International Journal of Molecular Sciences Article Induction of Innate Memory in Human Monocytes Exposed to Mixtures of Bacterial Agents and Nanoparticles Giacomo Della Camera 1,2 , Tinghao Liu 3,4 , Wenjie Yang 3,4 , Yang Li 3,4 , Victor F. Puntes 5,6,7 , Sabrina Gioria 2 , Paola Italiani 1,8,9 and Diana Boraschi 1,3,4,8,9, * 1 Institute of Biochemistry and Cell Biology (IBBC), National Research Council (CNR), 80131 Napoli, Italy 2 European Commission, Joint Research Centre (JRC), 21027 Ispra, Italy 3 Shenzhen Institute of Advanced Technology (SIAT), Chinese Academy of Sciences (CAS), Shenzhen 518055, China 4 China-Italy Joint Laboratory of Pharmacobiotechnology for Medical Immunomodulation (CNR, SIAT, SZN), SIAT, CAS, Shenzhen 518055, China 5 Institut Català de Nanociència i Nanotecnologia (ICN2), Consejo Superior de Investigaciones Científicas (CSIC) and The Barcelona Institute of Science and Technology (BIST), 08036 Barcelona, Spain 6 Vall d’Hebron Research Institute (VHIR), 08035 Barcelona, Spain 7 Institució Catalana de Recerca I Estudis Avançats (ICREA), 08193 Barcelona, Spain 8 Stazione Zoologica Anton Dohrn (SZN), 80121 Napoli, Italy 9 China-Italy Joint Laboratory of Pharmacobiotechnology for Medical Immunomodulation (CNR, SIAT, SZN), IBBC, CNR, 80131 Napoli, Italy * Correspondence: diana.boraschi@itb.cnr.it Abstract: We assessed whether concomitant exposure of human monocytes to bacterial agents and different engineered nanoparticles can affect the induction of protective innate memory, an immune mechanism that affords better resistance to diverse threatening challenges. Monocytes were exposed in vitro to nanoparticles of different chemical nature, shape and size either alone or admixed with Citation: Della Camera, G.; Liu, T.; Yang, W.; Li, Y.; Puntes, V.F.; Gioria, LPS, and cell activation was assessed in terms of production of inflammatory (TNFα, IL-6) and anti- S.; Italiani, P.; Boraschi, D. Induction inflammatory cytokines (IL-10, IL-1Ra). After return to baseline conditions, cells were re-challenged of Innate Memory in Human with LPS and their secondary “memory” response measured. Results show that nanoparticles alone Monocytes Exposed to Mixtures of are essentially unable to generate memory, while LPS induced a tolerance memory response (less Bacterial Agents and Nanoparticles. inflammatory cytokines, equal or increased anti-inflammatory cytokines). LPS-induced tolerance Int. J. Mol. Sci. 2022, 23, 14655. was not significantly affected by the presence of nanoparticles during the memory generation phase, https://doi.org/10.3390/ although with substantial donor-to-donor variability. This suggests that, despite the overall lack of ijms232314655 significant effects on LPS-induced innate memory, nanoparticles may have donor-specific effects. Academic Editor: Yoshiro Kobayashi Thus, future nanosafety assessment and nanotherapeutic strategies will need a personalized approach in order to ensure both the safety and efficacy of nano medical compounds for individual patients. Received: 30 September 2022 Accepted: 17 November 2022 Keywords: innate immunity; innate memory; nanoparticles; bacteria; LPS; monocytes; macrophages Published: 24 November 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Innate immune cells develop an innate memory that allows them to respond better to subsequent challenges [1–9]. The best-known agents that induce innate memory are infec- tious agents and microbial compounds. In particular, bacterial lipopolysaccharide (LPS) Copyright: © 2022 by the authors. induces a type of memory that leads to a less potent inflammatory response to a second Licensee MDPI, Basel, Switzerland. challenge, a mechanism aiming to protect the tissues/organs from inflammation-induced This article is an open access article damage while affording a sufficient response level [10–16]. The main cells that can develop distributed under the terms and innate memory are mononuclear phagocytes (monocytes and macrophages) [2,10,11,17,18], conditions of the Creative Commons as well as Natural Killer and other Innate Lymphoid Cells [5,9,19–22], although innate mem- Attribution (CC BY) license (https:// ory can also be observed with non-professional immune cells such as epithelial cells [23–28]. creativecommons.org/licenses/by/ Among mononuclear phagocytes, there is evidence that monocytes are more reactive 4.0/). than macrophages in memory generation [29], suggesting that the main memory cells Int. J. Mol. Sci. 2022, 23, 14655. https://doi.org/10.3390/ijms232314655 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 14655 2 of 15 are the effector blood monocytes (which extravasate and enter a point of inflammation in a tissue) that survive the inflammatory reaction and develop into monocyte-derived memory macrophages. Conversely, although there is evidence that naïve tissue-resident macrophages can develop memory in some circumstances [30], they may mainly act as sentinels that send alarm signals (e.g., chemokines). The relevance of assessing the possible effects of engineered nanoparticles (NPs) on