Iron oxide nanoparticles modulate lipopolysaccharide-induced inflammatory responses in primary human monocytes

Abstract

Co-stimulation of the immune system to more than one agent concomitantly is very common in real life, and considering the increasing use of engineered nanoparticles and nanomaterials, it is highly relevant to assess the ability of these materials to modulate key innate immune responses, which has not yet been studied in detail. We investigated the immunomodulatory effects of 10 nm and 30 nm iron oxide nanoparticles (IONPs) on primary human monocytes in the presence and absence of Toll-like receptor 4 agonist lipopolysaccharide (LPS). Prior to the cell studies, we characterized the physicochemical properties of the nanoparticles in cell culture medium and ensured that the nanoparticles were free from biological contamination. Cellular uptake of the IONPs in monocytes was assessed using transmission electron microscopy. Using enzyme-linked immunosorbent assay, we found that the IONPs per se did not induce the production of proinflammatory cytokines tumor necrosis factor-α, interleukin-6, and interleukin-1β. However, the IONPs had the ability to suppress LPS-induced nuclear factor kappa B activation and production of proinflammatory cytokines in primary human monocytes in an LPS and a particle dose-dependent manner. Using confocal microscopy and fluorescently labeled LPS, we showed that the effects correlated with impaired LPS internalization by monocytes in the presence of IONPs, which could be partly explained by LPS adsorption onto the nanoparticle surface. Additionally, the results from particle pretreatment experiments indicate that other cellular mechanisms might also play a role in the observed effects, which warrants further studies to elucidate the additional mechanisms underlying the capacity of IONPs to alter the reactivity of monocytes to LPS and to mount an appropriate cellular response.

Keywords:

• iron oxide nanoparticles
• primary human monocytes
• PBMCs
• cytokines
• LPS adsorption
• Toll-like receptors

Supplementary materials

Figure S1 Evaluation of biological contamination of the IONPs.

Notes: A luciferase reporter cell assay was used to assess contamination of the IONPs with (A) TLR2 and (B) TLR4 agonists. Human embryonic kidney (HEK293) cells were transiently transfected with a luciferase NFκB reporter plasmid (ELAM-luc) together with either vector only (TLR−), TLR2+/CD14, or TLR4+/MD2+/CD14 plasmids. The cells were treated with 10 nm and 30 nm IONPs (1 µg/mL, 10 µg/mL, 100 µg/mL) for 24 hours. TLR4 agonist LPS (100 ng/mL) and TLR2 agonist FSL-1 (100 ng/mL) served as positive controls for the activation of TLR4 and TLR2, respectively. Results are expressed as mean ± SEM (n=3).

Abbreviations: IONPs, iron oxide nanoparticles; TLR, Toll-like receptor; NFκB, nuclear factor kappa B; LPS, lipopolysaccharide; FSL-1, fibroblast-stimulating lipopeptide 1; SEM, standard error of the mean.

Figure S2 Cytokine adsorption to the IONPs.

Notes: Cell-free samples containing (A) 10 nm and (B) 30 nm IONPs (1 µg/mL, 10 µg/mL, and 100 µg/mL) were incubated with recombinant TNFα, IL-1β, and IL-6, respectively, for 6 hours, and cytokine concentration in the supernatant was measured using ELISA. Results from one representative experiment are shown.

Abbreviations: IONPs, iron oxide nanoparticles; TNFα, tumor necrosis factor α; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; Std, standard.

Figure S3 Cytokine response in IONPs-treated nonadherent monocytes and PBMCs in the presence of LPS.

Notes: (A) Nonadherent primary human monocytes and (B) PBMCs were treated with 10 nm IONPs (1 µg/mL, 10 µg/mL, and 100 µg/mL) and LPS (0.5 ng/mL) for 6 hours. The TNFα concentration released in the medium was measured using ELISA. Results are expressed as mean ± SEM (n=3). *Statistical significance (P<0.05) compared to LPS alone.

Abbreviations: IONPs, iron oxide nanoparticles; PBMCs, peripheral blood mononuclear cells; LPS, lipopolysaccharide; TNFα, tumor necrosis factor α; ELISA, enzyme-linked immunosorbent assay; SEM, standard error of the mean.

Figure S4 Influence of serum concentration in cell culture medium on cytokine response in IONPs-treated primary human monocytes in the presence of LPS.

Notes: The cells were treated with 10 nm IONPs (1 µg/mL, 10 µg/mL, and 100 µg/mL) and LPS (0.5 ng/mL) for 6 hours in cell culture medium containing varying concentrations of A+ serum. (A) RPMI 1640 with 5% A+ serum. (B) RPMI 1640 with 10% A+ serum. (C) RPMI 1640 with 20% A+ serum. The TNFα concentration released in the medium was measured using ELISA. Results are expressed as mean ± SEM (n=3). *Statistical significance (P<0.05) compared to LPS alone.

Abbreviations: IONPs, iron oxide nanoparticles; LPS, lipopolysaccharide; TNFα, tumor necrosis factor α; ELISA, enzyme-linked immunosorbent assay; SEM, standard error of the mean.

Figure S5 LPS adsorption to the IONPs.

Notes: Cell-free samples containing nanoparticles (100 µg/mL) and LPS (100 ng/mL and 500 ng/mL in two different setups) were centrifuged at 15,000× g for 2 hours (4°C). The fluorescence intensity of the resuspended pellets was compared to a sample containing only nanoparticles (100 µg/mL). A representative image of each sample is shown.

Abbreviations: LPS, lipopolysaccharide; IONPs, iron oxide nanoparticles.

Acknowledgments

The authors thank the Blood Bank at St Olav’s Hospital in Trondheim, Norway, for supplying the buffy coats and Doctor Syed Ali (US FDA) for providing the iron oxide nanoparticles. Furthermore, we thank Nan E Tostrup Skogaker and Bjørnar Sporsheim for their technical support with TEM and the fluorescence confocal microscopy, respectively, which was conducted at the Cellular and Molecular Imaging Core Facility, Norwegian University of Science and Technology (NTNU). JS and AMN are co-senior authors. SG was financed by NANOLAB, Norwegian University of Science and Technology (NTNU), where most of the particle characterisation was done.

Disclosure

The authors report no conflicts of interest in this work.