interaction interface, which is therefore a key element in of material, the exposure dose, route, and duration, the type of determining uptake and subsequent reaction. Uptake of NM in organism and the organism’s health conditions. The concept of plants is hindered by the cuticula in leaf tissue and the Cas- “safe-by-design” NM is therefore a chimera, and what we can parian strip, as well as the suberized endodermis in roots. Cell and should aim at is a nanotechnology that is “safer-by-design” walls are also a barrier, which only some NM can pass. Thus, in these different contexts. NM uptake by plants depends on many parameters, including Why do we think that nano-immunosafety studies should plant species, tissue, NM size, properties, and application focus on innate immunity, although adaptive immunity is such route. Uptake is reported for a number of nanoparticles (NP) an excellent and highly specific defense mechanism in man? in above-ground and underground tissues of specific plant spe- The reason is that innate immunity is the earliest and most cies, but it remains unpredictable, and no generalization can be potent type of immune reaction, thus the first to be engaged made on the nature of NM that are taken up, the plant species in the encounter with foreign materials. Most importantly, and the conditions under which internalization occurs.[4] innate immunity is the common type of immunity shared by Cells and tissues that form the interaction interface with the majority of living organisms, from plants to mammals, and NM in invertebrates represent the major animal recognition is the only type of immunity of over 95% of living eukariotic and defensive system in the many invertebrate phyla. They species (except higher vertebrates from bony fish to mammals are worldwide distributed, in each environment, and are that also display adaptive immune mechanisms). The high subjected to a wide range of threats even if their welfare is evolutionary conservation of innate immunity has produced overlooked compared to the attention devoted to vertebrates. a number of defensive mechanisms that are maintained with Invertebrate species are just as capable as vertebrates of expe- little variations across evolution. This may imply that, in the riencing stress and damage, and possess an evolutionarily recognition and response to NM, different organisms may conserved immune system acting as a first line of defense engage similar mechanisms. Eventually, it must be recog- against exogenous and endogenous threats to the host, such nized that in the development of innate immune mechanisms as pathogenic infection, tissue damage, or cancer. The inver- a key role is played by the host interaction with bacteria. The tebrate immune system encompasses a number of innate immune system does not develop in germ-free animals, and defensive mechanisms, such as cellular responses involved in this strongly suggests the interdependence and mutual coop- non-self recognition, phagocytosis, autophagy, cellular encap- eration of different living entities within an organism. sulation, and nodulation.[5] The most frequently used inver- When assessing nano-immunosafety, we should consider the tebrate model systems are those from the clade Protostomia organism in its entirety (including its symbiotic bacteria) and (e.g., cnidarians). Some other new model systems, such as also the environment in which it lives. In this review, we will the sea urchin (Echinodermata), are members of the Deu- try to address the interaction between NM and innate immu- terostomia, the same superphylum that includes vertebrates, nity across evolution by taking in consideration all the issues and thus have mechanisms that are likely to be more closely stated above. related to those occurring in humans. Unlike vertebrates, invertebrates lack classical antibody- based adaptive immunity, and key molecular and cellular actors 2. Nanomaterials and Innate Immune Effector Cells such as recombination-activating genes, B and T lymphocytes, except for some exceptions, are totally absent.[6] Notably, some 2.1. Innate Immune Cells across Living Organisms indications of alternative adaptive or anticipatory immune func- tions and memory-like responses characterized by challenge- In different living organisms, there are cells and mechanisms specific long-term protection were found in a few insect and that are able to recognize potential threats and react to them echinoderm species.[7,8] Recent findings blur traditional distinc- for defending and preserving the organism’s physical and func- tions between adaptive and innate immunity and emphasize tional integrity. that, throughout evolution, the immune system has used an Most interestingly, in plants there are no specialized immune unusually extensive selection of solutions to meet essentially cells. However, plants, as animals, can sense “danger” and react comparable requirements for protection.[6] If the adaptive to unfavorable conditions.[1] Danger signals are mainly external immune response is characterized by selective clonal gene factors such as pathogens, abiotic factors, and potentially also rearrangements from a broad repertoire of antigen-specific NM. As mentioned, plant defense does not depend on specific receptors on T and B lymphocytes, the innate immune response immune cells, as in principle all cells can sense threat. The is based mainly on different cell types recognizing invading response to challenge includes the release of preformed factors pathogens and activating antimicrobial immune responses such after cell wall damage, activation of danger receptors and the as phagocytic cells, granulocytes, macrophages, and mast cells.[9] Small 2020, 16, 2000598 2000598 (3 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com The use of invertebrate models for immunosafety investigation by neutral red uptake: after exposure, eleocytes started to die, requires a knowledge on the behavior of cells mediating immunity while amebocytes showed enhanced phagocytosis.[21] in each selected species, the degree of specificity (specific, quasi- Arthropods, including insects and crustaceans, use a range specific, or nonspecific) and memory in their immune strategies, of cellular and humoral immune defensive strategies to pro- together with information on particle behavior in the environ- tect themselves from damage. The main cellular defense mental medium in which animals live and the route of cell and mechanisms are phagocytosis, nodule formation (nodulation), organism exposure to NM. As a rule of thumb, the cells essen- and encapsulation. Both nodule formation and encapsulation tially involved in the invertebrate cellular and humoral defenses are responsible for the isolation and walling off of invading are the circulating and sessile blood cells (currently termed microbes and macroparasites.[22] In insects, there are circulating hemocytes or celomocytes) as well as other cell types, including blood cells, sessile hemocytes, and various other cell types like those residing in the fat body of insects, or in the hepatopancreas cells in fat body. Several types of hemocytes were described in and gills in crustaceans.[10] Regardless of the adopted cell names insects with the majority of species possessing only a few of of celomocytes and hemocytes, their defensive mechanisms are them.[23] For example, Drosophila hemocytes include three cell similar, and foreign material uptake and elimination is based on types:[24] plasmatocytes, which are involved in phagocytosis and phagocytosis, enzyme activation (e.g., lysozyme), reactive oxygen release of immune effector and signaling molecules; crystal species (ROS) formation, metabolic activation, and antimicrobial cells, which contain pro-phenoloxidase (PO) and other proteases protein (AMP) production (e.g., defensins).[11] of the pro-PO activating system;[25] and lamellocytes, which are In bivalve mollusks, hemocytes show both morphological rare in naïve animals but differentiate during infection and and functional features resembling those of the mammalian participate to encapsulation. Within the hemocoel of terrestrial monocyte/macrophage lineage.[12] As suspension feeders, isopods (such as Porcellio scaber), nongranular or hyaline hemo- bivalves have highly developed processes for the cellular inter- cytes are mainly responsible for phagocytosis.[26] Semigranular nalization of nano- and microscale particles in the key bio- cells also show some phagocytic ability but are apparently more logical functions of intracellular digestion and cell-mediated involved in encapsulation and nodulation. Granular cells are immunity. In this light, the hemocytes of different species predominately connected with the PO system[27] and, along (mussels, oysters, and clams) have been widely investigated as with semigranular cells, are thought to produce AMP and also a possible target for the effects of NM.[13,14] contribute to antioxidant defense.[28] In crustaceans, there are The coelomic fluid of the sea urchin Paracentrotus lividus reports that shrimp immune parameters (i.e., total hemocyte carries three different freely circulating cells, phagocytes, ame- count, respiratory bursts, PO, and superoxide dismutase (SOD) bocytes, and vibratile cells, which are morphologically and activity) were significantly affected by exposure or ingestion likely functionally distinct.[15] In the human body, macrophages of ZnO NP.[29] In a recent study on larvae, pupae, and adults recognize size and shape of their targets, facilitating inter- of Tenebrio molitor, transmigration of nanodiamonds through nalization via phagocytosis. The phagocytes of the sea urchin the insect cuticle was followed by inhibition of cellular and interact with NM (e.g., TiO2 NP) by internalizing them both humoral immune responses, presence of phagocytosed nano- in vivo and in vitro.[16–18] These cells have a dendritic-like mor- diamond aggregates mainly in hemocytes, and also in fat body phology and are the most abundant cell type present in the sea cells, but not in Malpighian tubule cells.[30] urchin body cavity fluid (>80%). Upon interaction with NM, sea Mammalian exposure to NM occurs at epithelial barriers urchin phagocytic cells potently respond with active phagocy- such as the epidermis, at mucosal sites if inhaled or ingested, tosis, becoming strongly adherent and metabolically active.[17,18] or directly in circulation, as in the case of nanomedical appli- Earthworm coelomocytes encompass two main subpopu- cations. Generally, NM do not cross the intact epidermal bar- lations, the amebocytes and the eleocytes, that differ for rier,[31] and therefore the contact with immune cells can more morphology, cytochemistry, and gene expression profile.[19] easily occur upon mucosal or intravenous exposure. Cells of the Eleocytes are large cells filled with a substantial amount of innate immune system can be abundantly found in the mucosal chloragosomes (large granules containing fluorophores). tissues and in circulation, have the task of recognizing foreign Although their main function is nutritional (e.g., storage of objects such as NM, and initiate an appropriate response. As glycogen and lipids), they also produce a number of bioactive already mentioned, the response can be adaptation/lack of reac- molecules.[11] Hyaline and granular amebocytes represent the tion (meaning that the foreign objects are considered harmless) main effector immunocytes and are specifically involved in or a defensive response. A successful response of these cells a broad range of defense functions including phagocytosis, will lead to sequestration and elimination or destruction of encapsulation, ROS production, and cytotoxicity,[11] corre- potential threats, including NM, while insufficient or overactive sponding to the expression of many immune-related molecules responses may lead to infection and tissue damage. found solely in amebocytes.