Nanoscale View Article Online PAPER View Journal | View Issue Gold nanoparticles (AuNPs) impair LPS-driven This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: Nanoscale, 2021, 13, 7648 immune responses by promoting a tolerogenic- Open Access Article. Published on 15 April 2021. Downloaded on 5/27/2021 1:45:14 PM. like dendritic cell phenotype with altered endosomal structures† Sara Michelini, a Francesco Barbero, b Alessandra Prinelli,c Philip Steiner,a Richard Weiss, a Thomas Verwanger,a Ancuela Andosch,a Ursula Lütz-Meindl,a Victor F. Puntes, b Damjana Drobne, d Albert Duschl a and Jutta Horejs-Hoeck *a Dendritic cells (DCs) shape immune responses by influencing T-cell activation. Thus, they are considered both an interesting model for studying nano-immune interactions and a promising target for nano-based bio- medical applications. However, the accentuated ability of nanoparticles (NPs) to interact with biomolecules may have an impact on DC function that poses an unexpected risk of unbalanced immune reactions. Here, we investigated the potential effects of gold nanoparticles (AuNPs) on DC function and the consequences for effector and memory T-cell responses in the presence of the microbial inflammatory stimulus lipopolysac- charide (LPS). Overall, we found that, in the absence of LPS, none of the tested NPs induced a DC response. However, whereas 4-, 8-, and 11 nm AuNPs did not modulate LPS-dependent immune responses, 26 nm AuNPs shifted the phenotype of LPS-activated DCs toward a tolerogenic state, characterized by downregula- Received 28th December 2020, tion of CD86, IL-12 and IL-27, upregulation of ILT3, and induction of class E compartments. Moreover, this DC Accepted 12th March 2021 phenotype was less proficient in promoting Th1 activation and central memory T-cell proliferation. Taken DOI: 10.1039/d0nr09153g together, these findings support the perception that AuNPs are safe under homeostatic conditions; however, rsc.li/nanoscale particular care should be taken in patients experiencing a current infection or disorders of the immune system. 1. Introduction cells (DCs) being considered the most important type of APC. APCs are usually located within tissues and are equipped with a The human immune system can be divided in two branches: broad spectrum of receptors which enables them to sense the the innate and the adaptive immune system. The innate presence of pathogens by recognizing conserved molecular pat- immune system represents the first line of defense against terns on their surface.2,3 One of these patterns is the endotoxin pathogens, damaged cells and non-self-objects (e.g. nano- lipopolysaccharide (LPS), located on the outer membrane of particles and microbes) and is characterized by a quick and con- Gram-negative bacteria and recognized by a receptor complex served set of responses carried out by innate immune cells.1 composed of Toll-like receptor 4 (TLR4), MD2 and CD14 on the Innate immune responses are usually followed by activation DC surface.4 Upon pathogen recognition, DCs become activated of the adaptive immune system, which is capable of mounting and begin a maturation process characterized by the upregula- a pathogen-specific response mediated by T cells and B cells. tion of co-stimulatory surface molecules, including CD86 and Antigen presenting cells (APCs) act at the interface between HLA-DR, and the release of specific cytokines. This maturation the innate and the adaptive immune systems, with dendritic process enables the DCs to activate naïve T cells, thereby initiat- ing adaptive immune responses. Based on signals provided by a Department of Biosciences, Paris-Lodron University Salzburg, Hellbrunner Str. 34, DCs (e.g. cytokines and surface receptors) during the DC–T-cell 5020 Salzburg, Austria. E-mail: jutta.horejs_hoeck@sbg.ac.at interaction, naïve T cells can polarize into pathogen-specific T b Insitut Català de Nanosciència i Nanotecnologia (ICN2), UAB Campus, Bellaterra, helper (Th) cell subsets, which are responsible for coordinating Barcelona 08193, Spain the elimination of the invading pathogen. Among the different c AvantiCell Science Ltd, Gibbsyard Building, Auchincruive, Ayr, KA6 5HW, UK d Th cell subsets, Th1 cells have the task of eliminating intracellu- Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia lar bacteria and viruses, whereas Th2 cells protect the body † Electronic supplementary information (ESI) available. See DOI: 10.1039/ against extracellular parasites. Th17 cells orchestrate the fight d0nr09153g against extracellular bacteria or fungi, while regulatory T cells 7648 | Nanoscale, 2021, 13, 7648–7666 This journal is © The Royal Society of Chemistry 2021 View Article Online Nanoscale Paper (Tregs) have the crucial role of suppressing excessive inflam- methods as described by Bastús et al.26 and by Piella et al. (for mation and autoimmunity.5–11 However, unbalanced Th cell the 4 nm particles)27 using tetrachloroauric(III) acid trihydrate polarization and activation is often linked to immune-related (99.9% purity) and sodium citrate tribasic dihydrate (≥99%) pathologies.12,13 Interestingly, each of these Th cell subsets, also (SC) purchased from Sigma-Aldrich. known as effector T cells, can be identified using specific mole- Since innate immune cells are extremely sensitive to endo- cular markers such as cytokines and surface markers.11 While toxin contamination, we applied an “LPS-free” synthesis proto- many effector T cells die during pathogen clearance, a specific col to prevent unspecific cell activation in our experiments. Th cell population termed “memory cells” remains poised for Briefly, the reagent solutions for the synthesis were prepared years and decades and is crucial for establishing long-term with endotoxin-free material and with LPS-free water (Cape This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. protection.14,15 Cod Incorporated) under a flow hood. Disposable plastic Because of their central role in shaping immune responses, materials and syringe needles were purchased LPS-free (B. Open Access Article. Published on 15 April 2021. Downloaded on 5/27/2021 1:45:14 PM. DCs are considered an important model to investigate poten- Braun), lab glassware was wrapped with aluminum foil and tial immunomodulatory properties of nanoparticles (NPs). depyrogenated in a lab oven at 200 °C overnight, and rubber Upon interaction of NPs with biomolecules, a biomolecular stoppers and stir bars were cleaned with ethanol and left to corona is formed, which has a significant influence on the fate dry in the flow hood. To quantify potential residual LPS con- and effects of NPs in tissues and cells.16–19 The interactions tamination, we performed both an NF-κB-luc reporter gene between NPs and cells can be intentionally exploited in assay and an EndoLISA assay. medical applications, like cancer treatment or diagnosis;20,21 however, unintended interactions can have undesired effects. 2.2 AuNP physicochemical characterization In this context, gold nanoparticles (AuNPs) have attracted NP post-synthesis characterization and analysis of protein particular attention due to their stability, biocompatibility, and corona formation in relevant culture medium were performed interesting optical properties, which make them not only suit- using four different techniques. The use of such techniques able for different applications in biomedicine, but also a for NP characterization was shown to be reliable in previous widely used “model particle” for studying the molecular basis studies.26,27 of nano-immune interactions.21,22 Electron microscopy. Size and shape of the AuNPs were Therefore, the aim of this work was to study the potential assessed via Scanning Electron Microscopy using an FEI effects of AuNPs on the ability of DCs to promote effector and Magellan XHR SEM in transmission mode (STEM) operated at memory T-cell activation and proliferation during LPS-induced 20 kV and an FEI Tecnai G2 F20 S-TWIN HR(S)TEM operated immune responses. at an accelerated voltage of 200 kV. After synthesis, 2 μL of the To achieve this goal, we first investigated the potential sample were drop-casted on a carbon-coated copper TEM grid immunomodulatory properties of endotoxin free-AuNPs of and dried at room