Artefactual nanoparticle activation of the inflammasome platform: in vitro evidence with a nano-formed calcium phosphate

Abstract

Aim: To determine whether in vitro experimental conditions dictate cellular activation of the inflammasome by apatitic calcium phosphate nanoparticles.

Material & methods: The responses of blood-derived primary human cells to in situ-formed apatite were investigated under different experimental conditions to assess the effect of aseptic culture, cell rest and duration of particle exposure. Cell death and particle uptake were assessed, while IL-1β and caspase 1 responses, with and without lipopolysaccharide prestimulation, were evaluated as markers of inflammasome activation.

Results: Under carefully addressed experimental conditions, apatitic nanoparticles did not induce cell death or engage the inflammasome platform, although both could be triggered through artefacts of experimentation.

Conclusion: In vitro studies often predict that engineered nanoparticles, such as synthetic apatite, are candidates for inflammasome activation and, hence, are toxic. However, the experimental setting must be very carefully considered as it may promote false-positive outcomes.

Keywords: IL-1β; apatite; caspase 1; experimental conditions; inflammasome; nanoparticle.

Figure 1. Physicochemical characterization of in situ-formed calcium phosphate nanoparticles

Following synthesis, calcium phosphate particles were analyzed (A–C) for particle size and structure by transmission electron microscopy, for size distribution in tissue culture medium by (D) DLS, (E) NTA and (F) static light scattering, and (G) for elemental composition by energy dispersive x-ray spectroscopy within the transmission electron microscopy (C and Cu signals are generated by the support film and grid). (H) Infrared analysis of hydroxyapatite standard (Sigma; 0–200 nm nanopowder) and (I) infrared analysis of the in situ-formed calcium phosphate particles in tissue culture medium with spectral features attributed as follows: (a) lattice vibrations, (b) phosphate vibration, (c) carbonate adsorption bands at 1465–1410 cm⁻¹, (d) amine adsorption bands from serum proteins at 1600–16,700 cm⁻¹, and (e) probable OH broadening from residual water with the main OH band at 3400 cm⁻¹. DLS: Dynamic light scattering; NTA: Nanoparticle tracking analysis; SLS: Static light scattering; T3: 3 h, T8: 8 h; T24: 24 h.

Figure 2. Influence of experimental conditions on IL-1β responses to apatitic nanoparticles

IL-1β secretion from peripheral blood mononuclear cells (1.10⁶ cells/ml) in experiments that were carried out using (A) unfiltered TCM or (B) 0.2-μm filtered TCM . In each setting, peripheral blood mononuclear cells were either used immediately after isolation (dark blue) or rested for 24 h (light blue) and subsequently stimulated for 24 h with apatite nanoparticles that were formed in situ by addition of CaCl₂ to TCM, in the presence or absence of LPS (10 ng/ml) as indicated in the figure. Data are represented as mean ± standard error of the mean (n = 2). *p < 0.05; **p < 0.01 and ***p < 0.001 versus control. AP: Apatite; C: Control; LPS: Lipopolysaccharide; TCM: Tissue culture medium.

Figure 3. Influence of duration of exposure to apatitic nanoparticles on IL-1β and caspase 1 secretion in peripheral blood mononuclear cells

Time course measurement for (A) IL-1β and (B) caspase 1 secretion from rested peripheral blood mononuclear cells (1.10⁶ cells/ml) following stimulation with LPS (10 ng/ml), apatitic nanoparticles in the presence or absence of 10 ng/ml LPS, or vehicle. Data are represented as mean ± standard error of the mean (n = 2, except at 8 h where n = 5). *p < 0.05 (AP + LPS vs control); **p < 0.01 (AP vs control); ***p < 0.001; ****p < 0.0001 (AP + LPS vs control). AP: Apatite; LPS: Lipopolysaccharide.

Figure 4. Apatitic nanoparticle-induced cytotoxicity

Flow cytometry measurement of cell death from the monocyte population within peripheral blood mononuclear cells (1.10⁶ cells/ml) that were stimulated with apatite nanoparticles or vehicle over time. Data are represented as mean ± standard error of the mean (n = 2). **p < 0.01; ***p < 0.001. AP: Apatite.

Figure 5. Apatitic nanoparticle uptake by monocytes

(A) Flow cytometric measurement in cells (CD14⁺ monocytes) that were stimulated with apatite or vehicle for (i) 1 h, (ii) 3 h and (iii) 24 h continuously. In each of the six panels only the viable CD14⁺ gated monocytes are imaged and thus occupy the top two quadrants. The colours represent density of cells in their plotted space (blue being the most dense and red the least). The right hand quadrants represent cells showing calcein positivity and thus intracellular calcium (‘unstimulated’) while the addition of apatite shows a marked and rapid increase in calcein positivity (‘AP’) and by 24 h few viable cells remain. (B) Imaging with a second independent technique, namely Image stream, showing three separate example images of CD14⁺ Calcein⁺ gated cells 3 h after challenge and showing internalization of AP particles. AP: Apatite.

Figure 6. Optimized innate cellular responses to apatitic nanoparticles

IL-1β responses from peripheral blood mononuclear cells (1.10⁶ cells/ml), (A) with or (B) without LPS prestimulation (3 h), and then challenged with vehicle, AP nanoparticles, soluble or crude peptidoglycan (Sol Pg and Pg, both at 10 μg/ml). IL-1β was measured after a further 3 h (i.e., between 3 and 6 h; dark blue) and 18 h after that (i.e., between 6–24 h; light blue). Data are represented as mean ± standard error of the mean (n = 4). ****p < 0.0001; ***p < 0.001 versus control. AP: Apatite; C: Control; LPS: Lipopolysaccharide; Pg: Peptidoglycan; Sol: Soluble.