[20] Cell viability is a meaningful In mammals, cells of the innate immune system are present immune marker in the earthworm, as it provides a general in the blood and in all body tissues. While many functions, overview of the organism´s immune capacity. Immunocytes such as pattern recognition, are conserved across innate cells, are reactive to a wide range of pollutants and may respond to each type contains specializations that benefit host defense. The NM in a distinct manner according to their immune reper- most abundant of innate cells are the short-lived phagocytic toire. In pollutant-spiked soils the amebocytes/eleocytes ratio and inflammatory cells called neutrophilic polymorphonuclear increases due to the higher mortality of eleocytes, which are leukocytes or neutrophils, which comprise between 50% and more susceptible to pollutants than amebocytes. The most vis- 70% of the white blood cells found in circulation. Their primary ible response difference is observed with metals, measured function is phagocytosis and destruction of foreign objects, Small 2020, 16, 2000598 2000598 (4 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com and they enter challenged tissues in large numbers following reaction macrophages participate to resolution of inflamma- a set of events known as the neutrophil recruitment cascade.[32] tion and tissue remodeling, thereby re-establishing the healthy Once in the tissue, they are able to engulf objects for phagocytic physical and functional status of the tissue.[38] DC are similar destruction, release factors and enzymes aimed at degrada- to monocytes and macrophages in their phenotype and func- tion of invaders, and release the so-called neutrophil extracel- tions, although they are poorly phagocytic, and are specialized lular traps (NET) that can capture and degrade objects in the in antigen presentation (a feature shared by other cell types surrounding environment.[33] Eosinophils are short-lived cells although less efficiently). DC are present in the circulation and mainly residing in tissues, involved in bacterial and parasite in tissues (immature DC), and are activated upon interaction clearance. They function through release of granules containing with foreign objects (mature DC), becoming able to efficiently different defensive molecules such as RNAses, peroxidases present antigens.[39] and soluble mediators (cytokines, chemokines, and growth fac- In conclusion, while in plants no specialized immune cells tors). Eosinophils are additionally known to be involved in the exist but all cells can sense and react to NM, in animals (from pathogenesis of allergies and asthma.[34] Mast cells are large and invertebrates to mammals) phagocytic cells are the major probably long-lived tissue-resident cells with several functions. effector of the innate immune defensive responses (Figure 1). Among them, they are innate effector cells involved in allergic These cells may adopt different morphologies in different reactions and inflammation but also in anti-inflammatory activi- microenvironmental conditions and can encompass subpopula- ties, upon activation and subsequent release of chemical media- tions with different names and more specialized roles in each tors by degranulation.[35] Within their defensive functions, mast organism, but they all have in common the capacity of rap- cells are involved in pathogen clearance in many bacterial, viral idly adapting to the presence of foreign materials by uptaking and parasitic infections, upon release of proteases, AMP, and and degrading/destroying them either directly or through the ROS production. Indeed, their primary evolutionarily conserved release of effector molecules. The modes of sensing and rec- role seems to be inflammation and pathogen killing, with a spe- ognition of foreign agents determines the type of reaction by cific capacity of toxin and venom detoxification. Innate lymphoid immune cells. A depiction of the interaction and uptake of NM cells (ILC) are a heterogeneous group of innate cells of lymphoid with innate immune cells is presented in Figure 2. origin classified into three subtypes according to their pheno- types, functions and cytokine expression profiles. ILC1 includes the classical natural killer cells, mainly secrete IFN-γ and can 2.2. Interaction of Nanomaterials with Innate Cells Depends induce T helper 1 cell activation. ILC2, also known as natural on the Microenvironment helper cells or nuocytes, secrete a number of interleukins (IL-4, IL-5, IL-9, and IL-13) in response to parasitic infections, and are The ability of NM to induce any type of reaction in immune involved in T helper 2 responses, including allergic reactions. cells strongly depends upon the previous interaction of ILC3 produces IL-22 and or IL-17 and are involved in the activa- microenvironmental factors with the highly reactive NM sur- tion of T helper 17 cells. As all innate cells, ILC are mainly acti- faces. NM entry in a body implies a rapid interaction with vated by stress signals, the cytokine milieu of the surrounding body fluids. Biomolecules adsorb to the surface and create tissue and microbial compounds.[36] a “biomolecular corona.” Thus, NM sensing is based on the Eventually, the mammalian innate immune system is highly biomolecular corona composition, and its particular spacing dependent on the multifunctional action of monocytes, mac- and structure, which depends both on the NM chemistry and rophages and DC. Monocytes are phagocytic cells found in size and on the biological tissue/fluid in which NM are pre- the blood, comprising ≈10% of the circulating white blood cell sent.[40] The corona can be a complex structure, sometimes population. Upon tissue damage, monocytes are recruited to 20–30 nm thick, consisting of soft and hard layers (soft and the tissue site and act as effective killer cells and initiators of hard corona), based on the affinity of the NM–biomolecule a more general defensive inflammatory reaction. Most of the interactions. Since the corona is composed by self-proteins inflammatory monocytes that enter a tissue probably die during and other self-molecules, these can mask the NM surface the local inflammatory reaction, but it is possible that some of and prevent immune recognition. However, the interaction them survive and differentiate into macrophages or DC within with the NM surface can alter the 3D structure and folding of the healing tissue. Macrophages are the tissue-resident mono- proteins, thereby making them signals for non-inflammatory nuclear phagocytes, mostly self-renewing tissue cells to which elimination by phagocytes or triggers for a defensive innate/ differentiated monocytes may contribute in different propor- inflammatory response.[41] Opsonization with complement tions (depending on the tissue).[37] Macrophages are mostly components, one of the most efficient innate defensive scavenging cells that keep the tissue clean by eliminating mechanisms against bacteria, can occur upon adsorption of senescent, dead or anomalous cells and denatured proteins. complement proteins in the NM biomolecular corona. Com- Macrophages are also sentinel cells that perceive foreign agents plement components can promote clearance by phagocytes (mostly invading microorganisms but also NM), can phagocy- through complement receptors, or trigger inflammation by tose and engulf them for destruction within a phagolysosome, activating the complement cascade. or sequester them for eventual elimination. Notably, mac- Evidence for the implications of NM biocorona in immune rophages are poor inflammatory effector cells but, if required, recognition and response is also emerging in non-mammalian they can initiate a defensive response by producing factors that vertebrate and invertebrate models. In zebrafish blood plasma, recruit and activate circulating effectors (such as neutrophils sex-specific coronas were identified around SiO2 NP,[42] with the and monocytes). Eventually, at the end of an inflammatory prevailing contribution of vitellogenins conferring a “female” Small 2020, 16, 2000598 2000598 (5 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Figure 1. Innate immune cells across living species. SEM images of A) a hemocyte of the mussel Mytilus galloprovincialis, B) a hemocyte of the earth- worm Eisenia andrei, C) a phagocyte of the sea urchin Paracentrotus lividus, and D) a hemocyte of the woodlouse Porcellio scaber. E) TEM image of a human monocyte-derived dendritic cell (after in vitro differentiation for 6 days with GM-CSF and IL-4). F) SEM image of a human blood monocyte after 7 days of culture on a collagen matrix. G) TEM image of a human monocyte-derived macrophage, differentiated for 7 days in culture with M-CSF. biological identity to the NM. Moreover, zebrafish lymphoid and precursor protein. Such biocorona promoted Ca2+-dependent myeloid blood cells preferentially accumulated those NP with a recognition by phagocytes, leading to uptake followed by tox- female corona profile. The capacity of leukocytes to remove such icity.[46] In contrast, the main constituents of the biocorona on NP–protein complexes from the bloodstream would prevent spe- the surface of TiO2 NP exposed to the supernatant of cultured cific targeting of vitellogenin-coated NP to developing oocytes, sea urchin immune cells were identified as a subset of adhe- and possible downstream consequences on reproduction.[42] sion and cytoskeletal proteins. Upon coating with these pro- NM can be coated with proteins and other molecules also teins, TiO2 NP aggregated on the outer cell surface of phago- in biological fluids of invertebrates. In earthworms, the bioco- cytes and were then internalized within well-organized vesicles rona formed around Ag NP contains a predominant protein, without eliciting harmful effects.[16] lysenin, that ultimately triggers higher uptake by immune Overall, data so far obtained in invertebrates indicate that, cells.[43] By analyzing gene transcription kinetics upon in vivo in each species, NM can be coated by different and peculiar exposure to Ag NP, the transcription of lysenin was, after tran- proteins, which makes the biological implications less straight- sient induction, gradually suppressed over time, with induction forward.[45] In addition, the results obtained in marine inverte- of a new secretome, suggesting a reshuffling of the NP bioco- brates suggest that the net surface charge retained by different rona that will change the interaction with immune cells.[44] In NM in biological fluids is an important factor in the formation the marine mussel Mytilus, the complement component C1q of a stable surface coating and for the consequent interaction and Cu,Zn-SOD represent the single protein types present in with immune cells. the biocorona that forms, upon in vitro interaction with mussel It should be noted that a corona can form on NM surface biological fluid, on different types of NM with positive (PS-NH2 even before entry into an organism, by surface adsorption of NP) or negative (CeO and TiO2 NP) charge, respectively.[45] environmental agents (such as bacterial components or aller- The formation of a distinct corona increased or decreased the gens). As in the case of the biomolecular corona mentioned immunotoxicity of either type of NM for hemocytes. above, the composition and steric/architectural features of In the celomic fluid of the sea urchin P. lividus, the bioco- the corona determines the subsequent interaction with living rona formed around PS-NH2 was dominated by the toposome organisms. We should underline the fact that no naked NM can Small 2020, 16, 2000598 2000598 (6 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Figure 2. NM uptake by innate immune cells across living species. A) Representative TEM image of a hyaline hemocyte of the earthworm Eisenia andrei showing internalized CuO NP (red arrows) after in vitro exposure (100 µg mL−1 CuO NP, diameter 5–15 nm) for 2 h. B) TEM image of a phagocyte of the sea urchin Paracentrotus lividus showing internalized TiO2 NP (red arrow) following in vitro exposure (1 µg mL−1 TiO2 NP, diameter 10–65 nm) for 24 h. C) TEM image of a human monocyte-derived macrophage exposed to Au NP (20 µg mL−1, 50 nm diameter) in vitro for 24 h. The white-framed area is magnified and displayed in (D), where red arrows indicate some of the internalized Au NP. be found in our environment, and therefore the biological sys- response that includes ROS production and MAPK activation, tems interact with “hybrid” NM that include environmentally but that can also lead to a hypersensitive response resulting in borne non-characterized molecules on their surface. cell death of the infected plant parts and restricting nutrition to the invading pathogen.[47] If and how plants use these mecha- nisms to sense and react to NM is poorly known at present. 