cells Article Gold Nanoparticles Modulate BCG-Induced Innate Immune Memory in Human Monocytes by Shifting the Memory Response towards Tolerance Benjamin J. Swartzwelter 1 , Francesco Barbero 2 , Alessandro Verde 1 , Maria Mangini 1 , Marinella Pirozzi 1 , Anna Chiara De Luca 1 , Victor F. Puntes 2 , Luciana C. C. Leite 3 , Paola Italiani 1 and Diana Boraschi 1, * 1 Institute of Biochemistry and Cell Biology, CNR, Via P. Castellino 111, 80131 Napoli, Italy; benjamin.swartzwelter@ibbc.cnr.it (B.J.S.); alessandro.verde@ibbc.cnr.it (A.V.); maria.mangini@ibbc.cnr.it (M.M.); marinella.pirozzi@ibbc.cnr.it (M.P.); annachiara.deluca@ibbc.cnr.it (A.C.D.L.); paola.italiani@ibbc.cnr.it (P.I.) 2 Institut Català de Nanociència i Nanotecnologia (ICN2), CSIC and The Barcelona Institute of Science and Technology (BIST), Campus UAB, Bellaterra, 08193 Barcelona, Spain; francesco.barbero@icn2.cat (F.B.); victor.puntes@icn.cat (V.F.P.) 3 Laboratório de Desenvolvimento de Vacinas, Instituto Butantan, São Paulo SP 05503-900, Brazil; luciana.leite@butantan.gov.br * Correspondence: diana.boraschi@ibbc.cnr.it  Received: 13 December 2019; Accepted: 8 January 2020; Published: 23 January 2020  Abstract: Innate immune memory is characterized by a modulation in the magnitude with which innate immune cells such as monocytes and macrophages respond to potential dangers, subsequent to previous exposure to the same or unrelated agents. In this study, we have examined the capacity of gold nanoparticles (AuNP), which are already in use for therapeutic and diagnostic purposes, to modulate the innate memory induced by bacterial agents. The induction of innate memory was achieved in vitro by exposing human primary monocytes to bacterial agents (lipopolysaccharide -LPS-, or live Bacille Calmette-Guérin -BCG) in the absence or presence of AuNP. After the primary activation, cells were allowed to return to a resting condition, and eventually re-challenged with LPS. The induction of memory was assessed by comparing the response to the LPS challenge of unprimed cells with that of cells primed with bacterial agents and AuNP. The response to LPS was measured as the production of inflammatory (TNFα, IL-6) and anti-inflammatory cytokines (IL-10, IL-1Ra). While ineffective in directly inducing innate memory per se, and unable to influence LPS-induced tolerance memory, AuNP significantly affected the memory response of BCG-primed cells, by inhibiting the secondary response in terms of both inflammatory and anti-inflammatory factor production. The reprogramming of BCG-induced memory towards a tolerance type of reactivity may open promising perspectives for the use of AuNP in immunomodulatory approaches to autoimmune and chronic inflammatory diseases. Keywords: innate immune memory; gold nanoparticles; monocytes; potentiation; BCG; inflammatory cytokines; anti-inflammatory cytokines 1. Introduction Innate immune memory is defined as the ability of innate immune cells to react differently to challenges based on previous stimulations [1–3]. While innate immune memory is the only type of immune memory in plants, invertebrates, and lower vertebrates that lack adaptive immunity, innate memory in higher vertebrates co-exists with adaptive immunological memory, which forms the basis of Cells 2020, 9, 284; doi:10.3390/cells9020284 www.mdpi.com/journal/cells Cells 2020, 9, 284 2 of 17 more effective and highly antigen-specific secondary responses [4–6]. In mammals, innate memory is non-specific, but in any case, aimed at developing a better-suited secondary response. The best-known phenomenon of innate memory in mammals is the so-called endotoxin tolerance, i.e., the decreased response to gram-negative bacterial endotoxin (lipopolysaccharide, LPS) upon repeated challenges, which aims at limiting the tissue damage that could be caused by a recurrent host inflammatory reaction [7–10]. More recent evidence has shown that previous challenge with some bacteria, such as the Mycobacterium bovis strain BCG, can induce an innate memory towards an enhanced and more protective secondary response (“trained” immunity) [11–14]. Among the innate immune cells that can develop memory, monocytes and macrophages are particularly important because of their evolutionarily conserved immune memory capacity and their role in modulating local