temperature (RT) using a vacuum drier. To different sizes (4, 8, 11 and 26 nm) in terms of cytokine avoid handling-dependent aggregation during sample prepa- secretion (IL-12, IL-27) and surface marker expression (CD86, ration, AuNPs were functionalized by adding 11-mercaptoun- HLA-DR, ILT3) in LPS-treated human monocyte-derived DCs decanoic acid (MUA) (Sigma-Aldrich) at a final concentration (moDCs). Next, we analyzed AuNP-induced ultrastructure and of 0.1 mM. The solution was then left for 2 h at RT under slow- morphological changes, which are of special interest because stirring conditions; subsequently, NPs were purified from the internalized NPs may affect the endosomal machinery of MUA excess by centrifugation. After image collection, particles mature DCs known as “endosomal sorting system required for were analyzed using ImageJ to obtain reliable size-distribution transport (ESCRT)”, which is involved in several processes rele- data. vant for cell-mediated immune responses.23–25 UV-vis spectroscopy. A Shimadzu UV-2400 spectrophoto- Subsequently, to investigate whether the identified changes meter was used to acquire the UV-vis spectra. Spectral acqui- in DC phenotype can alter T-cell polarization, we performed sition was performed at RT in the 300–750 nm range using dis- co-culture experiments of AuNP-treated, LPS-stimulated DCs posable plastic cuvettes. MilliQ water and complete culture with CD4+ Th cells and evaluated the production of Th1 cell- medium were used as a reference. specific cytokines. Size and zeta potential measurements. The hydrodynamic Finally, to study the effects of NPs in a more complex diameter and Z potential of AuNPs both in SC and after incu- model, we incubated human peripheral blood mononuclear bation in moDC medium were assessed at 37 °C using a cells (PBMCs) with AuNPs and/or LPS and assessed both Malvern Zetasizer Nano ZS by Dynamic Light Scattering (DLS), effector and memory T-cell responses using an advanced and Laser Doppler Velocimetry (LDV). Diameters were reported 13-color flow cytometry panel. as a distribution by intensity calculated by non-negative least squares (NNLS) analysis. Z potential is not an intrinsic value but depends on the conductivity of the solution in which NPs 2. Experimental section are dispersed. Consequently, to compare the Z potential of 2.1 AuNP synthesis different samples, their conductivity has to be similar. Thus, Sodium citrate-stabilized gold nanoparticles (AuNPs) were syn- after exposure to the medium the AuNPs were purified by cen- thesized using two kinetically controlled seeded growth trifugation and resuspended in SC (2.2 mM) to obtain a final This journal is © The Royal Society of Chemistry 2021 Nanoscale, 2021, 13, 7648–7666 | 7649 View Article Online Paper Nanoscale conductivity similar to that of the pristine AuNPs (about the synthetic method used in this paper generates particles 0.7 mS cm−1). that are more and more diluted after every growing step, in AuNP behavior in culture medium. To evaluate the stability order to apply 5 × 1011 NPs per well we added different of our NPs in moDC medium and to assess the formation of volumes of AuNP stock solution. The different volumes were the protein corona, we incubated the particles for 1, 24 and then equalized by the addition of SC 2.2 mM, which is used as 48 h, 5 × 1011 AuNPs per well with 900 μL of complete culture a stabilizer during the NP synthesis. LPS concentration was medium [moDC medium: RPMI-1640 (Sigma) supplemented based on previous studies.29 with 10% heat-inactivated (i.a.) pure fetal bovine serum (FBS) (PAA) (Thermo-Fisher), 2 mM L-glutamine (Sigma), 2.4 Quantification of LPS-contamination This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 100 U mL−1, penicillin/streptomycin (Sigma), 50 μM Two different methods were used to detect LPS contamination. β-mercaptoethanol (Gibco Laboratories)]. Differences in EndoLISA (Hyglos Gmbh). EndoLISA was used for quantitat- Open Access Article. Published on 15 April 2021. Downloaded on 5/27/2021 1:45:14 PM. volume between the AuNP batches were compensated by the ive determination of endotoxin contamination of AuNP prep- addition of LPS-free SC 2.2 mM up to a final volume of 1.2 mL. arations, according to the manufacturer’s recommendations. Since the absorption spectrum generated by the pH-sensing Samples were diluted 1 : 2 in LPS-free water and the binding molecule phenol red is comparable to the one obtained for step was carried out for 18 h. Acquisition was performed on a AuNPs, we used a phenol red-free RPMI [RPMI-1640 R7509]. microplate reader (Tecan Infinite M200Pro) with a fixed gain Sodium citrate, moDC medium and AuNPs in SC were used as of 100. Analysis was performed using a 4-Parameters Logistic controls. Regression Model. LPS contamination was then expressed in EU mL−1 (EU, endotoxin units). 2.3 Cell culture conditions NF-κB-luc TLR4 reporter gene assay. As described by Schwarz All cells were cultured at 37 °C, 5% CO2 and 95% humidity. The et al.,30 HEK293 cells (mycoplasma negative, culture passage incubation time of nanoparticles and cells was estimated on the 5–15) were seeded in DMEM [DMEM (Sigma-Aldrich) sup- basis of the findings of Casals et al.,28 who showed that protein plemented with 10% FBS, 2 mM MEM non-essential amino corona formation reaches a stability point (hard corona) after acids (PAA) (Sigma-Aldrich), 2 mM L-glutamine, 100 U mL−1 approximately 48 h of incubation in culture medium. penicillin/streptomycin] at a final concentration of 1.5 × 105 In order to exclude potential side effects of endotoxin con- cells per mL and incubated for 24 h. On day 2, cells were trans- tamination in AuNPs, we used a concentration of AuNPs which fected with both a reporter plasmid containing the NF-κB tran- was insufficient to activate immune cells. This concentration scription factor linked to a luciferase reporter gene, and a mix was calculated on the basis of the endotoxin content measured of 3 plasmids (TLR4, MD2 and CD14) encoding the LPS receptor by the EndoLISA assay. Since every batch possessed different components. After 24 h, supernatants were discarded and sub- levels of contamination, we used the highest possible amount stituted with 900 µL of fresh medium. Cells were then stimu- of 26 nm AuNPs as a reference (5 × 1011 NPs per well) and lated by the addition of LPS 30 pg mL−1 (E. coli lipopolysacchar- equalized the concentration of the other batches in terms of ide (LPS) 055:B5 (Sigma-Aldrich)), AuNPs of different sizes (5 × particle number. Because of the different sizes of the used 1011 NPs per well), LPS plus AuNPs, or left untreated. Volume AuNPs, maintaining the number of particles applied on cells differences were compensated by adding PBS to a final volume simultaneously increased the amounts of gold and surface of 1.2 mL. On day 4, supernatants were discarded, cells were area in a way that is directly proportional to the particle dia- lysed and lysates were read by a Tecan Infinite M200Pro instru- meter. Detailed information on corresponding concentrations ment. To exclude AuNP interference in signal absorbance, data of NPs in terms of µg mL−1 or surface area per µL can be calcu- are expressed in the form of a ratio between LPS or AuNPs + lated using Table 1 assuming a 100% spherical shape. Since LPS and the corresponding controls, untreated cells or AuNPs. Table 1 AuNP post-synthesis characterization Characteristics Batches Name 4 8 11 26 Material Au Au Au Au Stabilizer Sodium citrate Sodium citrate Sodium citrate Sodium citrate Diameter by STEM [nm] 4.0 ± 0.6 8.8 ± 1.5 11.8 ± 0.8 26.4 ± 3.0 Hydrodynamic Diameter [nm] 5.6 ± 1.4 12.8 ± 3.0 15.1 ± 3.7 36.8 ± 14.1 Absorption peak [nm] 504 519 516 523 Z potential [mV] Not detected. −49.3 ± 5.0 −47.0 ± 5.0 −35.6 ± 0.6 Concentration [NPs per ml] 5.0 × 1013 5.0 × 1012 6.0 × 1012 1.7 × 1012 Au concentration [mM] 0.167 0.166 0.500 1.650 Au concentration [mg mL−1] 0.033 0.033 0.100 0.325 Total surface area [nm2 mL−1] 2.5 × 1015 1.2 × 1015 2.6 × 1015 3.8 × 1015 Table summarizing the main characteristics of the batches used in this study. 