2.3. Innate Sensing Mechanisms in the Recognition and Uptake In invertebrates, the innate defense mechanisms also of Nanomaterials depend on sensing foreign material by several classes of immune receptors, which bind PAMP and damage-associated It is hypothesized that innate cells may sense NM upon inter- molecular patterns (DAMP) and activate rapid innate defensive action with surface receptors, mainly through typical innate reactions. As in plants, both cell surface receptors and intracel- immune pattern recognition receptors (PRR). lular sensing mechanisms can identify PAMP and initiate the Plant PRR are similar to animal toll-like receptors (TLR), innate immune response.[48] Echinoderms,[49] amphioxus[50] and can recognize microbe- or pathogen-associated molecular and, independently, also mussels[51] and sponges[52] have an patterns (M/PAMP), thereby triggering defense responses that expanded repertoire of PRR that include the intracellular NLR include ROS production, MAP kinase (MAPK) activation, and and the transmembrane TLR, while arthropods, nematodes, induction of defense genes, as well as immediate responses earthworms,[53,54] and tunicates have a more limited repertoire, such as phytohormone release and callose deposition to all resulting from independent but combinatorially constrained strengthen the cell walls. The sum of these responses leads to evolution of domain architectures.[55] The invertebrate immune enhanced resistance of the plant to invading pathogens, a phe- cells also express a wide range of inducible genes coding for nomenon known as pattern triggered immunity (PTI). Some extracellular recognition proteins, including lectins, pepti- pathogens can suppress PTI by injecting virulence factors into doglycan-recognition proteins, lipopolysaccharide (LPS) and the plant cells that are negatively affecting PTI signaling com- β1,3-glucan-binding proteins, and fibrinogen-related proteins ponents, leading to enhance susceptibility of the plant. Plants (FREP).[56–59] can recognize some of these virulence factors by NOD-like Despite the presence of PRR and their clear role in pathogen intracellular receptors (NLR) that can trigger a fast defense recognition and immune reaction to threats, recognition of NM Small 2020, 16, 2000598 2000598 (7 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Table 1. Cells and tissues interacting with NM across species. Species Cell/tissue Cell function NM type Refs. Zea mays L. (plant) Xylem Transport tissue in vascular plants CuO [3] Mytilus galloprovincialis (mollusc) Hemocytes Phagocytosis, opsonization, cytotoxicity, inflammation, humoral response, TiO2 [14, 45] ROS and NO production PS-NH2 CeO NP Eisenia fetida (earthworm) Amoebocytes eleocytes Phagocytosis, opsonization, cytotoxicity, inflammation, humoral response, Ag [14, 44] ROS and NO production, graft rejection Paracentrotus lividus (sea urchin) Phagocytes Phagocytosis, opsonization, clotting, cytotoxicity, inflammation, humoral TiO2 [16–18] response, graft rejection, ROS and NO production, clearance Penaeus semisulcatus (shrimp) Hemocytes Encapsulation, cytotoxicity, PO activity ZnO [29] Homo sapiens Macrophages Phagocytosis, clearance, inflammation TiO2 [41] by celomocytes through PRR sensing has been reported only endolysosomes. Elimination of endocytosed particles may be in the case of recognition and uptake of TiO2 NP by sea urchin achieved with lysosomal enzymes (if the NM are sensitive to phagocytes through TLR4.[18] Otherwise, these is no evidence of them) or by autophagic processes.[77–79] However, intracellular a direct receptor activation by NM with subsequent inflamma- degradation of several NM, for instance gold or tattoo pigment tory activation. particles, is not readily achieved. Consequently, many particles In vertebrates, both TLR and NLR are present, generally in that are engulfed by cells may remain as such for prolonged a smaller number than in invertebrates (possibly because of periods without inducing toxic effects, or can be passed from the presence in higher vertebrates of adaptive immunity).[55] macrophage to macrophage during time or upon cell death[74,80] On mammalian innate cells additional PRR include scavenger Innate immune cells can also capture and degrade NM without receptors (SR) and C-type lectin receptors, which can sense dif- engulfing them. Indeed, neutrophils, eosinophils, and mast cells ferent types of microbe-associated molecular pattern (MAMP), can react to the interaction with NM with degranulation and PAMP, and DAMP, allowing cells to react appropriately to release of proteases and other enzymes. Several studies have changes in their tissue conditions.[60,61] Upon activation, PRR shown that neutrophil and eosinophil peroxidases (EPO) can initiate a series of intracellular signals that lead to an immune degrade both multi-wall and single-wall carbon nanotubes.[81,82] response intended to eliminate the threat and restore tissue Another mechanism of particle capture and clearance that condition. Several NM have been reported as able to activate the does not imply their internalization is the formation of extracel- NLRP3 inflammasome,[62–66] thereby inducing caspase-1 activa- lular traps (ET).[83] Upon challenge resulting in cell death, cells tion and the production of inflammatory IL-1β. Mechanisms of release a net of chromatin fibers, which are used to immobilize inflammasome activation by NM include induction of K+ efflux extracellular particles, such as pathogens or non-self objects. and ROS generation and lysosomal membrane destabilization. The innate cells that can form ET include eosinophils, mono- Caspase-1 activation and pulmonary inflammation by silica cytes, macrophages, neutrophils, and mast cells.[34,84] This pro- particles has been shown depending on the presence of the cess is evolutionarily conserved, and ET have been described scavenger receptor SR-B1.[67] also in plants and invertebrates.[85,86] ET are often coupled with Generally, NM seem readily taken up by phagocytic cells. In local release of ROS and enzyme-containing intracellular gran- some cases it has been shown that PRR such as SR and TLR ules, which contribute to trapped particle degradation. Indeed, participate to the uptake,[68,69] and it is important to note that this mechanism acts efficiently also for NM, as shown in a the presence and characteristics of the biomolecular corona study describing the peroxidase-dependent extracellular deg- can dictate the type of interaction of NM with SR or other radation of single-walled carbon nanotubes trapped in NET.[87] innate receptors (e.g., complement receptors for complement- NM can trigger ET formation in vivo possibly through lyso- opsonized NM). After interaction with the cell membrane, NM somal leakage and ROS production.[88,89] uptake also depends upon factors such as shape, size, surface Based on these observations, we can generally conclude that charge and surface chemistry. It was shown that particles with NM are sensed by innate cells through different receptors/ a diameter ≤0.5 µm as well as rod/fiber-shaped NM were pref- mechanisms and either internalized and tagged for elimination, erentially taken up by DC compared to larger particles or those or can induce cell activation (mainly associated to cell death) with cubic or spherical shape.[70–73] Smaller NM (≤500 nm) that leads to ET formation and eventual particle handling. are mainly internalized via pino- or micro-pinocytosis and Table 1 summarizes the reported examples of NM–innate cell via clathrin- or caveolin-mediated endocytosis, while NM of interaction across species and their functional consequences. >500 nm are usually taken up by phagocytosis.[74–76] Thus, it appears that mechanical forces, as well as opsonizing proteins interacting with the various receptors, are responsible for the 3. Nanomaterials and Activation of Innate majority of NM endocytic uptake, with a major role for NM size Immune Genes/Factors and shape in determining the type of uptake mechanism. These NM internalized via endocytosis are encapsulated In addition to, and as part of, cell-based mechanisms of sensing into early endosomes that then fuse with lysosomes, to form and reaction to external challenges, a wide array of soluble Small 2020, 16, 2000598 2000598 (8 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Figure 3. Innate immune molecules and signaling across living species. Upper panels: comparative examples of evolutionary conserved innate sign- aling in plants (Arabidopsis), insects (Drosophila), sea urchins, and mammals. Signaling cascades initiate when pattern recognition receptors (PRR) on the cell membrane recognize specific pathogen-associated pattern (PAMP). Signal is transduced within the cytosol by a set of signaling proteins (mainly protein kinases), which in turn activate evolutionary conserved transcription factors that will orchestrate transcription of inflammatory related genes. Consequently, each organism produces a panel of soluble molecules (pathogenesis-related proteins, antimicrobial peptides, cytokines, and others) that contribute to coping with different foreign agents. Lower panels: comparative list of PRR and soluble molecules (pathogenesis-related proteins (PR), antimicrobial peptides (AMP), and cytokines) known in plants (Arabidopsis), insects (Drosophila), sea urchins, and mammals. Artwork by Andi Alijagic. molecules are produced that either directly exert defensive func- by producing ROS and phytohormones such as salicylic acid, tions or communicate with surrounding tissues for mounting a jasmonic acid, and ethylene.[90] NM are reported to activate defensive response. Figure 3 depicts the main molecules and some of these molecular defense responses, mainly ROS pro- pathways involved in the immune defensive reactions across duction and gene activation, suggesting that responses to NM species. may share similarities with immune responses.[91] Interestingly, Upon cell damage or PRR activation, plants can release pre- recent transcriptomic analysis in Arabidopsis thaliana revealed formed factors and phytocytokines, which function as endog- that exposure to different types of NP (e.g., ZnO, fullerene soot, enous danger signals that alert adjacent cells.[2] Most of the or TiO2) repressed a significant number of genes involved in defensive factors produced by plants are soluble molecules that phosphate starvation, pathogen and stress responses, with pos- reach all plant tissues using the same transport route as water sible negative effects on plant root development and defense and nutrients. Depending on the type of pathogen, plants react mechanisms. A recent systems biology approach, including Small 2020, 16, 2000598 2000598 (9 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com omics data from tobacco, rice, rocket salad, wheat, and kidney particles.[102] One of the mechanisms by which NM may provoke beans, confirmed that metal NM provoke a generalized stress cell toxicity is ROS generation. Oxidative stress is usually moni- response, with the prevalence of oxidative stress compo- tored by assessing lipid peroxidation and measuring antioxidant nents.[92,93] However, a general view on NM-induced immune enzymes.[103] Although exposing celomocytes to metallic NP can responses is difficult, as contradictory results have been lead to ROS production,[104] it is still controversial whether such obtained by different groups, possibly due to different experi- effect can be ascribed to NP or to released metal ions. Celomo- mental conditions. cytes exposed to metal-based NP (Ag and carbon) show modu- Invertebrate immune cells (hemocytes and celomocytes) are lated expression of oxidative stress genes and induction of ROS responsible for immune reactions through phagocytosis and production.[105–107] The antioxidant enzyme catalase is more the production of a number of cytotoxic factors, such as hydro- effective in ROS neutralization than superoxide dismutase in lytic enzymes, AMP, ROS, and nitric oxide (NO).[11,12,14,94,95] earthworms.[108] Upon catalase down-regulation, the accumula- Upon exposure to TiO2 NP, earthworm engulf NP by phagocy- tion of H2O2 was shown to accelerate NP dissolution.[109] tosis, and simultaneously modify mRNA expression of immune In arthropods (including crustaceans), lectins from the proteins and metallothioneins, thereby affecting the molecular hemolymph are important in immune recognition and phago- response of immune and detoxification systems.[96] Immune cytosis. A major defense molecule in the immune response cells also express many inducible immune-related genes that of crustaceans is the conserved Cu-containing enzyme PO. A are able to bind microbial patterns. Some of these are mem- number of pattern-recognition proteins (such as PGBP, LGBP, brane proteins involved in recognition, e.g., lectins and lectin- and BGBP) participate in immune defense by recognizing like molecules (see previous section), while others are released pathogen-associated carbohydrate-containing molecules, i.e., as soluble proteins, such as FREP and AMP.