immune responses, in addition to their direct ability to uptake foreign and endogenous agents that may pose a threat [15–18]. The protective scavenging role of monocytes/macrophages presents the question of whether and how foreign materials can impact innate immune memory in monocytes and macrophages. This is a critical issue that has to be considered in novel treatments and therapies that use biomedical materials. One class of materials that may have a particular impact on innate memory, because of their particulate nature, are nanomaterials, which have extensive clinical relevance and potential as metal-based imaging agents and as drug or vaccine carriers [19,20]. A wealth of studies on the immunological safety of medical nanomaterials have been conducted, leading to the design of nanoparticles (NP) that are immunocompatible, i.e., unable to trigger an immune/inflammatory reaction [21–23]. Gold NP (AuNP) are one of these immunosafe particles [24] and have already attained clinical relevance for uses such as specific cell targeting in photothermal and radiation-based treatments [25,26]. Beyond these, extensive potential exists for AuNP uses in a variety of diagnostic and therapeutic applications in humans [27]. For a more thorough assessment of the possible impact of AuNP on the host immune competence, it is important to investigate the effects of AuNP on innate memory, i.e., their capacity of altering or modulating the immune defensive reactivity to subsequent infectious or stressful agents/events. Some preliminary data suggest that this may be the case [28]. If indeed it could be possible to modulate innate immune/inflammatory reaction with AuNP, this would open the possibility of targeted interventions for limiting excessive inflammation in autoimmune, chronic inflammatory, and degenerative diseases, and likewise, to increase immune reactivity in situations of age- or disease-caused immunosuppression. The aim of the present study is to investigate whether AuNP are capable of inducing innate immune memory in human monocytes, and/or whether they may modulate memory induced by bacterial agents. We have used primary monocytes for studying the development of innate memory, thereby employing an in vitro model that reproduces a repeated exposure to foreign agents. Blood monocytes were chosen, as opposed to resident tissue macrophages, as they are the main inflammatory cells that engage with foreign materials during a tissue reaction and therefore those that most likely can develop memory of previous challenges. The use of human primary cells, rather than transformed cell lines or animals/animal cells, would ensure a higher predictivity and an improved relevance for the human situation in vivo. The results of this study confirm that AuNP do not activate innate immune/inflammatory reactions, being therefore in principle immunologically “safe”. However, we show here that AuNP can significantly modulate innate responses and innate memory induced by BCG, while remaining inactive the responses and memory induced by LPS. This suggests that AuNP can be used for regulating human innate reactivity and opens the way to future therapeutic applications. Cells 2020, 9, 284 3 of 17 2. Materials and Methods 2.1. Synthesis and Characterization of AuNP 2.1.1. AuNP Synthesis Synthesis of AuNP was conducted using wet chemistry methods as previously described by Bastús et al. [29]. Briefly, a 150 mL aqueous solution of sodium citrate (2.2 mM) was brought to a boil under reflux, after which 1 mL of 25 mM HAuCl4 was quickly injected into the citrate solution. Within several minutes the solution obtained a red hue, which is indicative of AuNP formation, resulting in ~10 nm NP seeds. This was followed by sequential growth-inducing steps, which consisted of adding further HAuCl4 to the sample solution, leading to the desired AuNP size. All reagents were obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA). 