Pro-inflammatory adjuvant properties of pigment-grade titanium dioxide particles are augmented by a genotype that potentiates interleukin 1β processing

Abstract

Background: Pigment-grade titanium dioxide (TiO₂) particles are an additive to some foods (E171 on ingredients lists), toothpastes, and pharma-/nutraceuticals and are absorbed, to some extent, in the human intestinal tract. TiO₂ can act as a modest adjuvant in the secretion of the pro-inflammatory cytokine interleukin 1β (IL-1β) when triggered by common intestinal bacterial fragments, such as lipopolysaccharide (LPS) and/or peptidoglycan. Given the variance in human genotypes, which includes variance in genes related to IL-1β secretion, we investigated whether TiO₂ particles might, in fact, be more potent pro-inflammatory adjuvants in cells that are genetically susceptible to IL-1β-related inflammation.

Methods: We studied bone marrow-derived macrophages from mice with a mutation in the nucleotide-binding oligomerisation domain-containing 2 gene (Nod2 ^m/m), which exhibit heightened secretion of IL-1β in response to the peptidoglycan fragment muramyl dipeptide (MDP). To ensure relevance to human exposure, TiO₂ was food-grade anatase (119 ± 45 nm mean diameter ± standard deviation). We used a short 'pulse and chase' format: pulsing with LPS and chasing with TiO₂ +/- MDP or peptidoglycan.

Results: IL-1β secretion was not stimulated in LPS-pulsed bone marrow-derived macrophages, or by chasing with MDP, and only very modestly so by chasing with peptidoglycan. In all cases, however, IL-1β secretion was augmented by chasing with TiO₂ in a dose-dependent fashion (5-100 μg/mL). When co-administered with MDP or peptidoglycan, IL-1β secretion was further enhanced for the Nod2 ^m/m genotype. Tumour necrosis factor α was triggered by LPS priming, and more so for the Nod2 ^m/m genotype. This was enhanced by chasing with TiO₂, MDP, or peptidoglycan, but there was no additive effect between the bacterial fragments and TiO₂.

Conclusion: Here, the doses of TiO₂ that augmented bacterial fragment-induced IL-1β secretion were relatively high. In vivo, however, selected intestinal cells appear to be loaded with TiO₂, so such high concentrations may be 'exposure-relevant' for localised regions of the intestine where both TiO₂ and bacterial fragment uptake occurs. Moreover, this effect is enhanced in cells from Nod2 ^m/m mice indicating that genotype can dictate inflammatory signalling in response to (nano)particle exposure. In vivo studies are now merited.

Keywords: E171; IL-1β; Muramyl dipeptide; NOD2; Nano; Particle; Peptidoglycan; TNF-α; TiO2.

Fig. 1

Transmission electron microscopy image of TiO₂ particles. Food- and pharmaceutical-grade anatase TiO₂ particles were suspended in distilled water with 0.5% BSA at a concentration of 1 mg/mL. The particle suspension was analysed with transmission electron microscopy at 80 kV. A representative image is shown; scale bar = 200 nm

Fig. 2

Size determination of TiO₂ particles. a Food- and pharmaceutical-grade anatase TiO₂ particles were suspended in distilled water with 0.5% BSA at a concentration of 1 mg/mL. The particle suspension was analysed with transmission electron microscopy at 80 kV. TiO₂ particle diameters were measured with image analysis software. The distribution of the particle diameters, grouped in sizes of 25 nm, is shown as a relative frequency histogram, n = 133. b Food- and pharmaceutical-grade anatase TiO₂ particles were suspended in RPMI 1640 medium with 10% FBS and 1% penicillin-streptomycin at a concentration of 1 mg/mL. TiO₂ particle sizes were determined with nanoparticle tracking analysis, and the size distribution of the particles is plotted as a line graph. Data represent mean ± SD from three independent experiments

Fig. 3

Viability of LPS-primed BMDMs after chasing with TiO₂ +/− peptidoglycan or MDP. BMDMs from WT (a) and Nod2 ^m/m (b) mice were pre-stimulated for 3 h with LPS (10 ng/mL). Then BMDMs were incubated for 3 h with the indicated concentrations of TiO₂ particles suspended in TCM alone (TCM), or TCM + 10 μg/mL MDP (MDP), or TCM + 10 μg/mL peptidoglycan (PGN). Cells were stained with PI and F4/80 antibody for murine macrophages and analysed with flow cytometry, and viability was determined with PI exclusion. Percentages of PI⁻ cells in relation to the total number of detected events are shown. Data represent mean ± SD from two independent experiments with three replicates each, n = 6