[11,97] peptidoglycans, lipopolysaccharides, and β-glucans.[110] In In the marine mussel Mytilus, hemocyte functional param- insects, the tracheae, epidermis, gonads, and gut epithelium eters and transcription of different immune-related genes are are involved in release of PO and AMP and in the production of affected by exposure to different NM types with a high degree ROS.[111] As in many other invertebrates, melanin synthesis is a of specificity.[13,98] Echinoderm gene sequencing reports over central mechanism of innate immunity and a major response 1000 immune genes falling into the innate immunity area,[55] to a variety of immune challenges.[112–116] Part of the melanin and exposure of P. lividus to NM induces selective changes in synthesis pathway is catalyzed by the enzyme PO. The PO cas- immunological pathways and molecules.[99] As an example, cade produces melanin, and also induces multiple potent bioac- exposing P. lividus to TiO2 NP activates suppressive mecha- tive agents, such as peroxinectin and ROS, that aid in phago- nisms by down-regulating the expression of genes encoding cytosis and cell adhesion. Proper PO modulation is crucial to immune-related and apoptotic proteins (e.g., NFκB, FGFR2, ensure survival of the organism. The majority of invertebrates JUN, FAS, VEGFR, and Casp8); elicits metabolic rewiring by activate the PO cascade from the pro-PO enzyme upon recog- boosting the immune cell antioxidant activity (e.g., pentose nition of foreign matter.[117] However, in a limited number of phosphate, cysteine-methionine, glycine-serine metabolism Crustacea, including P. scaber, the production of melanin is not pathways); and restores homeostasis by keeping at physiolog- initiated by pro-PO, and it has been suggested that hemocyanin, ical levels some key immune-related proteins (e.g., TLR4, IL-6, the oxygen transporter in isopod hemolymph, is responsible for MAPK, heat shock protein 70 (HSP70)).[17] initiating the cascade by undergoing a conformational change The earthworm celomic fluid also contains various antimi- upon interaction with non-self material.[118] crobial factors or peptides, such as lysozyme and lumbricin/ Reactive nitrogen species are chemically reactive species that lumbricin-related protein, and hemolytic molecules such as include NO, and are produced through normal metabolism, fetidin and lysenins. In Eisenia earthworms, lysenin turned out after an injury, during infections and disease and in response as a key protein in the earthworm secretome upon interaction to environmental pollutants and radiation. NO is known to with Ag NP, and it is possibly involved in opsonization-induced modulate many biological processes including inflammation cellular interaction.[43] The supplementation of culture medium and cytotoxicity.[119] In crayfish, the hemocyte-derived NO was with celomic fluid supports celomocyte survival during in vitro able to promote hemocyte-bacterial adhesion and increase the culture. Such protein-rich environment, present both in the hemocyte bactericidal activity.[120] earthworm body and in the supplemented culture medium, AMP are another major component of the invertebrate defen- leads to the formation of a biomolecular corona on the NM sur- sive system. They are small cationic, amphipathic molecules face during exposure. The biomolecular corona can function as effective against Gram-positive and Gram-negative bacteria, opsonin that promotes phagocytosis. Indeed, earthworms can yeasts, fungi, and some protozoa and enveloped viruses. AMP efficiently opsonize microbial particles by means of the soluble are found in the isopod hemolymph, function by disrupting the form of the receptor protein celomic cytolytic factor (CCF).[11,100] target cell membrane integrity via destabilization or pore for- As membrane protein CCF participates in the activation of mation, and there is some evidence that they may be translo- the pro-PO cascade, while its soluble form has opsonizing cated into the microorganisms’ cytoplasm where upon they can capacity.[101] Upon incubation of synthetic NM (HEMA particles) interact with specific intracellular targets to cause cell death.[121] with celomic fluid, CCF was adsorbed onto the particle surface In vertebrates, and more specifically in mammals, it is in a manner that was recognizable by CCF-specific monoclonal known that several types of NM (e.g., carbon, silica, polysty- antibodies (i.e., surface-adsorbed CCF retained its native confor- rene, and latex NM) can interfere with the immune system, mation). This adhesion capability of CCF is an early example of inducing oxidative stress, complement activation and release of pattern recognition-independent opsonization of non-biological soluble mediators such as cytokines, chemokines and growth Small 2020, 16, 2000598 2000598 (10 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com factors. Although an immunomodulatory effect has been TiO2, ZnO, CeO2, Mn3O4, and Ag NP, have been reported able shown for several types of NM, the extent of activation of the to induce oxidative stress signaling cascades in mammalian immune response is specific for every type of object, from cells.[131,132] During an inflammatory reaction, innate immune minimal (Au NM) to substantial (carbon nanotubes). When cells produce matrix metalloproteinases (MMP), which are fac- NM are used as biomedical products, they are often actively tors involved in the degradation and remodeling of the extra- injected in the bloodstream where they encounter a complex cellular matrix during wound healing. Recently it has been environment of plasma proteins and immune cells. NM can be reported that different NM (Ag, Au, Zn, and carbon-based sensed and come in contact with monocytes, platelets, polymor- NP) can regulate the synthesis of some MMP,[133,134] although phonuclear leukocytes, and DC and subsequently with tissue the variability of study models and of NM used does not yet resident phagocytes. The encounter between NM and immune allow full understanding. Overall, the possibility of using NM cells/factors at the site of injection can lead to several reactions, to modulate/control the immune cell activation and their ROS which could ultimately result in the complete clearance of the and MMP production opens promising perspectives for new NM. Hemolysis, thrombogenicity, and complement activation therapeutic approaches. are the main processes considered relevant, from a toxicological Also in the case of mammals, AMP are produced for con- point of view, when NM are injected in the bloodstream.[122] For trolling the interaction with bacteria. AMP, which include example, it has been reported that Ag NP at high concentra- defensins and cathelicidins, are involved in maintaining tions can induce hemolysis and affect lymphocyte viability in healthy commensal gastro-intestinal flora and in bacterial clear- human and rat blood in vitro, while unable to activate com- ance, with functions ranging from direct bactericidal activity plement and induce platelet aggregation, and also increase to initiation of innate immune responses.[135] Experimental sensitivity to thrombogenic factors in vivo in the rat.[123,124] evidence shows that different types of NM can promote and The effects obtained at very high doses used in these studies amplify the antibacterial capacity of AMP, as in the case of the do not appear at lower concentrations. Depending on their synergistic effect of Ag NP with the bacterial AMP polymyxin physico-chemical properties, NM may trigger the activation of B, likely caused by the polymyxin B-driven permeabilization of the complement system, which is crucial for the rapid detec- the bacterial outer membrane that enhances the intracellular tion and elimination of circulating particles and pathogens by antimicrobial activity of Ag NP.[136] Conversely, as in the case phagocytic cells. The complement system acts with three main of carbon NP and cathelicidin LL-37, NM may alter the AMP mechanisms. The first is the coating of pathogens/foreign structure, thereby hampering their bactericidal effect.[137] objects with proteins acting as opsonins (e.g., the complement A number of innate immune cells produce intracellular components C1q or C3b) that can be recognized by specific granules that store a range of soluble factors (e.g., proteolytic receptors on the surface of phagocytic cells thereby promoting enzymes, RNAses, peroxidases, histamine, and other media- clearance via phagocytosis. The second is the production of tors). Upon activation, these cells (eosinophils, neutrophils, chemotactic peptides (e.g., the complement component C5a) basophils, mast cells, monocytes, and macrophages) can for recruiting monocytes and neutrophils to the site of reaction. degranulate and release the active mediators in the surrounding Finally, the third mechanism is the formation of the membrane tissues. Proteins such as myeloperoxidase (MPO), EPO, MBP attach complex on the microorganism surface that leads to lysis (major basic protein) and ECP (eosinophil cationic protein) are of the pathogen.[125,126] Several studies reported the capacity very efficient in pathogen killing, although they can also induce of NM to activate the complement system, with NM surface toxicity in several tissues. MPO and EPO, mainly involved in charge playing a most prominent role. Charged NM (e.g., lipid bacterial and parasite clearance, were also found able to degrade and polycation-based NP) could easily bind human serum com- single-walled carbon nanotubes in vitro and in vivo, a fact that plement, compared to their neutral PEGylated counterparts.[127] underlines the role of leukocytes in NM clearance.[81,138] The fact Surface characteristics also are predictive of NM–complement that NM can promote NET formation, as bacteria do, further interactions. It has been shown that dextran coating in loop supports the notion that neutrophilic leukocytes are among the configuration increases cleavage of C3 into C3a and C3b upon central innate effectors in NM clearance.[88,89] incubation in human serum compared to polymers in brush Very important immune soluble factors are the cytokines, formation,[128] and that the switch of polymer configuration is chemokines, and growth factors, which regulate crucial associated with a shift from the classical to the lectin comple- immune and cellular processes such as proliferation, acti- ment pathway.[129] Although complement activation has been vation, differentiation and migration. The ability of NM to claimed as an important safety issue in relation to medicinal induce an immune response is, therefore, often character- NM, it seems that a major role is instead played by activation ized by their capacity to provoke the production or secretion of innate cells.[130] This stresses the need of examine in depth of inflammatory cytokines and chemokines. A comprehensive the complex interrelationships among innate immune mecha- study on the capacity of engineered NM to activate in vitro the nisms activated by exposure to NM, in particular in the design human monocytic cell line THP-1 to produce cytokines and and production of nano-medicaments. chemokines showed that, in response to 19 different NM, there As in invertebrates, NM can also induce ROS production and is little correlation between activation of innate immunity (i.e., oxidative stress in mammalian cells. Although ROS are natural production of innate cytokines and chemokines) and cytotoxic byproducts of cellular metabolism with an important role in cell effects.