7650 | Nanoscale, 2021, 13, 7648–7666 This journal is © The Royal Society of Chemistry 2021 View Article Online Nanoscale Paper 2.5 Generation of human monocyte-derived dendritic cells per well), LPS plus AuNPs, or left untreated. The NP dose (5 × (moDCs) 1011 NPs per well) was chosen because it is the highest concen- PBMCs were isolated from buffy coats of donors by density tration that can be added without inducing cell activation by gradient centrifugation using histopaque-1077 (Sigma) as LPS contamination and without diluting the cell culture described by Posselt et al.31 The buffy coats used in this medium to an extent that could result in reduced cell viability study were kindly provided by the blood bank of Salzburg, and NP aggregation. Austria. According to Austrian regulations, no informed Volume differences were compensated with SC 2.2 mM up consent is required if blood cells derived from anonymous to a final volume of 1.2 mL. After 48 h, supernatants were col- lected and tested for IL-6, IL-8/CXCL8, IL-12, TNF-α This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. healthy donors, discarded after plasmapheresis (buffy coats), are used. Therefore, no additional approval by the national (Peprotech) and IL-27 (R&D) release by ELISA. Cell surface markers and cell structures were analyzed via flow cytometry Open Access Article. Published on 15 April 2021. Downloaded on 5/27/2021 1:45:14 PM. ethics committee was required. Monocytes were then purified from PBMCs using the CD14+ MicroBeads UltraPure human or TEM. kit (Miltenyi Biotec) according to the manufacturer’s instruc- 2.8 CD4+ T-cell isolation and co-culture with moDCs tion. Subsequently, cells were cultured for 6 days in moDC medium (2.5 × 105 cells per mL, supplemented with 50 ng DCs were stimulated as described before. Briefly, 0.25 × 105 mL−1 GM-CSF and 50 ng mL−1 IL-4 (a generous gift from DCs per well were cultured for 48 h in 225 µL of moDC Novartis)). After 48 h, monocytes were fed 1 vol. medium medium supplemented with the previously stated stimuli in a containing fresh IL-4 and GM-CSF at a final concentration of 48-well plate (LPS 30 ng mL−1 final concentration, AuNPs 1.25 50 ng mL−1. To assess the efficacy of the differentiation pro- × 1011 NPs per well, LPS + AuNPs). Untreated cells served as a tocol, DC and monocyte lineage marker expression and mor- negative control. Again, volume differences were compensated phology were assessed by both flow cytometry and differen- with SC up to a final volume of 300 µL. After 48 h, allogeneic tial staining (Diff–Quik; Medion Diagnostics) following Pickl CD4+ T cells were isolated from buffy coats using a Human work.32 Residual CD14− CD1a− cells, most likely polymorpho- CD4+ T cell Isolation Kit from Miltenyi Biotec, added to nuclear granulocytes (PMNGs), which can be observed after moDCs at a ratio of 10 : 1 in 300 µL of moDC medium sup- monocyte isolation, are characterized by a limited lifespan plemented with IL-2 (100 U mL−1) and co-cultured for 6 days. and will therefore die during the process of moDC After 72 h, IL-2 50 U mL−1 was added to each well to promote differentiation. T-cell survival. On day 6, supernatants were harvested and tested for cytokine release by ELISA (Peprotech) and 2.6 Cytotoxicity and viability assessment LEGENDplex. 1 × 105 moDCs were plated in 900 µL of DC medium and 2.9 Generation and culture of human PBMCs stimulated for 48 h either with LPS (30 ng mL−1, final concen- PBMCs were isolated from buffy coats as described in section tration), different sizes of AuNPs (5 × 1011 NPs per well) or left 2.5 and seeded in a 24-well plate at a concentration of 1 × 106 untreated. Volume differences were compensated with sterile- in 900 µL, and stimulated for 6 days either with LPS (30 ng filtered LPS-free SC 2.2 mM to a final volume of 1.2 mL. To mL−1, final concentration), AuNPs of 4-, 8-, 11- and 26 nm (5 detect toxicity, two methods were used. × 1011 NPs per well), LPS plus AuNPs, or left untreated. The LDH assay (Promega). Cytotoxicity was assessed by quanti- final volume was then equalized by the addition of SC 2.2 mM fication of lactate dehydrogenase (LDH) release in 90 µL of up to a final volume of 1.2 mL. Subsequently, supernatants cell supernatant using a “CytoTox 96® Non-Radioactive were harvested and analyzed via ELISA (R&D), while cells were Cytotoxicity assay kit” following the manufacturer’s rec- stained and analyzed using a 13-color flow cytometry panel. ommendations. As a positive control, cells were lysed with The number of cells used in this experiment was calculated 0.10% (v/v) Triton X-100 for 30 min prior to harvesting to based on the composition of PBMCs in healthy individuals, achieve maximum LDH release. which presents an average amount of APCs (monocytes plus CellTiter-blue (CTB) (Promega). Cell viability was assessed DCs) ranging between 11% and 22% of the total population. via CTB assay following the manufacturer’s instructions. After By using 1 × 106 cells per well, we guaranteed the presence of 1 h of incubation, supernatants were analyzed. Cells treated at least 1 × 105 APCs in the preparation, which is in line with Triton X-100 served as a control for low viability. A micro- with the experiments performed with DCs (see sections 2.6 plate reader (Tecan Infinite M200Pro) was used to record and 2.7).33 sample absorbance (LDH) and fluorescence emission (CTB) following the manufacturer’s instructions. 2.10 Flow cytometry Surface marker expression on DCs was assessed via flow cyto- 2.7 Impact of AuNPs on LPS-stimulated moDCs metry by staining the cells with α-HLA-DR APC, Fixable To assess the potential impact of AuNPs of different sizes on Viability Dye eFluor506, α-CD1a BV421, α-CD86 PE LPS-induced immune responses, 1 × 105 DCs were plated in (eBioscience), α-CD14 PerCP-Cy5.5 (BD Bioscience) and α-ILT3 900 µL of DC medium and stimulated for 48 h either with LPS PerCP-Cy5.5 (Biolegend). Samples were subsequently acquired (30 ng mL−1, final concentration), AuNPs alone (5 × 1011 NPs using a FACS Canto II flow cytometer (BD Biosciences). The This journal is © The Royal Society of Chemistry 2021 Nanoscale, 2021, 13, 7648–7666 | 7651 View Article Online Paper Nanoscale shown MFI data were collected after exclusion of doublets and 2.14 Statistical analysis dead cells. Statistical analyses were performed using GraphPad Prism Surface marker expression on PBMCs was assessed via flow Software Version 6.01. P values for multiple groups were calcu- cytometry using a 13-color flow-cytometry panel generated by lated via repeated-measures ANOVA with a Tukey’s post-hoc using the Optimized Multicolor Immunofluorescence Panel- test. Data are shown as mean + SD. The Geisser-Greenhouse 030 (OMIP-030)34 as a reference: CD4 BV510, CD127 correction was not applied. P-values < 0.05 were considered AlexaFluor 700, CD8a PE-Cy5, CD3 PerCP-Cy5.5, CD45RA statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, BV605, CD25 PE, CD20 APC-Cy7, CD194 PE-Cy7, CD197 PE/ ****p < 0.0001). Dazzle594 (Biolegend), CCR10 APC (BD Biosciences), Ki-67 This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. BV650, CD196 BB151, CD183 BV421 (BD Horizon) Fixable 2.15 Reported information about the experiments Viability Dye APC-Cy7 (eBioscience). Samples were acquired Open Access Article. Published on 15 April 2021. Downloaded on 5/27/2021 1:45:14 PM. using a Cytoflex S flow cytometer (Beckman Coulter) and then The experiments were conducted and reported following analyzed using the gating strategy summarized in ESI Fig. 5.