2.1.2. STEM, UV-Vis, DLS, and Z-Potential Characterization STEM (scanning transmission electron microscopy) images were acquired using a FEI Magellan XHR scanning electron microscope (SEM) (FEI, Hillsboro, OR, USA), operated in transmission mode at 20 kV. AuNP samples stabilized with polyvinylpyrrolidone (55 kDa) to avoid aggregation were drop-cast (4 µL) onto a carbon-coated copper TEM grid and left to dry at room temperature. Images were acquired, and at least 500 particles from different regions of the grid were computer-counted using an in-house ImageJ macro. UV-Vis spectra of AuNP suspensions in sodium citrate were acquired using a Shimadzu UV-2400 spectrophotometer (SSI; Kyoto, Japan), reading a spectral range from 300 to 750 nm. Acquisitions occurred at room temperature with AuNP samples placed in 1 mL cuvettes, and milliQ water was used as a reference. The hydrodynamic diameter and Z-potential of AuNP in sodium citrate were acquired by dynamic light scattering and laser doppler velocimetry, using a Malvern Zetasizer Nano ZS instrument (Malvern Panalytical Ltd., Malvern, UK) equipped with a light source wavelength of 632.8 nm and a fixed scattering angle of 173◦ . Diameters were reported as distribution intensity calculated by non-negative least squares (NNLS) analysis. The software was arranged with the parameters of the refractive index and the absorption coefficient of AuNP and the solvent viscosity of water at 25 ◦ C. 2.2. LAL Assay Endotoxin contamination of AuNP was assessed using the chromogenic Pyrochrome LAL assay (Associates of Cape Cod, Inc.; East Falmouth, MA, USA), following a protocol that has been optimized for use with nanoparticulate samples [30]. Interference of AuNP with the LAL assay readout was assessed, and AuNP were tested at concentrations below the interference threshold. LAL contamination was expressed in terms of endotoxin units per milligram of AuNP (EU/mg). 2.3. Human Monocyte Isolation Blood was obtained from healthy donors. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Regional Ethics Committee for Clinical Experimentation of the Tuscany Region (Ethics Committee Register n. 14,914 of May 16, 2019). None of the subjects was vaccinated with BCG or was positive in the tuberculin test. Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll-Paque gradient density separation (GE Healthcare, Bio-Sciences AB, Uppsala, Sweden). CD14+ monocytes were isolated from PBMC using anti-CD14 antibody-bearing magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Cell viability was assessed by trypan blue dye exclusion and found to be > 95%. Monocyte purity was determined microscopically after cytocentrifugation and differential staining Cells 2020, 9, 284 4 of 17 with a modified Wright-Giemsa dye (Diff-Quik; Medion Diagnostics, Duedingen, Switzerland). Only preparations with >95% purity were used. 2.4. Monocyte Stimulation 2.4.1. Biocorona Formation on AuNP AuNP were incubated for 1 h at 37 ◦ C in heat-inactivated human AB serum (Sigma-Aldrich, Inc.) at a 1:1 ratio, allowing a protein bio-corona to form around the NP. Aggregation of particles upon addition to cell culture was thus avoided [31]. The AuNP-serum mixture was used directly for stimulation of monocytes, accounting for any serum added concurrently with the AuNP. 2.4.2. Primary Monocyte Response Freshly isolated monocytes were suspended in culture medium (RPMI 1640 + Glutamax-I; GIBCO by Life Technologies, Paisley, UK) supplemented (unless otherwise noted) with 50 µg/mL gentamicin sulfate (GIBCO), and 1 × 105 cells/well were seeded in 96-well flat bottom plates (Corning® Costar® ; Corning Inc. Life Sciences, Oneonta, NY, USA). For primary stimulation, monocytes were incubated, in a final volume of 0.2 mL, with culture medium (negative control), AuNP (10 µg/mL), LPS (1 ng/mL; from E. coli O55:B5; Sigma-Aldrich, Inc.), live BCG (Mycobacterium bovis BCG Moreau, at a multiplicity of infection (MOI) of 1; Instituto Butantan, São Paulo, Brazil), LPS + AuNP, or BCG + AuNP. Final AB serum concentration was adjusted at 5%. Priming in BCG experiments occurred in the absence of antibiotics. The MOI = 1 was selected from dose-response experiments as the lowest dose that yielded full cell activation in the absence of cell death (not shown). The LPS concentration was selected from dose–response experiments as a non-toxic concentration able to induce significant tolerance type memory (data not shown). After 24 h supernatants were collected and frozen at −20 ◦ C for subsequent cytokine analysis. By visual inspection, cell viability did not change in response to the different treatments. 