[139] Despite the significant limitation due to the use of survival, death, differentiation, signaling, and inflammation, an a highly proliferating tumor cell line instead of primary mono- imbalance in their generation and neutralization can result in cytes/macrophages, an interesting result is that NM can be oxidative damage, leading to cell death. Several NM, such as roughly grouped into those that can promote inflammation Small 2020, 16, 2000598 2000598 (11 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Table 2. Molecular defense responses activated by NM across species. Organism Exposure type Innate immune gene/factor activation NM type Refs. Plants In vitro (Tobacco BY-2 cells), Oxyradical production, gene activation (e.g., MAPK signaling, salicylic Ag, ZnO, Al2O3, TiO2 [91] in vivo (Arabidopsis thaliana, rice, acid signaling, and stress response), secondary metabolite release corn) Earthworms In vitro (primary immune cell Gene modulation (fetidin, metallothionein, coelomic cytolytic factor, and TiO2 [43, 96, 104, 106] culture) and in vivo superoxide dismutase), lysenin release, apoptotic signaling, oxyradical Ag production Mussels In vitro (short-term primary Oxyradical production, stress-activated MAPK signaling, apoptotic TiO2 [14] immune cell culture) and in vivo signaling, lysozyme release, AMP production SiO2 ZnO CeO2 Sea urchins In vitro (primary immune cell Gene and protein modulation (e.g., MAPK, NFκB, FGFR2, JUN, FAS, TiO2 [17, 18] culture) and in vivo VEGFR, and Casp8), metabolites rewiring (e.g., pentose phosphate, cysteine-methionine, glycine-serine metabolism pathways), cytokine production Mammals In vitro (primary immune cell Hemolysis, thrombogenicity, complement activation, NFκB and oxidative Ag, Fe3O4, TiO2, ZnO, CeO2, [81, 122–124, 127, culture) and in vivo stress signaling, production of oxyradicals, opsonins, chemotactic pep- Mn3O4, Au, carbon 131–136, 138–140] tides, MMP, AMP, proteolytic enzymes, RNAses, peroxidases, histamine, cytokines, chemokines, growth factors (innate immune activation) and those that can apparently down- pathological inflammation, but only a sign of an active defen- regulate inflammation/innate immune activation by blocking sive reaction. Most defensive reactions rapidly resolve, and the expression and production of inflammatory mediators. The only in rare cases these degenerate into chronic destructive induction of inflammatory mediators by NM correlates with anomalous inflammation (such as those observed in rheu- the block of peroxisome proliferator-activated receptor (PPAR)/ matoid arthritis or multiple sclerosis). Thus, when assessing LXR pathways. PPAR can antagonize the action of inflamma- the possible NM immunotoxicity, the induction of an innate/ tory transcription factors, such as nuclear factor κB (NFκB) inflammatory response must be considered as a normal effect, and activator protein-1 (AP-1), thereby inhibiting the produc- while only an anomalous reaction (too prolonged in time, of tion of many inflammatory mediators and cytokines.[140] PPAR excessive or insufficient extent) might be considered as poten- activation in monocytes/macrophages, obtained by targeting a tially harmful. PPAR agonist to phagocytes with a nanocarrier, achieved prom- Overall, the interaction of NM with the mammalian innate ising results in a preclinical model of myocardial infarction.[141] immune system can provoke a reaction, in terms of production Modulation of PPAR activity via NM may therefore be devel- of defensive factors, which mainly results in the re-establish- oped as a novel therapeutic strategy for anti-inflammatory and ment of tissue homeostasis. Only in rare cases were NM found pro-healing effects. able to provoke significantly harmful effects at the organism The maturation and secretion of IL-1β and IL-18, two key level. Conversely, the increasing body of data accumulated so mediators of inflammation, is triggered by a cytoplasmic mul- far shows great promise for NM as agents for modulating and tiprotein complex termed the inflammasome, of which the directing innate immune responses for future therapeutic appli- NLRP3 inflammasome is the most common in monocytes and cations. Table 2 summarizes the main immune pathways acti- macrophages. Since IL-1β is a potent pyrogen that can induce vated and defensive molecules produced in response to NM an inflammatory form of programmed cell death (pyroptosis), across species. but is also a key immune-amplifying factor in the development of defensive reactions, NLRP3 inflammasome activation and consequent production of mature IL-1β are factors that can 4. Nanomaterials and Global Defense either predict NM immunotoxicity or NM-mediated promo- to Infection/Damage tion of immune defensive capacity. Abundant evidence shows the ability of NM to activate the NLRP3 inflammasome and In all living organisms, NM represents foreign elements with promote the maturation of IL-1β by mechanisms that include their own physico-chemical properties, which therefore may ROS production, K+ efflux, lysosomal membrane destabiliza- interfere with the normal physiological defensive mechanisms tion and release of cathepsin G.[62,63,65,66] The stimulation of of the embryos, growing organisms, and adults against infec- monocytes or macrophages with by SiO2 and TiO2 NP could tions or other challenges. From the available information, it increase local production of adenosine, leading to an accumu- appears that, at environmentally relevant concentrations, NM lation of intracellular ATP that promotes/prolongs the activa- are unlikely to have a widespread detrimental effect on living tion of the NLRP3 inflammasome resulting in an enhanced organisms except under certain local release scenarios. How- release of IL-1β and IL-18.[142] It should be underlined that the ever, subtle effects of NM to immune pathways resulting from production of IL-1β is by no means a marker of detrimental/ exposures need to be understood. Small 2020, 16, 2000598 2000598 (12 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com In plants, several reports have addressed the activity of NM CeO2, and SnO2), which include reduction in the production of on plant immune defense. Field and lab studies have described the HSP70 protein, inhibition of the activity of cholinesterases the protective effects of different NM against a number of and decrease of the basal protein levels of glucose-regulated microbial plant pathogens. Protection of plant from fungal protein 78.[158] These changes correlate with global toxicity, and bacterial diseases has been reported for several inorganic with animals exposed to 10−2 g L−1 of NP surviving for only NP, such as ZnO, Cu, SiO2, TiO2, CaO, MgO, MnO, and Ag 1–2 days, while those exposed to a lower NP dose (10−4 g L−1) NP.[143,144] For example, ZnO NP can reduce infection by showed pathological signs (lack of spines, slow mobility) after Fusarium graminearum, Penicillium expansum, Alternaria alter- 5 days. On the other hand, immune cells exposed to TiO2 NP nata, Fusarium oxysporum, Rhizopus stolonifer, Mucor plumbeus, in vivo do not show altered HSC70 levels, and take up particles and Aspergillus flavus, as well as by phytopathogenic bacteria through a mechanism that involves the TLR4/p38 MAPK sign- such as Pseudomonas aeruginosa.[144–147] Cu NP were more effec- aling pathway without activating an inflammatory response.[18] tive against Phytophthora infestans infection compared to non- Notably, animals exposed to TiO2 NP do not display any patho- nano Cu formulations in tomato.[148,149] The best studied NM logical state, showing a normal locomotion capacity, regular for protection against infections are Ag NP. Their protective movement of the spines and tube feet, characteristic adhesion activity is likely due to the antimicrobial effect of Ag ions. The ability, no loss of spines, no excess of excretion, no loss of ecto- same applies to Cu NP, as Cu ions are active against fungal derm, and no signs of bald sea urchin disease (a bacterial infec- phytopathogens and are widely used in bioorganic farming as tion that affects several species of sea urchins).[95] fungicides. Cu in low doses is also essential for many plant In mammals, a recent study in mice showed that SiO2 NP processes as a micronutrient that supports the plant defensive increased mice susceptibility to P. aeruginosa. However, NP- mechanisms against infection. Other NM such as Zn, Ce, SiO2, loaded alveolar macrophages were fully efficient in bacterial and TiO2 NP also show suppressive effects on plant infections, clearing, highlighting that particle effects are related to altera- and might also function as micronutrients. Nano formulation tions of the alveolar-capillary barrier while not affecting the might make the elements more accessible to the plant and normal course of the inflammatory responses.[159] Metal and allow for lower-dose applications and potentially better uptake metal oxide NM, including Ag and ZnO NP, are highly effec- rates.[148] tive in bactericidal activity, and CuO and TiO2 NP can also In crustaceans, exposure to dietary NM enhances immune show microbicidal capacity against various microbes including functions. In the freshwater prawn Macrobrachium rosenbergii Klebsiella pneumoniae, Shigella dysenteriae, Vibrio cholerae, Bacillus postlarvae, improved survival, growth, total hemocyte counts subtilis, Escherichia coli, Enterococcus faecium, P. aeruginosa, and and digestive enzyme activity were reported after exposure to Staphylococcus aureus.[160] More recently, it was reported that dietary ZnO, MgO, or Cu NP.[150–152] Treatment of Pacific white vitamin C lipid NP, used to deliver antimicrobial peptide to shrimp Litopenaeus vannameinano with capsules containing the macro­phage lysosomes, show remarkable antimicrobial activities antioxidant lipoic acid induced positive response on immune in fighting multidrug-resistant bacteria, including S. aureus and parameters,[153] and Au NP exerted immunostimulatory and E. coli, even in immunocompromised septic mice.[161] protective effects against Vibrio parahaemolyticus.[154] In the It should be noted that the vast majority of the results crab Eriocheir sinensis, dietary CeO2 NP promote growth, relieve showing little toxic or immunomodulatory effects of NM was ammonia nitrogen stress, and improve immune protection obtained in healthy animals or under standard cell culture con- against infection with the pathogen Aeromonas hydrophila.[155] ditions. The possibility that NM may impact the innate immune In marine invertebrates, including bivalves, a number of defensive mechanisms of weaker, immunocompromised in vivo studies have shown that, although NM at predicted organisms, or under chronic inflammatory conditions is, there- environmental concentrations are generally not toxic, they fore, still possible and remains under-investigated. In a murine may induce sub-lethal responses both at the molecular level model of ovalbumin (OVA)-induced lung inflammation, SiO2 and at the overall organism level. In addition to the observed NP act as adjuvant of allergic airway inflammation, suggesting immunomodulatory effects on circulating hemocytes, dif- that they might worsen the disease in allergic individuals.[162] ferent NM have been shown to modulate inflammatory and Additional studies confirmed the allergy-promoting effects of stress responses systemically.[13,98] The overall impact on the SiO2 NP and report similar effects for metal oxide NP. Indeed, physiological defense responses can be positive, as shown in the particular individual conditions play a crucial role in deter- Mytilus for certain types of CeO2 NP, which upregulate the mining the immune reaction to NP; while in non-pregnant expression of several immune-related genes in hemocytes, and mice the allergy-promoting effect of TiO2 NP is only moderate, exert antioxidant properties at the tissue level as in mammalian OVA-induced lung inflammation is clearly exacerbated in preg- models.[156] Moreover, exposure of animals to TiO2 NP increases nant mice. This indicates that, possibly due to a pro-allergy lysozyme and ROS production and upregulates transcription reactive hormone/cytokine milieu in pregnant mice, inflamma- of AMP in hemocytes, thus resulting in increased bactericidal tory responses to NM may be enhanced during pregnancy.[163] capacity of whole hemolymph.[157] On the whole, the results Because NM have the ability to penetrate physiological barriers so far available on the effects of NM on innate immunity in including the placental barrier,[164] potential effects of accumu- bivalves indicate that, in realistic environmental exposure con- lating NP in embryonic tissues require attention. Many types ditions, NM are not immunotoxic but can even have a benefi- of NM, including Si, Ag, and TiO2 NP, were reported having cial effect on immune functions. effects on embryonic development in fish, amphibian, bird, and Changes in immune cell reactivity can be observed in the sea mammalian embryos, with reduced weight, deformities and an urchin P. lividus exposed in vivo to a range of NP (e.g., Fe3O4, increase in toxicity.