ORIGINAL RESEARCH published: 04 November 2021 doi: 10.3389/fimmu.2021.751683 Innate Memory Reprogramming Edited by: by Gold Nanoparticles Depends David Pozo, University of Seville, Spain on the Microbial Agents That Reviewed by: Umut Can Kucuksezer, Induce Memory Istanbul University, Turkey Mario M. D’Elios, Benjamin J. Swartzwelter 1,2†, Sara Michelini 2, Tobias Frauenlob 2, Francesco Barbero 3, University of Florence, Italy Alessandro Verde 1†, Anna Chiara De Luca 1†, Victor Puntes 3,4,5, Albert Duschl 2, *Correspondence: Jutta Horejs-Hoeck 2, Paola Italiani 1 and Diana Boraschi 1,6,7* Diana Boraschi diana.boraschi@ibbc.cnr.it 1 Institute of Biochemistry and Cell Biology (IBBC), National Research Council (CNR), Napoli, Italy, 2 Department Biosciences, Paola Italiani Paris Lodron University of Salzburg (PLUS), Salzburg, Austria, 3 Institut Català de Nanociència i Nanotecnologia (ICN2), paola.italiani@ibbc.cnr.it Consejo Superior de Investigaciones Científicas (CSIC) and The Barcelona Institute of Science and Technology (BIST), † Present address: Barcelona, Spain, 4 Vall d’Hebron Research Institute (VHIR), Barcelona, Spain, 5 Institució Catalana de Recerca I Estudis Benjamin J. Swartzwelter, Avançats (ICREA), Barcelona, Spain, 6 Stazione Zoologica Anton Dohrn, Napoli, Italy, 7 Shenzhen Institute of Advanced Department of Microbiology, Technology (SIAT), Chinese Academy of Sciences (CAS), Shenzhen, China Immunology & Pathology, Colorado State University, Fort Collins, CO, Innate immune memory, the ability of innate cells to react in a more protective way to United States Alessandro Verde, secondary challenges, is induced by exposure to infectious and other exogeous and Institute for Experimental endogenous agents. Engineered nanoparticles are particulate exogenous agents that, as Endocrinology and Oncology, “G. Salvatore” (IEOS), Second Unit, such, could trigger an inflammatory reaction in monocytes and macrophages and could Consiglio Nazionale Delle Ricerche therefore be also able to induce innate memory. Here, we have evaluated the capacity of (CNR), Napoli, Italy engineered gold nanoparticles (AuNPs) to induce a memory response or to modulate the Anna Chiara De Luca, Institute for Experimental memory responses induced by microbial agents. Microbial agents used were in soluble Endocrinology and Oncology, vs. particulate form (MDP and the gram-positive bacteria Staphylococcus aureus; “G. Salvatore” (IEOS), Second Unit, Consiglio Nazionale Delle Ricerche b-glucan and the b-glucan-producing fungi C. albicans), and as whole microrganisms (CNR), Napoli, Italy that were either killed (S. aureus, C. albicans) or viable (the gram-negative bacteria Helicobacter pylori). The memory response was assessed in vitro, by exposing human Specialty section: This article was submitted to primary monocytes from 2-7 individual donors to microbial agents with or without AuNPs Molecular Innate Immunity, (primary response), then resting them for 6 days to allow return to baseline, and eventually a section of the journal challenging them with LPS (secondary memory response). Primary and memory Frontiers in Immunology responses were tested as production of the innate/inflammatory cytokine TNFa Received: 01 August 2021 Accepted: 14 October 2021 and other inflammatory and anti-inflammatory factors. While inactive on the response Published: 04 November 2021 induced by soluble microbial stimuli (muramyl dipeptide -MDP-, b-glucan), AuNPs partially Citation: reduced the primary response induced by whole microorganisms. AuNPs were also Swartzwelter BJ, Michelini S, Frauenlob T, unable to directly induce a memory response but could modulate stimulus-induced Barbero F, Verde A, De Luca AC, memory in a circumscribed fashion, limited to some agents and some cytokines. Puntes V, Duschl A, Horejs-Hoeck J, Thus, the MDP-induced tolerance in terms of TNFa production was further Italiani P and Boraschi D (2021) Innate Memory Reprogramming by Gold exacerbated by co-priming with AuNPs, resulting in a less inflammatory memory Nanoparticles Depends on the response. Conversely, the H. pylori-induced tolerance was downregulated by AuNPs Microbial Agents That Induce Memory. Front. Immunol. 12:751683. only relative to the anti-inflammatory cytokine IL-10, which would lead to an overall more doi: 10.3389/fimmu.2021.751683 inflammatory memory response. These effects of AuNPs may depend on a differential Frontiers in Immunology | www.frontiersin.org 1 November 2021 | Volume 12 | Article 751683 Swartzwelter et al. Modulation of Innate Memory by Gold Nanoparticles interaction/association between the reactive particle surfaces and the microbial components and agents, which may lead to a change in the exposure profiles. As a general observation, however, the donor-to-donor variability in memory response profiles and reactivity to AuNPs was substantial, suggesting that innate memory depends on the individual history of exposures. Keywords: innate immunity, innate memory, nanoparticles, microbial agents, monocytes INTRODUCTION BCG, endotoxin (lipopolysaccharide -LPS-) and b-glucan, agents such as muramyl dipeptide (MDP) and Candida Immunological memory was long considered a distinctive trait of albicans have each demonstrated the capacity to alter the adaptive immunity, resulting in the capacity to mount a more secondary reactivity of monocytes or macrophages (18–20). rapid and more effective specific immune response to infectious Recently, several studies have examined whether engineered challenges (1). It is however evident that organisms that only nanoparticles are also capable of inducing or modulating display innate immunity, the most ancient non-specific innate immune memory. While pristine graphene could induce defensive system, can develop an immunological memory a potentiated status in murine macrophages (21), gold that allows them to resist better to various environmental nanoparticles (AuNPs) failed to independently induce a pathogens and stressful events (e.g., heat, wounds) (2–4). memory response in human monocytes, although they seem Higher