Annalisa
Pinsino
*a,
Neus G.
Bastús
b,
Martí
Busquets-Fité
c,
Laura
Canesi
d,
Paola
Cesaroni
e,
Damjana
Drobne
f,
Albert
Duschl
g,
Marie-Ann
Ewart
h,
Ignasi
Gispert
c,
Jutta
Horejs-Hoeck
g,
Paola
Italiani
ij,
Birgit
Kemmerling
k,
Peter
Kille
l,
Petra
Procházková
m,
Victor F.
Puntes
bno,
David J.
Spurgeon
p,
Claus
Svendsen
p,
Colin J.
Wilde
h and
Diana
Boraschi
*ij
aInstitute for Biomedical Research and Innovation, National Research Council, Palermo 90146, Italy. E-mail: annalisa.pinsino@irib.cnr.it
bCatalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST, Campus UAB, Bellaterra, Barcelona, Spain
cApplied Nanoparticles S.L., Barcelona, Spain
dDepartment of Earth Environment and Life Sciences, University of Genoa, Genoa 16126, Italy
eALTA S.r.l.u, Siena, Italy
fDepartment of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana 1000, Slovenia
gDepartment of Biosciences, Paris-Lodron University Salzburg, Salzburg 5020, Austria
hAvantiCell Science Ltd, Ayr KA6 5HW, UK
iInstitute of Biochemistry and Cell Biology, National Research Council, Napoli 80131, Italy. E-mail: diana.boraschi@ibbc.cnr.it
jStazione Zoologica Anton Dohrn, Napoli 80121, Italy
kCenter for Plant Molecular Biology – ZMBP, Eberhard-Karls University Tübingen, Tübingen 72076, Germany
lSchool of Biosciences, Cardiff University, Cardiff CF10 3AX, UK
mInstitute of Microbiology of the Czech Academy of Sciences, Prague 142 20, Czech Republic
nInstitució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona 08010, Spain
oVall d Hebron Institut de Recerca (VHIR), Barcelona 08035, Spain
pUK Centre for Ecology and Hydrology, Wallingford OX10 8BB, UK
First published on 14th October 2020
Understanding how engineered nanomaterials affect immune responses of living organisms requires a strong collaborative effort between immunologists, toxicologists, ecologists, physiologists, inorganic chemists, nanomaterial scientists and experts in law and risk management. This perspective aims to provide a new viewpoint on the interaction between engineered nanomaterials and the immune defensive systems across living species, gained within the EU Horizon 2020 project PANDORA. We consider the effects of nanoparticle exposure on immune functions in plants, marine and terrestrial invertebrates and their relation to the current state of knowledge for vertebrates (in particular humans). These studies can shed light on the broader perspective of defensive and homeostatic mechanisms (immunity, inflammation, stress responses, microbiota, stem cell differentiation) suggesting ways to: i) perform a comparative analysis of the nanoparticle impact on immunity across model organisms; ii) inspire best practices in experimental methodologies for nanosafety/nanotoxicity studies; iii) regroup and harmonise fragmented research activities; iv) improve knowledge transfer strategies and nano-security; v) propose innovative tools and realistic solutions, thereby helping in identifying future research needs and tackling their challenges.
Environmental significanceThis perspective is based on the experience gained within a collaborative work that compared the immune reaction to NPs in plants, invertebrates and vertebrates. Each species uses a shared number of core defensive mechanisms upon exposure to NPs (recognition, uptake, gene regulation, production of defensive molecules), and then adapts them and adds others to its own specific requirements. In general, an immune reaction to NPs is not detrimental for the organism and resolves rapidly with NP elimination, while in few cases it can produce damage or have beneficial effects. Each species has its own peculiarities, but the commonality of their basic defensive mechanisms becomes a valuable tool for NP-related environmental health risk assessment and management. |
One of the major shortcomings in our understanding of the effects of NPs relates to the impact of NP exposure on the immune system. The conserved nature of many immune mechanisms suggests that comparative studies on the immune responses of organisms covering different phyla and hierarchical levels of organization can together provide a knowledge base for understanding the mechanisms and consequences of NP exposure on human and environmental health. Key topics for comparative nano-immunosafety studies include: i) the identification of species differences and/or species similarities in the immunological mechanisms triggered by NPs; ii) the extrapolation of predictive markers of risk vs. safety by a cross-species comparison of innate immune defence capacity; iii) the design of predictive in vitro assays, in the practical and vigilant application of the 3R principle, to measure the nano-immunological risk to the environment and human health, for industrial and environmental nanosafety testing application.