[165] Small 2020, 16, 2000598 2000598 (13 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Figure 4. Effect of NM on innate immune defensive functions across living species. The figure depicts the effects of different NM, listed on the left, on innate immune defensive functions of different living species, i.e., plants (upper right), crustaceans and other invertebrates (center right), and mam- mals (lower right). Details are given in the text. Artwork by Paola Italiani. While most studies explain potential pathologic effects nanomicrobiology. It is notable that microorganisms naturally of NM by their ability to induce toxicity and inflammation, a synthesize NP as a mechanism to detoxify their environment growing body of evidence suggests that some NM, especially from toxic elements, normally heavy metals,[167] or use them as metal, carbon and CeO2 NP, can display immunosuppressive electrodes to assist direct interspecies electron transfer[168] or effects. This is through direct interference with inflamma- as reservoirs to release with molecular precision some essen- tory signaling pathways, preventing inflammasome assembly tial elements (upon NM dissolution) at the site of action, for or acting as radical scavengers, thereby suppressing oxidative instance bactericidal ions (Ag+)[169] or pro-proliferating ones stress and inflammatory responses. While potentially useful in (Fe2+).[170] The NM size and water solubility allow them to suppressing excessive inflammation (e.g., in chronic inflam- finely integrate into the biological machinery. The interaction matory diseases), a potential immunosuppressive effect due to of prokariota with inorganic natural elements is clearly more the NM should be considered in nanomedicine, in particular intense and effective when compared to eukariota. in nanoparticulate anticancer therapies, because the NM could Regarding the interaction of NM with the immune system reduce efficient antitumor responses and increase susceptibility through microbiota, one has to bear in mind that the immune to infections.[166] The multiple effects of NM and their dual system develops upon interaction with microbiota. The role in inflammation and immunosuppression thus further system reaches an equilibrium in which commensal micro- increases the complexity of understanding the contribution of biota populate different organs and fluids of the organism NM to immunity. Developing embryos, young immunologically without triggering an immune reaction, while only invading immature animals, damaged organisms or disease-affected microorganisms induce a defensive immune response. Thus, individuals (e.g., chronic inflammatory diseases, cancer, and NM can trigger immune responses only when they localize autoimmunity) may all react to NM in an inadequate manner, in some usually secluded tissues and present particular sur- failing to adapt or to respond by elimination, thereby risking face patterns that can be recognized by PRR or other innate pathological consequences. receptors.[171] Triggering immunity may be undesirable if the The effects of different NM on innate immune defenses of activation is excessive, prolonged, or inducing severe tissue living species, from plants to mammals, are summarized in damage, but may result into an increase of host defenses Figure 4. against potential infection. Effects can be even more basal because of the possible similarities between microorganisms and NM that mimic biological structures, since the immune 5. Nanomaterials and Interaction with Bacteria reactivity and competence develops as a consequence of the interaction with microorganisms.[172] The interaction between NM and microorganisms is signifi- NM may admix with microbial communities and inter- cant, and it is at the basis of an emerging discipline dubbed fere with their biology, and induce population shifts and Small 2020, 16, 2000598 2000598 (14 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com modifications in the different consortia by empowering or limited in number and have been reviewed in detail else- restraining different species. The possibility of shaping the where.[185,187,188] Many of these studies have used rodents, and microbiota communities with the use of NM can be used at only a few have used nonrodent vertebrates or invertebrates as our advantage, as increasing evidence indicates that our health model species. The outcomes are somewhat contrasting with status[173] and therapy response[174] are strongly dependent on regards to the capacity of biocidal NM to cause disturbance to the gut microbiota. NM can provide microbiota with different the host-associated microbiota. Some studies, for example, indi- essential elements, from iron to magnesium and zinc, to cate a clear negative effect of Ag NP on the relative abundance ultratrace ones such as selenium, cobalt or nickel,[175] or they of Firmicutes and Lactobacillus,[189,190] whereas other studies can provide a hostile environment to pathogens or undesirable indicate a positive or no impact of Ag NP on the relative abun- species colonizing our gut.[176] dance of these taxa.[191–193] The absence of clear effects of biocidal NM on the bacterial community structure, as reported by some,[193,194] is surprising 5.1. Host–Bacteria Interactions under Nanomaterial Exposure given the high activity of biocidal NM in in vitro exposure studies. This lack of toxicity on gut microbial communities has been linked A major application of NM is as an antimicrobial agent. NM to physical–chemical conditions in the gastric environment. At efficacy as antimicrobial agents is thought to be linked to the low pH, as in some gastrointestinal tracts, Ag NP can quickly dis- physical interaction with the bacterial walls, followed by forma- solve but can subsequently precipitate to form the much less toxic tion of lesions in the cell membrane and subsequent intracel- AgCl NP.[195] Intestinal anoxia is another possible driver of toxicity lular accumulation of toxic ions.[177,178] Over the last decade, of biocidal NM. In anoxic conditions, both Cu NP and Ag NP the increased availability and accessibility of high-throughput can quickly sulfidize,[196–198] a reaction that strongly reduces their sequencing methods has greatly enhanced our understanding toxicity.[199–201] In addition, gut microbiota may be physically pro- of the diversity and functionality of the microbial world and its tected from exposure to biocidal NM due to their embedment into role in animal health. The roles microbes can play in animal intestinal folds. Physical and chemical conditions in the intestine health can be diverse and include both beneficial as well as path- are thus likely modulating the toxicity of NM, as well as other ogenic interactions. Well described beneficial roles of microbes toxicants,[184] which could explain the discrepancy in outcomes include the provision of nutrients,[179] aid in digestion[180] and between in vitro and in vivo NP toxicity studies.[193] degradation of complex organic molecules.[181,182] Notably, com- Studies that have looked at disruption of mucosal integrity mensal microbiota play an important beneficial role in immune induced by NM have yielded contrasting results, with some defense by allowing for the adequate development of local and studies showing no evidence for any histological change under systemic immunity and by preventing the establishment of biocidal NM exposure[192,194] and others showing strong signs of microbial invaders.[172] The interaction between microbes and epithelium and crypt damage.[189] It is thus not fully understood host is highly dynamic, and it can switch from a beneficial or under which conditions NM induce histological damage to intes- commensal equilibrium to a pathogenic/invasive disequilib- tinal tissue. Recent studies showed that in the bivalve Mytilus gal- rium, for example when microbes move from their original loprovincialis the immunomodulatory effects of exposure to TiO2 niche to another.[183] The immune system of animals regulates NP is associated with a shift in hemolymph microbial compo- the host–microbe interaction at the interaction/barrier sur- sition, indicating the interplay between the microbiota and the faces (e.g., in the gut), maintaining microbes that are benefi- immune system.[157] Similarly, in rodents, NM-induced altera- cial to the host and preventing invasion and barrier breaches. tions of the gut microbiota is linked to altered expression of host Immune recognition of microbes through PRR does not really cytokines.[189,190] The co-occurring modulation by NM of the host- distinguish between pathogenic and non-pathogenic bacteria, associated microbiota and the host immune system, as reported and threat is associated to the capacity of invasion (breaches of in both invertebrates and vertebrates, thus reflects the integrated the barrier). This is the reason why most of the innate immune link between microbes and immunity, and indicates the need to cells that can sense microbial patterns are located right below include microbiota analysis in nano-immunosafety assessment. the barrier. Thus, in the case of damage of the barrier (as in some diseases, or mechanical or toxic) the underlying innate cells recognize and react also to commensal bacteria, because 5.2. Interaction of Bacterial Molecules with Nanomaterials of their entry into the body. Chemical exposure can lead to dis- turbance of the microbiota (dysbiosis),[184,185] which can nega- The effect of NM in the host–microbiota interaction is of course tively affect host immunity, thereby promoting susceptibility to not limited to direct effects on bacterial communities or on the infections. As an example, in the tsetse fly antibiotic-induced immune system. The interaction of microbial factors with NM loss of symbiotic bacteria severely compromises host immune can modulate immune reactions in a way that is different from functions making the host unable to control bacteria that are those achieved by microbial molecules or NM separately. In a non-pathogenic under normal conditions.[186] recent study, human DC were exposed in culture to SiO2 NP Since microbiota and immune functions are integrally together with supernatants from either commensal or patho- linked, assessment of the immunosafety of NM and chemicals genic bacterial cultures.[202] In this study, NP and bacterial should include studies on impact on the interaction between molecules together induced the production of inflammation- bacteria and host immunity especially for those agents that are related cytokines, with differences between bacterial molecules designed to target microbes (biocides). Studies on the impact (those from pathogenic bacteria could induce cytokines only in of biocidal NM on host-associated microbiota are still rather the presence of toxic NP concentrations) and depending on the Small 2020, 16, 2000598 2000598 (15 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com NP concentration (non-pathogenic bacterial molecules could that are present in the same environment. In plants and induce the production of different cytokines if in the presence invertebrates, which do not have adaptive immunity, the term of non-toxic vs toxic NP concentrations). The effect was truly “immune memory” is sufficient for defining the phenomenon, synergistic and not due to adsorption of bacterial molecules on as they display only one type of immunity, i.e., the innate defen- the NP surface. sive mechanisms. On the other hand, in higher vertebrates in It is an important issue in immunotoxicity studies the notion which adaptive and innate immunity coexist, the term “innate that bacterial molecules can be adsorbed on the NM surface immune memory” specifically defines the evolutionarily con- giving rise to hybrid entities with new functional characteris- served memory mechanisms of innate immunity, distinct from tics. Of particular importance is the interaction between NM the better-known adaptive immune memory. Innate memory and bacterial endotoxins, highly inflammatory and pyrogenic enables organisms to cope in a more efficient way with external lipopolysaccharidic molecules (i.e., LPS). Given the high abun- challenges, after a first encounter, thereby ensuring increased dance of endotoxin in the outer membrane of Gram-negative survival and fitness. How NM may induce innate memory or bacteria, efficient recognition of endotoxin by the immune modulate the establishment of memory following the interac- system is a key mechanism in host defense. Humans are par- tion of organisms with foreign agents is an open question that ticularly susceptible to LPS, which induces potent innate and needs attention, as it can have a significant long-term impact inflammatory reactions by activating cells mainly through TLR on the living organisms’ capacity to cope with/adapt to their receptors. It has been shown that LPS can adsorb on the sur- environment. face of citrate Au NP, stabilize them and reduce their ability Innate memory in plants corresponds to systemic acquired to form a biocorona in human plasma.