vertebrates maintain an efficient innate immunity, able to modulate in different directions the innate memory in parallel to adaptive responses, and it is now evident that induced by microbial agents (22–26). priming/exposure to microbial/stressful agents generates “innate In this context, here we have evaluated the capacity of AuNPs memory” in innate immune cells, such as monocytes to modulate the innate memory response of human primary and macrophages. The innate memory is at least partially non- monocytes primed with different microbial agents in soluble vs. specific and allows for a more protective reaction to subsequent particulate forms (MDP and the gram-positive bacteria challenges (2, 3, 5–7). Staphylococcus aureus; b-glucan and the b-glucan-producing The first type of innate memory described in mammals is fungi C. albicans) and with a live microbial agent (the gram- known as “endotoxin tolerance” and results in a less potent negative bacterium Helicobacter pylori), using a realistic in vitro secondary response to gram-negative endotoxin or other model based on human primary monocytes. The results show bacterial challenges, aiming at attaining sufficient protection that AuNPs are unable per se to induce an inflammatory reaction while avoiding the substantial damage to the host tissues and or to induce innate memory in monocytes, but can partially affect organs that can be caused by a full innate/inflammatory the stimulus-induced cell activation. response, which includes the deadly endotoxin shock (8–11). In other cases, e.g., in the case of exposure to the tuberculosis vaccine BCG or to the fungal b-glucan, the memory response results in a potentiated reaction (“trained immunity”) (6, 12). MATERIALS AND METHODS The innate memory responses, both tolerance and potentiation, are based on epigenetic and metabolic modifications, rather than AuNP Synthesis and Characterization in a general shift in gene transcription, and they should be AuNP Synthesis understood as a medium-term functional reprogramming aimed AuNPs were synthesized as previously described by Bastú s et al. at enhanced host defense (lasting several months to years in (27). Briefly, 150 mL of sodium citrate 2.2 mM was brought to a mammals) (6, 13–16). However, anomalous innate memory has boil under reflux, followed by rapid addition of 1 mL of HAuCl4 been proposed to contribute to the development of immune/ 25 mM. AuNP “seeds” were formed in this manner, and inflammatory diseases, such as autoimmune syndromes and sequential addition of HAuCl4 achieved the desired particle chronic inflammatory diseases (6, 17). Which substances size. All reagents were obtained from Sigma-Aldrich® (Merck activate innate immune memory, in which direction KGaA, St. Louis, MO, USA). (protective vs. detrimental, tolerance vs. potentiation) and how different agents might differentially modulate innate memory is AuNP Characterization still largely unexplored. Innate memory-inducing substances Transmission Electron Microscopy and Scanning Electron should be considered both from a safety perspective, in which Microscopy excessive inflammation or immune suppression can be NP characterization images were obtained by STEM (scanning detrimental, but also for their therapeutic potential, to down- transmission electron microscopy) using a FEI Magellan XHR regulate or up-regulate excessive or insufficient innate immunity microscope (FEI, Hillsboro, OR, USA) in transmission mode in different disease conditions. with an acceleration of 20 kV, as previously described (22). Several microbial stimuli have been described for their AuNP samples were stabilized with polyvinylpyrrolidone memory inducing capacity. In addition to the aforementioned (55 kDa) (28) and drop cast onto a carbon-coated TEM grid. Frontiers in Immunology | www.frontiersin.org 2 November 2021 | Volume 12 | Article 751683 Swartzwelter et al. Modulation of Innate Memory by Gold Nanoparticles After drying, samples were imaged and particle size was assessed purity by differential counting on Wright-Giemsa-stained using an ImageJ macro. Scanning Electron Microscopy (SEM) cytosmears (Diff-Quik; Medion Diagnostics, Düdingen, was conducted on a JEOL 6700F scanning electron microscope Switzerland) examined by optical microscopy. Cell viability (JEOL, Peabody, MA, USA) as described previously (29). was assessed by trypan blue dye exclusion. Only cell isolations with at least 95% purity and 95% viability were used. UV-vis Spectroscopy To assess particle stability and uniformity of size, UV-vis spectra of the AuNP suspensions were obtained using a Shimadzu UV- Human Monocyte Primary Stimulation and 2400 spectrophotometer (SSI, Kyoto, Japan) with a range of 300- Innate Memory Response 700 nm. Samples were measured at room temperature, and AuNP Biocorona Formation milliQ water was used as a reference. Before addition into cell culture, AuNPs were pre-incubated in 50% inactivated human AB serum (Sigma-Aldrich) at 37°C for Dynamic Light Scattering 1 h, in order to obtain the formation of a bio-corona of serum Particle z-potential and hydrodynamic diameter were proteins and other components on the particle surface thereby determined by laser doppler velocimetry and dynamic light ensuring particle stability in culture. However, being this a soft scattering (DLS), respectively, using a Malvern Zetasizer Nano corona, it still allowed for interaction of the reactive particle ZS instrument (Malvern Panalytical Ltd., Malvern, UK) with a surface with microbial agents and cells in culture (32, 33). The light source wavelength of 632.8 nm and a fixed scattering angle serum-AuNP mixture was added directly to culture wells (34), of 173° (at 25°C). adjusting particle and serum concentration to the desired values. Atomic Force Microscopy Monocyte Primary Innate Response AFM measurements were performed with XE-70 microscope Freshly isolated monocytes were suspended in culture medium (Park Systems, Suwon, South Korea). The instrument is (RPMI-1640 + Glutamax-I; GIBCO by Life Technologies, equipped with two flexure scanners (XY