Titanium dioxide nanoparticles exacerbate DSS-induced colitis: role of the NLRP3 inflammasome

Abstract

Objective: Western lifestyle and diet are major environmental factors playing a role in the development of IBD. Titanium dioxide (TiO₂) nanoparticles are widely used as food additives or in pharmaceutical formulations and are consumed by millions of people on a daily basis. We investigated the effects of TiO₂ in the development of colitis and the role of the nucleotide-binding oligomerisation domain receptor, pyrin domain containing (NLRP)3 inflammasome.

Design: Wild-type and NLRP3-deficient mice with dextran sodium sulfate-induced colitis were orally administered with TiO₂ nanoparticles. The proinflammatory effects of TiO₂ particles in cultured human intestinal epithelial cells (IECs) and macrophages were also studied, as well as the ability of TiO₂ crystals to traverse IEC monolayers and accumulate in the blood of patients with IBD using inductively coupled plasma mass spectrometry.

Results: Oral administration of TiO₂ nanoparticles worsened acute colitis through a mechanism involving the NLRP3 inflammasome. Importantly, crystals were found to accumulate in spleen of TiO₂-administered mice. In vitro, TiO₂ particles were taken up by IECs and macrophages and triggered NLRP3-ASC-caspase-1 assembly, caspase-1 cleavage and the release of NLRP3-associated interleukin (IL)-1β and IL-18. TiO₂ also induced reactive oxygen species generation and increased epithelial permeability in IEC monolayers. Increased levels of titanium were found in blood of patients with UC having active disease.

Conclusion: These findings indicate that individuals with a defective intestinal barrier function and pre-existing inflammatory condition, such as IBD, might be negatively impacted by the use of TiO₂ nanoparticles.

Keywords: IMMUNE RESPONSE; INFLAMMATORY BOWEL DISEASE; INFLAMMATORY MECHANISMS; INTESTINAL BARRIER FUNCTION; REACTIVE OXYGEN SPECIES.

Figure 1

Administration of titanium dioxide (TiO₂) nanoparticles aggravates colitis in the dextran sodium sulfate (DSS) mouse model of acute colitis through activation of the nucleotide-binding oligomerisation domain receptor, pyrin domain containing (NLRP)3 inflammasome. (A–E) DSS-treated wild-type (WT) and Nlrp3 ^−/− C57BL/6J mice received TiO₂ by oral gavage as indicated. Mice were sacrificed at day 8 and colon length was measured (A). Mucosal damage was assessed by colonoscopy (B). H&E staining of sections displayed severe barrier breakdown (C) with extensive infiltration reaching the lamina muscularis mucosae (D). Total histological score was calculated as the sum of epithelial damage and infiltration score (E). Results represent mean±SEM; WT mice: n=12, Nlrp3 ^−/− mice: n=10, *p<0.05; **p<0.01; ***p<0.001. (F) Cryosections of spleen from DSS-treated WT mice administered with TiO₂ or TiO₂-free water were fixed and nuclei were stained with DAPI (blue). The presence of crystals (red) was analysed combining confocal reflection microscopy with fluorescence microscopy. Results are representative of four experiments. Scale bars: 10 μm.

Figure 2

Caspase-1 downstream effectors are activated in macrophages and intestinal epithelial cells (IECs) on exposure to titanium dioxide (TiO₂). (A) THP-1 cells were treated for 24 h with indicated amounts of TiO₂ nanoparticles and microparticles, and the secretion of active interleukin (IL)-1β in cell culture supernatants was assessed by ELISA (n=3, **p<0.01, ***p<0.001). (B–D) Caco-2 and THP-1 cells were treated for 24 h with indicated amounts of TiO₂ nanoparticles and microparticles. The activation of caspase-1 (B) and IL-1β (C) in response to 5 μg/mL TiO₂ was detectable in both cell lines. Lysates were subjected to co-immunoprecipitation (Co-IP) using nucleotide-binding oligomerisation domain receptor, pyrin domain containing (NLRP)3-antibody prior to western blotting to detect caspase-1 and ASC (D). (E–G) Incubation of Caco-2 (E) with TiO₂ microparticles resulted in release of active IL-18 in cell supernatants (n=4). In THP-1 cells, release of both IL-18 (F) and IL-1β (G) was significantly increased on stimulation with TiO₂ microparticles (n=3, ***p<0.001), an effect that was dependent on the presence of inflammasome components. Small interfering RNA (siRNA)-mediated knockdown of ASC, NLRP3 or caspase-1 in THP-1 cells resulted in significantly lower levels of IL-1β on TiO₂ stimulation (G) (n=3, ***p<0.001).

