nanomaterials Review Cross-Species Comparisons of Nanoparticle Interactions with Innate Immune Systems: A Methodological Review Benjamin J. Swartzwelter 1 , Craig Mayall 2 , Andi Alijagic 3 , Francesco Barbero 4 , Eleonora Ferrari 5 , Szabolcs Hernadi 6 , Sara Michelini 7 , Natividad Isabel Navarro Pacheco 8 , Alessandra Prinelli 9 , Elmer Swart 10 and Manon Auguste 11, * 1 Institute of Biochemistry and Cell Biology, National Research Council, 80131 Napoli, Italy; swartzwe@colorado.edu 2 Department of Biology, Biotechnical Faculty, University of Liubljana, 1000 Ljubljana, Slovenia; craig_mayall@hotmail.co.uk 3 Institute for Biomedical Research and Innovation, National Research Council, 90146 Palermo, Italy; andialijagic@gmail.com 4 Institut Català de Nanosciència i Nanotecnologia (ICN2), Bellaterra, 08193 Barcelona, Spain; fra.barbero@gmail.com 5 Center for Plant Molecular Biology–ZMBP Eberhard-Karls University Tübingen, 72076 Tübingen, Germany; eleonora.ferrari2018@gmail.com 6 School of Biosciences, Cardiff University, Cardiff CF10 3AX, UK; hernadi222@gmail.com 7 Department of Biosciences, Paris-Lodron University Salzburg, 5020 Salzburg, Austria;  sara.michelini@sbg.ac.at  8 Institute of Microbiology of the Czech Academy of Sciences, 142 20 Prague, Czech Republic; Citation: Swartzwelter, B.J.; natividad.pacheco@biomed.cas.cz 9 AvantiCell Science, Ltd., Ayr KA6 5HW, UK; alessandra.prinelli@gmail.com Mayall, C.; Alijagic, A.; Barbero, F.; 10 UK Centre for Ecology and Hydrology, Wallingford OX10 8BB, UK; elmswa@ceh.ac.uk Ferrari, E.; Hernadi, S.; Michelini, S.; 11 Department of Earth Environment and Life Sciences, University of Genova, 16126 Genova, Italy Navarro Pacheco, N.I.; Prinelli, A.; * Correspondence: manon.auguste@edu.unige.it Swart, E.; et al. Cross-Species Comparisons of Nanoparticle Interactions with Innate Immune Abstract: Many components of the innate immune system are evolutionarily conserved and shared Systems: A Methodological Review. across many living organisms, from plants and invertebrates to humans. Therefore, these shared Nanomaterials 2021, 11, 1528. https:// features can allow the comparative study of potentially dangerous substances, such as engineered doi.org/10.3390/nano11061528 nanoparticles (NPs). However, differences of methodology and procedure between diverse species and models make comparison of innate immune responses to NPs between organisms difficult in Academic Editors: Diana Boraschi many cases. To this aim, this review provides an overview of suitable methods and assays that can and David M Brown be used to measure NP immune interactions across species in a multidisciplinary approach. The first part of this review describes the main innate immune defense characteristics of the selected models Received: 18 February 2021 that can be associated to NPs exposure. In the second part, the different modes of exposure to NPs Accepted: 7 June 2021 across models (considering isolated cells or whole organisms) and the main endpoints measured are Published: 9 June 2021 discussed. In this synergistic perspective, we provide an overview of the current state of important cross-disciplinary immunological models to study NP-immune interactions and identify future Publisher’s Note: MDPI stays neutral research needs. As such, this paper could be used as a methodological reference point for future with regard to jurisdictional claims in published maps and institutional affil- nano-immunosafety studies. iations. Keywords: environmental models; human cells; innate immunity; markers; NPs testing Copyright: © 2021 by the authors. 1. General Introduction: The Need for Studying Nanoparticle–Immune System Licensee MDPI, Basel, Switzerland. Interactions This article is an open access article distributed under the terms and Over the last twenty years, there has been a significant growth in the research, de- conditions of the Creative Commons velopment, and production of engineered NPs [1]. When materials are downsized to the Attribution (CC BY) license (https:// nanoscale, novel physical and chemical properties emerge, conferring them with new and creativecommons.org/licenses/by/ unique behaviors. Depending on their nature (e.g., composition, size, shape, surface state), 4.0/). these materials have remarkable optical, magnetic, electrical, catalytic, structural, and Nanomaterials 2021, 11, 1528. https://doi.org/10.3390/nano11061528 https://www.mdpi.com/journal/nanomaterials Nanomaterials 2021, 11, x FOR PEER REVIEW 2 of 32 Nanomaterials 2021, 11, 1528 2 of 32 these materials have remarkable optical, magnetic, electrical, catalytic, structural, and chemical properties, which can be exploited in many different sectors such as automotive, chemical properties, which can be exploited in many different sectors such as automotive, agricultural, pharmaceutical, and biomedical fields [2–5]. It is estimated that the global agricultural, pharmaceutical, and biomedical fields [2–5]. It is estimated that the global nanomaterialproduction nanomaterial productioninin2014 2014 was was between between 0.30.3 andand 1.6 million 1.6 million tons,tons, withwith SiO2 ,SiO TiO2,2 TiO and2 and ZnO ZnO nanomaterials nanomaterials beingbeing the most the most abundantly abundantly produced produced [6]. [6]. The wide utilization and increasing production of The wide utilization and increasing production of NPs has NPs has inevitably inevitably lead lead toto an an in- in- crease in humans and environmental exposure to these materials although crease in humans and environmental exposure to these materials although exposure routes exposure routes are not are not necessarily necessarily identical identical for different for different organisms. organisms. The expected The expected increased increased exposure expo- in sure in human and environmental organisms has given rise to concerns regarding human and environmental organisms has given rise to concerns regarding potential safety poten- tial safety risks. risks. exposure The main The mainroutes exposure routes to NPs to NPs in both in both humans andhumans and environmental environmental species are species are highlighted and summarized highlighted and summarized in Figure 1. in Figure 1. Figure 1. Figure 1. The Thedifferent differentexposure exposurepathways pathwaysofof engineered NPs engineered thatthat NPs cancan interact withwith interact human or or human environmental species. environmental species. In humans, In humans,the thefirst firstmain mainexposure exposure pathway pathway is via is via intentional intentional introduction introduction of NPs, of NPs, for for instance instance duringduring medical medical administration. administration. The ability The ability of someof some NPs toNPs to interact interact with mo- with molecular lecular and and cellular cellular processes processes and to beand to be target targetmakes specific specific makes their use intheir drug use in drugan delivery delivery an attractive attractive application. application. They haveThey have known long been long been known to play to play anrole an effective effective role in vaccination, in vaccination, acting not acting only asnot only carriers, antigen as antigen butcarriers, but also as also as adjuvants adjuvants that activate that innateactivate immunity innate andimmunity thereby increase and thereby the efficacy increase ofthe antigen presentation efficacy of antigen [7]. They can also presentation [7].beThey valuable toolsbe can also in valuable medical imaging and diagnosis, tools in medical imaging and andinnovative diagnosis, newandtherapies innovative [8].new Alongside therapies the [8]. potential benefits Alongside the of nanoparticle-based potential therapies, there is also benefits of nanoparticle-based a risk associated therapies, there is also with parenteral a risk associated introduction with par- of novelintroduction enteral substances, and thus there of novel is a need substances, andtothus ensure thatisNPs there a needwilltonot negatively ensure that NPsimpact will the not normal negatively functioning impact the of normal the immune systemof functioning [9–13]. Other interactions the immune system [9–13]. canOther arise inter- from passive actions canexposure such passive arise from as through cosmetic exposure suchproducts as through or food. cosmeticAlthough products NPs orwill food.likely Alt- first interact with epithelial and mucosal barriers, in some cases hough NPs will likely first interact with epithelial and mucosal barriers, in some casesthey are able to cross these barriers they areor potentially able causebarriers to cross these adverseor effects, for example potentially by interacting cause adverse effects,with the natural for example by gut microbiome [14]. interacting with the natural gut microbiome [14]. Although Although most most NPs NPs areare not not directly directly applied applied in in the the environment, environment, many many NPs NPs used used in in consumer consumer products products or or industry industry areare expected expected to to be be released released into into the the environment environment during during production, production, use use oror during during the the disposal disposal of of products products containing containing NPs NPs [15]. [15]. Over Over thethe past past decade, decade, an increasing number of products containing NPs have been introduced intoagri- an increasing number of products containing NPs have been introduced into ag- cultural ricultural practices practices with withthethe aimaim of increasing of increasing cropcropyield and and yield reducing reducingproduction productioncostscosts [16]. In addition, [16]. the usethe In addition, of use wastewater treatment of wastewater plant biosolids treatment as crop fertilizers plant biosolids can facilitate as crop fertilizers can release of NPs into the terrestrial environment leading to exposure facilitate release of NPs into the terrestrial environment leading to exposure in soil organ- in soil organisms [17]. NPs ismscan [17].also NPs reach aquatic can also environments, reach including seashores, aquatic environments, through landfill including seashores, through leachates, landfill or direct disposal of wastes (e.g., consumer products containing plastics) [18]. Once in Nanomaterials 2021, 11, 1528 3 of 32 the water, NPs can remain in suspension in the water column, interacting with planktonic organisms, or due to interactions with organic matter and/or their higher density, NPs can aggregate and deposit on the seafloor. This has been reflected by several models predicting NP concentrations within different regions which showed higher concentrations of NPs in sediments than surface water [19]. Therefore, benthic and sediment dwelling organisms are expected to be exposed to NPs, due to their feeding habit (e.g., filter, deposit feed- ers) [20]. In addition, some marine invertebrates possess an open (or semiopen) circulatory system, which is in direct contact with the external environment, eventually contributing to increased exposure. Considering the many possible exposure and entry routes of NPs, defining common parameters for assessing organism-NP interactions is fundamental for allowing compar- isons at different taxonomic levels. Innate immunity is a shared feature for every multicellu- lar organism and the effector mechanisms of the innate immune system are the first line of defense that detect and protect the body from