† MIRIBEL guidelines.37 2.11 LEGENDplex™ Supernatants were centrifuged in 96-well V-bottom plates for 3. Results and discussion 10 min at 1000 g to eliminate NPs which might interfere with 3.1 AuNP characterization both after synthesis and in the assay. The release of cytokines was assessed using the relevant culture medium LEGENDplex™ Human Th Panel (13-plex) (Biolegend) kit, As reported by other authors, the majority of immune according to the manufacturer’s recommendations. Sample acquisition was performed using a Cytoflex S flow cytometer responses to nanomaterials (NMs) are characterized by an (Beckman Coulter), and LEGENDplex v8.0 Analyst Software was adaptation process resulting in silent elimination of the NM or used for data evaluation. transient inflammation followed by full resolution.1 However, despite being regarded as reasonably safe when administered 2.12 Transmission electron microscopic (TEM) preparation in therapeutic doses and under homeostatic conditions, some of DCs NMs may have deleterious effects in cases where there is a less After stimulation, moDCs were transferred into sample balanced immune system, e.g. during acute inflammation. holders for subsequent high-pressure freeze fixation (HPF). Due to their elevated energy potential, NPs are highly reactive HPF was carried out in a Leica Empact HPF device (Leica objects that constantly interact with other molecules in order Microsystems), with a cooling rate of approximately 12 000 °C to achieve a more stable thermodynamic status. This might s−1 and a pressure value of at least 2040 bar. The cryo-substi- result in the binding and functional alteration of biomolecules tution was transacted with a Leica EM AFS (Leica and signaling pathways which play a critical role in defining Microsystems) at defined cycles.35 The substitution medium the outcome of an immune response, ultimately leading to the contained 2% osmium tetroxide (OsO4) and 0.05% uranyl establishment of undesired immunological side effects.17 acetate in anhydrous acetone. After the cryo-substitution, the Therefore, evaluation of immunomodulatory effects of NMs is samples were washed three times with anhydrous acetone and crucial to ensure patient safety. propylene oxide and were then embedded into epoxy resin Since the reactivity of NPs is strongly influenced by their (medium grade; Agar Scientific). The embedding of samples composition, size, charge, shape, and aggregation status, it is was carried out in Beem® capsules (Agar Scientific) to ensure of outmost importance to carry out a comprehensive character- that moDC samples would sink and gather at the narrow ization of NMs used in biological studies. In this work in par- bottom of the capsules for further preparation. Afterwards, ticular, characterization of our gold nanoparticles was per- samples were polymerized at +70 °C for 24 h. Ultra-thin sec- formed both after synthesis and in relevant culture conditions tioning (∼70 nm) was performed with a Leica UC7 following MIRIBEL (Minimum Information Reporting in Bio- Ultramicrotome (Leica Microsystems) and sections were col- Nano Experimental Literature) recommendations.37 lected on Formvar-coated copper grids. TEM preparation was To do so, we first generated four different batches of mono- conducted as in previous studies and adapted for DCs.35,36 dispersed citrate-stabilized gold NPs with an average diameter of 4.0 ± 0.6 nm, 8.8 ± 1.5 nm, 11.8 ± 0.8 nm and 26.4 ± 3.0 nm, 2.13 Transmission electron microscopic (TEM) imaging respectively, using two kinetically controlled seeded growth of DCs methods. Then, data regarding their Z potential, absorption TEM imaging of DCs was carried out by using a LEO 912 AB spectra and hydrodynamic diameter after synthesis were TEM with an in-column Omega energy filter (Zeiss) at 80 kV acquired via laser doppler velocimetry (LDV), transmission accelerating voltage. TEM images were recorded with a Tröndle electron microscopy (TEM), UV-vis spectroscopy and dynamic TRS Sharp Eye bottom-mounted 2 K CCD camera (Tröndle), fil- light scattering (DLS) and are summarized in Table 1 and tered at zero energy loss. The TEM implementation and Fig. 1A. Interestingly, all the physicochemical properties of the recording process were controlled by iTEM 5.0 software tested nanoparticles were in line with the ones obtained by (Olympus). Bastús et al.26 and by Piella et al.38 in their work, highlighting 7652 | Nanoscale, 2021, 13, 7648–7666 This journal is © The Royal Society of Chemistry 2021 View Article Online Nanoscale Paper This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 15 April 2021. Downloaded on 5/27/2021 1:45:14 PM. Fig. 1 AuNP post-synthesis characterization and behavior in culture medium. (A) Representative TEM images of AuNP samples overlaid with both the corresponding size distribution curve and a photo of the AuNP suspensions; (B) UV-vis absorption spectra (top) and zeta potential values (bottom) of AuNPs both after synthesis (AS) or incubation in moDC medium for 1, 24, and 48 h. Data are shown as mean + SD. Dashed line in panel B bottom represents the Z potential of FBS. the quality and reliability of the synthetic method used in this values after 24–48 h), suggests the formation of a protein study. corona which promotes a change in the local refractive index Subsequently, since NP aggregation is also known to be a on the NP surface (Fig. 1B top). This hypothesis was further critical factor able to modulate the in vitro response of supported by the data acquired via LDV, which show (at 1 h of immune cells, such as monocytes and DCs, in terms of NP incubation) an increase in the Z potential values comparable uptake and cell activation,39 we investigated NP stability and to that obtained for FBS (dashed line), suggesting that serum behavior in biologically relevant culture medium. proteins were effectively adsorbed on the NP surface, masking To do so, AuNPs were incubated for 1, 24 and 48 h in the particle’s negative charge (Fig. 1B bottom). Unfortunately, moDC medium and analyzed by UV-vis spectroscopy and LDV. due to the higher scattering intensity of the serum proteins, it Upon data evaluation, we did not detect profile changes of UV- was not possible to obtain meaningful data for the 4 nm vis spectra (e.g. a secondary peak) that would be representative AuNPs in culture medium. of NP aggregation. This indicates that the 8-, 11-, and 26 nm Taken together, these results suggest that the AuNP batches batches were colloidally stable over the tested period under used in this study are monodispersed, not aggregated, and these conditions. However, the presence of a red-shift in the stable under the tested culture conditions. Thus, we can expect UV-vis spectra, which increased over time (reaching stable that any potential immunomodulatory effects that these This journal is © The Royal Society of Chemistry 2021 Nanoscale, 2021, 13, 7648–7666 | 7653 View Article Online Paper Nanoscale AuNPs exert on human DCs will be due to the activity of single Factor C and ending in the activation of a pro-clotting particles rather than their aggregates. enzyme.43 However, the reliability of non-cellular assays can be affected by an “LPS-masking effect” caused by chelators or 3.2 AuNP characterization via cell-based assays: cytotoxicity detergents, which makes LPS undetectable in Factor C-based and LPS contamination assays.30 Therefore, we additionally applied an NF-κB reporter LPS is an important component of the bacterial cell wall and, gene assay using HEK293 cells transiently transfected with due to its great stability and broad distribution on all surfaces, plasmids encoding a functional LPS receptor together with an is frequently encountered as an impurity in pharmaceutical NF-κB-luciferase reporter plasmid. preparations, orthopedic implants and NPs even in the Using the EndoLISA assay, we analyzed the endotoxin This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. absence of viable bacteria.40–42 Because immune cells such as content in the AuNP stock solutions and found that 8- and DCs are extremely sensitive to exceedingly low endotoxin con- 26 nm particles contain a detectable amount of endotoxin, as Open Access Article. Published on 15 April 2021. Downloaded on 5/27/2021 1:45:14 PM. centrations (<20 pg mL−1),41 testing all NP suspensions for the indicated in ESI Table 1.