Interaction between Macrophages and Nanoparticles: In Vitro 3D Cultures for the Realistic Assessment of Inflammatory Activation and Modulation of Innate Memory

Abstract

Understanding the modes of interaction between human monocytes/macrophages and engineered nanoparticles is the basis for assessing particle safety, in terms of activation of innate/inflammatory reactions, and their possible exploitation for medical applications. In vitro assessment of nanoparticle-macrophage interaction allows for examining the response of primary human cells, but the conventional 2D cultures do not reproduce the three-dimensional spacing of a tissue and the interaction of macrophages with the extracellular tissue matrix, conditions that shape macrophage recognition capacity and reactivity. Here, we have compared traditional 2D cultures with cultures on a 3D collagen matrix for evaluating the capacity gold nanoparticles to induce monocyte activation and subsequent innate memory in human blood monocytes in comparison to bacterial LPS. Results show that monocytes react to stimuli almost in the same way in 2D and 3D cultures in terms of production of TNFα and IL-6, but that notable differences are found when IL-8 and IL-1Ra are examined, in particular in the recall/memory response of primed cells to a second stimulation, with the 3D cultures showing cell activation and memory effects of nanoparticles better. In addition, the response variations in monocytes/macrophages from different donors point towards a personalized assessment of the nanoparticle effects on macrophage activation.

Keywords: 2D cultures; 3D cultures; gold nanoparticles; in vitro models; inflammation; innate immunity; innate memory; macrophages; monocytes.

Figure 1

Main characteristics of AuNPs. (Left): size distribution by STEM; (Centre): STEM image; (Right): absorbance peak via UV-VIS.

Figure 2

SEM images of monocytes in 3D culture. Upper panels: monocytes exposed for 24 h to culture medium alone or containing AuNPs (showing identical morphology; (left)) or LPS (right). Lower panels: monocytes primed with either culture medium alone or containing AuNPs (left) or LPS (right) observed after 6 days of resting in the absence of stimuli.

Figure 3

Primary cytokine production by monocytes in 2D vs. 3D culture. Production of the inflammatory cytokines TNFα and IL-6 (upper panels), of the chemokine IL-8 (lower left panel) and of the anti-inflammatory cytokine IL-1Ra (lower right panel) by monocytes of six individual donors stimulated for 24 h with culture medium alone (medium) or containing 1 ng/mL LPS (LPS) or 1 μg/mL AuNPs (AuNP). Parallel 2D (blue) and 3D (purple) cultures were established. Results are expressed in ng/10⁶ cells. The violin plots represent the distribution of the individual donors’ values in each experimental condition. The red line indicates the median value, and the yellow lines the first and third quartiles. Individual donors are represented with different symbols: ◇ donor 1; ■ donor 2; Δ donor 3; ▼ donor 4; ☐ donor 5; ● donor 6. Statistical significance was assessed with the paired one-tailed non-parametric Wilcoxon signed-rank test. LPS stimulation induced a significant increase (p < 0.05) of TNFα, IL-6 and IL-8 vs. medium controls. All other differences, including the 2D vs. 3D comparisons, were not significant.

Figure 4

Secondary cytokine production by primed monocytes challenged with LPS in 2D vs. 3D culture. Production of the inflammatory cytokines TNFα and IL-6, the chemokine IL-8 and of the anti-inflammatory cytokine IL-1Ra by monocytes of six donors previously primed in vitro with culture medium alone (unprimed), LPS (LPS-primed) or AuNPs (AuNP-primed). After 24 h priming, cells were rested in culture for 6 days and then challenged with 10 ng/mL LPS for 24 h. Results obtained in 2D cultures are reported in the upper panels, while data from 3D cultures are reported in the lower panels. The violin plots represent the distribution of the individual donors’ values in each experimental condition. The red line indicates the median value, and the yellow lines the first and third quartiles. Individual donors are represented with different symbols: ◇ donor 1; ■ donor 2; Δ donor 3; ▼ donor 4; ☐ donor 5; ● donor 6. Statistical significance was assessed with the paired one-tailed non-parametric Wilcoxon signed-rank test, and show significant differences (p < 0.05) between unprimed and LPS-primed production of TNFα (2D and 3D), IL-6 (2D) and IL-1Ra (2D), while the differences between 2D and 3D were significant for IL-8 and IL-1Ra.