The full functioning of innate immunity is of central importance for the survival and fitness of organisms, because of the critical role of immunity in preserving tissue and cellular homeostasis. The particulate nature of NPs dictates a preferential interaction with those cells in the immune system that are deputed to recognition and elimination of foreign particulate matters.1 NPs can interact with the immune system in a number of ways, which in the majority of cases end in a defensive reaction that resolves with the elimination of the particles and the re-establishment of homeostasis.2,3 In some instances, however, reaction to NPs may trigger an anomalous immune response. This can be evident as a quantitatively exaggerated or insufficient response (immune hyperreactivity or immunodeficient response) and also as a response sustained over extended timescales, beyond those taking place under normal physiological conditions, again implying hyperreactivity.4 While such reactions may be at the basis of inflammatory and other pathologies, on the other hand the NP capability to activate, regulate or modulate immune responses is of great interest, because of potential developments into treatment strategies for numerous disorders, such as autoimmune and inflammatory diseases, and cancer.5 Thus, assessing the impact of NPs on the immune system not only is an issue of common immuno-nanosafety (reactive mechanisms shared among all organisms), but it can also provide an improved understanding of the impact of NP exposure on the nano-immune interaction that can be exploited for targeted immunomodulation, disease management and crop protection.
By studying and comparing humans, plants and invertebrates, we can learn much about the general and specific mechanisms of the NP effects on immunity. The interactions of NPs with cells and molecules of the innate immune system represents the best ground for understanding the common mechanisms of immune defence. Innate immunity includes natural barriers at the organism–environment interface that limit internal exposure to pathogens, and an array of different cellular, molecular and chemical effector and regulatory players participating to the defensive responses. The full operational capacity of innate immunity is a major determinant in the survival and fitness of all organisms. Therefore, the immunosafety of engineered NPs is a key element of environmental nanosafety. The fact that living species have been consistently exposed to potential pathogens and other threats through their extended evolutionary history has directed the evolution of innate immune protective signalling pathways and mechanisms towards diversification (mainly dependent on species and environmental characteristics) of specialized immune-related molecules, which mediate cellular responses.6 Ecological immunology attempts to explain variability in immune defence, and, in turn, shows that the optimal immune response changes with environmental changes, establishing an evolutionary ecology framework around immunity.7 Since the appropriate functioning of the immune system is important for maintaining individual health, an agent that compromises its function can have implications for survival, reproduction and, hence, population fitness. Here we discuss the potential of NPs to act as agents affecting the immune status in different environmentally relevant species, with a focus on the emerging experimental models launched by the EU Commission H2020 project PANDORA (probing safety of nano-objects by defining immune responses of environmental organisms).8
Fig. 1 Anatomical maps of some invertebrates, showing the location of immune cells relative to organs and barriers. A) Mussel; B) echinoderm; C) arthropod. |
Changes in cell/tissue/organ functions following NP exposure may indirectly affect immune functions. A relevant example is the effect of biocidal NPs on the structure and/or function of the gut microbiota.15 The resident microbiota plays a central role in the stimulation, training, and function of the host immune system. In healthy physiological conditions, the immune system and microbiota cooperate in inducing protective responses to foreign substances and in maintaining regulatory pathways.16 All organisms initiate immune reactions to maintain metabolic homeostasis and self-integrity after suffering from stress, injury or invaders. They require mechanisms to recognize damage, to discriminate between the molecular signatures of invading pathogens (“non-self” molecules) and cellular constituents that generally pose no health risk (“self” molecules), and to discriminate “self” from “damaged self”.17 An interesting question is whether it could be possible to predict innate defence responses against NPs based on how the immune system manages pathogens. NPs may (or may not) set the immune system in motion, communicate with cells, receptors and proteins, trigger signalling cascades, and generate unpredictable immune responses (activation or suppression) as well as other harmful outcomes,18 depending on their physico-chemical characteristics, especially their surface properties.19 In the case of nanomedical applications, it may be attractive to design NPs able to engage with the immune system in a specific manner, e.g., immunostimulation or immunosuppression, depending on the intended use. This has encouraged the development of NP and cell-based hybrid systems that cannot be detected/eliminated by the immune system, making them highly biocompatible and physiologically integrable within tissues in humans.11 For example, nanoporous silicon NPs coated with cellular membranes purified from leukocytes are able to elude the immune system, avoiding opsonization and phagocytosis, and to transmigrate across the endothelial barrier to enter a tissue.20
Notably, some NPs may selectively accumulate in organs with high adsorptive, filtering and detoxification capacity (e.g., spleen, liver, kidney, intestine and lung), engulfed by the mononuclear phagocytic system (MPS) that includes both resident macrophages and circulating blood monocytes.21 Phagocytic cells are responsible of several important processes, including clearance (engulfment/elimination of putative threats), biodistribution (selective localisation in organs and tissues) and balance between tolerance and toxicity (e.g., between production of reactive oxygen species –ROS– and antioxidant response). Each organ can count on the availability of an exclusive equipment of cells that coordinate and perform local innate immune reactions. The cells responsible of innate immune activities include both “professional” immune cells (such as mononuclear and polymorphonuclear phagocytes, mast cells and innate lymphoid cells) and non-professional defence cells, such as endothelial cells, epithelial cells, and fibroblasts.22
Experimental methodologies for nanosafety/nanotoxicity studies must allow us to capture the relevant details of a complex nano-immune interaction. While in vitro models facilitate the analysis of particle effects on isolated single cells and tissues, in vivo models allow for a clearer evaluation of the NP effects in whole organs or in the entire organism. While exposure time, route, and NP concentration help in determining the modes of interaction between NPs and the immune system (or any other system they come in contact with), there is a parameter that is of major importance in any kind of nano-bio interaction, i.e., the changes particles experience when coming in contact with biological fluids. Mimicking such interactions in the laboratory is always challenging because of the complexity and multiplicity of interactions taking place in real life environments. For instance, the presence of natural organic matter (including toxins) may play a role in the NP partition, which is difficult to control but that should be considered, as they are part of a natural environment. This is a critical point in the context of ecotoxicological assays and how and to which purpose we want to use them. Thus, given the advantages of the in vivo and vitro/ex vivo approaches in providing nanosafety information, future studies should aim to improving the exposure conditions of the ecotoxicological assays in order to better mimicking complex environments and provide realistic and valid nanosafety data.