nonself objects such as NPs [21–23]. As is the case for natural pathogens, NPs have the potential to induce an immune response. In cases where NPs can elicit an immune response, there is a need to study the type and degree of this response, and the NP-immune interaction mechanisms rather than remaining limited to only measurements of acute toxicity. Comparative immunology, by its multidisciplinary approach may unravel fundamental mechanisms activated by NPs and help further global understanding regarding the effects of NPs. Experiments in a laboratory are first necessary to allow the understanding of basic mechanisms under controlled conditions. However, carefully chosen models and as- sessment parameters are important with regard to future translocation to more realistic environmental exposure. To this end, models within this review have been selected which can be good indicators and representatives of their regional and global distribution and which are easy to maintain under laboratory conditions. Environmental models can be therefore compared across taxa and even to human cells, through both in vitro and/or in vivo approaches according to the model possibilities (Figure 2). Plant models are a com- pelling place to begin for assessment of NP-immune interactions. In particular Arabidopsis thaliana, a small flowering plant belonging to the Brassicaceae family, which is widely used in crop science studies and was also the first plant genome to be fully sequenced [24–26]. Among terrestrial invertebrates, earthworms belonging to the family Lumbricidae (Eisenia fetida) are abundant in the soil and play an essential role in soil formation, by facilitating nutrient cycling, fragmenting biomass and aeration of soil through bioturbation [27,28]. Similarly, terrestrial isopods, such as Porcelio scaber, are crustaceans which evolved to live on land, inhabiting the top-soil level. They are decomposers and play an important role in returning nutrients to the soil [29–31]. Their feeding habits makes it likely they will come into contact with environmental pollutants, including NPs, and thus represent interesting model species to study these interactions. The Mediterranean mussel Mytilus galloprovin- cialis and the sea urchin Paracentrotus lividus are both sessile marine invertebrates. Mussels are able to filter large quantities of water which they use for breathing and feeding, while sea urchins graze on the seafloor layer. These qualities, as well as the ease with which they can be harvested along seashores, make these good models in which to study invertebrate interactions with NPs [20,32–34]. This work is supported by the EU PANDORA project [35], which devoted effort to study the effects and mechanisms of action of NPs on the innate immunity of different models from across the tree of life. The general outcomes of the project were previously reported, summarizing the main findings but also to set future perspective and research direction in this field [21,36]. The remainder of this review will focus on the translatable aspects of experimental methodology, parameters and endpoints used, the suitably of the selected models when considering investigating NP effects, and the possibilities regarding research at the whole organism level (in vivo) or with isolated cells (in vitro). Nanomaterials 2021, 11, 1528 4 of 32 Nanomaterials 2021, 11, x FOR PEER REVIEW 4 of 32 Figure 2. The different models discussed in the current review, and their main experimental usage in Figure 2. The different models discussed in the current review, and their main experimental usage the laboratory: in vivo (whole organism experiments) and in vitro (isolated cells or cell lines). in the laboratory: in vivo (whole organism experiments) and in vitro (isolated cells or cell lines). Here we aim to: (i) give a short overview of the characteristics of various relevant This work is supported by the EU PANDORA project [35], which devoted effort to innate immune models from across tree of life; and (ii) provide a comparative analysis of study the effects and mechanisms of action of NPs on the innate immunity of different the methods used to study the interaction of NPs with these innate immune models. models from across the tree of life. The general outcomes of the project were previously reported, summarizingofthe 2. Short Description themain findings Innate Immune butSystem also to set for future perspective the Models and research of Interest direction 2.1. Generalities and Conserved Innate Immune Traits across the Selected Models translatable in this field [21,36]. The remainder of this review will focus on the aspects of experimental methodology, parameters and endpoints used, the suitably of the The ability to mount an immune response against external threats is a characteristic selected models when considering investigating NP effects, and the possibilities regarding of every living organism. While increasing levels of immune complexity are found in research at the whole higher organisms, atorganism every stagelevel of (in vivo) orthere evolution withisisolated presentcells (in vitro). a basic initial host defense that has been characterized as innate immunity. Innate immunity is of Here we aim to: (i) give a short overview of the characteristics various a fast, relevant standardized, innate immune models from across tree of life; and (ii) provide a comparative nonspecific response which includes multiple levels of defense mechanisms, beginning analysis of the methods used to study the interaction of NPs with these innate immune with physicochemical barriers (e.g., shell, mucosal or epithelial barrier) [37,38]. Further models. mechanisms of defense rely on dual components of the immune system, the immune 2.cells Short Description (e.g., monocytes, of the Innate Immune macrophages System fororthe for vertebrates, Models ofcoelomocytes hemocytes, Interest for in- 2.1. Generalitiesand vertebrates) andtheConserved productionInnateofImmune humoral Traits acrossInnate factors. the Selected immune Models cells found in the circulating fluids The ability to of invertebrates mount an immune can response have different names against and the external cellular threats is aportion of their characteristic innate immunity relies on these unique cells. These cells can of every living organism. While increasing levels of immune complexity are found in be subdivided into different cell populations, higher organisms, such as granular at every or hyalinethere stage of evolution cells,isand they ahave present basicdistinct rolesdefense initial host and can trigger a specific response upon encountering threatening nonself that has been characterized as innate immunity. Innate immunity is a fast, standardized, material. Only plants lack these specialized nonspecific response which immune cells,multiple includes but in plants levelsallofthe cells are defense believed tobeginning mechanisms, be able to mount a defensive response to foreign attack [39]. Complex machinery, with physicochemical barriers (e.g., shell, mucosal or epithelial barrier) [37,38]. Further including cells and humoral factors is involved in recognition of nonself material, mechanisms of defense rely on dual components of the immune system, the immune cells and especially in detecting domains (e.g., called pathogen/microbe monocytes, associated molecular macrophages for vertebrates, or hemocytes, patterns (PAMPs/MAMPs) coelomocytes for inverte-that are typically displayed on the surface of bacterial, fungal, and brates) and the production of humoral factors. Innate immune cells found in the circulat- parasitic organisms and virus-infected cells. Host recognition of nonself will involve a large range ing fluids of invertebrates can have different names and the cellular portion of their innate of cell membrane bound and immunity scattered relies pattern on these uniquerecognition receptors cells. These (PRRs). cells can Although the be subdivided intodistinctive differentPRRs cell can vary between populations, such as models, granulartheor main concept hyaline is and cells, consistent, they have anddistinct different PRRs roles andshare canatrigger similar role upon recognition of nonself particles. PRRs in humans, much like their invertebrate a specific response upon encountering threatening nonself material. Only plants lack these homologues, are responsible for initiating innate immune cell responses including the initi- specialized immune cells, but in plants all the cells are believed to be able to mount a ation of phagocytosis or endocytosis, cellular motility, and beginning the processes leading defensive response to foreign attack [39]. Complex machinery, including cells and hu- to inflammatory reactions [40]. Upon successful recognition, the pathogen detecting cell moral factors is involved in recognition of nonself material, and especially in detecting will initiate a process of destruction or sequestration to eliminate eventual danger, and later domains called pathogen/microbe associated molecular patterns (PAMPs/MAMPs) that repair the stress or damage caused by this unexpected material. Most invertebrate immune are typically displayed on the surface of bacterial, fungal, and parasitic organisms and cells, similarly to human macrophages or monocytes, are involved in phagocytosis, which virus-infected cells. Host recognition of nonself will involve a large range of cell mem- remains one of the most efficient mechanisms to clear nonself material. The induction of brane bound and scattered pattern recognition receptors (PRRs). Although the distinctive some global defense mechanisms can be easily observed across different models, such as PRRs can vary between models, the main concept is consistent, and different PRRs share the production of reactive oxygen species (ROS) and nitrogen radicals (RNS), synthesis and a secretion similar role upon recognition of antibacterial of nonself proteins, and antifungal particles. cytokine-like PRRs in humans, muchand proteins, likehydrolytic their in- vertebrate enzymes. Antimicrobial peptides (AMPs—small cationic, amphipathic molecules)in- homologues, are responsible for initiating innate immune cell responses are cluding very well the studied initiationandof phagocytosis highly involved or endocytosis, in invertebrate cellular motility,They immunity. and canbeginning the tag objects processes or induceleading direct to inflammatory destruction reactions [40]. by destabilizing Upon successful biological membranes, recognition, which make the patho- them gen detecting cell will initiate a process of destruction or sequestration effective against large range of unicellular organisms like bacteria, yeast, fungi, and also to eliminate even- tual danger, and later repair the stress or damage caused by this unexpected material. Nanomaterials 2021, 11, 1528 5 of 32 some protozoans and enveloped viruses [41]. Circulating fluid also contains a large panel of enzymes (released by immune cells) with hemolytic, proteolytic and cytotoxic roles (one of the most common being lysozyme). The encapsulation of foreign objects and activation of enzymatic cascades that regulate melanization and coagulation of hemolymph are also common defense mechanisms en- countered in invertebrates. Indeed, phenoloxidase is considered among the most important components of the invertebrate immune system, especially in insects and crustaceans. The phenoloxidase cascade