Fig. 4

Particle uptake by LPS-primed BMDMs after chasing with TiO₂ +/− peptidoglycan or MDP. BMDMs from WT (a) and Nod2 ^m/m (b) mice were pre-stimulated for 3 h with LPS (10 ng/mL). Then BMDMs were incubated for 3 h with the indicated concentrations of TiO₂ particles suspended in TCM alone (TCM), TCM + 10 μg/mL MDP (MDP), or TCM + 10 μg/mL peptidoglycan (PGN). Cells were stained with PI and F4/80 antibody for murine macrophages and analysed with flow cytometry, and median SSC intensities of PI⁻F4/80⁺ cells were recorded. Data represent mean ± SD from two independent experiments with three replicates each, n = 6

Fig. 5

Correlation of SSC intensity and dark spots in bright-field images by flow and imaging cytometry. PBMCs from human blood were incubated for 24 h with 0 μg/mL, 5 μg/mL, or 10 μg/mL TiO₂ particles in TCM and stained with either CD11c or CD14 antibodies for human monocytes/myeloid cells and analysed with conventional flow or imaging cytometry, respectively. a Correlation between increases in SSC MFI of CD11c⁺ myeloid cells identified using conventional flow cytometry and the percentages of CD14⁺ cells bearing dark spots in bright-field measured by imaging cytometry; Pearson correlation p < 0.01, r = 0.8424. b Representative images of cells designated ‘dark spot negative’ or ‘dark spot positive’ by imaging cytometry; scale bar = 10 μm

Fig. 6

IL-1β secretion by LPS-primed BMDMs after chasing with TiO₂ +/− peptidoglycan or MDP. BMDMs from WT and Nod2 ^m/m mice were pre-stimulated for 3 h with LPS (10 ng/mL). Then BMDMs were incubated for 3 h with the indicated concentrations of TiO₂ particles suspended in TCM alone (a) or suspended in TCM + 10 μg/mL MDP (MDP) or TCM + 10 μg/mL peptidoglycan (PGN) (b). Supernatant concentrations of IL-1β were analysed by ELISA. Data represent mean ± SD from two independent experiments with three replicates each, n = 6. a Results were analysed with one-way ANOVA and Tukey’s post-hoc test; **p < 0.01, ***p < 0.001 compared to respective WT or Nod2 ^m/m cells incubated without TiO₂. b Results were analysed with two-way ANOVA and Tukey’s post-hoc test; *p < 0.05, **p < 0.01, ***p < 0.001 for Nod2 ^m/m cells compared to WT cells cultured with the same TiO₂ concentration, ^††† p < 0.001 for WT and Nod2 ^m/m cells compared to respective WT or Nod2 ^m/m cells incubated without TiO₂, ^‡ p < 0.05 for Nod2 ^m/m cells compared to Nod2 ^m/m cells incubated without TiO₂

Fig. 7

TNF-α secretion by LPS-primed BMDMs after chasing with TiO₂ +/− peptidoglycan or MDP. BMDMs from WT and Nod2 ^m/m mice were pre-stimulated for 3 h with LPS (10 ng/mL). Then BMDMs were incubated for 3 h with the indicated concentrations of TiO₂ particles suspended in TCM alone (a) or suspended in TCM + 10 μg/mL MDP (MDP) or TCM + 10 μg/mL peptidoglycan (PGN) (b). Supernatant concentrations of TNF-α were analysed by ELISA. Data represent mean ± SD from two independent experiments with three replicates each, n = 6. a Results were analysed with one-way ANOVA and Tukey’s post-hoc test; *p < 0.05, ***p < 0.001 compared to respective WT or Nod2 ^m/m cells incubated without TiO₂

Fig. 8

IL-1β secretion by PBMCs following exposure to MSU crystals or SNPs. Secretion of IL-1β by PBMCs with (main figure) or without (inset) initial exposure to 10 ng/mL LPS for 3 h followed by exposure to MSU crystals (100 μg/mL) or SNPs (100 μg/mL) for a further 3 h. The supernatant was either collected immediately for analysis (3 h; open bars) or following a further 21 h of cell incubation in fresh (i.e. without added particles or MAMPs) TCM (3 + 21 h; black bars). All data are expressed as mean ± SEM (n = 4). Results were analysed by paired T test in comparison to baseline (B), i.e. non-particle-exposed cells; *p < 0.05 and **p < 0.01 versus respective baseline