Aggregation/agglomeration influences the fate of the NPs in the environment, particularly in aqueous environments. NP colloidal stability is influenced by the solution ionic strength, organic matter composition, pH and solvent composition. In some conditions NPs can agglomerate, and return in colloidal solution when the conditions change, while in some cases NPs can aggregate, forming irreversible larger clusters. Heteroaggregation/agglomeration, in which NPs are admixed with other environmental molecules/particles, is likely the most common form in which NPs are present in the environment and come in contact with living organisms. Such complexes can also associate with other environmental biological and non-biological contaminants, such as bacterial toxins (e.g. Gram-negative endotoxin) and chemicals.23 Since these complexes are the real-life forms in which NPs come in contact with living organisms, the future ecotoxicological assays should consider using complex environmental matrices to adequately reflect the exposure conditions in real life. Although nude particles can rapidly aggregate/agglomerate in a fluid, coated particles acquire chemical functions of the coat that modify the particle behaviour, for example by reducing the formation of aggregates/agglomerates. Within biological fluids (blood, haemolymph, coelomic fluid, mucus, etc.), which are rich in biomacromolecules (e.g., lipids, sugars, nucleic acids, and particularly proteins), NPs readily adsorb on their surface various organic molecules that form a complex biomolecular corona and lead to an increase in the NP hydrodynamic range.24 Large aggregates/agglomerates are transported in vesicles via different mechanisms of endocytosis/exocytosis (based on the size, shape and surface characteristics, and the cell internalization machinery).25 For example, NPs of <200 nm can be internalized via clathrin-coated pits by non-phagocytic cells, whereas at increasing size a shift towards caveolae-mediated internalization become predominant.26 NPs can also enter cells by a passive uptake, which can or not disturb the membrane depending the size of the NPs. Small NPs have a higher probability to be internalized by passive uptake than large ones.25
The bio-corona composition may vary among different NPs and upon contact with different biological fluids that contain different mixtures of biomolecules. The bio-corona evolves with time, with a progressive loss of molecules of lower avidity (soft corona) and the establishment of a hard corona of strongly interacting biomolecules. The structure of this macromolecular coating is most likely a key factor in making the NPs “visible” to cells for uptake, contributing to clearance from circulation and possibly reducing their interactions with other cells and tissues.27 On the other hand, functionalizing particles with some capping agents (e.g., polyethylene glycol) reduces accumulation of proteins on the NP surface, making them functionally “invisible” to the immune system in humans.28,29 The majority of the bio-corona studies have been performed in vitro, and have contributed to unravel the role of the bio-corona in the immune recognition of NPs. Conversely, the in vivo approach is important for a better understanding of the mechanisms of particle clearance vs. toxicity, which would be greatly improved by examining the differences in clearance kinetics and type of toxic effect across living species.