[203] LPS on the NP sur- resistance (SAR), a phenomenon that is induced in plants by face is still active and can induce an inflammatory response in local pre-infection with pathogens.[217] This local infection leads human monocytes comparable to that of free endotoxin.[203] to the activation of defense responses in the local tissue but also Similarly, the inflammatory effect of TiO2 NP on murine mac- in distant uninfected tissue and is effective not only against the rophages is significantly increased upon LPS adsorption.[204] pre-infecting pathogen but to a broad spectrum of diverse path- Compared to free LPS, LPS absorbed on TiO2 NP induces ogens. Such response is effective after a few hours and lasts similar levels of NOS, but higher expression of NOS2 pro- for several weeks, and it primes the plant for defense to sub- teins, whereas the effect of NP in combination with unbound sequent infection and thereby renders the locally pre-infected LPS is low.[204] Whether adsorption of LPS on NM increases, plant resistant in all parts and against multiple pathogens.[218] decreases or leaves unchanged the inflammatory effectiveness The activation of SAR is characterized by an accumulation of of LPS appears to strongly depend on the physical properties the phytohormone SA, which acts as a signaling molecule in of NM, the dose and timing of administration and the target the induction of pathogenesis-related (PR) proteins. Azelaic cell type.[205–209] We can in any case assume that LPS-coated NM acid is supposed to be a mobile signal that transports the behave differently from free LPS and uncoated NM, suggesting priming information to distant tissues.[217] that LPS-coated NM represent new nano-objects with different Only very few studies concerning the role of NM on the characteristics, which will have to be considered as such in induction of SAR have been conducted, and so far the mech- nanotoxicological studies. anisms of these effects on the induced resistance are mostly LPS is a widespread and thermostable bacterial molecule that unknown. Changes in the expression of A. thaliana genes represents a ubiquitous potential source of contamination even involved in SAR after treatment with Ag NP coated with poly- in the absence of viable bacteria. Humans are highly sensitive vinylpyrrolidone and silver ions (Ag+) were analyzed by Kaveh to LPS and respond to 1000× lower LPS concentrations com- et al.[219] The study shows that the flagellin pattern recognition pared to mice.[210,211] Careful and reliable detection of potential receptor FLS2 and the azaleic acid induced gene 1 (AZI1) are endotoxin contamination of NM is therefore crucial, especially downregulated after NM treatment, suggesting that Ag NP in the fields of immunology, drug development, and drug have a negative impact on innate memory in plants.[219] The delivery.[212,213] This also applies to NM synthesis.[214] It has been expression of the PR genes PR1, PR2, and PR5 was exam- shown that specific components, including citrate, Triton X-100, ined in A. thaliana after application of nanosized Ag–silica and bovine serum albumin hinder reliable LPS recognition by hybrid complexes and Ag NP. Genes implicated in SAR were commercially available LPS detection assays.[215] From these significantly upregulated after treatment with NM, but the data, we can conclude that effects of NM on immune cells must hybrid complexes caused a faster activation of PR genes.[220] be analyzed very carefully to distinguish potential contami- Another study examined the capacity of Ag NP to induce SAR nating LPS effects from real NM-mediated effects. However, against tomato mosaic virus and potato virus, and their direct due to their ability to interact with endotoxin and due to their ability to suppress viral infections on tomato plants. Plants optical and physical properties, novel LPS detection platforms treated with Ag NP and later inoculated with the virus showed involving NM may be developed in the future.[216] a lower disease rate. Indeed, Ag NP were able to bind virus particles and/or block viral replication in the case of many dif- ferent viruses in the absence of toxicity for the host cells.[221] In 6. Nanomaterials and Innate Memory contrast with the study described above, the expression of the SAR marker gene PR1 was repressed in Solanum lycopersicum Immune memory is a key defensive mechanism that allows plants treated with chitosan-polyvinyl alcohol (PVA) hydrogels living organism to adapt to their environmental conditions plus copper NP (Cs-PVA + Cu NP) and Cs-PVA alone. In both by mounting efficient defense against the threatening agents cases the expression of PR1 was repressed in normal condition Small 2020, 16, 2000598 2000598 (16 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com but also under saline stress.[222] Imada et al.[223] reported that observe a shift in granular hemocyte subpopulations, together nano-MgO treated roots rapidly generated ROS along with with re-establishment of basal functional parameters and of upregulation of PR1, jasmonic acid, ethylene, and systemic proliferation/apoptotic markers. Moreover, the hemolymph resistance-related genes.[148] bactericidal activity was increased, as well as transcription of The development of nanotechnology has opened up new immune related genes coding for secreted proteins. The results frontiers in order to solve technical problems in efficiently indicate both tolerance and potentiation as compensatory delivering plant immune inducers. In particular, mesoporous mechanisms to maintain immune homeostasis after a second silica NP, already used as anticancer drug delivery systems, are encounter with PS-NH2, representing a form of immune expected to be a promising carrier of resistance inducers.[224] memory.[233] To date engineered NP have been successfully used as phyto- The same forms of innate memory, “tolerance” and “trained hormone delivery systems[225] or like pesticide carriers in order immunity/potentiation,” are documented in mammals/ver- to target their activity and reduce the indiscriminate leaking of tebrates. Although the innate memory can be induced by any toxic compounds, alleviating the potential adverse effects on the type of exposure, most of our knowledge on the tolerance environment.[226] Functionalized mesoporous silica NP with a phenomenon comes from study on LPS exposure, while that redox-responsive gatekeeper system, able to specifically release on trained immunity/potentiation is mainly related to expo- the resistance inducer and avoiding premature leakage in the sure to Bacillus Calmette–Guérin (BCG) or to fungal cell wall delivery process, demonstrated their potential application in β-glucans. The concept of innate memory in higher vertebrates, agriculture. In fact, this system has proven to be an efficient known for decades, has recently experienced renewed interest, technique to introduce resistance inducers and reduce Phy- with a wealth of studies describing the effect of “priming,” tophthora infections in pineapple.[224] NM application in plants either in vivo or in vitro, on the subsequent reactivity of innate is still in its infancy but promising strategies are emerging. immune cells (especially macrophages and monocytes) to an Therefore, some NM might in the future offer applications to unrelated challenge. A typical experiment on the induction of stimulate innate immune responses in plants and help agron- human innate memory in vitro showed that human mono- omists to reduce yield losses caused by biotic factors without cytes primed with β-glucan from Candida albicans developed extensive use of conventional pesticides. an enhanced inflammatory cytokine production after restimu- Invertebrates can adapt and survive solely relying on innate lation with TLR1/2 agonists.[234] The molecular mechanisms immunity. This implies the presence of complex and sophisti- underlying the development of innate memory in mammals cated mechanisms of immune specificity, and the capacity to include metabolic changes (i.e., shift from oxidative phospho- mount a faster/more effective response upon re-exposure to rylation to aerobic glycolysis—“Warburg Effect)” and epige- threatening agents, which is the function of innate memory or netic reprogramming (e.g., DNA hypermethylation and histone “immune priming.” The innate memory of invertebrates is a modi­fications). Human monocytes and macrophages primed well-known phenomenon, observed and exhaustively described with LPS or β-glucan present specific histone modifications,[234] in practically all phyla as consequence of infection immunity, and β-glucans from C. albicans induce a metabolic reprogram- natural transplantation immunity, individual, and transgenera- ming of human macrophages toward aerobic glycolysis that is tional immune priming.[227] However, molecular and functional possibly involved in the development of innate memory.[235] The immune parameters responsible of the induction of innate interplay between cellular metabolism and chromatin remod- memory sensu stricto (as response to a challenge of a quies- eling associated with the development of innate memory has cent immune system that has been previously primed) remains been reviewed elsewhere.[236,237] largely unknown. Memory is usually induced by priming with Older evidence of the existence of the innate memory in infective microorganisms, and the effect is examined as survival humans in vivo comes from epidemiological studies showing to reinfection.[228–230] Notably, memory, which is based on epi- that vaccination with Mycobacterium bovis BCG (a live attenuated genetic changes, can be inherited by the progeny of primed ani- tuberculosis vaccine) provides protection against non-related mals, thereby enhancing the fitness of the progeny in the same infections.[238,239] A recent study highlighted the effect of BCG environmental challenges encountered by the parents.[228,231,232] vaccination on the development of the innate memory both in Despite the widespread studies on invertebrate immune vivo and in vitro. The authors observed that BCG vaccination memory, information on memory responses to NM is virtu- protects humans from viral challenges (simulated with the ally absent. The only available data come from an unpublished administration of Yellow Fever Vaccine in volunteers) through study in bivalves. The mussel Mytilus spp. has extremely pow- the involvement of IL-1β production, and confirm in vitro the erful immune defenses to cope with different potential patho- role of IL-1β in inducing epigenetic reprogramming of mono- gens and contaminant stressors, including NM. The possibility cytes.[240] The BCG-induced memory was shown to persists one that NM may participate in immune memory has been recently year after vaccination.[241] This prolonged persistence of BCG- investigated in M. galloprovincialis subjected to repeated expo- induced memory is hard to explain because of the shorter life- sure to nanoplastics (amino-modified polystyrene PS-NH2), by span of the innate immune cells that develop memory following examining the modulation of several functional and molecular priming (mostly monocytes and macrophages). To explain the parameters in hemocytes and hemolymph. First exposure indi- persistence of innate memory, recent evidence shows that BCG cates the occurrence of stress conditions in the hemocytes, can access the bone marrow and change the transcriptional which, however, did not result in changes in the overall bac- landscape (i.e., the areas of chromatin accessibility) of hemat- tericidal activity (suggesting a reaction that resolves without opoietic stem cells (HSCs) and multipotent progenitors, leading consequences). After second exposure, it was possible to to local cell expansion and enhanced myelopoiesis.