produces the antimicrobial molecule melanin, as well as inducing multiple potent bioactive agents such as peroxinectin and ROS, that aid in phagocytosis and cell adhesion. Melanization is essential in wound healing, encapsulation, and nodula- tion. Proper modulation of this enzyme is crucial to ensure survival of the organism. The majority of species activate the phenoloxidase cascade using the proPO enzyme [42–44]. Although general immune features are conserved across the previously described models and organisms, adaptations exist in each case that address the organism particular vulnerabilities and environmental stresses. These adaptations occur according to the organism’s lifestyle, habits and need, which might cause certain parameters to be more important than others in some species to deal with threats some models are more likely to encounter. In line with this, the next section aims to report the main mechanisms and characteristics of innate immune responses for the selected models, and in particular those known to be activated upon exposure to NPs. A summary is presented in Table 1. Table 1. Summary of the main defense mechanisms involved in innate immunity at different levels of the models discussed. Innate Immune Whole Organism Humoral/Extracellular Recognition Name Cellular Response Cell Types Level Defense Factors & Activation MAMP-triggered ROS production immunity PRRs: Cell wall Hormones Plant Effector triggered RLKs All cells Waxy epidermal (ethylene, JA, SA) Arabidopsis thaliana Immunity RLPs cuticle Antimicrobial secreted Hypersensitive NLRs peptides response AMPs (lumbricin I) Skin Phagocytosis Bacteriolytic enzyme PRRs: CCF Amoebocytes Mucus Agglutination- (lysozyme) (lectinlike Earthworm (granular and Expulsion by encapsulation Hemolytic, proteolytic domain) Eisenia fetida hyaline) dorsal pore ProPO cascade → and cytotoxic proteins TLR Eleocytes Autotomy melanization (fetidin and lysenins) LBP/BPI ROS production Phagocytosis Terrestrial Hemocyte Encapsulation AMPs PRRs: isopod Granular and Cuticle ProPO cascade → ROS/NO production TLR Porcelio scaber hyaline melanization AMPs (mytilin, myticin, mytimicin), Defensins PRRs: lectins Shell barrier Marine mussel Hemocyte Phagocytosis Complement system PGRPS Mucus layer Mytilus Granular and Encapsulation (C1qDC) TLR Pseudo- galloprovincialis hyaline ProPO Bacteriolytic C1qDC feces enzyme-Lysozyme FRED ROS /NO production Nanomaterials 2021, 11, 1528 6 of 32 Table 1. Cont. Innate Immune Whole Organism Humoral/Extracellular Recognition Name Cellular Response Cell Types Level Defense Factors & Activation PRRs: Macrophage-like TLRs Sea urchin phagocytes, Test ROS production, AMPs NLRs Phagocytosis Paracentrotus amoebocytes Gut barrier (strongylocins, centrocins, SRCR Encapsulation lividus (colorless, red); Faeces paracentrin 1), lysozyme domain- vibratile cells containing proteins Phagocytosis PRRs:TLRs, Monocytes Inflammation Complement NLRs, Macrophages Epithelial and Human Granulocyte antibodies, AMPs Scavenger DCs mucosal tissue recruitment NETs, ROS/NO Receptors, Granulocytes 1 Antigen presentation RLRs, CLRs, 1Other innate cell types exist that are not discussed, including natural killer cells and innate lymphoid cells. Refer to the main text for the meaning of the abbreviations. 2.2. Model Specific Immune System Characteristics 2.2.1. Plants Plants lack specialized mobile immune cells, and every cell is believed to be capable of initiating an immune defense against pathogens and invaders. Two layers of innate immune responses, i.e., pattern triggered immunity (PTI) and effector-triggered immunity (ETI), provide an efficient defense that keeps most pathogens and external attacks under control [39,45]. The activation of PTI relies on PRRs perceptions of MAMPs/PAMPs to trigger complex immune responses. PRRs are solely on the cell surface of plant cells and among them the most studied is the plant flagellin receptor-FLS2 [46–51]. The second line of defense, ETI, is mediated by nucleotide-binding domain leucine-rich repeat (NB- LRR) disease resistance proteins (NLR), which induce defense responses leading to a hypersensitive programmed cell death [52]. NLRs can detect effectors directly or by indirect surveillance of the effector action on other host target proteins [39]. Both lines of defense share parts of their defense signaling pathways [53,54]. After pathogen detection, multiple morphological and physiological responses are induced, such as ion fluxes over the plasma membrane, including Ca2+ - and H+ -influx; production of ROS and antimicrobial compounds (phytoalexins); activation of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs). In consequence, the transcriptome will be reprogramed by activation of a subset of transcription factors; callose deposition; stomatal closure; restriction of nutrient transfer from the cytosol to the apoplast and programmed cell death [55,56]. Phytohormones such as salicylic acid, abscisic acid, jasmonic acid, and ethylene have a critical role in the plant’s responses to specific pathogens [57]. Defense hormones can be transported within and between plants to alert distant tissues and confer systemic immunity [55,58]. 2.2.2. Earthworms Earthworms are protostome animals that have large coelomic cavities throughout the length of the animal. The coelomic cavity is typically nonsterile, open to the outer environment through dorsal pores, allowing the entrance of fungi, bacteria, and proto- zoans. Coelomocytes can be classified into two major cell types: amoebocytes and eleocytes. Amoebocytes (hyaline and granular) are involved in various immune responses including phagocytosis, encapsulation, and the production of antimicrobial molecules [59,60]. Eleo- cytes display more nutritive and accessory functions [59,61]. Three types of PRRs have so far been identified: coelomic cytolytic factor (CCF) [62], toll-like receptors (TLR) [63], and lipopolysaccharide-binding protein/bacterial permeability-increasing protein (LBP/BPI) [64]. CCF has two recognition domains that can interact with bacterial or fungal MAMPs, which in turn triggers the proPO cascade [42,65]. A range of antimicrobial molecules including Nanomaterials 2021, 11, 1528 7 of 32 lysozyme and the hemolytic proteins fetidins and lysenins are involved in the elimination of the microorganisms [66–69]. 2.2.3. Isopods The hemocytes of Porcellio scaber originate in the hematopoietic glands located along the animals dorsal vessel, and can be split into granular and hyaline hemocytes [70,71]. Hyaline (absence of granules) hemocytes are mainly responsible for phagocytosis [71]. Semigranular cells also show some phagocytic ability but seem more involved in encapsu- lation and nodulation. Granular cells are predominantly involved with the phenoloxidase system [72], and along with semigranular cells are thought to produce AMPs and be involved in antioxidant defense [73,74]. In P. scaber, the PO cascade is initiated by hemo- cyanin [44]. In addition, for defense they are able to produce, RNS, ROS, and AMPs like other invertebrates [75,76]. Genomic mining of the terrestrial isopod, Armadillidium vulgare, revealed genes for specific AMPs including anti-lipopolysaccharide factor (ALF) 1 and 2, crustin 1, 2, and 3, and I type lysozyme, and pathogen recognition genes C-type lectins 1, 2, and 3 and peroxinectin-like A and B [77]. 2.2.4. Mussels The innate effector cells of Mytilus, hemocytes, are composed of granulocytes and hyalinocytes. Mature granulocytes are among the first lines of cell defense for the elimina- tion of invaders via phagocytic processes [78]. In Mytilus, a large range of PRRs, anchored on the cell outer membrane and secreted are encountered, with lectins the most dominant group. Other classes of soluble PRRs are found, like C-terminal fibrinogen related domain- FReD-containing proteins, which have been shown to improve the rate of phagocytosis. TLRs and peptidoglycan recognition proteins-PGRPs, and others have been recently discov- ered but further study remains to be done to properly appraise their mechanisms of action (see [79] for more details). Moreover, Mytilus possesses the complement system pathway and relies on the involvement of C1qDC (C1q domain-containing) proteins [80]. Several signaling transduction pathways have been reported to be present in bivalves such as the mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), the complement component, the toll pathways, and the JAK-STAT pathway (reviewed in [79]). Additionally, as with other species, hemocytes can trigger the production and release of several factors such as ROS, nitric oxide-NO, hydrolytic enzymes (e.g., lysozyme), and AMPs. Several AMPs have been identified in Mytilus such as mytilin, myticin, mytimicin (with an antifun- gal and/or antibacterial role) and defensins [81]. In extreme cases and for larger objects, hemocytes can encapsulate foreign matter via the coordination of several hemocytes and the release of cytotoxic products (enzymes or ROS) to degrade the material, followed by cellular reabsorption of the debris [82]. Finally, the proPO cascade, while present, remains relatively unstudied in bivalves [83]. 2.2.5. Sea Urchins Sea urchins contain circulating immune cells called coelomocytes which can be sub- divided into four classes and are able to infiltrate into different tissues [84,85]. The macrophage-like phagocytes can encapsulate and internalize nonself particles, red amoebo- cytes release the bactericidal pigment echinochrome A, and white/colorless amoebocytes operate the cytotoxic/cytolytic response, while vibratile cells most probably degranulate and trigger immune cell aggregation [85]. Recently, their genome sequences have revealed the presence of a vast array of immune-related genes, including those coding for PRRs such as TLRs, NLRs or SRCR domain-containing proteins, and complement proteins (Com- plement C3 homologue) [86,87]. Moreover, lectins are also important in sea urchins and in addition to their role in opsonization, they show lytic functions, and are involved in wound repair [88]. Sea urchins contain several humoral factors including hemolysin and agglutinin which can be induced upon cell activation. ROS are also produced during im- mune responses. Echinoderms possess many different AMPs with various modes of action Nanomaterials 2021, 11, 1528 8 of 32 depending on the species, among them paracentrin 1 in P. lividus, showing an antimicrobial role [89]. Interestingly, the phagocytes can contain AMPs but they are not released into the extracellular medium, instead they play a role within the phagolysosome [88]. Finally, sea urchins belong to deuterostome lineage which makes them phylogenetically near to chordates, sharing several common traits with mammalians, especially with regard to cytokine production [90,91]. 