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Abstract

Background: The rapid advancement of nanotechnology has led to a wide use of engineered nanomaterials (ENMs). Ensuring their safety remains critical for successful clinical translation. It was previously reported that administration of ferumoxytol (an FDA-approved iron oxide nanoparticle formulation) resulted in fatal anaphylactic reactions. Importantly, previous studies demonstrated that ENMs can induce mast cell early-phase activation, with 20 nm citrate-coated silver nanoparticles (AgNPs) producing a robust response. Purpose: This study investigated late-phase activation of mast cells following exposure to AgNPs in comparison to a classical IgE activation. Research Design: The study used murine bone marrow-derived mast cells (BMMCs) exposed to 20 nm citrate-coated AgNPs (25 μg/mL). Results: AgNP exposure induced late-phase BMMC activation, though this response was delayed and lower in magnitude compared to IgE activation. Specifically, exposure to AgNPs induced lipid mediator release (1h), and in some cases, showed differential regulation, with an overall lower magnitude of lipid release compared to IgE activation. Furthermore, AgNP exposure activated several inflammatory genes, with a delayed onset and reduced intensity relative to IgE stimulation. These data were reflected in the activation of mitogen-activated protein kinases (MAPKs) with significant changes occurring at later time points (24h). Importantly, no cytotoxicity or elevated levels of intracellular reactive oxygen species were observed, although Lamp-2 expression remained upregulated compared to IgE stimulation. Conclusion: The results suggest that AgNPs induce late-phase BMMC activation, potentially via a non-IgE-mediated mechanism characterized by a delayed and overall attenuated response. These findings underscore the importance of understanding ENM–bio interaction, not only for nanosafety, but also for unraveling novel therapeutic applications.

Keywords

allergy anaphylaxis nanoparticles nanotechnology nanosafety

Introduction

The prevalence of type I allergic disease, such as asthma and food allergies, is increasing in developed and developing societies.¹ Such an increase is partially attributed to environmental exposure.^1,2 For instance, it has been previously demonstrated that exposure to specific environmental factors, such as diesel exhaust particles, can induce or exacerbate type I allergic disease.² However, our mechanistic understanding regarding which environmental exposures result in type I allergic reactions remains limited.

Mast cells are key effector cells in type I hypersensitivity reactions.^3,4 They are abundantly present at host-environment interfaces, such as the mucosal tissues of the gastrointestinal tract, skin, and respiratory system, and hence, they act as frontline defenders against foreign substances and pathogens.³ Activation of mast cells is implicated in a wide range of type I allergic diseases such as asthma, atopic dermatitis, and anaphylaxis.⁴ The severity of mast cell-driven type I allergic reactions ranges from mild hypersensitivity to life-threatening anaphylaxis. Mast cell activation via the high-affinity immunoglobulin E (IgE) receptor (FcεR1) occurs in response to antigen crosslinking of IgE molecules bound to surface FcεR1. This results in activation of an early-phase (degranulation) response involving a rapid release of preformed granules, and a late-phase response including synthesis and release of lipid mediators (i.e., arachidonic acid metabolites), and cytokines and chemokines.⁵ Such responses consequently result in recruiting other immune cell types, such as eosinophils and macrophages, leading to pathophysiological changes that eventually result in the development of allergic disease.⁵ Mast cell activation also occurs in response to non-IgE-mediated stimuli recognized via a variety of surface receptors such as G-protein-coupled receptors (GPCRs), Toll-like and NOD-like receptors.⁶

Nanotechnology has transformed various industrial sectors, driven by the rapid growth of consumer products and applications that incorporate engineered nanomaterials (ENMs) to enhance material properties and functionality.⁷ However, their long-term safety in humans remains poorly understood.⁸ One of the critical toxicological outcomes associated with ENM exposure is modulation of immune responses and immune cell function.⁹ Accumulating evidence suggests that exposure to even subtoxic levels of ENMs can alter immune responses.¹⁰ Notably, activation and modulation (activation or suppression) of type I hypersensitivity reactions have been reported following exposure to ENMs.¹¹ A significant clinical incident of fatal severe anaphylaxis has been reported following treatment with ferumoxytol, an iron oxide nanoparticle-based medication given to anemic patients with chronic kidney disease.¹² Other iron oxide nanoparticle-based medications have also been associated with the induction of allergic-like reactions.^13,14 This is not exclusive to metallic ENMs as micellar and polymeric nano-formulations have also been shown to induce hypersensitivity reactions.¹⁵