2.4.3. Secondary Monocyte Response Following primary stimulation, culture wells were replenished with fresh culture medium containing 5% AB serum, and cells were rested for 6 days, refreshing medium every 2 days. The resting period was selected as sufficient for achieving return to baseline production of inflammatory cytokines in cells previously activated for 24 h with LPS (0.1–10 ng/mL) or BCG (0.1–10 MOI). At day 7, cells were challenged with either culture medium or 5 ng/mL LPS (a 5× higher concentration than in the primary response) for 24 h, and then supernatants were collected and frozen at −20 ◦ C for subsequent cytokine analysis. Cell viability did not change in response to the different treatments (daily visual inspection of each well). 2.5. Transmission Electron Microscopy Cells in 6-well plates (1 × 106 cells/well) were exposed to AuNP (20 µg/mL) alone or in the presence of LPS (1 ng/mL) or BCG (MOI = 1) for 24 h. Cells were then fixed in 1% glutaraldehyde diluted in HEPES (0.2 M) for 1 h at room temperature, washed with PBS, placed in PBS + 1% BSA and carefully dislodged using a plastic scraper. Cells were washed and pelleted by centrifugation, stained with osmium tetroxide and uranyl acetate, and progressively dehydrated using 30%, 50%, 70%, 90%, and 100% EtOH, and then incubated in acetone. The cell pellet was embedded in an EPON resin and polymerized by baking at 60 ◦ C for 48 h, and ultrathin sections (70 nm) were obtained using a Leica EM UC7 ultramicrotome (Leica Microsystems). TEM images were obtained using a FEI Tecnai 12 transmission electron microscope. Cells 2020, 9, 284 5 of 17 2.6. Cytokine Analysis Cells 2020, 9, 284 5 of 17 The levels of the inflammatory cytokines TNFα and IL-6 and of the anti-inflammatory factors IL-1Ra and IL-10 were assessed by ELISA (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions. Absorbance of assay wavelength was measured at 450 nm (subtracting manufacturer’s instructions. Absorbance of assay wavelength was measured at 450 nm (subtracting background present at 550 nm) using a Cytation 3 imaging reader (BioTek, Winooski, VT, USA). background present at 550 nm) using a Cytation 3 imaging reader (BioTek, Winooski, VT, USA). 2.7. Statistical Analysis 2.7. Statistical Analysis Data from cytokine measurements have been analyzed using GraphPad Prism7 software Data from cytokine measurements have been analyzed using GraphPad Prism7 software (GraphPad Inc., La Jolla, CA, USA), and are presented in terms of ng/1066 plated monocytes. Results (GraphPad Inc., La Jolla, CA, USA), and are presented in terms of ng/10 plated monocytes. Results are reported as mean ± SEM of values from different donors/experiments. The use of SEM is meant are reported as mean ± SEM of values from different donors/experiments. The use of SEM is meant for showing the precision of the mean. In each experiment, two replicate wells were prepared for for showing the precision of the mean. In each experiment, two replicate wells were prepared for each experimental point, and two separate ELISA determinations were run on each well. Statistical each experimental point, and two separate ELISA determinations were run on each well. Statistical significance of differences is indicated by p values, calculated using a paired Student’s two tail t-test. significance of differences is indicated by p values, calculated using a paired Student’s two tail t-test. 3. 3. Results Results 3.1. Effect Effect of AuNP on Innate Memory Responses Induced by LPS In this this study, study,wewehave have used used AuNP AuNP with with an average an average size size of 25 of 25 nm ± 2.9 ± 2.9 nm dispersed dispersed in 2.2 in 2.2 mM mM sodium sodium citrate1). citrate (Figure (Figure 1).spectroscopy UV-Vis UV-Vis spectroscopy revealed arevealed maximum a maximum absorbance absorbance of 524 nm of 524no with nm with no additional additional peaks, that peaks, indicating indicating particlesthat particles were were free from free from DLS aggregation. aggregation. DLS ademonstrated demonstrated hydrodynamic a hydrodynamic diameter of 32.8diameter ± 10.4 nmof and 32.8a± Z-potential 10.4 nm andofa −31.3 Z-potential of −31.3 ± 0.6 mV. ± 0.6 mV.LAL By modified By modified assay, theLAL assay, endotoxin the endotoxin contamination of the AuNP batch used in this study was 9.36 EU/mg. contamination of the AuNP batch used in this study was 9.36 EU/mg. Pre-incubation