2.2.6. Human Cells Human and other mammalian immune responses are organized within two branches: the innate immune response which is characteristic of all eukaryotes, and additionally a highly specific adaptive immune response, individualized for distinct pathogens. As NPs do not display highly specified and unique surface patterns, it is the innate branch of human immunity that is tasked with responses to NP exposure. Nonself particles and pathogenic threats that enter human circulation may activate humoral components such as AMPs and complement alongside innate immune cells. Cells participating in the human innate immune response include granulocytes, such as neutrophils (which primarily function to overwhelm pathogenic invaders through large numbers and phagocytic mechanisms), and myeloid-derived cells, including monocytes, macrophages, and dendritic cells (DCs). Monocytes represent about 2–8% of the leukocytes in circulation at any given time [92] and generally patrol the circulatory system for signs of foreign particles or internal damage. They can be recruited in tissue via resident cells releasing chemokines such as CCL2 [93]. As monocytes attempt to engulf foreign particles by phagocytosis, simultaneous chemokines and cytokines are secreted that signal for a broader inflammatory and immune response. They can be further involved in the resolution of an inflammatory reaction, assisting in tissue repair [94]. Macrophages, are tissue resident and represent up to 15% of the cells in a given tissue [95]. Functionally two broad classes of macrophage exist, M1 which display a more inflammatory phenotype involved in early immune response (killing and defending); and later M2, which display more phagocytic and tissue repair oriented traits [96–98]. Finally, DCs are known as antigen-presenting cells, acting as the bridge between human innate and adaptive immunity. They play a role in the generation of pathogen-specific T-cells and B-cell antibodies. Of the PRRs in humans, toll-like receptors (TLRs) play the most prominent role in the detection of extracellular pathogens [99], where they recognize substances such as bacterially associated carbohydrate patterns or RNA sequences associated with viruses [100]. Other PRRs that can be found on the membrane of human innate immune cells include scavenger receptors, which detect various polymers and lipoproteins [101], and C-type lectin receptors including dectin-1, which recognizes B-glucan components of various fungi [102]. In addition, they are some intracellular PRRs found in cytosol, such as NOD-like receptors (NLRs) and rig-I-like receptors (RLRs), which recognize a large range of PAMPs [103–105]. The most notable difference with invertebrates, is the diversity and number of types of cells involved in the immune response. 3. Parameters Assessed: From NPs to Innate Immune Responses 3.1. NPs: What to Consider When You Use a Biological System? The physico-chemical characteristics of a NP and its behavior in different exposure media are fundamental considerations when attempting to understand the interactions of NPs within a biological model. It is important to take into account that the relatively large surface area to volume, the low coordination of atoms at the surface, and their colloidal nature cause NPs to display physical and chemical characteristics that differ from their bulk counterparts. It is also fundamental to understand the characteristics of the final object that living organisms will encounter and to correlate the pristine and final NP features with the potential effects on living organisms. The main NP characteristics to be considered are reported in Figure 3. Nanomaterials 2021, 11, 1528 9 of 32 Nanomaterials 2021, 11, x FOR PEER REVIEW 9 of 32 Figure3.3. The Figure The different different characteristics characteristicsofofNPs NPsand andparameters parametersto to investigate when investigate they when are are they in sus- in pension media suspension forfor media laboratory experiments. laboratory experiments. 3.1.1. 3.1.1.Primary PrimaryCharacterization Characterization The Thefirst firstdetermination determinationofofprimary primarycharacteristics includes characteristics thethe includes description descriptionof of thethe mate- ma- rial composition, the nominal size, shape, and surface charge (zeta potential). Moreover, terial composition, the nominal size, shape, and surface charge (zeta potential). Moreover, characterization characterization of of NP coating coatingandandother othersurface surface modifications modifications areare crucial crucial to consider to consider (Fig- (Figure 3 left ure 3 left panel). panel). 3.1.2. 3.1.2.Behavior BehaviorininMedium Medium InInaddition to addition to thethe known known properties propertiesof aofchosen NP following a chosen NP followingsynthesis, once exposed synthesis, once ex- toposed biological conditions (e.g., medium or circulating fluid), NPs can to biological conditions (e.g., medium or circulating fluid), NPs can display display unpredicted unpre- new characteristics. NPs can have a propensity to move towards a more stable thermody- dicted new characteristics. NPs can have a propensity to move towards a more stable ther- namic state via different means: aggregation (which can mean escaping from the nanoscale), modynamic state via different means: aggregation (which can mean escaping from the formation of a coating composed of various molecules, chemical transformations, particle nanoscale), formation of a coating composed of various molecules, chemical transfor- corrosion, and dissolution [1]. All these transformations can change the identity of the NP mations, particle corrosion, and dissolution [1]. All these transformations can change the or produce new chemical entities (e.g., reactive metal ions), modifying their behavior and identity of the NP or produce new chemical entities (e.g., reactive metal ions), modifying their potential associated risk and interactions (Figure 3 right panel). Therefore, the deter- their behavior and their potential associated risk and interactions (Figure 3 right panel). mination of NP characteristics in exposure medium needs to be assessed. This generally Therefore, the determination of NP characteristics in exposure medium needs to be as- includes the aggregation state (Z-average), the change in surface charge (zeta potential), sessed. This generally includes the aggregation state (Z-average), the change in surface and the dispersion index (PdI). Moreover, the evolution over time of these parameters can charge (zeta potential), and the dispersion index (PdI). Moreover, the evolution over time also be of value for a full appraisal of the NPs dynamic in the exposure medium. All these of these parameters can also be of value for a full appraisal of the NPs dynamic in the analyses are usually performed using DLS (dynamic light scattering) analysis, or electron exposure medium. microscopy (TEM and AllSEM) thesedepending analyses are usually on the performed material using DLS (dynamic being investigated. light Additionally, scattering) analysis, or electron microscopy (TEM and SEM) depending careful controls have to be performed in order to avoid artifacts due to the presence of on the material being investigated. chemicals, often usedAdditionally, to stabilize the careful controls particles haveor [106,107], tocontaminants, be performed such in order to avoid as bacterial artifacts due to the presence of chemicals, often used to stabilize lipopolysaccharide (LPS), that can cause false positives in an immune assay [108]. the particles [106,107], or contaminants, such as measured Another routinely bacterial lipopolysaccharide (LPS), that parameter is the presence and can cause false composition of positives the molec- in an immune assay [108]. ular biocorona, where components of biological fluids can be adsorbed by the NP, forming a corona Another on itsroutinely measuredthey surface. Usually, parameter is the to are believed presence be mostlyandconstituted composition byofproteins the mo- lecular biocorona, where components of biological fluids can be (protein corona -PC) but other macromolecules including lipids present in the medium can adsorbed by the NP, forming a corona on its surface. Usually, they are believed to be mostly also contribute to its formation. The presence of this supplementary layer on top of the NPs constituted by proteins can in turn(protein affect thecorona -PC) butand NPs behavior other macromolecules interactions with the including surrounding lipids present media. in the However, medium this corona can also contribute depends on both the to its formation. biological The(plasma, fluid presenceorofotherwise) this supplementary composition layer and on topproperties the of the NPsofcan thein turnincluding NPs, affect thesize, NPscurvature, behavior and interactions surface with the surrounding functionalization, and charge. media. The However, composition ofthis the corona is depends on both theoretically the biological divided fluidcorona into the soft (plasma, or otherwise) (weakly bound) composition and the properties of the NPs, including size, and the hard corona (tightly bound), but it is dynamic and the ligand on the top curvature, surface functionali- can be zation, and charge. The composition of the corona is theoretically divided into the soft Nanomaterials 2021, 11, x FOR PEER REVIEW 10 of 32 Nanomaterials 2021, 11, 1528 10 of 32 corona (weakly bound) and the hard corona (tightly bound), but it is dynamic and the ligand on the top can be exchanged and replaced over time, according to the affinity of exchanged and replaced the macromolecule over for the NPstime, according [109,110]. The to PCthe affinity is the of theidentity biological macromolecule of the NPforand therepresents NPs [109,110]. what The cellsPC is the “see” andbiological with which identity they ofwilltheinteract NP and[111–118]. represents what cells Consequently, “see” and withby recognition which immunetheycells willcaninteract [111–118]. be different andConsequently, specific from one recognition type of NPby to immune another, cells can be different and specific from one type of NP to another, which means that they will interact with the protein on the surface rather than thewhich means that theyNP will interact with the protein on the surface rather than the NP itself. itself. This results in the triggering of defense mechanisms different from those observedThis results in the triggering in medium of defense free of mechanisms proteins. This different from does not thoseonly apply observed in mediumplasma, to mammalian free of proteins. but it has This beendoes not apply only demonstrated to mammalian in the biological fluids plasma, but it has of different been demonstrated terrestrial and marine in the inverte- biological fluids of different terrestrial and marine invertebrates, including brates, including earthworms, bivalve, and sea urchins, in which the composition and ef- earthworms, bivalve, fects onand sea urchins, immune in which parameters appearedthe composition different for andeacheffects NP type on[119–122]. immune parameters For these rea- appeared sons, the PC is an important parameter to consider under laboratory PC different for each NP type [119–122]. For these reasons, the is an important conditions and needs parameter to considerwith to be characterized under laboratory precision conditions during and needs the exposure event. to be characterized with precisionAllduring the exposure the previously citedevent. characteristics can also be applied to environmental media [123–126]. The NPs will be subjected to othercan All the previously cited characteristics also like factors be applied to environmentalparam- abiotic physico-chemical me- dia [123–126]. The NPs will be subjected to other factors like abiotic eters (such as pH, ionic strength, temperature) which can influence their dispersion, ag- physico-chemical parameters (such as pH, ionic strength, temperature) which can influence their dispersion, gregation, agglomeration [127,128], interaction with molecules present in their environ- aggregation, agglomeration [127,128], interaction with molecules present in their environ- ment, and adsorption to macro-organic matter (e.g., eco-corona) [117]. Scientific literature ment, and adsorption to macro-organic matter (e.g., eco-corona) [117]. Scientific literature is being produced on the physico-chemical transformation of NPs due to their exposure is being produced on the physico-chemical transformation of NPs due to their exposure to to aquatic and terrestrial scenarios, correlating the environments and particle properties aquatic and terrestrial scenarios, correlating the environments and particle properties with with the observed changes. Consideration of this should be taken into account in future the observed changes. Consideration of this should be taken into account in future studies studies working with environmental scenario experiments [129–132]. working with environmental scenario experiments [129–132]. 