Importantly, previous studies have demonstrated that exposure to ENMs, depending on their composition and physicochemical properties, can activate mast cells and modulate their response to IgE-mediated stimulation.^16–19 Silver nanoparticles (AgNPs) are among the most widely used ENMs, with applications ranging from clothing and food packaging to paints and coatings for medical devices.²⁰ Despite their widespread use, previous studies have reported toxicological effects of AgNPs in both humans and experimental models, indicating that their safety profile is not fully understood.^21–23 We previously demonstrated that exposure to AgNPs can induce mast cell degranulation (an early-phase activation response) driven mainly by their physicochemical properties, including size, shape, and surface coating.²⁴ Notably, our previous studies demonstrated robust mast cell degranulation following exposure to 20 nm citrate-coated AgNPs, potentially mediated through an IgE-independent mechanism.^24–26 However, it remains unknown whether, and to what extent, exposure to 20 nm citrate-coated AgNPs could affect late-phase activation of mast cells. Therefore, the aim of this study was to assess whether exposure to AgNPs induces late-phase mast cell activation in comparison to a classical IgE-mediated activation.

Methods

Characterization of silver nanoparticles

20 nm citrate-coated AgNPs (1 mg/mL) were purchased from NanoComposix (San Diego, CA). Size and shape of the AgNPs were confirmed by transmission electron microscopy (TEM) (Figure S1). Hydrodynamic size and zeta potential were measured by Malvern DLS Nanosizer. The hydrodynamic diameter of AgNPs in RPMI (Roswell Park Memorial Institute) cell culture media was 250.63 ± 5.30 nm and the zeta potential was −18.63 ± 0.55 mV. It is worth noting that AgNPs formed larger aggregates and/or agglomerate when suspended in serum-supplemented RPMI. This is typically observed with different types of metal and metal-oxide ENMs when they are suspended in vehicle and biological fluids.^27–29 We used a concentration of 25 μg/ml of AgNPs because we have shown in the past that it produces robust early-phase responses (degranulation) without inducing any direct toxicity. In this work, we wanted to be consistent and to assess whether this concentration would induce a late-phase activation.

Cell isolation and differentiation

Bone marrow-derived mast cells (BMMCs) were differentiated from stem cells isolated from the femur bone of male C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME). Cells were cultured at 37°C and 5% CO₂ for 4 to 6 weeks in in RPMI cell culture media supplemented with 10% FBS, 100 μg/ml streptomycin, 100 U/ml penicillin, 100 μg/ml primocin (Invitrogen, San Diego, CA), 25 mM HEPES, 1.0 mM sodium pyruvate, nonessential amino acids (BioSource International, Camarillo, CA), 0.0035% 2-mercaptoethanol, and 300 ng/ml purified recombinant mouse IL-3 (PeproTech, Rocky Hill, NJ). Mature BMMC populations were routinely confirmed based on the surface expression of FcεRI and cKIT via flow cytometry as previously described.²⁴ Animal procedures were carried out in accordance with the National Institutes of Health guidelines and approved by the University of Colorado Denver Institutional Animal Care and Use Committee (IACUC protocol number 00281). Mice were housed in individually ventilated, micro-isolated cages (four animals per cage) under a 12-h light/dark cycle, with free access to food and water. Experiments were performed on mature mast cells from at least three individual batches of cells (n ≥ 3), with each batch grown from femoral bone marrow of two mice.

Assessment of apoptotic and necrotic cell death

Cells were evaluated using Annexin V staining to detect apoptotic cell death (BD Biosciences, San Jose, CA) and propidium iodide (PI) staining to detect necrotic cell death (Invitrogen, San Diego, CA). Briefly, cells (2 × 10⁵ cells per sample) were exposed to AgNPs (25 μg/ml) or dinitrophenylated human serum albumin (DNP-HSA) (100 ng/ml) (Sigma–Aldrich, St Louis, MO) (hereafter referred to as DNP) for 24h. Samples were washed twice with PBS, then resuspended in PI-containing binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4) at a final concentration of 3 μM. Annexin V (2.5 μl) was then added to samples. Following 15 min incubation at room temperature in the dark, fluorescence was measured by flow cytometry (BD Accuri™ C6, BD Biosciences, San Jose, CA). Cells were gated to exclude debris based on the control group by selecting the main cell population on the forward scatter area (FSC-A) versus side scatter area (SSC-A) plot (i.e., excluding events with low FSC/SSC signals in the bottom-left corner).