plane and Z) both for Paisley, UK) supplemented with 100 U/mL penicillin/ probe tips and samples. Scans were performed on an area up to streptomycin (Sigma-Aldrich). Cells (1x105/well) were added 15x15 µm2 with a topographic resolution below 1 nm (30). AFM to 96 well flat bottom plates (Corning® Costar®; Corning Inc. images were acquired after deposition of AuNPs (10 µL at 1022 Life Sciences, Oneonta, NY, USA). Cells were exposed to µg/mL) on a quartz slide by drop-casting. b-glucan (extracted from C. albicans; 2 µg/mL; a generous gift Evaluation of Endotoxin Contamination from Charles Dinarello, University of Colorado, Denver CO, The presence of endotoxin contamination in NP samples was USA), MDP (10 µg/mL; InvivoGen, San Diego, CA, USA), heat- assessed with the Limulus Amoebocyte Lysate assay (LAL). The killed S. aureus (ratio with monocytes 1:1; strain ATCC 6538, chromogenic Pyrochrome LAL assay (Associates of Cape Cod, InvivoGen), heat-killed Candida albicans (ratio 0.1:1; strain Inc.; East Falmouth, MA, USA) was conducted at NP ATCC 10231, InvivoGen), or live H. pylori (at MOI 0.2, 1, 5; concentrations determined be to below the threshold for WT strain p12, cultured in-house as described previously) (35). optical interference, following a protocol optimized for NPs H. pylori CFUs were determined by spectrophotometric (31), and sample absorbance was assessed using a Cytation 3 measurement of bacterial culture turbidity (OD 6 0 0 ; imaging reader (BioTek, Winooski, VT, USA). Endotoxin levels BioPhotometer plus, Eppendorf, Hamburg, Germany), were expressed as endotoxin units per milligram of AuNPs following an in-house CFU calibration curve. (EU/mg). Cell stimulation was performed in the presence or absence of 20 µg/mL AuNPs. The final serum concentration of each well Human Monocyte Isolation was adjusted to 5%. The primary monocyte response to stimuli Primary human monocytes were isolated from buffy coats of 20 was assessed as cytokine analysis in the 24 h supernatants. For healthy anonymous donors (provided by the blood bank of stimulation with H. pylori, antibiotics were absent during the Salzburg, Austria, following overnight refrigeration), with cells primary stimulation to ensure bacterial integrity during the from 4-8 buffy coats used for each primary stimulus. The study primary activation/memory induction phase. Antibiotics were was conducted in accordance with the Declaration of Helsinki, added into the culture medium for both the resting and challenge and under Austrian national guidelines. According to Austrian phases, to avoid unwanted activation by residual bacteria and to regulations, no informed consent is required if blood cells maintain the same culture conditions as for other stimuli. derived from anonymous healthy donors, discarded after plasmapheresis (buffy coats) are used, therefore no additional Monocyte Memory Innate Response approval by the national ethics committee was necessary. After the primary response and supernatant collection, cells were Peripheral blood mononuclear cells were obtained by Ficoll- rested in fresh culture medium for 6 days, with medium changes Paque gradient density separation (GE Healthcare, Bio-Sciences on days 4 and 6. A resting period of 6 days was sufficient for the AB, Uppsala, Sweden). Monocytes were further isolated by complete extinction of monocyte activation induced by the CD14 + magnetic microbead separation (Miltenyi Biotec, different stimuli, based on the production of inflammation- Bergisch Gladbach, Germany) following the manufacturer’s related factors. That monocytes were no longer activated was instructions. The resulting cell suspension was monitored for assessed by measuring cytokine production in the 6-day Frontiers in Immunology | www.frontiersin.org 3 November 2021 | Volume 12 | Article 751683 Swartzwelter et al. Modulation of Innate Memory by Gold Nanoparticles supernatant (representing the cytokine production from day 4 to 6 days after monocyte exposure to NPs for 24 h (Figure 1E). 6; data not shown) and by challenging the primed cells with Particles could be observed within endosomes, but not free in the culture medium alone (see first column on the left “challenged by cytosol or within nuclei. This is in agreement with the notion that medium” in all the figures reporting innate memory results). At particles are endocytosed and kept within vesicles for eventual day 7, cells were exposed to fresh culture medium alone of degradation, implying a mechanism of silent, non-inflammatory containing 5 ng/mL of LPS (from Escherichia coli O55:B5; elimination of foreign/anomalous materials. The absence of Sigma-Aldrich). Supernatants were collected after 24 h for AuNPs in the cytoplasm suggests that the particles are unable to cytokine analysis. destabilize the vesicle membrane and, consequently, to induce the activation of cytoplasmic inflammasomes by released lysosomal Cytokine Analysis enzymes and mitochondrial ROS. Importantly, despite the lack of Production of TNFa and IL-1Ra was measured in the culture inflammatory activation, the mechanism of silent elimination was supernatants by ELISA (R&D Systems, Inc., Minneapolis, MN, reported as able to “prime” macrophages and induce a protective USA). All other cytokines and chemokines were measured using a innate memory vs. subsequent challenges (40). At this timepoint, ProcartaPlex multiplex assay (Thermo Fisher Scientific, Waltham, no appreciable differences were noted in intracellular NP number, MA, USA). The lower detection limits for the assays used was: size and distribution across all experimental conditions (not TNFa, 15.6 pg/mL; IL-6, 9.4 pg/mL; IL-1Ra, 93.8 pg/mL; IL-10, shown), and no morphological alterations in monocytes were 7.1 pg/mL; MCP-1, 15.0 pg/mL; IL-1a, 9.5 pg/mL; MIP-1a, observed (Figure 1F). 