Vertebrates and invertebrates display common immune mechanisms but they have also developed divergent immune tools for attaining challenge-specific protection. As an example, whereas vertebrates use somatic rearrangement of immune receptors and immunoglobulins, invertebrates privilege alternative splicing of pattern-recognition genes.30 Whereas vertebrates have developed an array of different cell types as effectors of innate immune responses (e.g., monocytes, macrophages, mast cells, neutrophils, basophils, eosinophils, NK cells, ILC, epithelial cells, fibroblasts), invertebrates use a limited range of multifunctional cells. Plants do not have specialised immune cells, with all cells being able of innate immune reactivity and with systemically spreading signals emanating from injured sites.31
The innate immune system also exhibits adaptive traits, a feature that has been termed “innate immune memory” (or “trained immunity” in vertebrates).32 The innate immune memory is the ability of the immune system to record and recall information of previously met pathogens to activate a faster and more efficient protective response upon re-exposure to the same or different pathogens/stressful agents.33 The phenomenon is well known in plants and invertebrates, which also show some degree of specificity,34,35 and has been also observed in vertebrates (in particular mouse and man), in which however the memory is as non-specific as innate immunity itself.32 How NPs may induce/alter innate memory is an open issue that requires special attention, as alteration of immune memory can modify the capability of an organism to adapt to its environment. NPs can modulate stimulus-induced memory in human monocytes, mostly by shifting the memory response towards tolerance.36 Innate memory in plants is known as systemic acquired resistance (SAR), which leads to the activation of a better protective response (e.g., increase in the levels of pattern-recognition receptors, accumulation of dormant signalling enzymes, alterations in chromatin state) not only in the tissue re-exposed to the same or similar insult but also in an unexposed tissue.37 If found able to induce an epigenetic and metabolic reprogramming of immune responses, as they are able to promote root growth (B. Kemmerling, unpublished results), NPs would support the design of new agriculture strategies able to reduce yield losses, thereby allowing for reduction of conventional pesticides. Invertebrates show a high degree of memory in their immune responses, including recall immunity to infections, natural transplantation immunity, and individual/trans-generational immunological priming.34 How NPs can affect the establishment of innate memory in invertebrates is however still largely unknown. The only available results describing nanoplastics-induced innate memory and some underlying molecular and cellular mechanisms come from mussels.38 Upon a primary exposure to amino-modified nanopolystyrene, Mytilus haemocytes respond with changes in some immunological parameters (e.g., lysozyme release in the haemolymph, lysosomal acidification, membrane destabilization), whereas a second exposure induces an immunological memory by shifting the immune parameters towards a tolerant condition (recovery).
An important issue that should be considered when examining the interaction between NPs and immunity is the role of microbiota. This is particularly important for NPs that are ingested (as in the case of most aquatic and terrestrial/earth-dwelling invertebrates and vertebrates) but also for NPs to which organisms are exposed through the skin or inhalation, because symbiotic and commensal microbiota populate the gut and all the barrier surfaces or environmental organisms and it is well known that microbiota profoundly influence and shape immunity at the barrier level.39 However, little is currently known on how the interaction between NPs and commensal microbes can impact on immune responses, in particular in the long term (Fig. 2).
Fig. 2 Impact of NPs on the microbiota–immune axis in vivo: differences across environmental organisms. |
Recent data show that TiO2NPs impact the immune system of mussels and induce a shift in the microbiota composition of the haemolymph40 upon exposure in vivo, while CuONPs cause a shift in the earthworm gut microbiota despite the absence of an evident immune reaction.41 The only available data on impact of NPs on the human microbiota–immune axis come from in vitro studies, which underlines an essential a lack of information on the realistic impact of the NPs ingested via food in humans.39 Whether chronic dietary exposure to NPs may be viewed as a risk factor that facilitates disease development remains an open issue.
In the next section, we will provide an overview of the environmentally relevant organisms and approaches used, within the framework of the EU PANDORA project, to study nano-immune interactions and identify their common vs. divergent mechanisms.
In this section, we report and discuss recent studies aiming at integrating nanotoxicological evaluations with assessment of immune-related effects in plant, human and invertebrate animal models, in vivo and in vitro (Fig. 3). We will examine the similarities and differences in the defensive responses of each organism upon exposure to NPs. Comparison will contribute to increasing our knowledge of the commonalities in the immune reaction to NPs, and allow us to identify pathways and biomarkers of nano-risk that are shared across species. Notably, several features must be considered in order to correctly evaluate the NP interaction with the immune defences in the selected experimental models: experimental conditions (in vivo, in vitro/ex vivo); environmental factors; basal immunological state; immunological memory.