Mast cell degranulation (early-phase activation)

Mast cell degranulation was assessed based on the release of β-hexosaminidase and surface expression of Lamp-2. Briefly, BMMCs were seeded in 96-well flat-bottom plate at 50,000 cells per well overnight. Cells were treated with AgNPs (25 μg/ml) (NanoComposix, San Diego, CA) or DNP (100 ng/ml) (these cells were previously sensitized with mouse anti-DNP IgE antibody (100 ng/ml) overnight) at the indicated time points. P-nitrophenyl-N- acetyl-b-D-glucopyranoside (PNAG) (Sigma–Aldrich, St Louis, MO) (a chromogenic substrate of β-hexosaminidase) was then added to both cell supernatants and lysates (90 min at 37 C). Glycine (0.4 M) was then added to stop the reaction and optical density was read at 405 nm Synergy^TM Microplate Reader (BioTek Instruments Inc, Winooski, VT). Percent degranulation was calculated based on the following equation: [ (supernatant x 2/lysate x 4) x 100 ] (dilution factors). Mast cell surface Lamp2 expression was measured by flow cytometry based on staining with FITC-conjugated Lamp2 antibody. Briefly, BMMCs were seeded in 24-well plate at 300,000 cells per well overnight. Cells were then treated with AgNPs (25 μg/ml) or DNP (100 ng/ml) at the indicated time points. Cells were centrifuged (1200 rpm for 5 min) and pellets were gently washed 3 times in warm PBS and then re-suspended in PBS-containing FITC-conjugated Lamp2 antibody (eBioscience Inc., San Diego, CA, USA) at a dilution of 1:100 and incubated for 2h. Cells were washed 2 times with warm PBS. 10,000 events were measured using an Accuri^TM C6 flow cytometry (BD Biosciences, USA) and expression was plotted as fold change of mean intensity fluorescence relative to non-treated control. Cells were gated as described in the previous section.

Measuring intracellular reactive species levels

Reactive oxygen species formation was assessed by flow cytometry using H₂DCFDA (Invitrogen, San Diego, CA). Briefly, BMMCs (2 × 10⁵ cells per sample) were exposed to AgNPs (25 μg/ml) or DNP (100 ng/ml) for 24 h. Samples were washed twice with PBS, and resuspended in PBS-containing 5 μM H₂DCFDA. Cells were then incubated at 37°C in the dark for 30 min, after which fluorescence was measured using a flow cytometer (BD Accuri™ C6, BD Biosciences, San Jose, CA). Cells were gated as described in section 2.3.

Measuring lipid mediator release

BMMCs were seeded in a 24-well plate at 2 × 10⁶ cells per sample overnight. Cells were treated with AgNPs (25 μg/ml) or DNP (100 ng/ml) (these cells were previously sensitized with mouse anti-DNP IgE antibody (100 ng/ml) overnight) for 1h. Cells were then centrifuged at 1200 rpm at 4°C for 5 min and supernatants were collected (pellets were used to quantify protein). Lipid mediator release was measured as previously described.³⁰ Briefly, samples were diluted in methanol (9%)/ethanol (1%) and internal standard solution (1%) was added. The reconstituted extracts were loaded on a Strata-X 33 µm solid phase extraction (SPE) column (Phenomenex, Torrance, California). The SPE column was washed with 10% methanol and eluted into a reduced surface activity (recovery) glass autosampler vial with 1.0 ml of methyl formate. Using a steam of nitrogen, the methyl formate was evaporated and the SPE cartridge was eluted with methanol which was evaporated later. Samples were reconstituted with 20 μl of ethanol and analyzed or kept at 70°C until further analysis.