8.9 pg/mL; MIP-1b, 110.1 pg/mL; GROa, 3.0 pg/mL; IP-10, 23.4 pg/mL; IL-8, 31.2 pg/mL. Two ELISA replicates were run Effect of AuNPs on the Primary Innate for each sample, and each experimental condition was tested with Response Induced by Soluble vs. duplicate samples. Particulate Microbial Stimuli We aimed to determine whether the presence of AuNPs might Statistical Analysis interfere with the induction of innate immune memory by Cytokine levels are reported as ng/106 plated monocytes. microbial stimuli. In particular, we wanted to examine possible Graphical presentations and statistical analysis were obtained differential effects on innate memory induced by particulate using Graphpad Prism 9 (GraphPad Inc., La Jolla, CA, USA). stimuli (whole microorganisms) or by microbial molecules. Data are shown as averages of biological triplicates or as averages Freshly isolated human blood monocytes were exposed for of technical replicates of biological duplicates. Statistical analysis 24 h in vitro to culture medium alone or containing one of was conducted using one-way ANOVA with the Fisher’s LSD four microbial agents: the bacterial surface molecule muramyl post hoc test for multiple comparisons. The Shapiro-Wilk dipeptide (MDP, 10 µg/mL); heat-killed gram-positive normality test was conducted on each data set prior to Staphylococcus aureus (S. aureus, ratio 1:1 with monocyte); the ANOVA, to ensure normal distributions. fungal polysaccharide b-glucan (2 µg/mL) and heat-killed Candida albicans (C. albicans, at a ratio 0.1:1 with monocytes). Concentrations were selected based on preliminary experiments RESULTS AND DISCUSSION and literature data as able to induce a significant but suboptimal innate immune activation (19, 41, 42; data not shown). AuNP Characterization The direct, primary response of monocytes to microbial In this study, we used gold nanoparticles (AuNPs in 2.2 mM stimuli was evaluated in the absence or in the presence of sodium citrate) of an average diameter of 51 ± 4 nm AuNPs (20 µg/mL), and assessed in the 24-h supernatant as (Figures 1A–E). Particle size and uniformity was confirmed by production of innate/inflammatory cytokines and chemokines. UV-vis (a single peak found at 531 nm), while DLS revealed a The size of AuNPs was chosen based upon preliminary data hydrodynamic size of 59 ± 16 nm, and a z-potential of about -39 ± using AuNPs of different sizes, which suggested that 50 nm 3 mV. The stock concentration following synthesis was 278 µg/mL AuNPs were the best for observing effects on innate memory (corresponding to a particle concentration of 2x1011 NPs/mL, to (23). The concentration was selected as the highest non-toxic and 1.4 mM Au, and to a surface area of 1.7x103 mm2/mL), with endotoxin-free concentration (data not shown). an endotoxin contamination (determined by LAL assay) of Figure 2 shows the primary response of monocytes in terms 3.97 EU/mg (Figure 1A). Endotoxin may activate monocytes at of production of the inflammatory cytokine TNFa, measured in concentrations above 0.1 EU/mL; our preparation thus allowed for monocytes from 2-4 individual donors. As expected, cells a NP working concentration in culture of 20 µg AuNPs/mL, exposed to culture medium alone or containing the endotoxin- containing 0.079 EU/mL of endotoxin, which is below the free AuNPs did not produce appreciable levels of TNFa (<0.3 ng/ endotoxin activation threshold (36, 37). Prior to addition into 106 monocytes). Stimulation of monocytes for 24 h with bacterial culture, AuNP were incubated in 50% AB serum, to better mimic MDP or killed gram-positive S. aureus resulted in a substantial the physiological conditions of NP interaction with human production of TNFa, which was not overall significantly immune cells (38). Formation of a serum-dependent biocorona impacted by the presence of AuNPs, although a decrease in the on the NP surface avoided particle aggregation in culture medium response to S. aureus was evident for cells of 3 out of 4 donors (32, 39). The presence of AuNPs within cells was assessed by TEM (Figures 2A, B). Cells were also stimulated with C. albicans- Frontiers in Immunology | www.frontiersin.org 4 November 2021 | Volume 12 | Article 751683 Swartzwelter et al. Modulation of Innate Memory by Gold Nanoparticles A B C D E F FIGURE 1 | Gold nanoparticle characterization. (A) Summary of characteristics of the AuNP batch used in this study; (B) TEM image and size distribution (calculated via ImageJ); (C) Atomic Force Microscopy image; (D) UV-vis spectrum, hydrodynamic size distribution calculated by DLS, and z-potential; (E) TEM and (F) SEM images of human primary monocytes, pre-exposed to AuNP for 24 h and then cultured for 6 additional days. derived b-glucan and with the whole killed C. albicans can be observed on cells from many donors, these effects being organisms. Both fungal agents also induced TNFa production, variable (increase or decrease of the response) depending on the although this increase did not attain statistical significance for donor and irrespective of the inflammatory agent. Only in the b-glucan (only two subjects could be tested; Figure 2C). Co- case of C. albicans was a similar decrease observed in all donors, exposure of monocytes to C. albicans and AuNPs caused a thus reaching statistical significance (Figure 2D). significant suppression of TNFa production in cells from all donors (Figure 2D). To investigate whether the AuNP effect Effect of AuNPs on the Memory Innate observed for C. albicans-stimulated TNFa production was Response Induced by Soluble vs. common to other C. albicans-induced factors, we examined the Particulate Microbial Stimuli production of the inflammatory cytokine IL-6, the anti- Following primary activation, cells were rested for 6 days. This inflammatory factor IL-1Ra and the chemokine MCP-1/CCL2, allows monocytes sufficient time to return to quiescence, prior to and found that AuNPs did not affect the stimulus-induced restimulation. Cell number and morphology following resting production of any of them (Supplementary Figure 1). appeared consistent (by visual inspection) across wells from These results confirm previous observations that AuNPs do different primary conditions. After resting, cells were not have a substantial impact on the innate/inflammatory challenged with either medium alone or 5 ng/mL of LPS, in response