in human serum Pre-incubation in human serum prevented prevented aggregation uponaggregation subsequentupon subsequent addition addition to cell cultures to cell cultures [31]. [31]. Figure 1. Characterization of 25 nm AuNP. Size distribution profile (left panel), STEM image (central Figure 1. Characterization of 25 nm AuNP. Size distribution profile (left panel), STEM image (central panel), and UV-Vis profile (right panel) of AuNP following synthesis. panel), and UV-Vis profile (right panel) of AuNP following synthesis. We have investigated whether AuNP are capable of inducing innate memory in human monocytes. We have To this end, we investigated whether have used primary cellsAuNP isolatedare fromcapable of inducing the blood of healthyinnate donorsmemory in human in an in vitro model monocytes. To this end, we have used primary cells isolated from the blood of repeated challenges (Figure 2). Primary monocytes were exposed in culture to medium alone,of healthy donors in an in vitro model of repeated challenges (Figure 2). Primary monocytes were exposed AuNP, the bacterial agents LPS (lipopolysaccharide from E. coli) or BCG (live bacteria), or to the in culture to medium mixture ofalone, AuNP, bacterial thewith agents bacterial AuNP.agents MonocyteLPSactivation (lipopolysaccharide was measured from E. coli) in terms or BCG (live of production of bacteria), or to the mixture of bacterial agents with AuNP. Monocyte activation inflammation-related factors after 24 h. After a 6-day period of resting in culture, cells were was measured exposed into terms of production of inflammation-related factors after 24 h. After a 6-day period a secondary challenge (a higher concentration of LPS, i.e., 5 ng/mL) and their response again assessed. of resting in culture, cells wereofexposed The concentration to a secondary AuNP selected for thesechallenge experiments (a higher has been concentration chosen as theof LPS, i.e., highest 5 ng/mL) concentration and their response again assessed. The concentration of AuNP selected for at which the endotoxin contamination was below monocyte activation threshold. The AuNP size these experiments has been chosen as was selected thepreliminary from highest concentration experiments at which in whichtheNPendotoxin contamination of different was below size were tested monocyte (Supplementary activation threshold. The AuNP size was selected from preliminary experiments Figure S1). Uptake of AuNP was assessed by TEM and found comparable in the different groups in which NP by of different size were tested (Supplementary visual inspection (Supplementary Figure S2). Figure S1). Uptake of AuNP was assessed by TEM and found comparable in the different groups by visual inspection (Supplementary Figure S2). Cells 2020, 9, 284 6 of 17 Cells 2020, 9, 284 6 of 17 LPS or BCG ± AuNP LPS Primary response Resting Secondary response 24 h 6 days 24 h Figure 2. Figure Schematic depiction 2. Schematic depiction of of the the in in vitro vitro model model ofof innate innate immune immune memory. memory. Primary Primary activation activation was induced was induced by by stimulation stimulation for for 24 24 hh with with LPS LPS or or BCG, BCG, inin the the absence absence oror in in the the presence presenceofofAuNP. AuNP. Supernatants were collected and cells rested for six days. On day 7, a secondary response was Supernatants were collected and cells rested for six days. On day 7, a secondary response was induced induced by challenge by challenge with with LPS LPS for for 24 24 h h (control (control groups groups were were challenged challenged withwith medium alone), after medium alone), after which which supernatants were collected for evaluation of cytokine production. supernatants were collected for evaluation of cytokine production. Data in Data in Figures Figures33and and4 4show show thetheprimary primary andandsecondary secondary response response of monocytes of monocytes whenwhen LPS is used LPS is as stimulus. LPS is known as a potent inflammatory activator and as used as stimulus. LPS is known as a potent inflammatory activator and as a potent inducer of a a potent inducer of a tolerance type of memory tolerance [8,9]. type of Monocyte memory [8,9].activation Monocyte was assessed activation aswas production assessedofasthe inflammatory production