3.2. 3.2. Models, Models, Cell Cell Culture Culture andand Mode Mode of of Exposure Exposure InIn experimental experimental science, science, thethe useuseofof in in vitro vitro assays assays is being is being promoted promoted as as sustainable sustainable alternative for a large range of product testing, including NPs, following alternative for a large range of product testing, including NPs, following the 3R principlethe 3R principle (replacement, (replacement, reduction,and reduction, andrefinement). refinement). The The extraction extractionofofimmune immunecells, separation, cells, separation, cul- ture and feasibility to maintain such isolated primary cells varies across culture and feasibility to maintain such isolated primary cells varies across models. To models. To illus- trate, a summary illustrate, a summary of the different of the methods different methods of cell harvesting of cell harvestingandand exposure for both exposure inver- for both tebrates and human primary cells are reported in Figures invertebrates and human primary cells are reported in Figures 4 and 5. 4 and 5. Figure 4. The different in vitro approaches and NPs exposure parameters encountered across the selected models. Nanomaterials 2021, 11, 1528 11 of 32 3.2.1. Nonmammalian In Vitro Assays In many invertebrates, coelomocytes/hemocytes act as the first line of defense against nonself objects. The induction of functional responses with these cells is often rapidly observed, helping to counteract the limitation of the relative short-term lifespan of cells in cultures (ranging from a few hours to a few days depending on the model). As a natural defense mechanism, earthworms can extrude their coelomic fluid, through dorsal pores. Therefore, coelomocytes can be extracted by mild electrical stimulation or by exposing the animals to an irritative substance [133,134]. Immediately upon collection, the coelomocytes need to be stabilized in a culture medium in order preserve cell viability. Recent studies show that RPMI 1640 medium is the optimal medium for earthworm coelomocytes cultur- ing, as well as the assessment of NP toxicity towards coelomocytes [135,136]. Critical for the successful culturing of coelomocytes is the adjustment of osmolality of the medium so that it reflects that of the coelomic fluid [137–139]. Exposure time for in vitro assays depends on cell viability and may range from between 2 to 72 h, with 24 h being the optimum time for cell cultures in RPMI 1640, according to some investigations [136,137,140]. For terrestrial isopods, the culturing of hemocytes did not show hopeful results yet. Hemolymph can be collected by puncturing through the intersegmental membrane on the dorsal side of the isopod with a sterile needle and collecting the hemolymph with a micropipette. With the use of ringers solution and a MAS (mitochondrial assay solution) buffer, cells appeared to hardly survive for even a few hours outside the body. The selection of a suitable medium is still needed to be identified and adapted for keeping hemocytes alive without showing excessive levels of stress [141]. In marine invertebrates, in vitro experiments are much more abundant, in particular, experiments using hemocytes of the marine mussel M. galloprovincialis extracted via a non- invasive method. This method can provide a first line of investigation for testing several types of substances, including NPs [142–145]. Mytilus hemolymph is easy to collect via the adductor muscle and fluid quantities are sufficient (depending on the season, volume can be as high as several ml per animal) to perform various experiments [146]. Short-term exposures (≤1 h) have shown rapid activation of hemocyte functional parameters but longer exposure times (up to 24 h) have shown the induction of further immune or stress parameters. For short experiments, hemocytes can be maintained in a natural hemolymph or seawater suspension, in tubes or as monolayers on glass slides. Longer culture times were more successful when modified synthetic basal medium (Basal Medium Eagle) was used in microwell plates. These in vitro experiments are possible due to the cells ability to quickly adhere to supports (<20 min) [147,148]. Ex-vivo tissue explant has also been used (e.g., gills) to study the first interactions and potential uptake of NPs [149]. The coelomocytes from sea urchin once extracted are placed in cell culture plates and kept in EGTA-containing cell culture medium and artificial seawater [84,85,150]. The coelomocytes can be kept for a long period of time (over two weeks), with regular medium replenishment and without the addition of the special growth factors or nutrients [90,151]. Finally, as plants do not possess specialized mobile immune cells, in vitro research is not typically suitable/realizable, and the main experiments in laboratories are made on the full plant or tissues excisions (see next section). Each piece of tissue should respond upon exposure as each single cell is able to launch an effective immune response [152]. 3.2.2. Human Cell Models The study of human cells offers a wide range of possibilities not currently developed for invertebrate models. Multiple cell types, coculture conditions, and cell maturation or differentiation programs exist to define more precisely the interactions with NPs. Several important models used in mammalian systems to test NP–immune system interactions are listed below. Nanomaterials 2021, 11, Nanomaterials 2021, 11, 1528 x FOR PEER REVIEW 12 of 32 12 32 Figure 5. The different in vitro NP exposure possibilities available for human primary cells and the procedure of cell Figure 5. The different extraction in vitro NP and preparation exposurewith (Reprinted possibilities permissionavailable for human from Michelini primary et al. (2021) cells [153].and the procedure Copyright of cell extraction 2021, Copyright Royal and preparation (Reprinted with Society of Chemistry). permission from Michelini et al. (2021) [153]. Copyright 2021, Copyright Royal Society of Chemistry). In vitro modelling of human innate immunity is usually conducted using monocytes, macrophages, or dendritic cells, as these cells are responsible for directing the innate im- mune response from pathogen recognition to phagocytosis to inflammation, inflammation, and even even to to eventual antigen presentation and induction of adaptive immunity. immunity. Cell lines for each lines for each of of these cell types exist and are frequently used due to their easy experimental repeatability and scalability, scalability,with with THP-1 THP-1 (monocytes) (monocytes) andand U937 (macrophages) U937 beingbeing (macrophages) the mostthefrequently most fre- reported [154]. However, quently reported cell linescell [154]. However, arelines trulyare limited truly to the representative limited phenotype to the representative pheno-ob- served at the time type observed of culture, at the time of and evenand culture, thiseven is susceptible to mutations this is susceptible that do notthat to mutations represent do not the true reactivity represent the trueof healthy human reactivity cells.human of healthy More robust models cells. More of innate robust models immune respon- of innate im- siveness utilize primary cells, which are collected directly from donors mune responsiveness utilize primary cells, which are collected directly from donors and and may be isolated using may be techniques that select isolated using for the desired techniques cellfor that select type. thePrimary desiredcells cell are representative type. Primary cells of are an individual’s current in vivo condition, and lack the altered metabolic and representative of an individual’s current in vivo condition, and lack the altered metabolic epigenetic profile inherent to cellprofile and epigenetic lines. Furthermore, inherent to cellaslines. monocytes are found Furthermore, abundantly as monocytes areinfound circulation, abun- and since they can be precursors for both macrophages and DCs, the dantly in circulation, and since they can be precursors for both macrophages and DCs, the differentiation of monocytes in culture into primary differentiated macrophages or DCs differentiation of monocytes in culture into primary differentiated macrophages or DCs is an effective tool to create is an models effective tooloftoinnate createimmune models of responses in vitro.responses innate immune However,inmodels utilizing primary vitro. However, models cells must contend with individual variability as the immune utilizing primary cells must contend with individual variability as the immune experience and capacity experience is different between donors [155]. and capacity is different between donors [155]. The The whole whole blood blood assay assay is is one one of of the the most most simple simple andand rapid rapid tests tests for for assessing assessing the the immune activating capacity of novel substances within a human system. Typically, blood is immune activating capacity of novel substances within a human system. Typically, blood drawn from healthy donors and immediately exposed to the substance under investigation, is drawn from healthy donors and immediately exposed to the substance under investi- with 250 µL of blood typically diluted in 750 µL of RPMI plus the tested material and gation, with 250 µ L of blood typically diluted in 750 µ L of RPMI plus the tested material incubated for 24–48 h [156]. Peripheral blood mononuclear cell (PBMCs) can also be and incubated for 24–48 h [156]. Peripheral blood mononuclear cell (PBMCs) can also be Nanomaterials 2021, 11, 1528 13 of 32 isolated from the whole blood using Ficoll–Paque density gradient centrifugation [157]. Magnetic cell separation using some CD-4 beads can be used to isolate monocytes, and later growth factors can be added to differentiate macrophages such as macrophage colony stimulating factor (M-CSF) or DC GM-CSF and IL-4 [94,158]. The monocyte activation test (MAT) models (or using macrophage and DCs) can assess the exposure and response of monocytes to NPs, and many parameters may be assessed following activation [159–161]. Usually cells (in the range of ~500,000 cells/mL) in plate culture can be directly exposed to NPs added to the wells, and tests on mono- cyte/macrophage/DC activation are typically completed within 24 h. Oftentimes, PBMC culture is conducted in round bottom wells, which simulate a lymph node in which com- munication