Figure 3

Aggregates of titanium dioxide (TiO₂) accumulate intracellularly in macrophages and intestinal epithelial cells (IECs). (A) After 24 h incubation with 100 µg/mL TiO₂, THP-1 cells were fixed and stained with eosin. Arrows indicate intracellular TiO₂ aggregates. Scale bars: 100 μm. (B and C) BMDM were stimulated with 20 μg/mL TiO₂, without (B) or with cytochalasin D (C) for 24 h, and images of live cultures were taken. TiO₂ aggregates can be found in all cells, and they show a distinct distribution pattern (arrows). Inhibition of endocytosis with cytochalasin D did not abolish the uptake of TiO₂. (D and E) HT-29 (D) and Caco-2 (E) cells were grown on glass cover slips and incubated with 100 µg/mL TiO₂ for 24 h. Intracellular aggregates of TiO₂ were found in both cell types, indicated by arrows. Scale bar: 100 µm.

Figure 4

Aggregates of titanium dioxide (TiO₂) accumulate intracellularly in Caco-2 cells in a dose-dependent manner. (A and B), Caco-2 cells grown on transwells were incubated with 100 µg/mL TiO₂ for 24 h. Cross-sections were stained with H&E (A) or DAPI to visualise nuclei and the same frame was observed in phase-contrast view to identify TiO₂ aggregates (B). Scale bar: 25 µm. TiO₂ aggregates were located intracellularly in aggregates of various sizes, indicated by arrows. (C–F) Cross-sections were processed as described above, and TiO₂ aggregates and nuclei were counted. Incubation of Caco-2 for 24 h with indicated amounts of TiO₂ resulted in a dose-dependent increase of intracellular TiO₂ (C). The size of TiO₂ aggregates also increased in a dose-dependent manner. Electron dense regions with a diameter larger than 3.5 µm were considered as large aggregates (D). Cross-sections were subjected to transmission electron microscopy, revealing electron-dense aggregates indicated by arrows (E). The elemental analysis of a selected region (indicated as asterisk in panel E) confirmed that aggregates contained titanium, which appeared as main peak at 4.5 keV (indicated by arrow) (F).

Figure 5

Titanium dioxide (TiO₂) triggers production of reactive oxygen species (ROS) and influences epithelial permeability in human intestinal epithelial cells (IECs). (A and B) HT-29 showed significantly increased production of ROS at the highest dose of 100 μg/mL of TiO₂ (A). In Caco-2 cells, a significant increase of ROS was detected at TiO₂ concentrations of 20 and 100 μg/mL (B). (C) Monolayers of Caco-2 cells were treated with indicated amounts of TiO₂ for 24 h, and incubated apically with FITC-labelled beads for 1 h. An increase of basolateral fluorescence was observed in TiO₂-treated cells as compared with non-treated cells (n=5, **p<0.05).

Figure 6

Patients with IBD present elevated titanium levels in blood. (A and B) Titanium levels in blood of healthy donors and patients with IBD are shown as measured by inductively coupled plasma mass spectrometry (ICP-MS) (A) (n=28 control, n=28 IBD, °depicts outliers). Titanium levels in subgroups of patients with IBD; significantly increased levels were detected in samples from patients with active UC (B) (n=28 control, n=5 UC active, n=6 UC remission, n=8 Crohn's disease (CD) active, n=9 CD remission; ***p<0.001).