innate immunity stems from the fact that, with the explosion of nanotechnological applica- tions, NPs are extensively produced and used in many different applications and products, which has raised concerns about their safety for human and environmental health. TiO2 NPs are among the most produced types of NPs worldwide [31], with applications in manufacturing and construction (e.g., car tires, concrete, sports equipment), food addi- tives, paintings and sunscreens among others [32–34]. TiO2 NPs are generally considered safe [35,36], although their safety is currently being reconsidered [37]. CeO2 NPs are largely used in solar cells [38], chemical mechanical planarization [39], corrosion protection [40], fuel oxidation catalysis [41], car exhaust treatment [42] and biological sensors [43]; they are also being explored for future therapeutic applications in a range of inflammation-based diseases based on their potent anti-oxidant activity [44–49]. NPs can enter the human body by different routes, mainly inhalation and ingestion but also by skin contact (if the skin is damaged) [50,51] or, in the case of nanomedical products, by injection. Some inhaled or ingested NPs have been shown to cross biological barriers (alveolar or intestinal ep- ithelium) and reach the blood and inner tissues [52], where they can readily interact with innate immune cells, including monocytes and macrophages [31,53]. Whether NPs can be considered immunologically safe depends on many factors, including the NP’s size, shape and composition and the concomitant presence of other bioactive agents (e.g., LPS), which can induce and modulate innate immune activation [54]. The direct effects of different NPs on innate immunity have been extensively stud- ied [53,55,56], while less is known regarding their capacity to induce innate memory, although some studies have proposed that they can act as “priming” agents able to induce memory in innate immune cells [57–63]. In addition to a direct memory-inducing capacity, whether NPs may affect the generation of innate memory by microbial agents is an issue that still needs investigation. Even in the absence of a direct inflammatory effect, the capacity of NPs to induce or modulate innate memory is not only relevant in the general immunosafety evaluation of nanomaterials present in the human environment, but also in terms of the immunosafety of nanomedical products deliberately administered to patients. To examine the generation of human innate memory, we have used an in vitro model that mimics the encounter between blood monocytes (representing the effector cells re- cruited into an inflamed tissue) and bacteria (represented by the main inflammatory molecule of gram-negative bacteria, i.e., LPS). Monocyte activation by LPS was assessed in the absence or in the presence of different types of NPs in order to examine whether NPs may have a direct effect on monocyte activation or affect LPS-induced activation. The NPs examined here include differently shaped CeO2 (spherical, stamp-like) and seed-like TiO2 . The capacity of these NPs to directly induce innate memory or to affect the tolerance-type memory generated by exposure to LPS was assessed by examining cell activation in terms of alterations to the balance between inflammatory and anti-inflammatory cytokines. The results obtained with these three types of NPs were compared with previously published results obtained in the same experimental conditions (endotoxin-free NPs pre- coated with human serum, human monocytes, LPS as a challenge) and overall show that metal NPs alone, independent of chemical composition, shape or size, have little capacity to induce innate memory and little capacity to affect the tolerance memory induced by LPS, thereby proving to be generally safe. However, some effects are detectable, which seem to be donor-dependent and exist irrespective of NP characteristics. coated with human serum, human monocytes, LPS as a challenge) and overall show that metal NPs alone, independent of chemical composition, shape or size, have little capacity to induce innate memory and little capacity to affect the tolerance memory induced by Int. J. Mol. Sci. 2022, 23, 14655 LPS, thereby proving to be generally safe. However, some effects are detectable, which 3 of 15 seem to be donor‐dependent and exist irrespective of NP characteristics. 