of human monocytes to microbial stimuli, but that a order to observe whether the previous exposure to inflammatory partial reduction of the response to whole microorganisms can agents resulted in development of an innate immune memory be observed in the majority of donors. Notably, individual effects (an increased or decreased response compared to control Frontiers in Immunology | www.frontiersin.org 5 November 2021 | Volume 12 | Article 751683 Swartzwelter et al. Modulation of Innate Memory by Gold Nanoparticles A B C D FIGURE 2 | Effect of AuNPs on the primary response of monocytes exposed to soluble or particulate microbial stimuli. CD14+ monocytes were exposed to MDP (10 µg/mL) (A), S. aureus (ratio 1:1; S.a.) (B), b-glucan (2 µg/mL; b-g) (C) or (C) albicans (ratio 0.1:1; C.a.) (D) for 24 h in the presence or absence of AuNPs (20 µg/mL, indicated by blue bars). The inflammatory response is reported in terms of production of TNFa (ng/106 monocytes). Values from individual donors are depicted concurrent with mean cytokine production. Relevant p values are indicated when < 0.05. n = 4 (A, B, D), n = 2 (C). unprimed cells). The in vitro model adopted for assessing the need for an individual profiling of innate and memory memory induction is depicted in Figure 3. Challenge with responses, in order to predict reactivity to future challenges. medium alone resulted in TNFa levels below the detection Such profiling should include the production of a number of limit of the assay for all primed cells, indicating that cells had inflammatory and anti-inflammatory factors in response to returned to baseline TNFa production (Figure 4; medium- different microbial challenges, in order to assess the overall challenged cells are grouped into one bar that includes every balance between inflammation and anti-inflammation (26). priming condition tested). Restimulation with LPS induced The memory response of MDP-primed cells was of tolerance significant production of TNFa, which was comparable type, relative to TNFa production, with a decreased production between medium- and AuNP-primed cells, suggesting that pre- of the inflammatory cytokine compared to medium-primed cells. exposure to AuNPs was unable to induce a consistent memory Also in this case, the global difference was not statistically response. It should be however noted that, while the average significant (p=0.0956), due to the interindividual variability production is not statistically different between control and (with monocytes from 1 out of 4 donors showing no change). AuNP-primed monocytes, at the individual level there are Cells primed with MDP + AuNPs showed a significant tolerance cases in which AuNP-primed cells respond to challenge with at restimulation, compared to cells primed with either stimulus an increased TNFa production, others in which there is a alone, confirming the tendency to tolerance observed with single decrease, and others in which there is no change (see for priming agents (Figure 4A). Restimulation of cells primed with instance the four donors in Figure 4C). This again underlines S. aureus (which contains MDP as part of its surface structure) Frontiers in Immunology | www.frontiersin.org 6 November 2021 | Volume 12 | Article 751683 Swartzwelter et al. Modulation of Innate Memory by Gold Nanoparticles FIGURE 3 | The time course of an in vitro model of innate immune memory. Fresh naïve monocytes are activated by exposure in culture to different stimuli for 24 h (primary response). After elimination of stimuli, cell activation subsides with time (in our in vitro model 6 days are sufficient), during which period cells return to a resting status. Upon restimulation, cells that were not previously exposed to activating agents (unprimed) develop a secondary response of a given intensity. Conversely, cells that were previously primed and activated can react to restimulation with a secondary “memory” response, either more powerful (training/ potentiation) or reduced (tolerance), compared to unprimed cells. demonstrated that, similar to MDP, S. aureus is a potent inducer CCL3 and MIP-1b/CCL4; and three CXC chemokines: GROa/ of innate memory in the direction of tolerance (Figure 4B). CXCL1, IP-10/CXCL10 and IL-8/CXCL8) (Supplementary Figure Unlike the memory effect upon MDP priming, AuNPs had no 2). Two of these factors (IL-1Ra and CCL2/MCP-1) were effect on S. aureus-driven tolerance. The difference in the effect of spontaneously produced at high levels, and their levels were not AuNPs on memory induced by MDP vs. the entire S. aureus increased in response to the LPS challenge. Similar to TNFa, priming bacteria might be ascribed to the different mechanisms of with C. albicans did not induce memory (either potentiation or primary cell activation (which then initiate the epigenetic and decrease of the secondary response) in terms of production of any of metabolic reprogramming responsible for the establishment of these factors. Likewise, the presence of AuNPs at priming did not memory), MDP mainly acting through NOD2 in the cytoplasm have any significant effect (Supplementary Figure 2). after receptor-independent endocytosis/transport through In all cases, again it should be noted that the interindividual membrane channels (43, 44), while the whole bacteria variability is high and that, while the average values are not principally interact with the plasma membrane through statistically different, the individual effects can be substantial lipoteichoic acid activation of TLR2, thereby initiating an both as decrease and increase of the memory response in the MyD88-dependent signaling pathway (45, 46). The metabolic presence of AuNPs. cost and pathway involvement of bacterial phagocytosis compared to uptake of soluble factors most likely also Effect of AuNPs on Primary and contribute to the different memory profiles generated by Memory Innate Responses Induced S. aureus and MDP, although this remains unstudied to date by Live Bacteria (47). Thus, the presence of AuNPs, which are endocytosed, may Previous data suggest that the impact of