of the cytokines inflammatoryTNFα and IL-6. As expected, when using endotoxin-free AuNP [30,32], particles cytokines TNFα and IL-6. As expected, when using endotoxin-free AuNP [30,32], particles did not did not induce inflammatory cytokineinflammatory induce production, while cytokineLPSproduction, potently induced while TNFα and IL-6induced LPS potently production TNFα (Figure 3). Co-exposure and IL-6 production to LPS and AuNP did not change the reactivity of monocytes (Figure 3). Co-exposure to LPS and AuNP did not change the reactivity of monocytes to LPS (differences between LPS and LPS to LPS + AuNP groups: p > 0.05), confirming that AuNP do not directly activate (differences between LPS and LPS + AuNP groups: p > 0.05), confirming that AuNP do not directly human monocytes, nor do they interfere activate human with LPS-induced monocytes, nor do inflammatory they interfere activation [32]. with LPS-induced inflammatory activation [32]. After primary activation, cells were rested in culture for 66 days, After primary activation, cells were rested in culture for days, during during which which timetime they they returned returned to aa quiescent to quiescentstatus, status,measured measured as as thethereturn return to baseline of inflammatory to baseline of inflammatory cytokine production. cytokine By visual production. By inspection, cell number in all treated groups was comparable to that visual inspection, cell number in all treated groups was comparable to that in the control wells, in the control wells, with nowith sign of increased no cell deathcell sign of increased in death groupsintreated groupswith LPS with treated or BCG. LPSPrimed or BCG. quiescent Primed cells were then quiescent cells exposed were then to culture medium exposed to culture alone or containing medium alone oracontaining higher LPSa concentration (to mimic exposure higher LPS concentration (to mimic to aexposure more potentto a adverse event). Restimulation with medium confirmed that, in more potent adverse event). Restimulation with medium confirmed that, in all primed cells, all primed cells, cytokine production cytokine had returned production to returned had baseline, tothus achieving baseline, thusa achieving true resting status a true (Figure resting 4, the status identical (Figure lack 4, the of response identical lack in all primed cells exposed to medium alone is reported as a single of response in all primed cells exposed to medium alone is reported as a single Ctrl zero value). Ctrl zero value). Restimulation with LPS induced Restimulation withaLPSsignificant inducedproduction a significant of inflammatory production of cytokines inflammatory in cells primedinwith cytokines cellsmedium primed alone or with AuNP, indicating that AuNP did not induce any change with medium alone or with AuNP, indicating that AuNP did not induce any change in the secondary in the secondary response and are therefore unable to induce memory (as far as these two cytokines response and are therefore unable to induce memory (as far as these two cytokines are concerned). are concerned). Confirming previous notions, Confirming restimulation previous notions, of cells previously restimulation of primed with LPS resulted cells previously primed in significantly with LPS resulted reducedin production of both TNFα and IL-6 compared to medium-primed significantly reduced production of both TNFα and IL-6 compared to medium-primed cells, cells, indicating the induction by LPS priming indicating theof a “tolerance” induction by LPS type of innate priming of a memory. “tolerance” Priming type ofwith innateLPS in the presence memory. Priming with of AuNP LPS induced a tolerance type of response to the LPS challenge that in the presence of AuNP induced a tolerance type of response to the LPS challenge that isis indistinguishable from that induced by LPS alone, indicating indistinguishable that induced from that AuNP doby notLPSinterfere alone,with or modulate indicating the LPS-induced that AuNP innate memory do not interfere with or modulate the LPS-induced innate memory (Figure 4). While this experimental design both (Figure 4). While this experimental design includes the use of the same stimulus (LPS) for primary includes the and secondary response, the LPS-induced tolerance memory is non-specific, use