between myeloid cells and lymphocytes occurs. Finally, NPs can also interact with other cells present in blood, such as DCs, which link with adaptive immunity. Similar principles of the MAT test can also be applied for testing NPs, but also cocultures with T-cells of self or foreign origin [153]. These types of test can mimic autoimmunity or the mixed lymphocytes reaction and are of interest for the use of NPs in vaccines and immunotherapy [162]. Experiments considering primary isolated cells offer other advantages by representing a simplified model, limiting interfering factors, which could help to spotlight NPs mecha- nisms of action before performing further experiments; e.g., coculture, tissue models, or even using whole-organism in vivo experiments. Moreover, these in vitro experimenta- tions allow easier comparison between models, particularly, with human cells. In addition to the commonly known pros for in vitro assessment such as cheap cost, fewer animals used and relatively fast results, a list of the more important pros and cons for each model, with special input for in vitro assays, is presented in Figures 4 and 5, lower panel. Although they provide a simplified set up and can be used to try and understand some of the basic mechanisms, they do not represent the true exposure pathway. Additionally, large varia- tions in the exposure time and the culture methods between different models persist. In invertebrates, the immune cells are usually easy to collect, except for isopods, and in large quantity. There are some species-specific difficulties in experimentation, such as molt cycles in isopods or seasonality with reproductive period in mussels that can impact immune measurements. Moreover, some cells are more sensitive and fragile to handle compared to others. Usually, the immune cells from invertebrates are viable for shorter times in culture, as basal parameters are quickly impacted. For humans, in addition to regulatory hurdles, donor availability is restricted for the obtaining of primary immune cells. Each donor is usually considered independently, which can reflect stronger variabilities in responses. However, from one whole blood sample many cells can be collected and offer a large range of possible assays after purification. 3.3. Whole Model Exposure Experiments In vivo experiments allow for evaluation of the effects and mechanisms of action of NPs in organisms at different levels of biological organization (molecular, cellular, tissue level). They provide a realistic scenario of the exposure pathways as encountered under natural conditions. For controlled laboratory experiments, the mode of exposure to NPs needs to be adapted for each model; a summary is presented in Figure 6. These tests are usually conducted in environmentally relevant mediums (soil or water), through feeding experiments or through breathing and filtering experiments for aquatic species. As the selected models are usually easy to maintain in laboratory conditions for long periods of time, requiring little space and maintenance (e.g., feeding), the exposure time (acute, semichronic and chronic exposure) can range from hours, to days and weeks. Nanomaterials 2021, 11, 1528 14 of 32 Nanomaterials 2021, 11, x FOR PEER REVIEW 14 of 32 Figure 6. NP exposure approaches using the whole organism with the different exposure pathways across the Figure 6. NP exposure approaches using the whole organism with the different exposure pathways across the selected selected models. models. Plants can germinate and grow directly in the presence of the NP in the growth medium, Plantsi.e.,can germinate soil, hydroponicand grow nutrientdirectly in theor solutions presence of the NPagents, agar-solidified in the growth or theyme- can be dium, i.e., exposed at soil, hydroponic subsequent nutrient solutions development stages.orDespite agar-solidified agents,oforcell the presence they can be walls, ex-can that posed at subsequent represent development a barrier preventing NPsstages. entering Despite the presence into the plant cellofand cellcytoplasm, walls, that canNPsrep- might beresent a barrier absorbed preventing through root orNPsleafentering and beinto the planttransported potentially cell and cytoplasm, NPs might to the shoot or to be other absorbed through root or leaf and be potentially transported to the points through the phloem (vascular system) [163–166]. NPs can be also dispensed onto shoot or to other points through the plantsthe phloem surface (vascular by foliar spraysystem) [163–166]. application [167].NPs Aftercan be alsointo entrance dispensed the leaf onto tissue,theNPs plants surface by foliar spray application [167]. After entrance into can diffuse into the intercellular space, the apoplast, or membranes and cause secondary the leaf tissue, NPs can diffuseMoreover, effects. into the intercellular temporality space, the apoplast, is important, andorit membranes is necessaryand cause secondary to understand ef- the course fects. Moreover, temporality is important, and it is necessary to understand the course of of plant growth and development, from seed germination to root elongation and shoot plant growth and development, from seed germination to root elongation and shoot emer- emergence, in relation to NP exposure [168]. The following investigations can assess the gence, in relation to NP exposure [168]. The following investigations can assess the NP NP uptake by cells and further nanophytotoxicity, focusing on the toxicity symptoms uptake by cells and further nanophytotoxicity, focusing on the toxicity symptoms of of plants. plants. In vivo earthworm exposures are typically conducted in soils following well-described In vivo earthworm exposures are typically conducted in soils following well-de- and standardized procedures (e.g., [27]) that can also be applied for NPs [140,169]. How- scribed and standardized procedures (e.g., [27]) that can also be applied for NPs [140,169]. ever, care must be taken when it comes to the mixing of NPs with soils, adjusting the However, care must be taken when it comes to the mixing of NPs with soils, adjusting the parameters parameters depending depending on onthe theform form (i.e., (i.e., as solution as solution dispersion dispersion or asor as powder) powder) in which in which the the NPs are supplied [170]. Furthermore, coexposures with infectious microbes are alsoare NPs are supplied [170]. Furthermore, coexposures with infectious microbes im-also important portant inin order order toto establish establish whether whether anan exposure exposure to NPs to NPs hashasan an effect effect on on thethe ability ability of aof a host hosttotomaintain maintainimmunity immunity[171]. [171].AAmethodological methodologicalapproach approachtotoinvestigate investigatethe theimpact impactNPs have NPs onhavetheonearthworm’s the earthworm’s ability to maintain ability to maintain immunity immunity whenwhen coexposed coexposedwith withinfectious infec- bacteria has been tious bacteria has recently established been recently [140].[140]. established AAmajor major benefit of working benefit of workingwith withthetheterrestrial terrestrialisopod, isopod, P. P. scaber scaber is that is that theythey areareableable totobe be exposed to the NPs in a manner similar to how they would be exposed in nature.nature. exposed to the NPs in a manner similar to how they would be exposed in NP NP suspensions suspensions can can be spread be spread on leaves on leaves that P.that P. scaber scaber eat,both eat, and and theboth the leaf leaf and and animal animal are are then placed in a petri dish. During the experiment, feeding rate, defecation rate, and mortality can be monitored. This also allows for modelling of real-world impacts of NPs on the organism from behavioral changes, like feeding avoidance and mortality, Nanomaterials 2021, 11, 1528 15 of 32 to cellular immune responses. However, the gut of P. scaber is covered in a thick cuticle which is believed to stop the translocation of NPs from the gut into the hemocoel where the hemocytes are, so the immunological effects of the NPs might not be seen when ingested [172]. There is the possibility for an alternative exposure scenario, with injection experiments delivering substances directly into the animals hemocoel, allowing for the study of the direct interaction of a known concentration of NPs with the hemocytes. This ensures NP and immune cell interaction [141,173]. Mussels are suspension feeders and are able to filter large amounts of water (up to 3 L per hour) implying that, in a short period of time, they can easily uptake the NPs present in the seawater of experiment tanks. For this reason, the NPs can be directly added to the seawater and the ventilation system allows for constant movement of water within the tank. To study the first immune defense response, short term experiments (24 h to 96 h), have been shown to be sufficient to induce the activation of the immune system [142]. Moreover, the use of artificial seawater (ASW) implies the absence of organic matter or other substances that could interact with the NP suspension; together with a constant salt content and as such are reproducible for all periods of the year. The experiments are mainly conducted in the spring and summer periods where mussels are at their healthiest. During experiments, mussels are not fed and can readily survive several days without feeding [146]. This is necessary for NP experiments, as the presence of microalgae could interact with the NPs. In this context, the study relies only on the uptake of the NPs in seawater. To mimic a more realistic exposure, other studies have performed longer- period experiments to consider the interactions between food intake and the NPs, but this generally focused more on physiology and tissue changes and not strictly the immune response [148,174]. Biological uptake routes are dependent on NP properties and may occur as direct uptake in gill tissues and/or through transference from the cilia to the digestive system. Moreover, the agglomeration of particles in seawater has been shown to facilitate NP ingestion by suspension feeding bivalves, and their potential translocation from the gut to the circulatory system [175,176]. However, this internalization pathway seems to vary according to the NPs and some can be captured and excreted in pseudofaeces (mixture of mucus and undigested particles) before arriving to the stomach, resulting in lower tissue accumulation and higher depuration [177]. The existing in vivo studies utilizing sea urchins mainly focus on the immune status of the animal after exposure to NPs via the injection of the NP suspension into the mouth or directly into the coelomic cavity (through the soft peristomal membrane surrounding the mouth). Consequently, NPs injected orally partially cross the intestinal epithelium, invade the coelomic fluid and are then engulfed by phagocytes, while the remaining particles pass the digestive system and can be excreted [178]. On the other hand, NPs injected into the coelomic cavity can directly interact and be recognized by phagocytes [85,179]. In general, the in vivo passive exposure experiments consider more realistic exposure pathways (feeding, breathing) of NPs. However, for more simplistic set up and to be sure that NPs encounter immune cells, NP suspensions can be also injected into the animal. Results obtained from in vivo tests can provide a good proxy of interactions of immune cells in situ and thereby in vivo tests are crucial to resolve the issue of whether NPs pose an immune threat to living organisms. These models have been shown to be easy to maintain in the laboratory, and exposure experiments allow for the effects of NPs to be studied at different levels of the organism. For future experiments, mesocosms will help to mimic environmental scenarios before further studies in the environment. 