Fig. 3 Overview of the experimental models for investigating the interactions of nanomaterials with the immune system used within the EU Commission H2020 project PANDORA: from the plants to humans. |
Plants have a well understood immune system able to detect and react to danger, and are easily accessible to genetic and biochemical analyses and therefore provide a valuable tool for NP-related risk assessment.45 Plant immunity is an exceptional example of sensing capability based on networks of immune receptors that act as sensors of invading pathogens.46 Receptors evolve rapidly to keep up with the pathogens' variability, and interact with each other creating systemic signals emanating from infection sites that induce an effective immune response. The task of mounting an immune reaction is not dependent on specialized immune cells, but each plant cell is able to react against invaders. The plant immune system is organised in three layers, the pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI) (preliminary defence), the effector triggered immunity (ETI) (secondary defence), and the exosome-mediated cross-kingdom RNA interference (CKRI) system (additional defence).47 Resistance against a non-adapted pathogen involves the rapid production of cell wall appositions (physical barriers), and inducible-specialized metabolites at the infection sites via PTI and ETI signalling.48,49
The growth and metabolic functions in response to NPs vary among plants.50 Reports reveal contradictory effects of NPs depending on the experimental conditions (field or lab studies), medium of plant growth, duration of exposure and species.51 For example, TiO2NPs improve light absorption efficiency and promote the activity of Rubisco activase, accelerating growth in spinach;52 whereas they reduce the photosynthetic rate thereby decelerating growth in Ulmus elongata.53Arabidopsis thaliana leaf cells exposed to large particles of single-walled carbon nanotubes form endocytosis-like structures in the plasma membrane, whereas the same particles fail to penetrate the cell walls in other plants.54 Notably, the interaction of plant cells with NPs leads to the modulation of plant gene expression and related biological pathways, which in turn influence plant growth and development.55 For example, exposure of A. thaliana to gold NPs induces the expression of a few key regulatory molecules and microRNAs (miRs) thereby stimulating seed germination, growth, and antioxidant metabolic activity.56A. thaliana is a small model plant with a short life cycle, which allows for easy manipulation and study. The observations made in this species for studying NP uptake and metabolism encourage more relevant and significant research in this field, due to commonalities between Arabidopsis and edible plants. Well controlled and field studies will allow us to get an overview about the potential of plants to recognize and react to NPs, keeping in mind that NPs bear also the potential to improve plant yield and health and reduce crop loss by specific, localized and efficient application of nano-fertilizers, pesticides or immunostimulating agents.51
The mechanisms of interaction of NPs interact with different components of the earthworm immune system are still not well defined. There are data showing that AgNPs, both in vivo and in vitro on Eisenia fetida coelomocytes, can transiently affect the expression of a number of immune-related genes, such as those involved in oxidative stress (CAT, SOD), energy metabolism (aspartate aminotransferase, glucose-6-phosphate isomerise, phosphoglucomutase) and immune response (lysozyme, MyD88, PKC, MEKK1).64,65 The intracellular signalling pathway involved in the AgNP-induced modulation of earthworm immunity apparently involve the myeloid differentiation factor 88 (MyD88) (encoding a central adaptor protein of toll-like receptors), and the mitogen activated protein kinase (MAPK) cascades.65 This upstream signalling cascade induces activation of nuclear factor (NF)-κB, resulting in transcription of genes encoding inflammatory cytokines in humans.66 Cytokine production is necessary for defence, but it can also induce to tissue damage by inducing the generation of ROS, enzymes and other toxic defensive molecules.67 These findings underline the presence of conserved innate immune activation mechanisms and pathways between earthworm and human immunity. In agreement, the 1H NMR-based metabolomic analysis of Eisenia fetida earthworms exposed to TiO2NPs in soil showed significant changes in the metabolic profile consistent with oxidative stress as the main mechanism of immune-related inflammation/toxicity.68 Upon exposure to another type of particle, CuO2NPs, in soil, the earthworm Metaphire posthuma showed an immune metabolic impairment in term of nitric oxide generation, superoxide dismutase, phenoloxidase, catalase and alkaline phosphatase activity.69
Since earthworms ingest soil and their associate microbiota, the NP effects on the soil microbial communities70–72 will affect the composition of the microbiota in the earthworm gut. This is particularly important, in terms of effects on immune functions, because the interaction between immunity and microbiota at the gut level defines the defensive capacity of an organism (from earthworms to human beings), and any variation of microbiota can affect immune competence. Unfortunately, little is known on how NPs might interact with earthworms' bacterial communities and even less on how this might affect their immunity. Earthworms capture NPs from contaminated soil using mucus glycoproteins/glycans of gut/microbial origin.73 Exposing the oligochaete Enchytraeus crypticus to 50–100 nm plastic particles caused a significant shift in the gut microbiota, including a significant decrease in the relative abundance of the families Rhizobiaceae, Xanthobacteraceae and Isosphaeraceae,74 but how these changes may affect immunity is unknown. The earthworm Eisenia fetida can survive in soil containing high ZnONP concentrations, and accumulate zinc ions in the body. ZnONPs enhance the activity of cellulolytic bacteria (Bacillus and Pseudomonas sp.) in the gut, contributing to bioconversion (digestion) of lignocellulosic waste.75 The metagenomic analysis of Eisenia fetida earthworms exposed to ZnNPs in soil shows a decrease in biodiversity of the gut microbiota, mainly due to a significant decrease in the relative abundance of the phylum Firmicutes.76 Reduction in the abundance of these bacteria correlates with inflammatory/autoimmune diseases in mammals.77 Although there is no strong evidence that this type of correlation occurs in earthworms, we can speculate that NPs impacting the gut microbiota can consequently affect the earthworm immunity upon in vivo exposure. This is based on the consideration that amoebocytes, the most active immune cells in the earthworm, are involved in a broad range of defensive functions in the gut78 reflecting the abundance of immune cells in the gut of vertebrates. However, recent data show that in vivo exposure to CuONPs can cause significant changes in the gut microbiota and microbiome of earthworms, but it does not affect the expression of immune-related genes (e.g., coelomic cytolytic factor, lysenin/fetidin, lysozyme) and resistance/susceptibility to a bacterial infection.41 Future studies of gene expression based on a greater time resolution of immune responses may aid the understanding of whether and how NPs could impact on the dialogue between gut commensals and immune cells in the earthworms.