[242,243] The Small 2020, 16, 2000598 2000598 (17 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Figure 5. Innate immune memory across living species. Upper panels: main mechanisms and effects of innate immune memory in plants, inverte- brates, and vertebrates. Lower panels: effects of NM on the innate immune memory mechanisms in plants, invertebrates, and vertebrates. SA, salycilic acid; AzA, azaleic acid; FLS2, flagellin pattern recognition receptor; AZl1, azaleic acid induced gene 1; PR, pathogenesis-related. See text for detailed description. Artwork by Paola Italiani. authors demonstrate that BCG-educated HSC generate epige- exposure to Au NP could change the subsequent response to netically modified macrophages able to protect mice against LPS and that changes could differ in different individuals.[244] virulent Mycobacterium tuberculosis infection, and that BCG- Another study has proven the significant effect of Au NP in induced HSC reprogramming occurs in vivo. reducing the BCG-induced memory in human primary mono- Despite the growing number of studies on innate immune cytes.[247] Overall, the putative capacity of NM to interact with memory in mammals/humans, and the large body of litera- innate immune processes at the level of innate memory has ture on the interaction of NM with the mammalian innate very interesting implications, opening the way to the med- immune system,[62] NM effects on the development or mod- ical use of NM for controlling subsequent innate/inflamma- ulation of the innate memory to date remains practically tory responses for a significant period of time, with benefi- unstudied. The hypothesis that NM may have an effect on cial effects in autoimmunity, infections and immune-related innate immune memory is possible and supported by a series diseases. of considerations arising from the fact that NM can have an A schematic depiction of the mechanisms of innate memory effect on the molecular mechanisms underlying the develop- and of the effects of NM on its establishment and effects is pro- ment of innate memory,[244] which are mainly based on epi- vided in Figure 5. genetic and metabolic reprogramming. Indeed, NM are able to induce both epigenetic[245] and metabolic changes.[246] Thus, it is reasonable to presume that NM may be able to induce or 7. Conclusions and Future Challenges modulate innate memory and, therefore, affect the capacity of innate cells to react to a second exposure. Indeed, preliminary When assessing the immunosafety of NM, we mainly need studies on human primary monocytes in vitro showed that to examine the interaction of exogenous NM with the innate Small 2020, 16, 2000598 2000598 (18 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Figure 6. Common immune mechanisms induced by NM across living species. Schematic depiction of the common mechanisms of immune reaction of living species (plants, green silhouette; invertebrates, purple silhouette; vertebrates, yellow silhouette). Upon interaction with the microenvironment, biomolecules adsorb to the NM surface and create a “biomolecular corona.” NM–biomolecule interactions signal for non-inflammatory clearance (opsonization and endocytosis) or trigger a defensive innate/inflammatory response through an expanded repertoire of PRR initiating an appropriate signaling cascade. Immune responses include ROS, MAPK activation, defense gene induction, and production of cytokines, chemokines, and growth factors. The combination of a multitude of cellular effects have, as result, the preservation of cell/tissue integrity and function. Artwork by Andi Alijagic. immune system of living organisms. Innate immunity is, for and fitness. There are essentially three ways to cope with NM. over 95% of living eukaryotic organisms, from plants to mam- Ignoring them (e.g., in marine filter feeders, they are largely mals, the only type of immunity displayed. This fact makes expelled with water), adapting to them (e.g., taking and elimi- adaptive immunity (coexisting with innate immunity in higher nating/storing them with non-inflammatory non self-damaging vertebrates) a less relevant actor in the nano-immune interac- mechanisms), or reacting to them with an active innate/inflam- tion. Indeed, innate immunity is the first, most powerful, and matory reaction. We should consider as immunosafe those NM fastest line of immune defense. The mechanisms of innate that fall in each of the three cases, including those that trigger immunity are fundamentally similar across species and evo- an innate/inflammatory reaction. Only when such reactions lution, despite the fact that they are to some extent the result turn out to be anomalous (too prolonged in time or too short/ of parallel and challenge-specific evolution and that there are inefficient, excessive or too weak), then they could cause patho- species- and environment-specific differences. Natural and logical consequences. A summary view of the innate immune anthropogenic NM are one of the many foreign challenges mechanisms engaged in the interaction with NM is presented present in the environment that come in contact with living in Figure 6, which underlines the similarities across species. organisms and their immune system. For NM as for all foreign In light of the above considerations, we should re-think agents, based on sensing and sorting molecules and mecha- immuno-nanosafety by critically examining the validity and nisms, the immune system decides the best way to cope with predictivity of the data produced by the scientific community, them with the ultimate goal of preserving the organism’s health and by identifying the future challenges and most promising Small 2020, 16, 2000598 2000598 (19 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com avenues. One of the drawbacks for understanding relevant immune response to subsequent challenges. Immune effects is the use of high concentrations of NM in experimental memory is the mechanism by which organisms “remember” settings that do not represent real life situations. Cell death previous exposure to foreign agents, so that they respond in vitro upon exposure to high concentrations of NM may better (with a more effective immune response) to recurring not reflect toxicity for the same cells in vivo within a complex challenges. While the phenomenon of innate memory is well organism and at realistic concentrations. Also, experimental known in plants, invertebrates, and also in vertebrates, very studies still suffer of many limitations that hamper their little is known on the possibility that NM may alter the estab- reliability and that may not provide useful information. For lishment of protective memory or even induce an anomalous instance, studies on human innate reactivity to NM are usu- memory. This is an area that needs addressing, as NM that ally restricted to in vitro systems that, however complex they have no direct effect and are therefore considered immunosafe may be, still cannot reproduce the in vivo situation. The use of could nevertheless modify immune memory with unpredict- tumor cell lines instead of primary cells further decreases the able consequences. possibility that the results obtained may be valid for normal As we have summarized in this review, examining the cells. In addition, studies of chronic exposure are not fea- interaction of NM with the innate immune defensive mecha- sible with in vitro models. In vivo experimentation in rodents nisms in plants and animals leads to the overall feeling that is also not ideal because of a number of significant differ- NM in general do not pose significant threats to the organ- ences, including the geometry of exposure (for instance how isms’ survival and fitness, as they rarely induce direct effects inhaled NM will deposit in the lungs of animals based on gait) or interfere with ongoing innate reactions under realistic con- and some critical innate immune mechanisms (e.g., comple- ditions. Thus, it seems that the major immune responses to ment). Therefore, one of the future challenges for human NM is silent elimination (adaptation) or successful immune nano-immunosafety is that of identifying reliable experimental elimination (innate/inflammatory reaction with full resolu- models that could realistically represent the in vivo immune tion). Of course, it is not possible to generalize, and caution reactions to NM and the true risk for health. Stepping from should always be taken, and the new challenges opened by short-term to long-term exposure models will be a particular the advancements in our knowledge and technology should be challenge. An interesting approach is to focus on invertebrates confronted. Some of them, already discussed above, are listed and plants, in which the global response to NM exposure can hereafter: be assessed much more easily and over a long time, although 1) Realistic and predictive experimental models. even among invertebrates we should identify representative 2) Organ/tissue restriction of NM immune effects. and predictive models. 3) NM effects in immunologically frail organisms. Another challenge that we will have to face in assessing 4) NM effects on organism-associated microbiota. nano-immunosafety is the fact that different cells can have 5) NM effects on innate immune memory. different sensitivity to NM challenge. Indeed, as mentioned before, some highly proliferating cells can be more suscep- Overall, we should aim at defining NM immunosafety not in tible to chemicals or other compounds, while quiescent cells absolute terms, but relative to specific conditions of use/expo- can be more resistant. In the context of a particular organ (e.g., sure (organism, tissue/organ, exposure features, health condi- the lung) some cells can die while others are untouched, but tions). While the “safe-by-design” concept seems unfeasible, we what counts is the final outcome, i.e., how the organ succeeds can successfully target a “safer-by-design” nanotechnology. in eliminating the foreign potentially dangerous agents and re- establishing the organ physical and functional integrity. This means that the susceptibility or resistance to toxicity of one Acknowledgements single cell type may not reflect the events in real life conditions. Notably, the reaction in one organ may be limited spatially to All authors were supported by the EU H2020 project PANDORA (GA 671881). The authors gratefully acknowledge the following additional that organ (or part of the organ, or some cells within the organ), financial support: EU H2020 project ENDONANO (GA 812661; PI, AD, without affecting survival or fitness. VFP); the Italian Ministry of Research Flagship project InterOmics The immunosafety of NM should also consider the pre- (DB, PI); the Austrian FWF Doctoral Program W1213: Immunity in existing or ongoing health conditions of the organisms. Plants Cancer and Allergy (ICA; AD, JHH); Catalan Agència de Gestió d’Ajuts or animals that are infected or damaged or senescent can Universitaris i de Recerca (2017-SGR-1431; VFP, NGB), Ramon y Cajal be less able to adequately react to new challenges. Thus, in program (RYC-2012-10991; NGB); Severo Ochoa program from Spanish MINECO (Grant No. SEV-2017-0706; VFP, NGB); CERCA Programme/ human nano-immunosafety, the effects of NM that are harm- Generalitat de Catalunya (NGB). All authors are particularly grateful less in healthy conditions can still be potentially harmful in to Giuliana Donini (EU Commission, Bruxelles, Belgium) and Paola immunologically frail conditions. As health in invertebrates Cesaroni (ALTA Srl, Siena, Italy) for the relentless and expert support and vertebrates also depends on the composition of the in all the stages of the project development. The authors wish to associated microbiota, an additional very important area of acknowledge the contribution of Rita Marino (Stazione Zoologica Anton investigation will be the NM–microbiota interaction, and the Dohrn, Napoli, Italy), Daniela Melillo, Maria Mangini, and Alessandro health-related consequences of such interaction for the whole Verde (IBBC-CNR, Napoli, Italy), Anna Chiara De Luca and Marinella Pirozzi (Euro-BioImging Facility, IBBC-CNR, Napoli, Italy), Ursula Lütz- organism. Meindl and Ancuela Andosch (University of Salzburg, Austria), and the Finally, a very interesting new area of investigation in EM Facility of CAS (Prague, Czechia; supported by the project LO1509 of nano-immunosafety is the possible effect of NM in shaping the Ministry of Education, Youth and Sports) for providing the electron innate memory, i.e., in determining changes in the protective microscopy images. Small 2020, 16, 2000598 2000598 (20 of 25) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 16136829, 2020, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202000598 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Conflict of Interest [28] Y. 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