2. Results 2. Results 2.1. Nanoparticle Characterization 2.1. Nanoparticle Characterization Three types of NPs were used in this study: CeO2 spherical NPs of very small size Three types of NPs were used in this study: CeO2 spherical NPs of very small size (CeO2 SPH, 3.5 nm), stamp‐like CeO2 NPs that shared one dimension with the spherical (CeO 2 SPH, NP (CeO 3.5 nm), stamp-like CeO2 NPs 2 STA, 3 × 19 nm) and seed‐like TiO that shared one dimension with the spherical 2 NPs that shared the other dimension with NPstamps the (CeO2(TiO STA, 3 × 19.6 2 SEE, 19 nm) nm)and seed-like (Figure 1). TiO2 NPs that shared the other dimension with the stamps (TiO2 SEE, 19.6 nm) (Figure 1). Size Figure1.1.Size Figure and and morphology morphology ofNPs of the the NPs used used in thisin this study. study. TEM (left TEM images images (leftand panels) panels) TEM and TEM number-weighted number‐weighted size size distribution distribution (right (right panels) panels) of spherical of spherical CeO CeO2 NPs 2 NPs (CeO (CeO 2 SPH, 2 SPH, upper upper panels), panels), CeO CeO2 2stamp‐shaped stamp-shaped NPsNPs(CeO 2 STA, middle panels) and TiO2 seed‐shaped NPs (TiO2 SEE, lower (CeO STA, middle panels) and TiO seed-shaped NPs (TiO 2 2 2 SEE, lower panels). For CeO2 STA, the distribution of both dimensions (side, thickness) is shown. panels). For CeO2 STA, the distribution of both dimensions (side, thickness) is shown. The NP morphological characteristics are summarized in Table 1. The NP The NP morphological characteristics are summarized in Table 1. The NP hydro- hydrodynamic features are reported in Table 2 and show that NPs are monodispersed dynamic features are reported in Table 2 and show that NPs are monodispersed when when suspended in their buffer (TMAOH 10 mM), except for CeO2 SPH that presents two suspended in their additional peaks buffer (TMAOH of aggregation. 10 mM), Suspension in except culturefor CeO2 SPH medium leadsthat presents two addi- to significant aggregation in all cases, whereas preincubation of NPs with human serum allows foraggregation tional peaks of aggregation. Suspension in culture medium leads to significant a in all level good cases,ofwhereas preincubation dispersion and shows aof modest NPs with human increase in serum allows for a size, NP hydrodynamic good level of dispersion attributed to and shows a modest the biocorona of serumincrease proteins in onNP the hydrodynamic NP surface (Tablesize, attributed 2). The number‐to the bio- coronasize based of serum proteins distribution on the shows NP surface a similar (Table profile, with 2). The number-based serum coating affordingsize distribution good dispersion (Figureprofile, shows a similar S1). with serum coating affording good dispersion (Figure S1). Table 1. Nanoparticle morphology. Parameter CeO2 SPH CeO2 STA a TiO2 SEE (nm) 3.5 19.1, 2.7 19.6 σd (nm) 0.4 1.5, 0.4 0.8 σd / 9% 13%, 6% 23% Shape quasi-sphere stamp seed Stokes diameter (nm) b 2.52 38.74 20.52 The mean diameter is reported along with the standard deviation of the diameter σd and the coefficient of variation σd /. a Side and thickness b by analytical ultracentrifugation. Int. J. Mol. Sci. 2022, 23, 14655 4 of 15 Table 2. Nanoparticle hydrodynamic features. Parameter CeO2 SPH CeO2 STA TiO2 SEE Dh a in buffer (nm) b 4.2 (37.8) (142) c 91.3 68.1 Dh in H2 O (nm) d 4.2 (28.2) (122) 106 58.8 (5560) Dh in PBS 1x (nm) 4.2 (28.2) (122) 91.3 58.8 (5560) Dh in medium (nm) e 1480 1110 1480 Dh in medium plus HS (nm) f 10.1 (58.8) (459) 190 (5560) 164 (5560) ζ potential (mV) g −23.60 −40.20 −35.40 Endotoxin activity (EU/mg) h 25.0 9.8 4.4 a b Dh is the intensity-based hydrodynamic size measured by DLS. Buffer is TMAOH 10 mM for all NP types. c Between parentheses is the Dh of additional agglomerate peaks in DLS. d Endotoxin-free ultrapure water. e RPMI-1640 culture medium. f NPs precoated with human serum (HS) in RPMI-1640 medium. g In PBS 1x. h Endotoxin contamination of NP preparations is expressed as activity (endotoxin units, EU) per mg of NPs. All measurements were carried out in triplicate at 25 ◦ C. A single NP concentration was used for the innate memory experiments. The highest possible endotoxin-free concentration was selected as the working NP concentration. This would allow us to assess NP effects without the confounding presence of strongly inflam- matory/activating endotoxin contamination, which would lead to misinterpretation of results. Endotoxin contamination of the NP preparations was measured with an optimized detection assay (Table 2), and NP concentration for the innate memory experiments was adjusted to be at or below 0.01 EU/mL of endotoxin in order to avoid endotoxin-dependent monocyte activation. In fact, human monocytes are highly sensitive to bacterial endo- toxin, and even minute amounts of endotoxin (inactive in vivo or on other cells in vitro) can induce substantial activation. In addition, NP concentration was calculated in or- der for it to be the same for the three NPs. NP concentration was normalized to total exposed surface area, knowing that, generally, NP interactions with biological systems depend on NP surface area and characteristics. Based on these criteria, the concentration of 2.7 × 107 µm2 of total NP surface area/mL was selected, corresponding to 0.5 µg/mL CeO2 SPH (corresponding to 5.9 × 1012 NPs/mL, with 0.011 EU/mL endotoxin), 1 µg/mL CeO2 STA (corresponding to 1.5 × 1011 NPs/mL, with 0.010 EU/mL endotoxin) and 1.6 µg/mL TiO2 SEE (corresponding to 1.2 × 1011 NPs/mL, with 0.007 EU/mL endotoxin). At these concentrations, none of the NPs showed any toxic effect on human monocytes; toxic effect was measured as LDH release after 24 h of exposure and was visually monitored by optical microscopy throughout the entirety of experimental procedures. 