Quantitation of lipid mediators was performed using 2D reverse-phase HPLC tandem mass spectrometry (LC/MS/MS). The HPLC system consisted of an Agilent 1290 autosampler, an Agilent 1200 binary SL loading pump (pump 1), an Agilent 1290 binary analytical pump (pump 2), and a 6-port switching valve (Agilent Technologies, Santa Clara, California). Pump 1 buffer consists of 0.1% formic acid in water (solvent A) and 9:1 v:v acetonitrile: water with 0.1% formic acid (solvent B). Pump 2 buffer consists of 0.01% formic acid in water (solvent C) and 1:1 v: v acetonitrile: isopropanol (solvent D). 5 μl of extracted sample was injected onto an Agilent SB-C18 (2.1 × 5 mm 1.8 μm) trapping column using pump 1 at 2 ml/min for 0.5 min (97% solvent A: 3% solvent B), then at 0.51 min, the switching valve changed the flow to pump 2. The trapped lipid mediators were eluted onto an Agilent Eclipse Plus C-18 (2.1 × 150 mm 1.8 um) analytical column using the following gradient (at a flow rate of 0.3 ml/min): 75% A: 25% D (from 0 to 0.5 min), then a linear gradient from 25% to 75% D (over 20 min), then an increase from 75% to 100% D (from 20 to 21 min), then holding at 100% D for 2 min. During the analytical gradient, pump 1 washed the injection loop with 100% B (22.5 min at 0.2 ml/min). Both columns were re-equilibrated at starting conditions for 5 min before the next injection. Mass spectrometric analysis was performed on an Agilent 6490 triple-quadrupole mass spectrometer in negative ionization mode (drying gas was 250°C at 15 ml/min; sheath gas was 350°C at 12 ml/min; nebulizer pressure was 35 psi; capillary voltage was 3500 V). Data were acquired using experimentally optimized collision energies obtained by flow injection analysis of authentic standards. Calibration for each lipid mediator was analyzed at a range of concentrations (0.25 – 250 pg). Calibration curves were constructed using Agilent Masshunter Quantitative Analysis software.

Measuring gene expression

Measuring protein expression

BMMCs were seeded in 24-well plate at 1x10⁶ cells per well overnight. Cells were treated with AgNPs (25 μg/ml) or DNP (100 ng/ml) (these cells were previously sensitized with mouse anti-DNP IgE antibody (100 ng/ml) overnight) for the indicated time points. Cells were washed three times with ice-cold PBS and then lysed in ice-cold lysis buffer (80 mM Tris HCl, 1% SDS) containing phosphatase and protease inhibitors (Sigma–Aldrich, St Louis, MO) for 45 min at 4 ̊C. Samples were then briefly sonicated on ice and centrifuged at 10,000xg for 10min at 4 ̊C. Bradford reagent (Bio-Rad Laboratories Inc., Hercules, CA) was used for protein assay. 20 μg of samples were mixed with 5% of 2x Laemmli Sample Buffer (Bio-Rad Laboratories Inc., Hercules, CA) containing 2-mercaptoethanol and boiled (5 min). Samples were loaded into and ran through 12% SDS-polyacrylamide gels (Invitrogen, San Diego, CA) and then electrophoretically transferred to nitrocellulose membranes overnight. Membranes were first blocked with 5% BSA in TBS-T for 1h at 4°C. Primary antibodies (1:1000; phospho AMPK, Cat No. 9910, total MAPK, Cat No. 9926, Cell Signaling, Beverly, MA, USA) were incubated overnight (4°C). Membranes were washed three times with ice-cold PBS and then horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000; β-Actin, Cat No. 4967, Cell Signaling, Beverly, MA, USA) were incubated for 1 h at room temperature. After washing three times with ice-cold PBS, membranes were developed using Pierce Chemiluminescent Substrate (Thermoscientific, Waltham, MA). Relative densitometry (phospho-to total-protein to loading control) was normalized to average non-treated control using ImageJ software (NIH, Bethesda, MD, USA).

Statistical analysis

All graphs and analyses were performed using GraphPad Prism 5 (GraphPad, San Diego, CA). Data are presented as mean ± standard error of the mean (SEM). Statistical comparison and differences between treatment groups were performed by a one-way analysis of variance (ANOVA) and Bonferroni post hoc test. Differences between two treatment groups were performed by student’s t-test. Differences were considered statistically significant at p ≤ 0.05. Independent experiments were performed at least three times. All experiments were independently repeated at least three times (n ≥ 3). A p-value less than 0.05 (p < 0.05) was considered statistically significant between control and treatment groups (*).

Results

Early-phase activation (degranulation) following exposure to silver nanoparticles

Previous studies have reported that exposure to 20 nm citrate-coated AgNPs can induce robust mast cell degranulation.^17,24,25 Here we compared AgNP-induced BMMC degranulation over time in comparison with an IgE-mediated degranulation using two different end points based on the release of β-hexosaminidase (supernatant) and surface expression of lysosome-associated membrane protein 2 (Lamp-2) (expressed in the exterior membrane of the preformed granules).²⁴ As observed in previous studies, exposure to AgNPs induced a robust BMMC degranulation that was faster and higher in magnitude (i.e., release of β-hexosaminidase from preformed granules) compared to an IgE-mediated activation (Figure 1(a)). These results were also confirmed by measuring the surface expression of Lamp-2 protein (Figure 1(b)).