The interaction of NPs with the mussel biological fluids, in particular the haemolymph, allowed for the identification of specific proteins forming a stable biocorona on the NP surface, and to assess the features of the haemocyte interaction with the biocorona-coated NPs. Depending on the NPs and its bio-corona, this interaction can lead to distinct responses (recognition, internalization, activation/inhibition of immune responses), and therefore results in distinct immunomodulatory pathways.83 These findings underlie the strong similarities with the features of the nano-immune interactions in mammalian cells, suggesting that this model could be suitable for investigating the mechanistic basis and the biological outcomes of such interactions in conditions that reflect those in both mammals and environmental organisms. Notably, recent data showed that the immunomodulatory effects of different types of NPs are linked with shifts in the microbiota composition of the haemolymph, underlying a relationship between innate immunity and host microbiota in mussels, as it occurs in mammals.40
Current studies of the interaction of NPs with the immune system of the sea urchin Paracentrotus lividus show that, as in humans, a number of sea urchin extracellular proteins (glycoproteins, cytoskeletal proteins) form a biocorona on the particle surface in vitro.93 The protein-coated TiO2NPs are internalised more efficiently, transiently down-regulating the expression of genes encoding for proteins involved in immune response (NF-κB, FGFR2, JUN, p38MAPK), and increasing a few antioxidant metabolic pathways (pentose phosphate, cysteine–methionine, glycine–serine).2 Conversely, studies on sea urchin phagocytes exposed to PVP-AuNPs in vitro underline the transient involvement of the TLR4/ERK signalling pathway based on increased protein levels.94 The notion that these NPs do not cause a pathological immune response and associated irreversible damage in sea urchins may prompt us to consider them immunologically safe also for other environmental species and human beings, given the commonality of involved defensive mechanisms.
Thus, the combined use of in vivo and in vitro investigations, its proximity to mammalian reactivity as well as the similar features with invertebrate immune responses, make the sea urchin a valuable model for the study of the nano-immune interactions across evolution.
Representative images of the invertebrate models described in this section and their immune cells are shown in the Fig. 4.
An efficient and predictive method that can be applied for assessing the general impact of NPs on human blood is the whole blood assay,97,98 in which an aliquot of anti-coagulated blood is challenged with NPs. This assay encompasses NP coating with serum proteins and interaction with all types of circulating cells, without however allowing for the identification of specific cell types affected by NPs. Since phagocytes are the first defence cells that confront particulate matter, we can consider as the best models of innate immunity those based on monocytes isolated from blood of healthy donors (MAT, monocytes activation test), and on macrophages and dendritic cells differentiated in vitro from fresh monocytes.99 These models allow us to observe not only the direct activation of immune cells by NPs but also the capacity of NPs to interfere with the physiological course of an inflammatory response4 or with the induction of innate memory.36 The in vitro systems based on human primary cells present the main disadvantage of all in vitro systems, i.e., they do not reflect the complexity of an in vivo system, and, in addition, they suffer of donor-to-donor variations. Such variations are however sufficiently limited in the case of innate immune responses, with a general homogeneity of qualitative reaction and variability evident only in quantitative terms.100 Conversely, in the case of innate memory responses the variability is much higher, as also observed in adaptive immune responses, as memory depends on the individual history of exposure. This notion underlines the need of using human primary cells in immunoassays for assessing nano-risk, since they are more predictive evaluation of the NPs effect in vivo. Also, it warrants a personalised evaluation of the NP effects on immunity, as each individual subject may react differently to an NP challenge.