2.2. Primary Response of Human Monocytes to LPS and Nanoparticles Results in Figure 2 show the primary response of human monocytes to NPs alone, to LPS and to LPS admixed with NPs. Production of the inflammatory cytokines TNFα and IL-6 and anti-inflammatory cytokine IL-10 was undetectable in unstimulated cells (exposed for 24 h to culture medium alone; Figure 2A–C), whereas monocytes showed a substantial constitutive production of anti-inflammatory IL-1Ra (light green dotted column in Figure 2D) as expected. Exposure to NPs did not induce production of TNFα, IL-6 and IL- 10 and did not change the constitutive IL-1Ra levels. Exposure to LPS induced measurable levels of TNFα, IL-6 and IL-10, while it did not substantially increase the production of IL-1Ra. A quantitative donor-to-donor variability was observed in the response to LPS; thus, the induction of IL-10 by LPS did not reach statistical significance. substantial constitutive production of anti‐inflammatory IL‐1Ra (light green dotted column in Figure 2D) as expected. Exposure to NPs did not induce production of TNFα, IL‐6 and IL‐10 and did not change the constitutive IL‐1Ra levels. Exposure to LPS induced measurable levels of TNFα, IL‐6 and IL‐10, while it did not substantially increase the Int. J. Mol. Sci. 2022, 23, 14655 production of IL‐1Ra. A quantitative donor‐to‐donor variability was observed in5 the of 15 response to LPS; thus, the induction of IL‐10 by LPS did not reach statistical significance. Figure Figure2.2. Primary innate innateimmune immuneresponse response of human of human monocytes monocytes to NPs to LPS, LPS,orNPs theirormixture. their mixture. Human Human blood monocytes blood monocytes were exposed were exposed in cultureintoculture mediumto alone medium alonegreen (dotted (dotted green or columns) columns) or containing containing 0.5 μg/mL CeO 2 SPH (dotted orange columns), 1.0 μg/mL CeO 0.5 µg/mL CeO2 SPH (dotted orange columns), 1.0 µg/mL CeO2 STA (dotted pink columns), or 2 STA (dotted pink columns), 1.6 µg/mLorTiO1.6 μg/mL TiO2 SEE (dotted light blue columns), or to 1 ng/mL LPS alone (red columns) 2 SEE (dotted light blue columns), or to 1 ng/mL LPS alone (red columns) or together or together with NPs (brown, orange and light‐yellow columns). The levels of inflammatory (TNFα, with NPs (brown, orange and light-yellow columns). The levels of inflammatory (TNFα, IL-6; (A,B)) IL‐6; (A,B)) and anti‐inflammatory cytokines (IL‐10, IL‐1Ra; (C,D)) were measured in the 24 h and anti-inflammatory cytokines (IL-10, IL-1Ra; (C,D)) were measured in the 24 h supernatants by supernatants by ELISA. The columns represent the average value + SEM from three individual ELISA.Statistical donors. The columns represent***the significance: p 95% viable and >95% pure (assessed by trypan blue exclusion and cytosmears). Monocytes isolated with this method were not activated, based on analysis of the expression of inflammation-related genes (IL1B, TNFA) and the release of encoded proteins, compared to monocytes within PBMC (i.e., after Ficoll-Paque separation) and whole blood (i.e., after withdrawal with anticoagulants). Monocytes were cultured in culture medium (RPMI 1640 + Glutamax-I; GIBCO by Life Technologies, Paisley, UK) supplemented with 50 µg/mL gentamicin sulfate (GIBCO) and 5% heat-inactivated human AB serum (Merck Sigma-Aldrich® ). Cells (5 × 105 ) were seeded at a final volume of 1.0 mL in the wells of 24-well flat bottom plates (Corning® Costar® ; Corning Inc. Life Sciences, Oneonta, NY, USA) at 37 ◦ C in moist air with 5% CO2 . Monocyte stimulation was performed after resting overnight. 4.3. Human Monocyte Activation and Induction of Innate Memory For assessing the primary response to stimulation, monocytes were exposed for 24 h to culture medium alone (medium/negative control) or medium containing 1 ng/mL LPS (positive control; from E. coli O55:B5; Merck Sigma-Aldrich® ), serum pre-coated NPs or LPS together with NPs. For memory experiments, after the first exposure to stimuli for 24 h and supernatant collection, cells were washed and cultured with fresh culture medium for 7 additional days (one medium change after 4 days). During this period, the activation induced by previous stimulation subsides, and cells return to their baseline status (as determined by evaluation of inflammation-related cytokines in the supernatant). After the resting phase, the supernatant was collected, and cells were challenged for 24 h with fresh medium alone or medium containing 5 ng/mL LPS (at a 5× higher concentration than in the primary stimulation to mimic a stronger secondary challenge). All supernatants (collected after the first stimulation, after the resting phase and after the challenge phase) were frozen at −20 ◦ C for subsequent cytokine analysis. By visual inspection, cell viability and cell number did not substantially change in response to the different treatments. 