Figure 1.

Mast cell degranulation following exposure to silver nanoparticles (AgNPs). (a) Release of β-hexosaminidase from BMMCs following exposure to IgE/DNP (100 ng/ml) (blue line) or AgNPs (25 μg/ml) (black line) over several time points. (b) Cell surface expression of Lamp-2 protein following exposure to IgE/DNP (100 ng/ml) (blue line) or AgNPs (25 μg/ml) (black line) over several time points. All experiments were independently repeated at least three times (n ≥ 3). A p-value less than 0.05 (p < 0.05) was considered statistically significant between control and treatment groups (*).

Release of lipid mediators following exposure to silver nanoparticles

Release of lipid mediators represents a key late-phase response in activated mast cells.⁵ Because their synthesis and release occur relatively rapidly, we assessed BMMC lipid mediator release following a 1-h exposure to AgNPs (25 μg/ml) or IgE/DNP (100 ng/ml). Using a mass spectrometry-based approach, we measured a range of lipid mediators.³⁰ Our results show that exposure to 20 nm citrate-coated AgNPs induces BMMC mediator release across multiple pathways, including cyclooxygenases (COX) and lipoxygenases (LOX) (Figures 2 and 3 and S3); however, compared to IgE/DNP activation, the response was overall lower, in both magnitude and range of released mediators (Figures 2 and 3 and S3). Additionally, certain mediators, particularly those derived from the lipoxygenase pathways, appeared to be differentially regulated in response to AgNP exposure (Figure 2). We generated a heatmap to provide an overall view of the changes observed across all quantified lipids (Figure S4).

Figure 2.

Mast cell lipid mediator release via the cyclooxygenase pathways following exposure to silver nanoparticles (AgNPs). (a–f) BMMC lipid mediator release following exposure to IgE/DNP (100 ng/ml) or AgNPs (25 μg/ml) for 1 h. All experiments were independently repeated at least three times (n ≥ 3). A p-value less than 0.05 (p < 0.05) was considered statistically significant between control and treatment groups (*). Pg: prostaglandin; thromboxane (Tx).

Figure 3.

Mast cell lipid mediator release via the lipoxygenase pathways following exposure to silver nanoparticles (AgNPs). (a-f) BMMC lipid mediator release following exposure to IgE/DNP (100 ng/ml) or AgNPs (25 μg/ml) for 1 h. All experiments were independently repeated at least three times (n ≥ 3). A p-value less than 0.05 (p < 0.05) was considered statistically significant between control and treatment groups (*). LT: leukotriene; HETE: hydroxyeicosatetraenoic; HEPE: hydroxyicosapentaenoic.

Activation of inflammatory cytokines following exposure to silver nanoparticles

We measured gene expression of key inflammatory cytokines at multiple time points (1, 3, 6, and 24 h) following exposure to 20 nm citrate-coated AgNPs, compared to IgE/DNP-induced activation (1 h). The data indicate that exposure to AgNPs results in a significant increase in cytokine gene expression, however, compared to IgE/DNP activation, the responses were delayed and at a lower magnitude (Figure 4). This was evidenced across all measured inflammatory mediators, including TNFα, IL-1β, IL-6, MCP-1, and MIP-1α, compared to the rapid and strong response observed with IgE-mediated activation (Figure 4). Furthermore, AgNP-induced gene activation increased over several hours, peaking at 6 h and declining by 24 h. Unlike gene activation induced by IgE/DNP, it appears that exposure to AgNPs has differential gene activation with some genes (e.g., MCP-1) being induced more than others (e.g., IL-6).

Figure 4.

Mast cell inflammatory gene expression following exposure to silver nanoparticles (AgNPs). (a–e) Gene expression of inflammatory cytokines and chemokines following exposure to AgNPs (25 μg/ml) over several time points or IgE/DNP (100 ng/ml) after 1 h of exposure. All experiments were independently repeated at least three times (n ≥ 3). A p-value less than 0.05 (p < 0.05) was considered statistically significant between control and treatment groups (*).