† It is well documented that even the presence of residual LPS is essential to ensure that possible very low LPS concentration of 0.1 EU mL−1 is capable of acti- effects of NPs on immune cells are triggered by the NPs them- vating human immune cells, including DCs.41 Since the aim of selves and not by undetected LPS contamination. For this this study is to investigate the effects of AuNPs under inflam- purpose, we performed a cellular TLR4-NF-κB reporter gene matory conditions, we calculated the maximum number of assay and a non-cellular EndoLISA test. particles for all batches that can be applied to DCs without Non-cellular assays such as the EndoLISA are based on inducing an immune response induced by contaminating LPS, factors involved in the blood coagulation system of the horse- and found this NP-dose to be equal to 5 × 1011 NPs per well shoe crab Limulus polyphemus. These assays take advantage of (correspondent to 4.16 × 1011 NPs per mL). In this context, a coagulation cascade, starting with the binding of LPS to Fig. 2A top summarizes the estimated endotoxin content in Fig. 2 AuNP characterization via cell-based assays. Graphs represent (A) assessment of LPS contamination via EndoLISA (top) and via TLR4/NF-κB- luc reporter gene assay (bottom); here the ratio was calculated between the LPS-containing samples (30 pg mL−1 LPS) and the respective controls (AuNPs alone or untreated samples). The dashed line in panel A top represents the minimum amount of LPS capable of activating immune cells. The conversion rate from EU mL−1 to ng mL−1 is 10 : 1. (B) Cytotoxicity assay based on detection of LDH release and resazurin conversion (CTB) per- formed on moDCs after 48 h of stimulation with the stated stimuli (5 × 1011 NPs per well, 30 ng mL−1 LPS) (two individual experiments, n = 4). Statistical analysis was performed using repeated-measures ANOVA combined with the Tukey’s post test. ****P < 0.0001. Data are shown as mean + SD. CTB, CellTiter-Blue, EU, Endotoxin Unit, LDH, lactate dehydrogenase, LPS, lipopolysaccharide, MFI, mean fluorescence intensity, NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells, NP, nanoparticle, TLR4, toll-like receptor 4. 7654 | Nanoscale, 2021, 13, 7648–7666 This journal is © The Royal Society of Chemistry 2021 View Article Online Nanoscale Paper the culture medium after addition of 5 × 1011 NPs per well, surface marker expression by differential staining (Diff-Quik) which is below 0.1 EU mL−1 for all AuNP batches used. and flow cytometry, respectively. In line with previous To confirm that the endotoxin contamination present in studies,32 moDCs were characterized by high expression of the chosen dose of particles is indeed unable to activate the CD1a, the loss of CD14 and the presence of a round nucleus LPS receptor complex composed of TLR4, MD2 and CD14, we surrounded by several cytoplasmic projections, and therefore performed an NF-κB-luc reporter gene assay using transiently were considered to be fully differentiated. In contrast, mono- transfected HEK293 cells. As shown in Fig. 2A, lower panel, cytes were found to be CD1a−, CD14high cells with a lobular the TLR4/MD2/CD14-NF-κB reporter gene assay showed that nucleus (Fig. 3A).32 NF-κB activation is increased in the presence of LPS, both with To assess if AuNPs have an impact on LPS-induced immune This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. or without AuNPs, while none of the tested batches was able to responses, the obtained moDCs were incubated for 48 h either activate NF-κB when applied alone at the dose of 5 × 1011 NPs with LPS, AuNPs of different sizes (4, 8, 11, and 26 nm), AuNPs Open Access Article. Published on 15 April 2021. Downloaded on 5/27/2021 1:45:14 PM. per well (Fig. 2A, bottom). Since HEK293 cells expressing the plus LPS or left untreated (Fig. 3B). Upon LPS stimulation, DCs LPS receptor complex are as sensitive to LPS as DCs are,41 we are known to become activated and secrete several soluble conclude that AuNPs applied at a dose of 5 × 1011 NPs per well mediators, which are crucial for Th cell differentiation. As are not sufficient to activate human DCs via their residual shown in Fig. 3B and ESI Fig. 1,† and in line with previous endotoxin contamination. We thus used this NP concentration reports,29,41,47,48 the DCs produced high amounts of cytokines for all the following experiments. (IL-12, IL-27, IL-6, TNF-α) and chemokines (IL-8/CXCL8) in The next important step was to quantify, and eventually response to LPS stimulation. However, when LPS-treated DCs exclude, NP-induced toxicity, as this can deeply impair the were co-incubated with 26 nm AuNPs, the secretion of IL-12 ability of DCs to upregulate surface markers and release and IL-27, which are cytokines involved in promoting the soluble mediators, thus possibly masking immunomodulatory differentiation of Th1 cells,32,47–50 was significantly reduced properties of NPs. To this end, we performed both lactate (Fig. 3B). Other cytokines, such as TNF-α, IL-8/CXCL8 and IL-6, dehydrogenase (LDH) and CellTiter-Blue (CTB) assays in which are not clearly associated with Th1 cell development, moDCs that had been treated with AuNPs for 48 h. The LDH were not significantly affected by the treatment (ESI Fig. 1†). test measures cell death by quantifying LDH, a cytoplasmic Interestingly, 4, 8 and 11 nm AuNPs did not impair LPS-depen- enzyme released upon cell membrane lysis, whereas CTB pro- dent DC-activation and none of the tested batches were vides information on cellular metabolic activity by assessing capable of inducing the release of cytokines in the absence of the conversion of the substrate resazurin into resorufin by LPS (Fig. 3B and ESI Fig. 1†). viable cells. As shown in Fig. 2B, no significant cytotoxicity to moDCs was induced by exposing the cells to 5 × 1011 NPs per 3.4 26 nm AuNPs affect LPS-dependent surface marker well, which further supports the use of this dose in the follow- expression in moDCs ing experiments. Interestingly, these results are in line with Another hallmark of DC maturation is the expression of the comprehensive study by Khlebtsov et al.,44 who found that surface markers such as CD86, ILT3 and HLA-DR. Like soluble AuNPs are generally nontoxic when applied at concentrations mediators such as cytokines, these cell-surface proteins play a lower than 1012 NPs per mL. The high background observed in crucial role in determining the nature and intensity of an the LDH assay can be explained by LDH naturally present in immune response. Surface molecules facilitate the physical FBS, which is used as a supplement in the cell culture interaction of DCs and T cells during the process of antigen medium.45 presentation, whereby HLA-DR, for example, has the function of presenting antigenic peptides to antigen-specific naïve T 3.3 26 nm AuNPs decrease the LPS-dependent release of cells. In combination with signals provided by the costimula- IL-12 and IL-27 in DCs tory molecule CD86, which is upregulated upon LPS stimu- Monocyte-derived DCs are a subset of DCs which differentiate lation, T-cell activation can be induced. In contrast, the co- from monocytes in tissues under inflammatory conditions and inhibitory molecule ILT3 counteracts T-cell activation by pro- play a crucial role in host defense.46 These cells can also be viding immunoregulatory signals.51 Therefore, we assessed the generated in vitro by culturing monocytes with a cocktail of expression of CD86, HLA-DR and ILT3 on moDCs treated with polarizing cytokines. Therefore, they are an interesting model AuNPs/LPS by using flow cytometry. for investigating nano-immune interactions during acute In line with previous studies, LPS promoted significant inflammation.32 upregulation of both HLA-DR and CD86 expression on the DC To generate moDCs, we first purified human PBMCs from surface in comparison to untreated control cells (Fig. 4A).29,52 the blood of healthy donors via density gradient centrifugation Interestingly, 4, 8, and 11 nm AuNPs did not modulate the DC (Fig. 3A). Subsequently, we incubated these cells with beads response to LPS (Fig. 4A). However, treatment with 26 nm coated with anti-CD14 antibodies and extracted a population AuNPs/LPS caused significant downregulation of CD86 of CD14+ monocytes via magnetic cell separation. Monocytes (Fig. 4A), accompanied by robust upregulation of ILT3 were then differentiated into moDCs in the presence of IL-4 (Fig. 4B), while HLA-DR remained unchanged. Fig. 4C shows and GM-CSF for 7 days.31 To confirm the efficiency of the the relative expression of CD86 and ILT3 on cells of one repre- differentiation protocol, we investigated cell morphology and sentative donor. This journal is © The Royal Society of Chemistry 2021 Nanoscale, 2021, 13, 7648–7666 | 7655 View Article Online Paper Nanoscale This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 15 April 2021. Downloaded on 5/27/2021 1:45:14 PM. Fig. 3 26 nm AuNPs hamper LPS-induced cytokine secretion in moDCs. (A) Schematic representation of the protocol used. Morphological changes occurring during the