3.4. Innate Immune Parameters of Interest As highlighted in Figure 7, a variety of endpoints can be used to compare immune system–nanoparticle interactions between different models. This includes functional re- sponses, which comprise the biochemical assessment of cellular and humoral responses, and molecular responses that aim to evaluate changes in the expression of immune-related genes. Because there are many methods available to quantify functional responses, here Nanomaterials 2021, 11, x FOR PEER REVIEW 16 of 32 As highlighted in Figure 7, a variety of endpoints can be used to compare immune Nanomaterials 2021, 11, 1528 system–nanoparticle interactions between different models. This includes functional re- 16 of 32 sponses, which comprise the biochemical assessment of cellular and humoral responses, and molecular responses that aim to evaluate changes in the expression of immune-re- lated genes. Because there are many methods available to quantify functional responses, we here we provide provide a comparative a comparative overviewoverview of theseof these methods methods to showtowhich showare which mostare most ap- appropriate propriate for the purpose of NP testing and cross-species comparisons for the purpose of NP testing and cross-species comparisons (see Table 2).(see Table 2). Figure 7. Summary of the different endpoints measured in immune cells after exposure to NPs. Figure 7. Summary of the different endpoints measured in immune cells after exposure to NPs. Table 2. Overview of studies demonstrating the use of cellular and humoral parameters to characterize the immune Table 2. Overview of studies demonstrating the use of cellular and humoral parameters to characterize the immune re- responses of organisms. sponses of organisms. Sea Sea Plants Plants Earthworms Earthworms Isopods Isopods Mussels Mussels Human Human Urchins Urchins 1.Wholecell 1. Whole cell Cell viability ✔ ✔ ✔ ✔ ✔ ✔ Cell viability LDH or ATP release 4 [180] 4 [181] 4 4 4 4 [153,182] LDH or ATP Fluorescent release probes (FDA or PI) [180] [183,184] [181] [138,139] [185,186] [178] [153,182] [187] Metabolic activity (MTT or CTB) [188] [181] [189] [153] Fluorescent probes (FDA or PI) [183,184] [138,139] [185,186] [178] [187] Blue tryptan [190] [191] [173] [192] [193] [194] Metabolic activity(Annexin-V, (Pre)-apoptosis (MTT or CTB) DAPI, PI) [188] [195,196] [181] [138,139] [141] [189] [197] [121] [153] [198] Cell subpopulation or polarization [139,199] [141,173] [200,201] [179] [153,202] Blue tryptan [190] [191] [173] [192] [193] [194] NP internalization ✔ ✔ ✔ ✔ ✔ ✔ (Pre)-apoptosis (Annexin-V, TEM/SEM DAPI, PI) [195,196] [203,204] [138,139] [181,205] [141] [141] [197] [189,206–208] [121] [90,151,178] [198] [153,209] Organelles ✔ ✔ ✖ ✔ ✔ ✔ Cell subpopulation or polarization [139,199] [141,173] [200,201] [179] [153,202] Neural red uptake/ release [210,211] [212] [185,189] [150,179] [213] NP Lysosome internalization acidification 4 [214,215] 4 4 4 [200] 4 [179] 4 Other organelles integrity (Trans-Golgi ap- [189,206– [216] [178,179] TEM/SEM paratus, Mitochondria) [203,204] [181,205] [141] [90,151,178] [153,209] 208] 2. Phagocytic activity Organelles Phagocytosis 4✖ 4✔ ✖ 6 ✔4 ✔4 ✔4 Phagocytic activity release Neural red uptake/ (index, rate) [210,211] [139,181] [212] [174,216] [185,189] [150,217] [150,179] [218] [213] Lysosome acidification [214,215] [200] [179] Other organelles integrity (Trans-Golgi apparatus, [216] [178,179] Mitochondria) Nanomaterials 2021, 11, 1528 17 of 32 Table 2. Cont. Sea Plants Earthworms Isopods Mussels Human Urchins 2. Phagocytic activity Phagocytosis 6 4 6 4 4 4 Phagocytic activity (index, rate) [139,181] [174,216] [150,217] [218] 3. Cytotoxic factors Oxygen and nitrogen radicals 4 4 4 4 4 4 [146,208, ROS production [219,220] [139,205] [151,222] [223,224] 221] Lipid peroxidase activity [220,225] [138,139] [226] [227] RNS (including NO) production [228,229] [230] [173] [185] [231] Hydrolytic enzymes 4 4 6 4 4 4 Lysozyme [232] [233] [234] [235,236] [237] lysenin Other species specific enzymes [119] 4. Humoral factors Cytokines 6 6 6 6 4 4 IL, TNF, IF secretion [151] [94,238,239] Melanization 6 4 4 4 4 6 Phenoloxidase activation [230,240] [44] [83] [241] 5. Gene expression Oxidative stress genes 4 4 6 4 4 6 Antioxidant defense and [242] [140,195] [176,200] [243] detoxification genes (e.g., CAT, SOD) Circulating protein genes 6 4 6 4 6 4 Lysenin/Fetidin mytilin, Signal transduction protein, enzymes, [141,170,192] myticin, [231] AMPs (general and species-specific) CCF EPp [181,244] [176,200] Receptor protein genes 4 4 6 4 4 4 TLR [245] [244] [177] [151,179] [246] LBP/BPI (LPS-binding protein/bacterial [247] [64] [243] permeability-increasing protein) 3.4.1. Whole Cell Response Parameters looking at the whole immune cell concerns the immune cells viability, the membrane integrity, all the different types of interactions they can have with NPs, and their potential changes in morphology (Figure 7, first point). The first important cellular responses that can be used to investigate nanoparticle—immune system interac- tions is (immune) cell viability. Although cell death and apoptosis are part of a normal immune response, studies have shown that NPs are able to cause excessive mortality in immune cells with possible adverse effects on immunocompetence. There are several methods available that can measure cell viability (Table 2). A common method used in human cell lines is the measurement of the release of LDH or ATP through biochemical assays [153,182]. An alternative is the staining of living or dead cells using fluorescent probes (e.g., fluorescein diacetate–FDA or propidium iodide–PI) for observation using flow Nanomaterials 2021, 11, 1528 18 of 32 cytometry fluorescent microscopy. In some models, cell viability may also be studied by measuring metabolic activity through cell-permeable fluorescent reduction such as CTB or the colorimetric MTT. In some models the use of counter stain dye such as trypan blue or nigrosine [141,192] or the use of DNA-binding florescent dyes are alternative methods for assessing cell viability [150]. There are several methods available to measure the number of cells that are in the process of dying (apoptosis). Apoptosis and preapoptosis evaluation methods, which are available for several models, can be used as early markers for cell viability through the use of specific fluorescent dyes (e.g., annexin V binding, apostain, tetramethylrhodamine, ethylester perchlorate-TMRE or DAPI labelling) [139,197]. Cell via- bility is probably the best described immune parameter in most species (Table 2); therefore, this parameter is one of most relevant to assess in cross-species comparison. In addition to measuring the overall immune cell viability, quantifying changes in the ratios of different subpopulations of immune cells (e.g., total hemocyte counts-THC) can often give a more detailed view of the impact of NPs on immune cell viability [141,179,199,200]. In human cells, fluorescence-activated cell sorting (FACS) is a common method in which fluorescent antibody-tags can be used to determine a large range of parameters but also to discriminate sub-cell populations [153]. The subcellular effects of NP exposure can be identified via assessment of the in- tegrity of organelles, membranes, and other cellular compartments. Lysosomal functional integrity is an evolutionarily conserved marker of stress (including NPs) and of an in- dividuals’ health status, and is commonly evaluated by measuring neural red retention or uptake [185,189,248]. Other approaches that can be used to assess the effects of NPs on organelles include methods measuring trans-golgi apparatus integrity and internal membrane polarization [178,179,200]. Another crucial step in the characterization of NP–immune system interactions is assessing whether immune cells are able to internalize NPs. The internalization of NPs has been observed for different types of NPs across the selected models and was recently reviewed in [36]. There are several techniques available to detect the internalization of NPs. These include transmission electron microscopy (TEM) which can image internalized particles and scanning electron microscopy (SEM) which helps to visualize membrane- bound particles and can give a direct image of the particles and cells following contact. They can also provide details on how the interaction occurs as well as the state of the NPs (e.g., agglomeration, aggregation, precipitation). These techniques when coupled to an EDX system (energy dispersive X-ray) can be used to perform chemical characterization of the NPs’ surfaces. TEM and SEM are descriptive techniques that can provide valuable information but makes quantification difficult between models. Some research has reported the use of fluorescently labelled particles to help to measure particle uptake, although the use of such labelled particles requires additional controls to rule out any effects linked to the leakage of fluorescent dyes [249]. In general, NP internalization in human cells has been well reported but for invertebrate, similar methods often require adjustments to be made (as for example, the salt or osmotic concentrations during fixation) [90,141,153,205,206,209]. For plants, TEM can be used to verify the entry of NPs into the cells [203]. In addition, these kinds of techniques can reveal the change in cell morphologies and subcellular structures (e.g., vacuoles, phagosomes, endosomes) upon NP exposure and give hints regarding the general activation or damages that the cell has undergone [203,207]. 3.4.2. Phagocytic Activity While an immune response towards NPs could be part of normal immune func- tioning, overstimulation of the immune system resulting in damage or suppression lead- ing to a compromised immune functioning may pose a threat to the organism. Such suppression of immune functioning caused by NPs could be studied via the assess- ment of the immune cells capacity to phagocytose and the consequent changes on index and rates (Figure 7, second point, Table 2). In earthworms, mussels, and sea urchins, Nanomaterials 2021, 11, 1528 19 of 32 phagocytosis can be evaluated by using fluorescence beads or yeast (using neutral red stained zymosan) [139,145,150,181,216,250]. 