Activation of the MAPK pathway following exposure to silver nanoparticles

IgE-mediated activation triggers rapid phosphorylation of a plethora of proteins, particularly at tyrosine residues, leading to both early- and late-phase BMMC activation.⁵ The mitogen-activated protein kinase (MAPK) pathways, comprising extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 MAPK, play critical roles in late-phase BMMC responses, including the synthesis and release of lipid mediators and cytokines.³¹ However, it remains unclear whether, and to what extent, exposure to 20 nm citrate-coated AgNPs affects MAPK signaling in BMMCs. We conducted a preliminary study to examine MAPK activation at early time points (0.5 and 1 h) following AgNP exposure but observed minimal or no changes in MAPK phosphorylation (Figure S5). Based on previous studies where changes in total tyrosine phosphorylation following exposure to AgNPs were only manifested at delayed time points, we decided to assess the phosphorylation of the MAPKs over longer time points.^25,32 Specifically, we measured the MAPK phosphorylation following exposure to AgNPs over multiple time points (i.e., 2, 6, 24 h) in comparison with IgE/DNP (Figure 5). Our results indicate that AgNP exposure induces a phosphorylation pattern similar to IgE/DNP in terms of the specific MAPK phosphorylation and the overall extent of phosphorylation (Figure 5). However, AgNP-induced phosphorylation was delayed compared to IgE activation, only reaching similar phosphorylation levels after 24 h (Figure 5(c)).

Figure 5.

Mitogen-activated protein kinases (MAPK) activation following exposure to silver nanoparticles (AgNPs). (a) Representative Western blot of extracellular signal-regulated kinase 1/2 (ERK 1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38) following exposure to IgE/DNP (100 ng/ml) or to AgNPs (25 μg/ml) over several time points. (b and c) Quantification based on densitometry of ERK 1/2, c-Jun and JNK following exposure to IgE/DNP (b) or AgNPs (c). All experiments were independently repeated at least three times (n ≥ 3). A p-value less than 0.05 (p < 0.05) was considered statistically significant between control and treatment groups (*).

Cytotoxicity, Lamp-2 expression and reactive species generation following exposure to silver nanoparticles

We measured cellular apoptosis and necrosis in BMMCs exposed to AgNPs (25 μg/ml) or IgE/DNP (100 ng/ml) to exclude any cytotoxicity. Our data confirmed that neither exposure to AgNPs nor IgE/DNP for 24 h was associated with increased apoptotic or necrotic cell death (Figure 6(a), Figure S2). We also measured surface Lamp-2 expression at 24 h post-exposure to AgNPs or IgE/DNP and found that AgNP-treated cells had higher expression, compared to IgE/DNP-treated cells which returned to baseline control levels (Figure 6(b)). Yet, both treatment to AgNPs or IgE/DNP resulted in similar reactive oxygen species levels after 24 h (Figure 6(c)).

Figure 6.

Cytotoxicity, Lamp-2 expression and intracellular reactive species levels following exposure to silver nanoparticles (AgNPs). (a) Quantification of apoptotic and necrotic cell death was measured following exposure to IgE/DNP (100 ng/ml) or AgNPs (25 μg/ml) for 24 h. Hydrogen peroxide (H₂O₂) was used as a positive control. (b) Cell surface expression of lamp-2 protein 24-h post-exposure to AgNPs (25 μg/ml) or IgE/DNP (100 ng/ml). (c) Intracellular reactive species levels 24-h post-exposure to AgNPs (25 μg/ml) or IgE/DNP (100 ng/ml). All experiments were independently repeated at least three times (n ≥ 3). A p-value less than 0.05 (p < 0.05) was considered statistically significant between control and treatment groups (*).

Discussion

Previous studies have shown that exposure to ENMs can induce type I allergic reactions.^3,4 Notably, mast cell activation has been reported in response to a wide range of ENMs, with 20 nm citrate-coated AgNPs eliciting a robust early-phase activation, comparable to, or even exceeding, that of classical IgE-mediated stimulation.^16–19 In this study, we investigated whether exposure to 20 nm citrate-coated AgNPs also triggers a late-phase mast cell response, including the synthesis and release of lipid mediators and cytokines. This study demonstrates that these AgNPs can activate late-phase mast cell responses, including lipid mediator release, cytokine gene expression, and MAPK activation, however, it is substantially weaker and delayed compared to classical IgE-mediated mast cell activation. These findings underscore the complex nature of ENM-immune cell interactions and emphasize the importance of characterizing the cellular and molecular responses to ENMs.