moDC-differentiation process after MACS-purified monocytes have been incubated with GM-CSF and IL-4 are summarized in the rectangular insets. Monocytes are characterized by a U-shaped nucleus and high CD14 expression, whereas DCs exhibit several cell-protrusions, high CD1a expression and the loss of CD14 on their surface. Cells were stained with Diff-Quik stain and imaged by optical microscopy. (B) To assess the influence of AuNPs on LPS-induced cytokine release, moDCs were cultivated for 48 h with the stated stimuli (5 × 1011 NPs per well, 30 ng mL−1 LPS) and subsequently supernatant was collected and analyzed for IL-12 and IL-27 secretion via ELISA. Statistical analysis was performed using repeated-measures ANOVA combined with Tukey’s post test **P < 0.01; ****P < 0.0001 (4 individual experiments, n ≥ 7). Data are shown as mean + SD. Significance is shown as indicated. RBC, red blood cell, DC, dendritic cell, LPS, lipopolysaccharide, NP, nanoparticle, PML, polymorphonuclear leukocyte; PBMC, peripheral blood mononuclear cell. To exclude that our findings are batch-specific, we repeated Taken together, the results described in Section 3.3 and 3.4 all experiments using a batch of fully characterized 25 nm demonstrate that 26 nm AuNPs, but not smaller AuNPs, exert AuNPs produced as described for the first batch and obtained regulatory effects capable of impairing LPS-dependent DC similar results (ESI Table 2 and Fig. 2†). maturation, potentially affecting the capacity of DCs to 7656 | Nanoscale, 2021, 13, 7648–7666 This journal is © The Royal Society of Chemistry 2021 View Article Online Nanoscale Paper This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 15 April 2021. Downloaded on 5/27/2021 1:45:14 PM. Fig. 4 26 nm AuNPs downregulate LPS-induced CD86 expression while promoting ILT3 upregulation. To assess the influence of AuNPs of different sizes on LPS-induced surface marker expression, moDCs were cultivated for 48 h with the stated stimuli (5 × 1011 NPs per well, 30 ng mL−1 LPS), subsequently the cells were harvested and analyzed via flow cytometry. Graphs represent expression in terms of MFI of (A) pro-inflammatory markers and (B) anti-inflammatory markers on moDCs after stimulation. (C) Dot-plots depicting ILT3 and CD86 expression in control DCs and DCs treated with LPS, 26 nm AuNPs, or 26 nm AuNPs/LPS. Samples are from of one representative donor. Statistical analysis was performed using repeated-measures ANOVA combined with the Tukey’s post test. ****P < 0.0001 (three individual experiments, n = 6). Data are shown as mean + SD. LPS, lipopolysaccharide, MFI, mean fluorescence intensity, NP, nanoparticle. promote T-cell activation. Interestingly, AuNPs exerted these which are important mechanisms for eliminating alloreactive immunomodulatory effects only in the presence of a concomi- T cells and regulating the intensity and duration of inflamma- tant microbial stimulus (LPS). The fact that immunomodulat- tory immune responses.59 In this scenario, ILT3, which is a ing effects of AuNPs on DCs are rare is well documented in the characteristic marker of tDCs,60 plays a crucial role in the sup- literature53–55 and explains why AuNPs are generally con- pressor capacity of T cells and induces T-cell anergy.61 sidered to be safe and suitable for biomedical applications. In It is worth noting that, to our knowledge, this is the first the context of ongoing inflammation, however, we found that study to report that citrate-stabilized AuNPs can affect IL-27 our largest AuNPs did hamper DC activation, promoting the and ILT3 production. Effects of AuNPs on CD86 and IL-12 were acquisition of a semi-mature phenotype characterized by previously reported by Villiers et al. and Tomić et al., who reduced expression of the activation marker CD86, diminished observed similar immunomodulatory effects.54,55 However, in secretion of the pro-inflammatory cytokines IL-12 and IL-27, our study, the effects were induced exclusively by 26 nm AuNPs and concomitant upregulation of the anti-inflammatory recep- (and not 10 nm AuNPs). Moreover, we saw no evidence of tor ILT3. This phenotype differs substantially from that com- effects on HLA-DR expression. This discrepancy might be due monly induced by LPS10 or other bacterial stimuli,56 to several factors, including the doses of the stimulants, NP suggesting that the activation of anti-bacterial Th1 cells, which purity and NP concentrations. are usually generated upon LPS stimulation, and whose differ- Interestingly, in our study, only factors involved in Th1 entiation relies on CD86, IL-12 and IL-27 secretion,50,57,58 may polarization (CD86, IL-12, IL-27) were affected by AuNP treat- be significantly impaired by the application of 26 nm AuNPs. ment, while the ability of DCs to express HLA-DR, IL-8/CXCL8, A semi-mature phenotype is a typical feature of tolerogenic DC TNF-α and IL-6 was not impaired. These findings suggest that (tDCs). tDCs, by presenting antigens in the context of reduced DCs may retain their ability to promote antigen presentation costimulatory capacity (CD86low) and a significantly altered (HLA-DR), neutrophil recruitment (IL-8/CXCL8),62 and Th17 cytokine profile, can promote tolerance and T-cell anergy, differentiation (IL-6).11 This journal is © The Royal Society of Chemistry 2021 Nanoscale, 2021, 13, 7648–7666 | 7657 View Article Online Paper Nanoscale 3.5 Ultrastructure of the endosomal system and intracellular compartments.68,69,71 The ESCRT machinery plays a role not localization of NPs only in MVB maturation, but also in different steps of the In our attempt to understand the molecular mechanisms autophagic pathway. In fact, the depletion of ESCRT proteins underlying the effects of 26 nm AuNPs, we performed uptake in cells is commonly associated with the accumulation of studies and ultrastructure analysis of our untreated autophagosomes, similar to what we observed in our double- treated samples.72 Interestingly, AuNPs were shown by (Fig. 5A-Aii), LPS-treated (Fig. 5B-Bii), AuNP-treated (Fig. 5Cii Ma et al. to be able to block autophagy and alter the activity and ESI Fig. 3A, B†) and AuNP/LPS-treated DCs (Fig. 5D-Dii of the endosomal compartment by increasing the internal pH and ESI Fig. 3C, D†) via transmission electron microscopy of these vesicles and de facto blocking their fusion with lyso- This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. (TEM) after cryofixation. The obtained data show that the somes.73 In line with these findings, ESCRT components internal structure and, more precisely, the endosomal system have been found in fungi to be part of a pH-sensing Open Access Article. Published on 15 April 2021. Downloaded on 5/27/2021 1:45:14 PM. of LPS-activated DCs (Fig. 5B-Bii) differ substantially from complex which, under alkaline conditions, changes its local- those of untreated control cells (Fig. 5A-Aii).63 In particular, ization from the endosomal compartment to the plasma LPS induced an increase in the size (up to 2 µm) of electron- membrane,74 suggesting that pH changes might indeed play lucent late endosomes,64 some of which are characterized by a crucial role in the functionality of the DC endocytic the presence of intraluminal “buds” representing the early compartment. steps of formation of intraluminal vesicles (ILVs, black aster- This altered ultrastructural phenotype might partially isks),65 which are indicative of the endosomal transition to explain our finding at the cytokine level. In fact, LPS signaling multivesicular bodies (MVBs).66 These endosomes were often is known to rely on two different pathways: the MyD88-depen- accompanied by the appearance of autophagosomes (with dent cytosolic pathway, and the TRAF-TRIF-dependent intact cytoplasm) and autophagic compartments ( partly pathway, which relies on receptor endocytosis.4,66,75 Once acti- degraded material) in large numbers (white asterisks).64 vated, both pathways promote the expression of several genes When DCs were treated with AuNPs instead, a clear uptake/ linked to inflammation. However, while it has been reported internalization was visible, both in the absence (Fig. 5C-Cii that TNF-α and IL-6 are mainly regulated by MyD88, IL-12 and ESI Fig. 3A, B†) or presence of LPS (Fig. 5D-Dii and ESI secretion depends