of the same stimulus (LPS) for both primary and secondary response, the LPS-induced tolerance as similar results can be obtained using different agents at challenge (e.g., zymosan) [33]. memory is non-specific, as similar results can be obtained using different agents at challenge (e.g., zymosan) [33]. Cells 2020, 9, 284 7 of 17 Cells 2020,9,9,284 Cells2020, 284 77 of of 17 17 A4 B15 p <0.0001 A4 p <0.0001 B15 p <0.0001 p <0.0001 p = 0.0002 3 p =0.0007 6 monocytes) monocytes) 10 p = 0.0002 3 p =0.0007 6 monocytes) 6 monocytes) 10 2 (ng/10(ng/10 6 IL-6 (ng/10 2 IL-6 (ng/10 TNFαTNFα 5 1 5 1 0 0 medium AuNP LPS LPS+AuNP medium AuNP LPS LPS+Au NP 0 0 medium AuNP LPS LPS+AuNP medium AuNP LPS LPS+Au NP Figure 3. Primary response of human monocytes to LPS and AuNP. CD14+ monocytes were exposed for 24 h3.toPrimary LPS (1 ng/mL) in of the absence or in the presence of AuNP. AuNP + monocytes AuNP.(10 μg/mL). Releasewere of TNFα (A) Figure response human monocytes to LPS and CD14 exposed Figure 3. Primary response of human monocytes to LPS and CD14 + monocytes were exposed for and24IL-6 h to(B) LPS was(1 measured ng/mL) in the absence in the or in the Results supernatants. presenceareof reported AuNP (10asµg/mL). mean ± Release SEM of of TNFα data from(A) 4 for 24 h to LPS (1 ng/mL) in the absence or in the presence of AuNP (10 μg/mL). Release of TNFα (A) and IL-6 (B) individual was measured donors, tested in in theseparate four supernatants. Results experiments. p are reported values are as mean indicated ± SEM when p < of data 0.05. from 4 Although and IL-6 (B) was measured in the supernatants. Results are reported as mean ± SEM of data from 4 individual statistically donors, testedwe significant, in four separate experiments. p values are indicated < 0.05. Although when pAuNP individual donors, tested inhave fournot reported separate in the figure experiments. the differences p values between are indicated when p < 0.05.and LPS (p Although statistically < 0.0001 for significant, TNFα, and pwe have not = 0.0002 for reported in the figure IL-6) or between AuNP the differences and LPS + AuNP between AuNPfor (p < 0.0001 and LPS TNFα, statistically significant, we have not reported in the figure the differences between AuNP and LPS (p (p and< 0.0001 p = for TNFα, 0.0007 for and p = 0.0002 for IL-6) or between AuNP and LPS + AuNP (p < 0.0001 for TNFα, IL-6). < 0.0001 for TNFα, and p = 0.0002 for IL-6) or between AuNP and LPS + AuNP (p < 0.0001 for TNFα, and p = 0.0007 for IL-6). and p = 0.0007 for IL-6). p = 0.0011 A 10 p = 0.0121 p = 0.0011 B 3 p = 0.0010 A 10 p = 0.0121 B 3 p = 0.0004 p = 0.0010 p = 0.0004 8 6 monocytes) 8 6 monocytes) 2 6 monocytes) 6 monocytes) 6 2 6 (ng/10(ng/10 IL-6 (ng/10 4 IL-6 (ng/10 TNFαTNFα 1 4 1 2 2 0 0 Ctrl medium AuNP LPS LPS+Au NP Ctrl medium AuNP LPS LPS+Au NP 0 LPS 0 LPS Ctrl medium AuNP LPS LPS+Au NP Ctrl medium AuNP LPS LPS+Au NP Figure 4. Secondary response of human LPS monocytes primed with LPS and AuNP. LPS Following primary Figure 4. Secondary response of human monocytes primed with LPS and AuNP. Following primary activation (in x-axis: medium, AuNP, LPS, LPS + AuNP), cells were rested for six days and then activation (in x-axis: response Figure 4. Secondary medium,ofAuNP, human LPS, LPS + AuNP), cells were rested for Following six days and then challenged with LPS (5 ng/mL) for 24 h.monocytes Supernatants primed werewith LPSfor tested and AuNP. TNFα (A) and IL-6 (B).primary Ctrl challenged with LPS (5 ng/mL) for 24 h. Supernatants were tested for TNFα (A) and IL-6 (B). Ctrl activation the represents (in response x-axis: medium, AuNP, LPS,with of cells challenged LPSmedium + AuNP), cells (the lackwere rested for of response wassix days and identical then in cells represents previously the challengedprimed response with LPS of cells challenged with(5medium, ng/mL) for AuNP,24 h. with mediumwere Supernatants LPS, (the tested or LPS + AuNP). lack of response Resultsfor areTNFα wasas (A) reported identical and in cells IL-6±(B). mean SEMCtrlof previously data primed from 4 individual with medium, donors, tested AuNP, LPS, or in four separate LPS + AuNP). experiments. Results Further, are reported p values as mean areidentical indicatedin±when SEM represents the response of cells challenged with medium (the lack of response was cells pof