4.4. Cytotoxicity Evaluation The direct toxicity of NPs on monocytes was evaluated through the release of lactate dehydrogenase (LDH). Briefly, monocytes (1.2 × 105 cells/well of 96-well plates; Corn- Int. J. Mol. Sci. 2022, 23, 14655 12 of 15 ing Inc., Corning, NY, USA) were incubated for 24 h in 200 µL of culture medium alone or medium containing serum-coated NPs in triplicate. Positive control wells received 200 µL of 0.1% Triton-X 100. At the end of the incubation, release of the cytoplasmic en- zyme LDH was measured in the supernatant using a colorimetric assay (LDH-Cytotoxicity Colorimetric Assay Kit; BioVision, Inc., Milpitas, CA, USA). Validation of the LDH cytotox- icity results was obtained by visual inspection of cells in culture over the entire course of the experiments. Cell density (variations in the number of cells in culture), cell refractivity, morphology and adherence (associated to live/dead cells) were assessed by phase contrast optical microscopy at 24 h (after the first stimulus), 8 days (after the resting period) and at 9 days (after the challenge). 4.5. Assessment of Endotoxin Contamination Endotoxin contamination in NPs was assessed with a commercial chromogenic Limu- lus Amoebocyte Lysate (LAL) assay (Pyros Kinetix® Flex; Associates of Cape Cod, Inc., East Falmouth, MA, USA) following a protocol adapted for use with particulate agents [71]. Preliminary controls were run to assess the possible interference of NPs in the assay readout. These encompassed direct optical reading at 405 nm (the OD of the assay readout product paranitroaniline, pNA) and interference with detection of different concentrations of syn- thetic pNA. NPs were used in the LAL assay at concentrations that did not interfere with the assay readout. Additional controls included assessment of the possible interference of NPs with the assay components/reagents, performed by spiking the NP samples with a known amount of LPS (0.5 EU/mL) and assessing the recovery of spiked endotoxin. A recovery rate between 80 and 120% was considered acceptable. The endotoxin contamination was therefore reliably assessed at NP dilutions that did not interfere with the 405 nm readout and in which 80–120% spiked endotoxin could be recovered. The LAL assay was run with Glucashield® (Associates of Cape Cod, Inc.) using a dedicated tube reader and software (Associates of Cape Cod, Inc.) to eliminate possible false positives due to the presence of glucans. Assay sensitivity was 0.001 EU/mL. 4.6. Evaluation of Cytokine Production The levels of the human inflammatory cytokines TNFα and IL-6 and anti-inflammatory factors IL-10 and IL-1Ra produced by cultured monocytes were assessed in cell supernatants by ELISA (R&D Systems, Minneapolis, MN, USA) using a Cytation 3 imaging multi-mode reader (BioTek, Winooski, VT, USA). The presence of IL-1β was not measured because, from preliminary experiments, the production of this inflammatory cytokine in cells challenged with LPS after primary activation and 7 days of resting was below detection even in controls. Each sample was tested in duplicate in ELISA. 4.7. Statistical Analysis Data were analyzed using GraphPad Prism6.01 software (GraphPad Inc., La Jolla, CA, USA). For cytokine production, results are presented as ng of produced cytokine/106 plated monocytes. Results are reported as the mean of values from three donors, each tested with two to six replicates. The statistical significance of differences is indicated by p values, which were calculated with unpaired and two-tailed Student’s t-tests. Supplementary Materials: The supporting information can be downloaded at: https://www.mdpi. com/article/10.3390/ijms232314655/s1. Author Contributions: D.B. wrote the manuscript; G.D.C. performed the experimental work and the analytical NP characterization; T.L. and W.Y. analyzed the data; V.F.P. synthesized and characterized the NPs; D.B., Y.L., S.G. and P.I. contributed to the experimental design; all authors critically revised the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the EU Commission H2020 projects PANDORA (GA671881) and ENDONANO (GA 812661) (PI, DB), the Italian MIUR InterOmics Flagship projects MEMORAT and MAME (DB, PI), and the Presidential International Fellowship Program (PIFI) of the Chinese Int. J. Mol. Sci. 2022, 23, 14655 13 of 15 Academy of Science (2020VBA0028) (DB). Part of this work was carried out in the context of the JRC Visiting Scientist agreement no. 05/JRC.F.2/2019 (Directorate F—Health, Consumers and Reference Materials, Consumer Products Safety, Nanobiotechnology Lab). Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki and approved by the Regional Ethics Committee for Clinical Experimentation of the Tuscany Region (Ethics Committee Register n. 14,914 of 16 May 2019). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Data supporting reported results can be found in the supplementary material and are available from the authors upon request. Acknowledgments: We are grateful to Giuliana Donini, who has continuously encouraged and supported our studies. We thank Paola Migliorini (University of Pisa) for the ethical monitoring of our studies with human blood cells, Jessica Ponti (JRC) for TEM support, and Dora Mehn (JRC), Ben Swartzwelter (University of Colorado, Denver, CO, USA) and Jutta Horejs-Hoeck (Paris-Lodron University of Salzburg, Austria) for helpful discussion. DB also wishes to thank Wang Rui for her relentless efforts in facilitating our scientific work. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. References 1. Kurtz, J. Specific memory within innate immune systems. Trends Immunol. 2005, 26, 186–192. [CrossRef] [PubMed] 2. Foster, S.L.; Hargreaves, D.C.; Medzhitov, R. Gene-specific control of inflammation by TLR-induced chromatin modifications. Nature 2007, 447, 972–978. [CrossRef] [PubMed] 3. Netea, M.G.; Quintin, J.; van der Meer, J.W. Trained immunity: a memory for innate host defense. Cell Host Microbe 2011, 9, 355–361. [CrossRef] [PubMed] 4. Netea, M.G.; Joosten, L.A.; Latz, E.; Mills, K.H.; Natoli, G.; Stunnenberg, H.G.; O’Neill, L.A.; Xavier, R.J. Trained immunity: a program of innate immune memory in health and disease. Science 2016, 352, aaf1098. [CrossRef] 5. Sun, J.C.; Ugolini, S.; Vivier, E. Immunological memory within the innate immune system. EMBO J. 2014, 33, 1295–1303. [CrossRef] 6. Milutinovic, B.; Kurtz, J. Immune memory in invertebrates. Semin. Immunol. 2016, 28, 328–342. [CrossRef] 7. Reimer-Michalski, E.M.; Conrath, U. Innate immune memory in plants. Semin. Immunol. 2016, 28, 319–327. [CrossRef] 8. Melillo, D.; Marino, R.; Italiani, P.; Boraschi, D. Innate immune memory in invertebrate metazoans: a critical appraisal. Front. Immunol. 2018, 9, 1915. [CrossRef] 9. Pradeau, T.; Du Pasquier, L. Immunological memory: what’s in a name? Immunol. Rev. 2018, 283, 7–20. [CrossRef] 10. Beeson, P.B. Development of tolerance to typhoid bacterial pyrogen and its abolition by reticulo-endothelial blockade. Proc. Soc. Exp. Biol. Med. 1946, 61, 248–250. [CrossRef] 11. Dubos, R.J.; Schaedler, R.W. Reversible changes in the susceptibility of mice to bacterial infections. I. Changes brought about by injection of pertussis vaccine or of bacterial endotoxins. J. Exp. Med. 1956, 104, 53–65. [CrossRef] 12. Fan, H.; Cook, J.A. Molecular mechanisms of endotoxin tolerance. J. Endotoxin Res. 2004, 10, 71–84. [CrossRef] [PubMed] 13. Cavaillon, J.-M.; Adib-Conquy, M. Bench-to-bedside review: endotoxin tolerance as a model of leukocyte reprogramming in sepsis. Crit. Care 2006, 10, 233. [CrossRef] 14. Buckley, J.M.; Wang, J.H.; Redmond, H.P. Cellular reprogramming by Gram-positive bacterial components: a review. J. Leukoc. Biol. 2006, 80, 731–741. [CrossRef] [PubMed] 15. Morris, M.C.; Gilliam, E.A.; Li, L. Innate immune programing by endotoxin and its pathological consequences. Front. Immunol. 2014, 5, 680. [CrossRef] [PubMed] 16. Seeley, J.J.; Ghosh, S. Molecular mechanisms of innate memory and tolerance to LPS. J. Leukoc. Biol. 2017, 101, 107–119. [CrossRef] 17. Boehme, D.; Dubos, R.J. The effect of bacterial constituents on the resistance of mice to heterologous infection and on the activity of their reticuloendothelial system. J. Exp. Med. 1958, 107, 523–536. [CrossRef] 18. Bistoni, F.; Vecchiarelli, A.; Cenci, E.; Puccetti, P.; Marconi, P.; Cassone, A. Evidence for macrophage-mediated protection against lethal Candida albicans infection. Infect. Immun. 1986, 51, 668–674. [CrossRef] 19. Bowdish, D.M.E.; Loffredo, M.S.; Mukhopadhyay, S.; Mantovani, A.; Gordon, S. Macrophage receptors implicated in the “adaptive” form of innate immunity. Microbes Infect. 2007, 9, 1680–1687. [CrossRef] 20. Sun, J.C.; Beilke, J.N.; Lanier, L.L. Immune memory redefined: characterizing the longevity of natural killer cells. Immunol. Rev. 2010, 236, 83–94. [CrossRef] 21. O’Sullivan, T.E.; Sun, J.C.; Lanier, L.L. Natural killer cell memory. Immunity 2015, 43, 634–645. [CrossRef] [PubMed] Int. J. Mol. Sci. 2022, 23, 14655 14 of 15 22. Martinez-Gonzalez, I.; Math, L.; Steer, C.A.; Takei, F. Immunological memory of group 2 innate lymphoid cells. Trends Immunol. 2017, 38, 423–431. [CrossRef] [PubMed] 23. Serafini, N.; Jarade, A.; Surace, L.; Goncalves, P.; Sismeiro, O.; Varet, H.; Legendre, R.; Coppee, J.Y.; Disson, O.; Durum, S.K.; et al. Trained ILC3 responses promote intestinal defense. Science 2022, 375, 859–863. [CrossRef] [PubMed] 24. Naik, S.; Larsen, S.B.; Gomez, N.C.; Alaverdyan, K.; Sendoel, A.; Yuan, S.; Polak, L.; Kulukian, A.; Chai, S.; Fuchs, E. Inflammatory memory sensitizes skin epithelial stem cells to tissue damage. Nature 2017, 550, 475–480. [CrossRef] [PubMed] 25. Dai, X.; Medzhitov, R. Inflammation: memory beyond immunity. Nature 2017, 550, 460–461. [CrossRef] 26. Nasrollahi, S.; Walter, C.; Loza, A.J.; Schimizzi, G.V.; Longmore, G.D.; Pathak, A. Past matrix stiffness primes epithelial cells and regulates their future collective migration through a mechanical memory. Biomaterials 2017, 146, 146–155. [CrossRef] 27. Cassone, A. The case of an expanded concept of trained immunity. mBio 2018, 9, e00570-18. [CrossRef] 28. Bigot, J.; Guillot, L.; Guitard, J.; Ruffin, M.; Corvol, H.; Chignard, M.; Hennequin, C.; Balloy, V. Respiratory epithelial cells can remember infection: a proof-of-concept study. J. Infect. Dis. 2020, 221, 1000–1005. [CrossRef] 29. Madej, M.; Töpfer, E.; Boraschi, D.; Italiani, P. Different regulation of interleukin-1 production and activity in monocytes and macrophages: innate memory as an endogenous mechanism of IL-1 inhibition. Front. Pharmacol. 