on signaling along both pathways, Fig. 3C, D†). In all the examined images, AuNPs were always suggesting that its production might be affected by NP- found enclosed within vesicles, and when the DCs were treated mediated changes in receptor endocytosis and trafficking exclusively with NPs, the ultrastructure of the DC endosomal toward different endocytic compartments such as the endo- compartment was not significantly different from that of somes or MVBs.66,76–78 Additionally, it has been suggested by control cells. Interestingly, in this case, NPs were found to be Gangloff that endosomal pH and curvature (which is size- mainly localized in tubule-vesicular structures of the endo- dependent) may also play a role in the capacity of TLR4 to somal system (Fig. 5Cii and ESI Fig. 3A, B†) and occasionally recruit TRAM and activate the downstream signaling in late endosomes containing ILVs (black arrowhead) (ESI pathways.75 Fig. 3A†). Overall, the tubule-vesicular structures where com- Therefore, we propose that deviations in the endocytic posed of both electron-light (low buoyant density) and elec- pathway may partially explain the observed effect of AuNPs on tron-dense (high buoyant density) vesicles, with the latter DC activation; however, further studies are needed to confirm resembling classic lysosomes.67 this hypothesis. In contrast, when DCs were co-exposed to LPS and AuNPs (Fig. 5D-Dii), we observed dramatic changes in cell ultrastruc- 3.6 26 nm-treated DCs are less proficient at promoting Th1 ture and NP localization in comparison to both LPS-treated activation and AuNP-treated DCs. Upon microbial infection, DCs quickly initiate adaptive In the doubly treated cells, there was reduced formation of immune responses by activating T lymphocytes. For this large electron-lucent late endosomes, and the NPs were found reason, it is important to assess whether the changes we to be mainly segregated within autophagic compartments. detected in the DC phenotype, including the expression of Interestingly, only the combination treatment promoted the surface molecules, cytokine secretion, formation of endocytic formation of multilamellar cisternae organized in stacks structures and intracellular localization of AuNPs, affect the (black stars) (Fig. 5Dii) or U-shaped invaginated vesicles DC’s ability to induce T-cell activation. A well-accepted model observed in cross-section (white arrowheads) (Fig. 5Dii and ESI for studying such aspects is the allogeneic co-culture system. Fig. 3C, D†) resembling the E class compartments described In this study, we first pre-treated moDCs for 48 h and then by Russel et al. and other authors, which are considered a assessed their stimulatory capacity by co-culturing them with form of “stressed” MVBs.65,68–70 allogeneic CD4+ Th-cells for 6 days (Fig. 6A). During this The formation of class E compartments and “stressed” mul- period, DCs provide important signals to Th cells, which tivesicular bodies is strongly dependent on the activity of the instruct the latter to secrete cytokines indicative of their ESCRT machinery. When ESCRT is dysfunctional, endosomes specific function. and MVBs stop their correct maturation process, do not To confirm the quality of the APCs used for the co-culture produce ILVs, become “frustrated” and cluster to form class E experiment, the activation state of moDCs was fully character- 7658 | Nanoscale, 2021, 13, 7648–7666 This journal is © The Royal Society of Chemistry 2021 View Article Online Nanoscale Paper This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 15 April 2021. Downloaded on 5/27/2021 1:45:14 PM. Fig. 5 AuNP intracellular localization. To assess if 26 nm AuNPs are phagocytosed by DCs and if they can change the cellular phenotype at the ultrastructural level, moDCs were treated with the following stimuli and subsequently prepared for TEM inspection. (A-Aii) Untreated control, (B-Bii) LPS control; (C-Cii) 26 nm AuNPs; (D-Dii) 26 nm AuNPs + LPS. Black asterisks, endosomes; white asterisks, autophagic structures; black star, class E-like compartment; white arrowheads, “frustrated” MVBs. This journal is © The Royal Society of Chemistry 2021 Nanoscale, 2021, 13, 7648–7666 | 7659 View Article Online Paper Nanoscale This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 15 April 2021. Downloaded on 5/27/2021 1:45:14 PM. Fig. 6 26 nm AuNPs downregulate the expression of Th1-related cytokines. (A) To assess the influence of 26 nm AuNPs on the ability of moDCs to induce T-cell polarization, especially toward the Th1 phenotype, pre-treated moDCs (A1) were co-cultured with allogenic CD4+ T cells, obtained via magnetic cell separation (A2), for 6 days (A3). Afterwards, supernatants were analyzed by ELISA or LegendPLEX to investigate the secretion of Th-specific soluble mediators, which are shown in (B). Statistical analysis was performed using repeated-measures ANOVA combined with the Tukey’s post test (five individual experiments, n ≥ 8). Data are shown as mean + SD. *P < 0.05. AuNP, gold nanoparticle; LPS, lipopolysaccharide; NP, nanoparticle; n.s., not significant; PBMC, peripheral blood mononuclear cell; Th, T helper cell. ized by flow cytometry and enzyme-linked immunosorbent whereas other cytokines which are not associated with Th1 assay (ELISA) prior to co-incubation with T cells (ESI Fig. 4†). activation (e.g., TNF-α and IL-17) were not affected by 26 nm As mentioned above, LPS-stimulated moDCs would be AuNPs. expected to induce a specific T-cell subpopulation known as In fact, the secretion of IL-27 by DCs is of crucial impor- Th1 cells, which mainly secrete IFN-γ and IFN-γ-related tance for the initial commitment of naïve T cells to the Th1 factors.11,79 Consistent with this, LPS-treated moDCs promoted lineage, because this cytokine is able to promote the induction the release of IFN-γ as well as the IFN-γ-inducible protein of the Th1-specific transcription profile, which is subsequently CXCL10 in corresponding Th cells (Fig. 6B). However, the pro- stabilized by sustained IL-12 signaling.32,47–50,80 Since IL-27 duction of these factors was significantly lower in Th cells co- was significantly downregulated by 26 nm AuNPs (Fig. 3B) and cultured with 26 nm AuNP-treated LPS-stimulated DCs, the IL-27/IL-12-induced transcription profile is also directly 7660 | Nanoscale, 2021, 13, 7648–7666 This journal is © The Royal Society of Chemistry 2021 View Article Online Nanoscale Paper responsible for the secretion of IFN-γ in Th1 cells, it was not Because Th1 cells and their lead cytokine IFN-γ play a surprising to find IFN-γ significantly reduced in T cells incu- crucial role in macrophage activation and the recruitment of bated with LPS/AuNP-treated DCs. In accordance with this, we cytotoxic cells, the observed loss of Th1 cells might have a also observed a lower secretion of the IFN-γ-dependent chemo- negative effect on both pathogen clearance and elimination of kine CXCL10, which is known to attract many cell types to cancerous cells.81–85 However, Th1 cells also seem to be sites of infection.79 involved in the pathogenesis of autoimmune diseases such as This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 15 April 2021. Downloaded on 5/27/2021 1:45:14 PM. Fig. 7 26 nm AuNPs downregulate total numbers of TCMc and Th1 cells and T-cell proliferation. To assess the activity of 26 nm AuNPs in a more realistic model, human PBMCs were incubated, after resting, for 6 days with the stated stimuli (5 × 1011 NPs per well, 30 ng mL−1 LPS), and sub- sequently the supernatants were analyzed via ELISA and the cells were analyzed via flow cytometry (two individual experiments, n = 4). (A) Graphic summary of the protocol. (B) Representative dot plot depicting the distribution of the T cells within the different CD4+ T cell populations (Naïve, Effector, TEMc, TCMc) upon PBMC stimulation with 26 nm AuNPs ± LPS. Untreated PBMCs serve as a control. Graphs showing (C) total memory (TCMc and TEMc), (D) total Th1, (E) total proliferating CD4+, and (F) proliferating TCMc and Th1 cells expressed in terms of cells per µL. (G) IL-27 secretion from PBMCs. Statistical analysis was performed using repeated-measures ANOVA combined with the Tukey’s post test. *P < 0.05; **P < 0.005. Data are shown as mean + SD. DC, dendritic