Our previous RNA-sequencing data revealed that exposure to AgNPs (1 h) induced far less changes in gene expression compared to IgE activation.³³ These findings support the hypothesis that AgNPs may elicit a significantly lower late-phase activation relative to IgE stimulation. Upon IgE activation, mast cells release arachidonic acid via cytosolic phospholipase A2, leading to the production of eicosanoids, such as prostaglandins (via COX) and leukotrienes (via LOX).³⁴ These lipid mediators are critical in allergic inflammation. Mast cells can also be activated by non-IgE stimuli (e.g., TLR ligands, complement factors, neuropeptides, etc.), leading to differential lipid mediator release.^35–37 Unlike IgE stimulation, data on lipid mediator release under non-IgE stimulation remains limited with the majority of studies only measuring a small subset of lipid mediators.³⁸ Some studies suggest this response is stimulus- and phenotype-dependent (e.g., mucosal vs connective tissue mast cells).^35–37,39 Our findings indicate that while IgE stimulation triggers a robust production of lipid mediators including those via the COX, LOX and CYP450 pathways, AgNP exposure results in a markedly reduced lipid mediator response, with several mediators not being produced following AgNP exposure. Furthermore, certain lipid mediators, particularly those generated via the COX pathway, appear to be differentially activated (compared to IgE stimulation) with some mediators being reduced even compared to baseline control levels (e.g., prostaglandin D2 (PgD₂), thromboxane B2 (Tx_B2)), possibly reflecting the anti-inflammatory properties of AgNPs.⁴⁰ Importantly, these responses could, at least in part, be attributed to the release of silver ions, which have previously been shown to induce leukotriene release.⁴¹ Taken together, these findings not only confirm previous observations but also provide additional evidence supporting the hypothesis that AgNPs activate mast cells via distinct, non-IgE-mediated mechanism(s).

Since our previous studies suggest lower activation of gene expression following 1h exposure and also because the induction of gene expression could take a longer time (compared to the release of lipid mediators), we decided to measure gene expression of common cytokines and chemokines over several time points.³³ Consistent with previous observations, our study shows that AgNP exposure induces a lower (magnitude) and delayed expression of several inflammatory genes, including TNFα, IL-1β, IL-6, MCP-1, and MIP-1α, which peak at around 6 h and diminish by 24 h, with some mediators (e.g., MCP-1 and MIP-1α) appeared to be induced more than others (e.g., IL-1β and IL-6). This may indicate a differential impact of AgNPs on cytokine mediator release. Notably, a previous study reported that exposure to silver ions in Rat Basophilic Leukemia (RBL-2H3) cells (a mast cell model) did not induce the release of TNFα or IL-4, suggesting the potential role of the particulate form of AgNPs in eliciting mast cell activation.⁴² It is also important to mention that such a pattern is not easily explained within the context of other non-IgE stimuli, such as complement proteins, TLR ligands, and neuropeptides, which trigger varying levels of cytokine release depending on the specific stimulus.^6,39,43–45

The MAPKs regulate key signaling cascades involved in diverse cellular processes such as cellular proliferation, differentiation and apoptosis.⁴⁶ In mast cells, MAPKs are critical for late-phase activation, including the release of lipid mediators and cytokines.⁴⁷ While both IgE- and non-IgE stimuli are capable of activating MAPKs, previous studies have shown that IgE stimuli induce rapid and robust phosphorylation of MAPKs, while non-IgE stimuli often result in a stimulus-specific activation, typically weaker in magnitude.^{43–45,47,48} Since we observed delayed activation of inflammatory genes and our preliminary data showed no apparent changes in MAPK phosphorylation at early time points (0.5 and 1 h), we decided to assess MAPK expression at later time points, including 2, 6, and 24 h, in direct comparison with the response to IgE activation. Our findings indicate that AgNP exposure leads to a delayed MAPK response, reaching comparable phosphorylation levels to those induced by an IgE stimulation, although these occurred at later time points (i.e., 24 vs 1 h). The biological significance of this delayed activation remains unclear. Additionally, such responses are also distinct from other known non-IgE stimuli, which generally induce a rapid MAPK phosphorylation (i.e., within seconds to minutes) and result in differential cytokine release.^6,39,43–45 It is worth noting that sustained activation of MAPKs, including ERK1/2, has been previously observed in response to various ENMs across different cellular models, typically in association with oxidative stress and cytotoxicity.^49–51 In the context of our findings, where AgNP exposure induced minimal oxidative stress or cytotoxicity (Figure 6(a)) and elicited a more pronounced activation of ERK1/2 compared to p38 and JNK, this activation may potentially be associated with cell growth and differentiation rather than cellular stress or apoptotic cell death.^52–55 Nonetheless, the possibility of low-level cellular stress, potentially triggered by the release of silver ions, cannot be excluded.⁵⁶ Such stress may engage adaptive metabolic responses aimed at promoting cell survival and maintaining homeostasis, potentially reflected in the delayed and sustained activation of MAPKs.⁵⁷