In light of the notion that an unbalanced immune response underlies many diseases including autoimmunity, inflammatory diseases and inflammation-related cancers, the potential effects of NPs on the human immune responses requires extensive attention. To obtain a realistic overview on the effects of NPs on human immune responses, it is necessary to examine both the direct effects of NPs on innate immune effector cells (capacity to induce an innate/inflammatory reaction that does not resolve and become chronic) and their interference with an ongoing defensive innate/inflammatory response (capacity to exacerbate or inhibit a defensive response). In the latter context, the effects of NPs on the development of innate memory could be of great interest. NPs could be appropriately designed for modulating innate memory to treat two opposing conditions, i.e., diseases where an innate/trained memory should be inhibited (autoimmune, inflammatory and cardiovascular diseases) and those in which memory responses should be potentiated (cancer, sepsis, immunosuppression). Recent work describes that AuNPs could interfere with the induction of BCG-induced memory by shifting the human memory monocytes towards a tolerant phenotype.36 Another recent study shows that pristine graphene can directly prime mouse macrophages towards a potentiated secondary response to TLR-activating stimuli, by increasing the production of inflammatory cytokines and decreasing the production of regulatory factors.101 The effect of NPs on innate memory is a new field with great therapeutic potential. A representative in vitro model for assessing the capacity of NPs to induce innate immune memory is shown in the Fig. 5.
Which are the models and tools that we can use to assess nano-immune interactions, which information will they provide, and how can we use them optimally to answer to different questions?
We have focused on protostomes, including the earthworm Eisenia, the isopod P. scaber, the blue mussel Mytilus, and the plant A. thaliana for in vivo studies, while deuterostomes (including human models and the sea urchin P. lividus) have been used directly in vitro/ex vivo, as discussed previously.
In plants, uptake and response to NPs is dependent on the plant species and the capacity of NPs to cross the mechanical barriers. While in some cases NPs can induce stress responses, it is notable that in several instances the presence of NPs showed positive effects on growth and yield. This opens novel opportunities in the use of NPs in increasing the fitness of plants of agronomic interest. Whether such effect includes an increased resistance to pests, in terms of induction of immune memory, is currently under investigation.
From earthworms and isopods, we learned the major importance of gut and resident microbiota in determining the immune response to foreign particles (including NPs). A disturbed host-microbiota interaction, in particular in the gut, can impact on the immune system, increasing susceptibility to infections. NPs may have significant effects in altering the microbiota balance, indirectly affecting the host immune defensive competence. Thus, some protostomes become a practical choice to study the effect of ingested NPs on the gut microbiota interaction with the host immunity and its ability to overcome infections, a study that cannot be easily performed in human beings.
Mytilus shows, in response to exposure to high NP concentrations, a stress response that is very similar to that observed in humans, with activation of phagocytes and release of ROS. This warrants the use of Mytilus as easy in vivo model for evaluating the phagocyte response to NPs.
The comparison of the sea urchin and human genomes allows for discriminating between conserved immune genes and new human-specific genes. The possibility of exploiting long-term in vitro cultures of sea urchin immune cells makes it possible to compare in vivo effects of NPs with the evaluation of cell-specific molecular mechanisms, and to focus on conserved molecules across evolution. Table 1 reports the main features and mechanisms involved in the nano-immune interaction across the selected species.
Parameter | Organism | |||||
---|---|---|---|---|---|---|
Plant | Earthworm | Mussel | Terrestrial isopod | Sea urchin | Human | |
Exposure route | Soil | Soil | Seawater | Soil | Seawater | Skin, mucosal cells in gastrointestinal and respiratory tract |
Contact level | Root cells | Gut and skin epithelial cells, coelomocytes, mucus, microbiota | Gills, gut epithelial cells, haemolymph, microbiota, haemocytes | Microbiota. No direct contact with gut epithelial cells and haemocytes | Coelomic fluid, free immune cells, gut epithelial cells | Gut and skin and mucosal epithelial and immune cells |
Biocorona | ND | Lysenins | Complement component C1q, haemolymph components | ND | Depends on NP surface characteristics (several proteins) | Depends on NP surface characteristics and route of exposure (several proteins) |
Uptake by immune cells | Endocytosis-like | Endocytosis | Endocytosis | No uptake | Endocytosis | Endocytosis |
Immune-related effects/activation | Regulatory molecules, miRs, PRRs, MAPKs, energy metabolism, upregulation Rubisco activase | PRRs, MyD88, MAPKs, NF-κB, coelomic cytolytic factors, lysozyme, energy metabolism, antimicrobial peptides | PRRs, MAPKs, NF-κB, lysozyme, energy metabolism, antimicrobial peptides | Changes in protein, lipid, nucleic acid and carbohydrate levels. No immune response | PRRs, MAPKs, NFκB, cytokines, growth factors, energy metabolism | PRRs, MAPKs, NF-κB, energy metabolism, cytokines, chemokines, complement system, growth factors, etc. |
Oxidative stress and apoptosis | ROS production, antioxidant metabolism, Rubisco activase, apoptotic enzymes | ROS production, antioxidant metabolism, apoptotic enzymes | ROS production, antioxidant metabolism, apoptotic enzymes | ND | Antioxidant metabolism | ROS production, antioxidant metabolism, apoptotic enzymes |
Immune memory | ND | ND | Compensatory recall responses to maintain immune homeostasis | ND | ND | Innate immune memory |