AuNPs on innate memory have interfered with the MDP-dependent mechanism of memory induced by bacteria may vary depending on whether bacteria (BCG generation, while unable to affect the memory mechanisms in this specific case) are viable or not (23). We have therefore also initiated extracellularly by S. aureus. tested the effect of AuNPs on responses induced by a live Upon challenge, cells primed with the fungal agents b-glucan and microorganism, the gram-negative H. pylori, so as to compare C. albicans did not demonstrate an innate memory response, as their such effect with those induced by killed microorganisms (S. TNFa production did not differ from that medium-primed aureus and C. albicans). Monocytes were primed in vitro for 24 h (Figures 4C, D). In both cases, the presence of AuNPs at priming by live H. pylori at three concentrations (at MOI 0.2, 1 and 5) in the did not have any effect on the secondary response at challenge. To absence or presence of AuNPs. Primary stimulation with H. pylori make sure that the lack of memory induction was not restricted to the revealed a potent dose-dependent induction of TNFa production production of a single inflammatory factor, in the case of C. albicans (Figure 5), which was significantly suppressed by the presence of priming we also assessed the production of another inflammatory AuNPs, with the most robust suppression present at the lowest H. cytokine (IL-6), two anti-inflammatory factors (IL-10 and IL-1Ra) pylori dose (MOI 0.2). Thus, similar to what was observed with live and six chemokines (three CC chemokines: MCP-1/CCL2, MIP-1a/ BCG (23) and, to a lower extent, with killed whole bacteria Frontiers in Immunology | www.frontiersin.org 7 November 2021 | Volume 12 | Article 751683 Swartzwelter et al. Modulation of Innate Memory by Gold Nanoparticles A B C D FIGURE 4 | Effect of AuNPs on the memory response of monocytes primed by soluble or particulate microbial stimuli. CD14+ monocytes were primed with MDP (A), S. aureus (B), b-glucan (C) or C. albicans (D) for 24 h in the presence or absence of AuNPs (20 µg/mL, indicated by blue bars), then washed and rested for 6 days. Cells were then challenged with LPS (5 ng/mL) for 24 h, and supernatants collected for cytokine measurement. The inflammatory response is reported in terms of production of TNFa (ng/106 monocytes), and individual donor values are depicted concurrent with mean cytokine production. p values are indicated when < 0.05 for the comparisons between unprimed and primed groups and priming with and without NPs, n = 4 (A, C, D), n = 8 (B). (Figures 2B, D), AuNPs are capable of interfering with the primary primed cells, although again different behaviors were evident innate response induced by live H. pylori. between donors. The memory response of H. pylori-primed cells Induction of innate immune memory by H. pylori was assessed revealed a potent induction of an innate immune tolerance in using the same in vitro model described previously for soluble and terms of TNFa, IL-6, IL-10 and the CXC chemokine IP-10, particulate microbial stimuli. Following 6 days of resting, control though not for IL-1a, IL-1Ra and in all the other chemokines and primed cells were challenged with 5 ng/ml of LPS for 24 h. To tested (Figure 6 and Supplementary Figure 3). The presence of better assess the memory induction by H. pylori and the AuNP AuNPs during priming with H. pylori did not alter the H. pylori- impact, in addition to TNFa we have examined several other induced memory effect on any of the cytokines and chemokines important inflammation-related cytokines and chemokines. tested, with the exception of the anti-inflammatory cytokine IL 10. Results in Figure 6 show the memory response of H. pylori- In this case, the H. pylori-induced tolerance was significantly primed cells in terms of production of two key inflammatory enhanced by AuNPs (Figure 6D), an effect evident at all H. factors, TNFa and IL-6, and of two anti-inflammatory cytokines, pylori priming concentrations (data not shown). IL-1Ra and IL-10. Additional factors are reported in Thus, monocytes exposed to live H. pylori can mount a potent Supplementary Figure 2. Only the results at H. pylori MOI 0.2 inflammatory response that primes cells towards a generally less are shown, since no substantial differences were observed at higher potent secondary memory response, in terms of production of two concentrations. Upon challenge with LPS, cells primed with inflammatory factors (TNFa and IL-6), an anti-inflammatory medium exhibited elevated production of all cytokines and cytokine (IL-10) and the chemokine IP-10. That H. pylori chemokines measured, except IL-1Ra and MCP-1, whose priming may affect the production of four different cytokines baseline levels were already high (Figure 6 and Supplementary suggests different levels of epigenetic/metabolic reprogramming, Figure 3). Overall, the secondary response of cells primed with likely dependent on the multiplicity of its cell activation modes. AuNPs was not significantly different from that of medium- In fact, H. pylori can interact with TLR2 on the cell membrane, Frontiers in Immunology | www.frontiersin.org 8 November 2021 | Volume 12 | Article 751683 Swartzwelter et al. Modulation of Innate Memory by Gold Nanoparticles CONCLUSIONS The aim of this study was to assess whether engineered AuNPs, a nanomaterial with wide applications in many fields including medicine and generally considered safe, are able to modulate the innate immune/inflammatory responses of human subjects. This would contribute on one side to the implementation of a more thorough safety evaluation of AuNPs and, on the other hand, it could open the way to a targeted use of this nanomaterial for the therapeutic modulation of innate immunity/inflammation in several immune-related and inflammatory diseases. In particular, this study has addressed innate memory, i.e., the ability of monocytes/macrophages to activate a more protective reaction to a challenge when previously exposed to the same or a different infectious agent (6, 7, 9–17). Following