2017, 8, 335. [CrossRef] 30. Feuerstein, R.; Forde, A.J.; Lohrmann, F.; Kolter, J.; Ramirez, N.J.; Zimmermann, J.; de Agüero, M.G.; Henneke, P. Resident macrophages acquire innate immune memory in staphylococcal skin infection. eLife 2020, 9, e55602. [CrossRef] 31. Boraschi, D.; Duschl, A. Nanoparticles and the Immune System: Safety and Effects; Academic Press: Oxford, UK, 2013. 32. Mohajerani, A.; Burnett, L.; Smith, J.V.; Kurmus, H.; Milas, J.; Arulrajah, A.; Horpibulsuk, S.; Abdul Kadir, A. Nanoparticles in construction materials and other applications, and implications of nanoparticle use. Materials 2019, 12, 3052. [CrossRef] [PubMed] 33. Shi, H.; Magaye, R.; Castranova, V.; Zhao, J. Titanium dioxide nanoparticles: a review of current toxicological data. Part. Fibre Toxicol. 2013, 10, 15. [CrossRef] [PubMed] 34. Smolkova, B.; El Yamani, N.; Collins, A.R.; Gutleb, A.C.; Dusinska, M. Nanoparticles in food. Epigenetic changes induced by nanomaterials and possible impact on health. Food Chem. Toxicol. 2015, 77, 64–73. [CrossRef] [PubMed] 35. SCCS. Opinion on Titanium Dioxide (Nano Form) as UV-Filter in Sprays, Preliminary Version of 7 March 2017, Final Version of 19 January 2018; SCCS/1583/17; SCCS: Brussels, Belgium, 2018. 36. Dréno, B.; Alexis, A.; Chuberre, B.; Marinovich, M. Safety of titanium dioxide nanoparticles in cosmetics. J. Eur. Acad. Dermatol. Venereol. 2019, 33, 34–46. [CrossRef] 37. ESFA. Titanium Dioxide: E171 No Longer Considered Safe when Used as a Food Additive. 2021. Available online: https: //www.efsa.europa.eu/en/news/titanium-dioxide-e171-no-longer-considered-safe-when-used-food-additive (accessed on 3 September 2022). 38. Corma, A.; Atienzar, P.; Garcia, H.; Chane-Ching, J.-Y. Hierarchically mesostructured doped CeO2 with potential for solar-cell use. Nat. Mater. 2004, 3, 394–397. [CrossRef] 39. Reed, K.; Cormack, A.; Kulkarni, A.; Mayton, M.; Sayle, D.; Klaessig, F.; Stadler, B. Exploring the properties and applications of nanoceria: is there still plenty of room at the bottom? Environ. Sci. Nano 2014, 1, 390–405. [CrossRef] 40. Ivanov, V.K.; Shcherbakov, A.; Usatenko, A. Structure-sensitive properties and biomedical applications of nanodisperse cerium dioxide. Russ. Chem. Rev. 2009, 78, 855–871. [CrossRef] 41. Jung, H.; Kittelson, D.B.; Zachariah, M.R. The influence of a cerium additive on ultrafine diesel particle emissions and kinetics of oxidation. Combust. Flame 2005, 142, 276–288. [CrossRef] 42. Campbell, C.T. Oxygen vacancies and catalysis on ceria surfaces. Science 2005, 309, 713–714. [CrossRef] 43. Charbgoo, F.; Ramezani, M.; Darroudi, M. Bio-sensing applications of cerium oxide nanoparticles: advantages and disadvantages. Biosens. Bioelectron. 2017, 96, 33–43. [CrossRef] 44. Chen, J.P.; Patil, S.; Seal, S.; McGinnis, J.F. Rare earth nanoparticles prevent retinal degeneration induced by intracellular peroxides. Nat. Nanotechnol. 2006, 1, 142–150. [CrossRef] [PubMed] 45. Hirst, S.; Karakoti, A.; Tyler, R.; Sriranganathan, N.; Seal, S.; Reilly, C. Anti-inflammatory properties of cerium oxide nanoparticles. Small 2009, 5, 2848–2856. [CrossRef] [PubMed] 46. Colon, J.; Hsieh, N.; Ferguson, A.; Kupelian, P.; Seal, S.; Jenkins, D.W.; Baker, C.H. Cerium oxide nanoparticles protect gastroin- testinal epithelium from radiation-induced damage by reduction of reactive oxygen species and upregulation of superoxide dismutase 2. Nanomedicine 2010, 6, 698–705. [CrossRef] [PubMed] 47. DeCoteau, W.; Heckman, K.; Estevez, A.; Reed, K.; Costanzo, W.; Sandford, D.; Studlack, P.; Clauss, J.; Nichols, E.; Lipps, J.; et al. Cerium oxide nanoparticles with antioxidant properties ameliorate strength and prolong life in mouse model of amyotrophic lateral sclerosis. Nanomedicine 2016, 12, 2311–2320. [CrossRef] [PubMed] 48. Kim, J.; Hong, G.; Mazaleuskaya, L.; Hsu, J.C.; Rosario-Berrios, D.N.; Grosser, T.; Cho-Park, P.F.; Cormode, D.P. Ultrasmall antioxidant cerium oxide nanoparticles for regulation of acute inflammation. ACS Appl. Mater. Interfaces 2021, 13, 60852–60864. [CrossRef] [PubMed] 49. Casals, G.; Perramón, M.; Casals, E.; Portolés, I.; Fernández-Varo, G.; Morales-Ruiz, M.; Puntes, V.; Jiménez, W. Cerium oxide nanoparticles: a new therapeutic tool in liver diseases. Antioxidants 2021, 10, 660. [CrossRef] [PubMed] 50. Pelclova, D.; Navratil, T.; Kacerova, T.; Zamostna, B.; Fenclova, Z.; Vlckova, S.; Kacer, P. NanoTiO2 sunscreen does not prevent systemic oxidative stress caused by UV radiation and a minor amount of nanoTiO2 is absorbed in humans. Nanomaterials 2019, 9, 888. [CrossRef] Int. J. Mol. Sci. 2022, 23, 14655 15 of 15 51. Baroli, B.; Ennas, M.G.; Loffredo, F.; Isola, M.; Pinna, R.; Lopez-Quintela, M.A. Penetration of metallic nanoparticles in human full-thickness skin. J. Investig. Dermatol. 