Based on the above consideration, we can state that the realistic evaluation of nanosafety risk demands a ‘toolbox’ of cell-based models able to reproduce with fidelity the functions of barrier tissues and of the immune system. This is true for both environmental and human health risk. An important issue in this context is the accessibility of tests and assays, which would allow their use across several institutions, companies, agencies and private labs, without the need of specialised instrumentation and personnel. Within the PANDORA consortium, we have contributed to this ‘toolbox’ through adaptation of human immune cell models to be readily-available and user-friendly, whilst retaining the analytical reproducibility and predictive value obtainable only with primary cells (that retain the functions of their tissue of origin under test conditions in culture). To this end, human blood monocytes were extracted from blood transfusion by-products, distributed in multiwell plates, either used immediately after isolation or differentiated into macrophages or dendritic cells, frozen with a novel protocol in analysis-ready format and, in the case of poorly-adherent cells such as dendritic cells, anchoring them on bio printable scaffolds in NP-receptive 3D culture arrangement. Upon thawing the different types of cells were able to react to NPs in a fashion that is qualitatively and quantitatively superimposable to that of cells that did not experience freezing–thawing. The reproducibility of immune cell response, or more often the absence of response to NP challenge, translated to user-friendly cryopreserved cell models offers a robust platform against which effects of materials of uncharacterized or non-specific nano-composition can be tested with confidence.
The next-generation in vitro human models, including co-culture with multiple cell types, 3D spheroids, 3D tissue sections, microfluidic organ-on-a-chip, air–liquid interface systems, are showing great capacity in helping to bridge the gap between in vitro and in vivo exposure/effect. Notably, 3D organoids are self-organizing, multi-cellular aggregates that closely resemble function and architecture of human tissues.102 Furthermore, microfluidic technologies can model fluidic properties in organ-like tissues, making it possible to study small fluidic volumes in motion.103,104 Despite the availability of outstanding organoid models representing the lungs, gastrointestinal tract and many other organs, one of the greatest challenges for the coming years will be to further improve organoid-like tissues to include the interaction of tissues/organoids with the immune cells that in real life reside in the organ and with those that enter it during an inflammatory reaction.
The RRI strategy (“Science with and for Society”) is the approach that the PANDORA partners have tried to use to increase the researchers' capacity to think and act towards a more responsible, acceptable, and ethical science. The EU-funded project PANDORA encompassed five Academic Institutions, four Research Centres, and two small and medium-sized enterprises (SMEs) distributed within seven European countries (Italy, Austria, Czech Republic, Germany, Slovenia, United Kingdom, Spain), all with proven experience in higher education and training. Eleven PhD students have been involved in an overarching training programme including training-by-research, joint courses of technical, scientific and transferrable skills, completed with an intense inter-sectorial networking exchange plan. PANDORA merged scientists, NP producers and experts in law and risk management into a broader approach to environmental nanosafety. Within this complex framework, the younger scientists have been encouraged to critically reflect106 on the research process within, around and outside the laboratory, and to aim at collaboration and quality by privileging care and sustainability in their research planning and conduction.107 Such attitude/behaviour was particularly suited to the goals of their studies, dealing with environmental nanosafety, and helped them in progressing towards the fundamental dimensions of RRI: courage, rigor, safety, sustainability, inter- and trans-disciplinarity, critical creativity and elegance.
Based on these findings, what should we do in the future? Abandoning nanosafety research, only because in general NPs do not seem to be detrimental, is not a good idea because there are too many special situations that could increase risk. Just to make an example, the immune system of elderly or chronically ill individuals is weakened, and therefore we should expect that agents that are innocuous in healthy people may cause significant damage. Another case: even when exposure to NPs or other individual agents does not cause a damaging effect, co-exposure to several agents at the same time may synergize and provoke unexpected detrimental reactions.
Thus, we may foresee that future research of nano-immune interactions may explore these directions:
• Development and refinement of in vivo/in vitro models that can realistically predict the nanorisk associated to acute and chronic exposure to NPs. Goals will be having models that discriminate between a normal immune response and a pre-pathological reaction, models that predict risk for many different environmental species and human beings, models that predict realistic risk upon exposure to combined challenges, models that predict risk in frail organisms.
• Exploiting the potential of NPs in modulating some immune functions (such as innate memory) for designing new immunotherapeutic or immunopreventive approaches for human beings and crop production in agriculture.
Building on the environmental impact of nanosafety research and its impact on society for reshaping all these studies along the principles of RRI, contributing at the same time in building a new generation of researchers that privilege rigor, cooperation and sustainability.
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