previous studies showing that endotoxin-free Au and other NPs are essentially unable to induce innate/inflammatory responses and innate memory per se, but could at least in part modulate the memory induced by microbial agents (21–26, 72), here we have examined if the nature of the memory-inducing microbial agents could determine the FIGURE 5 | Effect of AuNPs on the primary response of monocytes to live H. pylori. CD14+ monocytes were stimulated with medium alone or containing capacity of AuNPs to interfere with the development of innate live H. pylori (MOI: 0.2, 1, 5) for 24 h in the presence or absence of AuNPs memory. To study innate memory, we have taken advantage of a (20 µg/mL, indicated by blue bars). The inflammatory response is reported in realistic in vitro model, based on human primary monocytes terms of production of TNFa (ng/106 monocytes). Values from individual exposed to microbial agents and to AuNPs coated with human donors are depicted concurrent with mean cytokine production. p values are serum. Based on our preliminary findings, we can draw the indicated when < 0.05 for the comparison between stimulation with or without NPs, n = 4. following conclusions and formulate the following hypotheses: 1. AuNPs generally decrease the inflammatory activation of possibly through Hsp60 (48–50), while its LPS has very limited monocytes induced by whole microorganisms, both killed inflammatory activity and does not trigger a significant TLR4- (C. albicans, S. aureus) and viable (H. pylori in this study, mediated inflammation (51–53). Of note, the cag pathogenicity BCG in ref. 23). Conversely, monocyte activation induced by island (cagPAI), which mediates H. pylori pathogenesis in gastric microbial molecules (b-glucan and MDP in this study, LPS in epithelial cells, is less important in macrophages and dendritic cells refs. 22, 23, 25, 26) is not consistently affected by co-exposure (50, 54). H. pylori can induce inflammatory activation of innate cells to AuNPs. This may be ascribed to a possible interference of (53, 55–57), possibly through the non-enzymatic interaction of AuNPs (which are readily and abundantly taken up by secreted urease with receptors/acceptors on the cell membrane (55, monocytes and stored in endosomal vesicles) with the 56), promote the macrophage M1 inflammatory phenotype through intracellular trafficking of phagocytosed microorganisms NOD1 (58), and survive for at least 24 hours within and the phagocytosis-dependent signaling pathways (47). phagolysosomes after ingestion thereby inducing potent ROS 2. The capacity of AuNPs to modulate innate memory induced by production (59–61). By inducing phosphorylation of the NFkB microbial agents seems to be specifically restricted to some agents p65 subunit at Ser-537, also the integrin-like kinase (ILK) promotes and to some of the memory response parameters (production H. plyori-induced TNFa production (62). All these mechanisms of levels of different cytokines), and appears to be independent of the inflammatory activation will likely induce a multitude of different effect on the primary response. In fact, AuNPs increase the metabolic and epigenetic changes resulting in a complex innate tolerance memory effect induced by MDP on the inflammatory memory profile. The tolerance memory response observed for cytokine TNFa, whereas no effect of the primary response to TNFa and IL-6, two cytokines mainly dependent on the MDP could be observed. In the case of H. pylori, while AuNPs activation of the NFkB pathway, suggests that the TLR2- could significantly decrease the TNFa primary response, no effect dependent priming may be principally involved. Conversely, the on the tolerance memory response was evident for the same effects on IL-10 and IP-10, two factors that largely depend on cytokine, while a significant decrease of the IL-10 memory interferon activation, are more likely mediated by other response was observed. Since IL-10 is an anti-inflammatory mechanisms, including the NOD1 pathway through the TRAF3- factor, its decrease would result in an overall increase of dependent induction of IRF3 and 7 and the production of type I inflammation. The circumscribed effects of AuNPs on the IFN (63–70), which in turn activates the JAK/STAT signaling memory production of some cytokines in response to some pathway (71). In this perspective, the capacity of AuNPs to microbial agents could be explained as interference with interfere with H. pylori-induced memory, which is evident only in distinct mechanisms of cell activation and reprogramming, the case of IL-10, suggests that AuNPs may increase the H. pylori although experimental evidence is currently missing. The effect through production of type I IFN. physical interaction between AuNPs and microbial agents at Frontiers in Immunology | www.frontiersin.org 9 November 2021 | Volume 12 | Article 751683 Swartzwelter et al. Modulation of Innate Memory by Gold Nanoparticles A B C D FIGURE 6 | Effect of AuNPs on the memory response of monocytes primed by live (H) pylori. CD14+ monocytes were exposed to medium or H. pylori (at MOI 0.2) for 24 h in the presence or absence of AuNPs (20 µg/mL, indicated by blue bars), then washed and rested for 6 days. After resting, cells were challenged with LPS (5 ng/mL) for 24 h, supernatants were collected and examined for the production of TNFa (A), IL-6 (B), IL-1Ra (C) and IL-10 (D). Values from individual donors are depicted concurrent with mean cytokine production. p values are indicated when < 0.05 for the comparisons between unprimed and primed groups and priming with and without NPs, n = 4. . priming may lead to a different recognition/activation profile and AUTHOR CONTRIBUTIONS trigger distinct epigenetic or metabolic pathways responsible of memory establishment. VP and FB synthesized and characterised the nanomaterials. BS, 3. The most striking observation made in this study, which confirms SM, TF, AV, and AL contributed to the experimental work. BS previous reports, is that opposite innate memory responses can be and SM planned the study. AD, JH-H, PI, and