2007, 127, 1701–1712. [CrossRef] 52. Brun, E.; Barreau, F.; Veronesi, G.; Fayard, B.; Sorieul, S.; Chaneac, C.; Carapito, C.; Rabilloud, T.; Mabondzo, A.; Herlin-Boime, N.; et al. Titanium dioxide nanoparticle impact and translocation through ex vivo, in vivo and in vitro gut epithelia. Part. Fibre Toxicol. 2014, 11, 13. [CrossRef] 53. Cronin, J.G.; Jones, N.; Thornton, C.A.; Jenkins, G.J.S.; Doak, S.H.; Clift, M.J.D. Nanomaterials and innate immunity: a perspective of the current status in nanosafety. Chem. Res. Toxicol. 2020, 33, 1061–1073. [CrossRef] 54. Himly, M.; Geppert, M.; Hofer, S.; Hofstaetter, N.; Horejs-Hoeck, J.; Duschl, A. When would immunologists consider a nanomaterial to be safe? Recommendations for planning studies on nanosafety. Small 2020, 16, e1907483. [CrossRef] [PubMed] 55. Boraschi, D.; Italiani, P.; Palomba, R.; Decuzzi, P.; Duschl, A.; Fadeel, B.; Moghimi, S.M. Nanoparticles and innate immunity: new perspectives on host defence. Semin. Immunol. 2017, 34, 33–51. [CrossRef] [PubMed] 56. Boraschi, D.; Alijagic, A.; Auguste, M.; Barbero, F.; Ferrari, E.; Hernadi, S.; Mayall, C.; Michelini, S.; Navarro Pacheco, N.I.; Prinelli, A.; et al. Addressing nanomaterial immunosafety by evaluating innate immunity across living species. Small 2020, 16, e2000598. [CrossRef] [PubMed] 57. Italiani, P.; Boraschi, D. Induction of innate immune memory by engineered nanoparticles, a hypothesis that may become true. Front. Immunol. 2017, 8, 734. [CrossRef] 58. Italiani, P.; Della Camera, G.; Boraschi, D. Induction of innate immune memory by engineered nanoparticles in mono- cytes/macrophages: from hypothesis to reality. Front. Immunol. 2020, 11, 566309. [CrossRef] 59. Lebre, F.; Boland, J.B.; Gouveia, P.; Gorman, A.L.; Lundahl, M.L.E.; Lynch, R.I.; O’Brien, F.J.; Coleman, J.; Lavelle, E.C. Pristine graphene induces innate immune training. Nanoscale 2020, 12, 11192–11200. [CrossRef] 60. Swartzwelter, B.J.; Barbero, F.; Verde, A.; Mangini, M.; Pirozzi, M.; De Luca, A.C.; Puntes, V.F.; Leite, L.C.; Italiani, P.; Boraschi, D. Gold nanoparticles modulate BCG-induced innate immune memory in human monocytes by shifting the memory response towards tolerance. Cells 2020, 9, 284. [CrossRef] 61. Swartzwelter, B.J.; Michelini, S.; Frauenlob, T.; Barbero, F.; Verde, A.; De Luca, A.C.; Puntes, V.; Duschl, A.; Horejs-Hoeck, J.; Italiani, P.; et al. Innate memory reprogramming by gold nanoparticles depends on the microbial agents that induce memory. Front. Immunol. 2021, 12, 751683. [CrossRef] 62. Barbosa, M.M.F.; Kanno, A.I.; Paiva Farias, L.; Madej, M.; Sipos, G.; Sbrana, S.; Romani, L.; Boraschi, D.; Leite, L.C.; Italiani, P. Primary and memory response of human monocytes to vaccines: role of nanoparticulate antigens in inducing innate memory. Nanomaterials 2021, 11, 931. [CrossRef] 63. Della Camera, G.; Madej, M.; Ferretti, A.M.; La Spina, R.; Li, Y.; Corteggio, A.; Heinzl, T.; Swartzwelter, B.J.; Sipos, G.; Gioria, S.; et al. Personalised profiling of innate immune memory induced by nano-imaging particles in human monocytes. Front. Immunol. 2021, 12, 692165. [CrossRef] 64. Hussain, S.; Al-Nsour, F.; Rice, A.B.; Marshburn, J.; Ji, Z.; Zink, J.I.; Yingling, B.; Walker, N.J.; Garantziotis, S. Cerium dioxide nanoparticles do not modulate the lipopolysaccharide-induced inflammatory response in human monocytes. Int. J. Nanomed. 2012, 7, 1387–1397. [CrossRef] [PubMed] 65. Franceschi, C.; Salvioli, S.; Garagnani, P.; de Eguileor, M.; Monti, D.; Capri, M. Immunobiography and the heterogeneity of immune responses in the elderly: a focus on inflammaging and trained immunity. Front. Immunol. 2017, 8, 982. [CrossRef] [PubMed] 66. Pan, Y.; Li, J.; Xia, X.; Wang, J.; Jiang, Q.; Yang, J.; Dou, H.; Liang, H.; Li, K.; Hou, Y. β-glucan-coupled superparamagnetic iron oxide nanoparticles induce trained immunity to protect mice against sepsis. Theranostics 2022, 12, 675–688. [CrossRef] [PubMed] 67. Zhou, X.D.; Huebner, W.; Anderson, H.U. Room-temperature homogeneous nucleation synthesis and thermal stability of nanometer single crystal CeO2 . Appl. Phys. Lett. 2002, 80, 3814–3816. [CrossRef] 68. Chen, H.I.; Chang, H.Y. Synthesis and characterization of nanocrystalline cerium oxide powders by two-stage non-isothermal precipitation. Solid State Commun. 2005, 133, 593–598. [CrossRef] 69. García, A.; Delgado, L.; Torà, J.A.; Caslas, E.; González, E.; Puntes, V.; Font, X.; Carrera, J.; Sánchez, A. Effect of cerium dioxide, titanium dioxide, silver, and gold nanoparticles on the activity of microbial communities intended in wastewater treatment. J. Hazard Mater. 2012, 199–200, 64–72. [CrossRef] [PubMed] 70. Pottier, A.; Cassaignon, S.; Chanéac, C.; Villain, F.; Tronca, E.; Jolivet, J.-P. Size tailoring of TiO2 anatase nanoparticles in aqueous medium and synthesis of nanocomposites. Characterization by Raman spectroscopy. J. Mater. Chem. 2003, 13, 877–882. [CrossRef] 71. Li, Y.; Italiani, P.; Casals, E.; Tran, N.; Puntes, V.F.; Boraschi, D. Optimising the use of commercial LAL assays for the analysis of endotoxin contamination in metal colloids and metal oxide nanoparticles. Nanotoxicology 2015, 9, 462–473. [CrossRef]