To gain further insights, we also measured Lamp-2 expression and intracellular reactive oxygen species levels 24 h following AgNP exposure. Our data has demonstrated that cells exposed to AgNPs were associated with sustained Lamp-2 expression, whereas in IgE activation, expression levels returned to baseline, potentially indicating an altered cellular homeostasis. This may contribute to priming cells for subsequent IgE activation, including both early- and late-phase activation as previously observed, or potentially to non-IgE stimuli including subsequent AgNP exposure.^19,32 On the other hand, exposure to both AgNPs or IgE/DNP for 24 h resulted in similar intracellular reactive oxygen species levels, significantly lower than control levels. Future mechanistic studies are warranted including to investigate whether repeated or chronic exposure could elicit cumulative or exaggerated consequences, particularly in vivo, and whether and how these interactions influence crosstalk with other innate immune cells.^58,59

In conclusion, our findings, together with previous reports, indicate that 20 nm citrate-coated AgNPs potentially activate mast cells through a novel, non-IgE-mediated mechanism.^25,26,32,33 While these AgNPs induce a robust early-phase activation, their impact on late-phase responses is delayed and attenuated compared to classical IgE-mediated stimulation. This distinction raises important questions about the biological significance of such a delayed response and whether it reflects an adaptive, regulatory mechanism or altered cellular homeostasis.^11,60 Supporting evidence, including the suppression of AgNP-induced degranulation via blockade of scavenger receptor B1 (SR-B1), delayed and weaker protein tyrosine phosphorylation, and distinct transcriptomic and metabolic profiles compared to IgE stimulation, collectively suggest a novel mechanism of mast cell activation induced by AgNPs, distinct from both IgE-mediated and known non-IgE-mediated activation.^25,26,32,33 Although our data support the involvement of non-IgE mechanism, the precise mechanisms remain unclear. It is possible that AgNPs modulate membrane receptors, and/or interact with subcellular components.^25,26,32,33 Our findings underscore the complex nature of type I allergic reactions and mast cell biological responses to ENMs.¹¹

Further research is warranted to gain more insights on the underlying molecular mechanisms. It is also important to emphasize that this work was conducted in vitro using bone marrow-derived mast cells, and future studies should explore tissue-specific mast cells of different phenotypes (e.g., mucosal vs connective tissue mast cells) and also validate these findings in relevant in vivo experimental models.⁶¹ Additional limitations that should be addressed in future studies include evaluating the effects of AgNPs with diverse physicochemical properties, examining a broader range of AgNP concentrations, and distinguishing between the effects of particulate silver and released silver ions.

Supplemental material

Supplemental material - Silver nanoparticles trigger delayed inflammatory responses in mast cells

Supplemental material for Silver nanoparticles trigger delayed inflammatory responses in mast cells by Nasser B. Alsaleh and Jared M. Brown in Human & Experimental Toxicology.

Footnotes

Acknowledgement

We would like to acknowledge the mass spectrometry core facility at the Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus for conducting the lipidomics analysis.

ORCID iD

Nasser B. Alsaleh

Ethical considerations

All use of animals in this study was approved by the University of Colorado Anschutz Medical Campus Institutional Animal Care and Use Committee.

Author contributions

Conceptualization, experimental work, data analysis, and original manuscript draft preparation were carried out by NBA. Funding acquisition and reviewing/editing manuscript were performed by JMB.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by the National Institute of Environmental Health Sciences Grant R01 ES019311 (JMB).

Declaration of conflicting interests

The corresponding author is the Lead Guest Editor of the Special Collection “Safety of Smart and Advanced Nanomaterials” to which this article was submitted. The corresponding author recused from all editorial process and decisions. Blinded peer review was maintained throughout the process.

Data Availability Statement

All data are included as part of this manuscript and associated supplements.*

Statements and declarations

ChatGPT (GPT-4o and GPT-5, OpenAI, ) was used to assist in improving grammar and sentence structure throughout the writing and revisions of the manuscript. All content was reviewed and edited by the authors.

Supplemental material

Supplemental material is available online.

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