DB evaluated the induced by the same agents and in the same conditions in results and monitored the experimental work. BS and DB wrote monocytes from different subjects. Thus, the same microbial the manuscript. BS prepared the figures and performed the agent, alone or in combination with AuNPs, can cause statistical analysis. All authors contributed to the article and potentiation, tolerance or no effect on cells from different approved the submitted version. donors. This suggests two considerations: first, it is not possible to classify the effects of NPs on innate memory in general terms; second, in order to know whether some NPs (to be used in medical applications) may have a detrimental effect on a patient, it FUNDING is necessary to obtain a personalized innate memory profile. This work was supported by the EU Commission H2020 projects PANDORA (GA 671881) and ENDONANO (GA 812661), the Italian MIUR InterOmics Flagship projects MEMORAT and DATA AVAILABILITY STATEMENT MAME, the Italian MIUR/PRIN-20173ZECCM, the Priority program ACBN (Allergy Cancer BioNano Research Centre) of The original contributions presented in the study are included in the University of Salzburg, the Cancer Cluster Salzburg, the the article/Supplementary Material. Further inquiries can be Research Grant from the University of Salzburg, and the directed to the corresponding author. Austrian Science Fund (FWF) Grant Nr. P 29941. Frontiers in Immunology | www.frontiersin.org 10 November 2021 | Volume 12 | Article 751683 Swartzwelter et al. Modulation of Innate Memory by Gold Nanoparticles production of IL-6 (A), IL-1Ra (B) and MCP-1/CCL2 (C) and expressed as ng/106 ACKNOWLEDGMENTS monocyte. Values from individual donors are depicted concurrent with mean cytokine production. Relevant p values are indicated when < 0.05. n = 4. We thank Marinella Pirozzi (EuroBioImaging facility at CNR) for the TEM images of NP uptake, and the Bioimaging Unit of Supplementary Figure 2 | Effect of AuNPs on the memory response of the Stazione Zoologica Anton Dohrn for providing technical monocytes primed by C. albicans. CD14+ monocytes were exposed to medium or assistance for SEM images. We thank Charles A. Dinarello C. albicans (ratio 0.1:1) for 24 h in the presence or absence of AuNPs (20 µg/mL, (University of Denver) for generously providing b-glucan, and indicated by blue bars), then washed and rested for 6 days. After resting, cells were Iris Gratz (Paris-Lodron University of Salzburg) for generously challenged with LPS (5 ng/mL) for 24 h. Supernatants were collected and cytokine providing heat-killed C. albicans. production measured: IL-6 (A), IL-10 (B), IL-1Ra (C) MCP-1/CCL2 (D), MIP-1/ CCL3 (E), MIP-1b/CCL4 (F), GROa/CXCL1 (G), IP-10/CXCL10 (H), IL-8/CXCL8 (I). Individual donor values are depicted concurrent with mean cytokine production (ng/106 monocytes). n = 4. SUPPLEMENTARY MATERIAL Supplementary Figure 3 | Effect of AuNPs on the memory response of The Supplementary Material for this article can be found online monocytes primed by H. pylori. CD14+ monocytes were exposed to medium or H. at: https://www.frontiersin.org/articles/10.3389/fimmu.2021. pylori (at MOI 0.2) for 24 h in the presence or absence of AuNPs (20 µg/mL, 751683/full#supplementary-material indicated by blue bars), then washed and rested for 6 days. After resting, cells were challenged with LPS challenge (5 ng/mL) for 24 h, and supernatant were collected Supplementary Figure 1 | Effect of AuNPs on the primary response of for evaluation of cytokines and chemokines: IL-1a (A), IL-8/CXCL8 (B), MCP-1/ monocytes to killed C. albicans. CD14+ monocytes were stimulated with medium CCL2 (C), MIP-1a/CCL3 (D), MIP-1b//CCL4 (E), IP-10/CXCL10 (F). 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Type I IFN Induces IL-10 Production in an IL-27–Independent Conflict of Interest: The authors declare that the research was conducted in the Manner and Blocks Responsiveness to IFN-g for Production of IL-12 and absence of any commercial or financial relationships that could be construed as a Bacterial Killing in Mycobacterium Tuberculosis–Infected Macrophages. potential conflict of interest. J Immunol (2014) 193:3600–12. doi: 10.4049/jimmunol.1401088 68. Fan Y-H, Roy S, Mukhopadhyay R, Kapoor A, Duggal P, Wojcik GL, et al. Publisher’s Note: All claims expressed in this article are solely those of the authors Role of Nucleotide-Binding Oligomerization Domain 1 (NOD1) and its and do not necessarily represent those of their affiliated organizations, or those of Variants in Human Cytomegalovirus Control In Vitro and In Vivo. Proc the publisher, the editors and the reviewers. Any product that may be evaluated in Nat Acad Sci USA (2016) 113:E7818–27. doi: 10.1073/pnas.1611711113 this article, or claim that may be made by its manufacturer, is not guaranteed or 69. Neuper T, Ellwanger K, Schwarz H, Kufer TA, Duschl A, Horejs-Hoeck J. endorsed by the publisher. NOD1 Modulates IL-10 Signalling in Human Dendritic Cells. Sci Rep (2017) 7:1–12. doi: 10.1038/s41598-017-00691-x Copyright © 2021 Swartzwelter, Michelini, Frauenlob, Barbero, Verde, De Luca, 70. Ernst O, Glucksam-Galnoy Y, Bhatta B, Athamna M, Ben-Dror I, Glick Y, Puntes, Duschl, Horejs-Hoeck, Italiani and Boraschi. This is an open-access article et al. Exclusive Temporal Stimulation of IL-10 Expression in LPS-Stimulated distributed under the terms of the Creative Commons Attribution License (CC BY). Mouse Macrophages by cAMP Inducers and Type I Interferons. Front The use, distribution or reproduction in other forums is permitted, provided the Immunol (2019) 10:1788. doi: 10.3389/fimmu.2019.01788 original author(s) and the copyright owner(s) are credited and that the original 71. Majoros A, Platanitis E, Kernbauer-Hölzl E, Rosebrock F, Müller M, Decker T. publication in this journal is cited, in accordance with accepted academic practice. No Canonical and Non-Canonical Aspects of JAK–STAT Signaling: Lessons use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Immunology | www.frontiersin.org 13 November 2021 | Volume 12 | Article 751683