cell; LPS, lipopolysaccharide; NP, nanoparticle; PBMC, peripheral blood mononuclear cell; TCMc, T central memory cell; TEMc, T effector memory cell; Prolif. proliferating. This journal is © The Royal Society of Chemistry 2021 Nanoscale, 2021, 13, 7648–7666 | 7661 View Article Online Paper Nanoscale rheumatoid arthritis,86 which, interestingly, was treated in the number of cells is often linked to limited proliferation capacity past using gold-based therapeutics. This suggests that the in the tested subpopulations. We therefore labeled our observed effects of AuNPs on Th1 differentiation might be samples with antibodies directed against the proliferation advantageous in the presence of autoimmune diseases, but marker Ki-67 and found that LPS-dependent proliferation of potentially harmful in the context of pathogen clearance. total, TCMc and Th1 CD4+ lymphocytes was indeed signifi- Our observation that AuNPs do not alter the maturation or cantly reduced by 26 nm AuNPs (Fig. 7E and F). activation of DCs in the absence of a concurrent microbial Several soluble mediators, such as IL-27, are known to regu- stimulus is consistent with a previous study by Malachin et al., late the ability of these cells to proliferate. Thus, we tested our who showed that silica NPs (SiNPs) are able to potentiate the cell supernatant and found a positive correlation between the This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. ability of DCs to activate T cells in response to PAMPs, while loss of proliferative potential and reduced IL-27 secretion SiNPs alone were unable to activate DCs, unless administered (Fig. 7G). This is of particular importance because this cyto- Open Access Article. Published on 15 April 2021. Downloaded on 5/27/2021 1:45:14 PM. in combination with microbial components.87 Taken together, kine also plays a crucial role in Th1 differentiation.89,90 these studies further support the importance of investigating Taken together, these experiment on autologous cells NP immunomodulatory properties during ongoing immune further support the findings generated in the allogeneic co- responses and not only under homeostatic conditions. culture on Th1 activation both in terms of number and prolif- erative potential of this population. Additionally, utilizing 3.7 26 nm AuNPs affect Th1 and TCMc numbers and flow cytometry, we proved that the presence of AuNPs dimin- proliferation ished not only Th1 cells but also the ability of TCMc to pro- When NPs are used for medical applications, they are often liferate in response to LPS stimulation. This fact may be injected intravenously into the bloodstream, where they come linked to the reduced costimulatory capacity of DCs character- into contact with a pool of immune cells called peripheral ized by both the upregulation of ILT3, which promotes T-cell blood mononuclear cells (PBMCs). Within this pool, both DCs anergy,61 and the attenuated expression of CD86 as well as and T cells are present; therefore, blood-derived PBMCs are the reduced secretion of IL-27 and IL-12. Interestingly, only considered a good model to investigate the effects of NMs on central and not effector memory cells reacted to LPS and homologous cell cultures.33 Here, we first extracted human were consequently affected by AuNPs. This fact might depend PBMCs from the blood of healthy donors, and incubated them on several differences that characterize these two subsets. For for 6 days in the presence of the respective stimuli (26 nm example: (1) TCMc are more prone to proliferate and might AuNPs, LPS, and LPS plus 26 nm AuNPs). Finally, we assessed therefore be more sensitive to reduced production of IL-27; the effects of LPS and 26 nm AuNPs on the activation of both (2) TCMc seem to be characterized by a lower activation Th1 and memory cells via flow cytometry (Fig. 7A) using an threshold than TEMc, which might make them more sensi- advanced 13-color flow cytometry panel (the gating strategy is tive to LPS stimulation; and finally, (3) TCMc tend to interact summarized in ESI Fig. 5†). more with professional APCs in comparison to TEMc, and In human blood, we find mainly two types of memory cells: therefore might be more impacted by the presence of tDCs.8 central memory T cells (TCMc) and effector memory T cells These results suggest that AuNPs negatively affect not only (TEMc).33 These populations can be distinguished based on Th1 functions, but also the activation and proliferation of the expression of distinct homing receptors which grant them lymphocytes within the central memory compartment, poten- different migration capacities. TCMc express CD197, which tially affecting the outcome of immune responses in the long allows their recruitment to lymphatic tissues, whereas TEMc term. express mainly markers that direct them to the periphery. Memory cells do not express the surface marker CD45RA and therefore can be easily distinguished via flow cytometry from 4. Conclusions CD45RA+ naïve and effector cells.88 As shown in Fig. 7B, by using these markers, we could Here we provided experimental evidence to show that, in the demonstrate that memory T cells (including TCMc and TEMc) absence of pro-inflammatory stimuli, AuNPs of different sizes represent the majority of the CD4+ Th cell populations in can be considered to be immunologically safe, since they show PBMCs,33 with TCMc being the most abundant subset. Upon neither immunomodulatory properties nor the ability to sig- treatment, memory cells responded differently to stimulation. nificantly affect, upon internalization, the ultrastructure of We observed an increase in the total number of TCMc, but not DCs up to a concentration of 5 × 1011 NPs per well. However, of TEMc, in LPS-treated samples. Interestingly, this expansion when 26 nm AuNPs (but not smaller ones) are combined with was significantly hampered by 26 nm AuNPs in TCMc, while LPS, they can direct DCs toward a tolerogenic-like phenotype there was no clear effect on LPS-stimulated TEMc (Fig. 7C). (CD86low, IL-12low, IL-27low, ILT3high, class E compartments+), We then investigated the amount of Th1 cells present in which is less proficient in promoting LPS-dependent Th1 acti- our samples, as they are the subset most responsive to LPS vation and TCMc proliferation, both in allogeneic co-culture and other bacterial products. As shown in Fig. 7D, 26 nm systems and in autologous PBMC culture settings. We con- AuNPs caused a decrease in LPS-dependent cell expansion, sig- clude that especially under pro-inflammatory conditions, nificantly lowering the total number of Th1 cells. A reduced 26 nm AuNPs may have immunomodulatory capacities. For 7662 | Nanoscale, 2021, 13, 7648–7666 This journal is © The Royal Society of Chemistry 2021 View Article Online Nanoscale Paper further studies, we would therefore recommend investigating 5 N. Hafsi, P. Voland, S. Schwendy, R. Rad, W. Reindl, the effects of NMs in both non-activated as well as activated M. Gerhard and C. Prinz, Human Dendritic Cells Respond immune cells to better understand their immunomodulatory to Helicobacter pylori Promoting NK Cell and Th1-Effector functions and to predict potential effects of NMs on ongoing Responses In Vitro, J. Immunol., 2004, 173, 1249–1257. immune responses. 6 K. Assani, M. F. Tazi, A. O. Amer and B. T. Kopp, IFN-γ Taken together, these findings further support the idea that Stimulates Autophagy-Mediated Clearance of Burkholderia AuNPs are safe under homeostatic conditions; however, par- cenocepacia in Human Cystic Fibrosis Macrophages, PLoS ticular care should be taken when deploying them in the One, 2014, 9, e96681. course of ongoing immune responses. 7 M. C. Greenlee-Wacker and W. M. Nauseef, IFN-γ targets This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. macrophage-mediated immune responses toward Staphylococcus aureus, J. Leukoc. Biol., 2017, 101, 751–758. Open Access Article. Published on 15 April 2021. Downloaded on 5/27/2021 1:45:14 PM. Author contributions 8 F. Sallusto, J. Geginat and A. Lanzavecchia, Central Memory and Effector Memory T Cell Subsets: Function, Generation, Conceptualization: S.M., F.B., A.D., D.D, V.P., U.L., J.H.H. and Maintenance, Annu. Rev. Immunol., 2004, 22, 745–763. 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