3.4.3. Cytotoxic Factors Upon contact with NPs, cells can be activated and produce cytotoxic factors inside the cells in order to help to remove internalized foreign particles (Figure 7, third point). Among them, the oxidative burst, which involves the production of several radicals from oxygen (ROS) and nitrogen (RNS) derivatives. To quantify ROS, several methods, including the use of fluorescent probes (e.g., DCF or calcein), UV-vis spectroscopy (e.g., cytochrome C reduction) or histochemical staining, can be used and many of which have been adjusted for use across the model organisms (Table 2). Moreover, lipid peroxidation can be measured as a proxy for the damages caused by oxidative stress to the membranes, even if it is more frequently analyzed in tissues than in individual cells [138,139]. As for the quantification of RNS and more commonly nitric oxide (NO), in isopods, mussels, and earthworms, NO levels in the hemolymph can be measured spectrophotometrically from hemolymph samples using Griess reagent [141,234,250]. Lysozyme is an evolutionary conserved enzyme that catalyzes the hydrolysis of peptidoglycan and plays a role in the innate immunity of many organisms including earth- worms, mussels, sea urchins, and plants [67,234,251,252]. The quantification of the release of lysozyme into the extracellular medium is based on the lysis activity of Micrococcus lysodeikticus which can be determined spectrophotometrically. Fluorescent probes can be also used to monitor the evolution of the lysosomal compartment and acidification in the cell upon exposure to NPs [200]. 3.4.4. Humoral Factors Humoral immune responses play a crucial role in immunity by facilitating communi- cation between immune cells and directing the extracellular destruction of foreign objects. Upon activation of the immune cells, some factors can be released into the extracellular medium (Figure 7, fourth point, Table 2). In mammals, cytokine production is a key driver of cellular immune responses [238]. Analyzing the extra- and intracellular concentration of cytokines secreted by (human) immune cells is a well-established method to test the effects and safety of NPs [253]. Many techniques have been developed to detect single or multiple cytokines and factors secreted by cells. These include classic methods such as western blot, ELISA, and bio-chemiluminescence assays, and many commercial possibilities for multi- plex assays including legendplex or Ella multiplex technology [153,209,254]. Interestingly, in sea urchins, cytokine IL-6 can be detected in immune cells and secretome using western blot analysis after exposure to NPs [151]. Lysozyme and radicals can also be released by immune cells in the extracellular medium, to directly destruct foreign particles in close proximity to the cell. In addition, there are species specific released factors such as the hemolytic protein lysesin found in the coelomic fluid of earthworms. Lastly, an important immunological parameter often analyzed in invertebrate mod- els is the measurement of phenoloxidase (PO) activity. This enzyme, produced via the pro-PO cascade, is involved in the production of melanin [43,141]. Upon detecting a melanized pathogen or object, immune cells quickly encapsulate the material resulting in the elimination of the threat. PO activity can be assessed by monitoring the forma- tion of a reddish-brown pigments in the hemolymph from an individual organism using spectrophotometry [44,173,240]. In bivalves, the presence of PO has been reported but its basal levels, the variation across species and especially its response to NP exposure remain poorly understood [83,255]. In the sea urchin Strongylocentrotus nudus coelomocytes, three proteins with PO-like activities have been identified using electrophoretic methods [241]. Nanomaterials 2021, 11, 1528 20 of 32 3.4.5. Molecular Response Increasingly, humoral responses can be measured through genetic or omics approaches (e.g., quantitative PCR or full-transcriptome sequencing) (Table 2). A main advantage of using these approaches over biochemical ones is their high-throughput potential and increased specificity. Furthermore, genetic or omics approaches allow for the assessment of entire immunological pathways instead of focusing on specific biochemical endpoints. However, major limitations of these methods are that they require species specific primers and the availability of transcriptomes, which are currently lacking for many invertebrate species. Moreover, gene expression is highly regulated and time-dependent, so careful consideration must be given to the experimental model in terms of stimulation/exposure time, and cell collection technique. Genes involved in different immune-related functions, such as oxidative stress re- sponse, humoral factors (e.g., AMPs), and receptor proteins, are available for some organ- isms and the main immune related genes known to be activated upon NP exposure are reported in Table 2. A whole genome transcript is under development and will be available for P. scaber. Using this and genes previously annotated in other more commonly used crustacean species, primers specific for P. scaber immune-related genes can be designed (Hernadi, Mayall personal communication). Moreover, gene expression offers alternative possibilities to study several proteins involved in the immune response that biochemical tests that evaluate their activity or functionality are not feasible, such as the effects of NPs on AMP modulation. In plants, microarray-based studies are good tools to monitor the expression of candidate genes involved in the plant defense responses after interaction with NPs [204]. Additionally, an important future issue for environmental molecular biology is to establish whether an up- or downregulation of a certain gene correlates to a modification in the levels of related proteins [256]. The study of transcriptomic changes in cells, tissues, or full invertebrate organisms after exposure to NPs is now emerging but is still in its early phase. Transcriptome analysis can highlight pathways being activated but it should also be accompanied by the study of functional parameters for a fuller, deeper understanding [228,257,258]. In addition, changes in protein repertoires (proteomics approach) have shown interesting outcomes; however, studying of the combined immune response to NP exposure remains in its infancy [259–261]. 4. Proposal for Future Cross-Species Evaluations and Conclusions During the PANDORA project [35], several studies were conducted on the innate immune response of different models exposed to a large range of different NPs. Based on the outcomes of these studies, several conclusions can be made which may help to guide future (comparative) studies on nanoparticle–immune system interactions (Figure 8). Because chemical conditions of mediums strongly affect the form and state of NPs and thereby the behavior of NPs, it is crucial to characterize the physico-chemical properties of NPs in the exposure medium as well as in their pristine form (e.g., after production). As the behavior and the interaction of NPs with immune systems are also time-dependent, experimental design will need to critically consider exposure duration as well. In vitro models can be considered as the prime focus for studying nanoparticle–immune system interactions. However, in vitro models are not available for all immune model species (e.g., isopods, plants), limiting comparative studies based on in vitro testing. In vivo experiments are crucial to study nanoparticle–immune system interactions under more realistic conditions. Studies using invertebrates, which are well-established and can be conducted on a routine basis, may serve as good alternatives to in vivo mammalian testing models such as mice or rats. Here, exposure route and exposure concentration will need to be critically considered as these factors are likely to significantly affect immune cells and their interaction with the NPs. Nanomaterials Nanomaterials 2021, 2021, 11, x FOR PEER REVIEW 11, 1528 2121 of 32 of 32 Figure Figure 8. Proposal 8. Proposal template template for for translatable translatable NPNP experiments experiments across across thethe models models of interest of interest (plants, (plants, terrestrial terrestrial andand marine marine invertebrates, and human cells). invertebrates, and human cells). In Because chemical this review, conditions we provided of mediums an overview of thestrongly methodsaffect used the form and state to characterize of NPs nanopar- and ticle therebyresponses immune the behavior of NPs, itorganisms in various is crucial to characterize across the treetheof physico-chemical life (Table 2). Among proper- ties ofparameters, cellular NPs in the exposure it appearsmedium as welltoasassess that methods in their pristine cell form viability (e.g., after (including production). assessments of As the behavior and subpopulations) andtheNPs interaction of NPs by internalization with immune immune systems cells are also are well time-dependent, described in most experimental organisms. design activity Phagocytic will need is to critically a crucial considertoexposure parameter duration be evaluated as well. In for immune vitro cells, models some however, can bemodels considered lack as thethe prime focus methods for studying to study nanoparticle–immune this parameter (e.g., isopods) orsystem do notinteractions. rely on this However, type of response in vitro(e.g., plants). models are Moreover, not available as methods for microscopy for all immune are model species universally available, (e.g., isopods, measurements plants), of the change limiting comparative in morphology studies based on in and external vitro interactions testing. In vivo ex- of periments NPs can beare studied crucialin to most immune study models. nanoparticle–immune system interactions under more re- Dueconditions. alistic to a lack ofStudies generalusing knowledge on the composition invertebrates, and functioningand which are well-established of humoral can be con- immunity ducted on in amost organisms routine basis, mayother thanasmammal/human serve good alternativesmodels, to in vivo it remains mammalian difficult testing to models identifysuch the most relevant as mice or rats. humoral parameter for cross-species evaluations. The ex- ceptionHere, beingexposure oxidativeroute (andandnitrosative) stress, for which methods are well described exposure concentration will need to be critically considered in most species. as these factors are likely to significantly affect immune cells and their interaction with theFurther NPs. work is needed to identify the interorganism comparability of otherwise species-specific markers, especially in invertebrates. In order to fully characterize NP-immune responses across species from the tree of life, there is a need for the iden- Nanomaterials 2021, 11, 1528 22 of 32 tification of markers indicative for both pro- and anti-inflammatory responses, as are currently already available for human models (e.g., [253]). The development of such mark- ers will require fundamental research on the innate immune systems of organisms other than human models. Thorough investigations in species from across the tree of life will help to understand how NPs interact with the innate immune system under different conditions and environments which may guide the future development of NPs that are immunologically safer-by-design. Author Contributions: Conceptualization, B.J.S., C.M. and M.A.; writing—original draft preparation, C.M., B.J.S., A.A., F.B., E.F., S.H., S.M., N.I.N.P., A.P., E.S., M.A.; writing—review and editing, B.J.S., C.M., E.S., M.A.; supervision, M.A. All authors have read and agreed to the published version of the manuscript. Funding: All authors were supported by the EU H2020 project PANDORA, grant number 671881. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Acknowledgments: The authors warmly acknowledge the project coordinator Diana Boraschi for her strong support during the whole project period, and all the PIs (L. Canesi, D. Drobne, A. Duschl, M.A. Ewart, J. 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