Pro-inflammatory adjuvant properties of pigment-grade titanium dioxide particles are augmented by a genotype that potentiates interleukin 1β processing
Riedle et al. Particle and Fibre Toxicology
Pro-inflammatory adjuvant properties of pigment-grade titanium dioxide particles are augmented by a genotype that potentiates interleukin 1β processing
Sebastian Riedle 3 4 5
Laetitia C. Pele 2
Don E. Otter 5 6
Rachel E. Hewitt 1 2
Harjinder Singh 4
Nicole C. Roy 0 4 5
Jonathan J. Powell 0 1 2
0 Equal contributors
1 Department of Veterinary Medicine, Biomineral Research Group, University of Cambridge , Madingley Road, Cambridge CB3 0ES , UK
2 Biomineral Research Group, MRC Human Nutrition Research, Elsie Widdowson Laboratory , 120 Fulbourn Road, Cambridge CB1 9NL , UK
3 Present address: Conreso GmbH , Neuhauser Str. 47, 80331, München , Germany
4 Riddet Institute, Massey University , Private Bag 11222, Palmerston North 4442 , New Zealand
5 Food Nutrition & Health Team, Food & Bio-based Products Group, AgResearch, Grasslands Research Centre , Tennent Drive, Private Bag 11008, Palmerston North 4442 , New Zealand
6 Present address: Center for Dairy Research, University of Wisconsin-Madison , 1605 Linden Drive, Madison, WI 53706-1565 , USA
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 (Nod2m/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 Nod2m/m genotype. Tumour necrosis factor α was triggered by LPS priming, and more so for the Nod2m/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 Nod2m/m mice indicating that genotype can dictate inflammatory signalling in response to (nano)particle exposure. In vivo studies are now merited.
Nano; Particle; TiO2; E171; NOD2; IL-1β; TNF-α; Muramyl dipeptide; Peptidoglycan
Background
Potential toxicological effects following exposure to
titanium dioxide (TiO2) are of current interest [
1, 2
].
TiO2 is a mineral pigment which, when used in a
particulate form, is valued for its properties as a whitening
or brightening agent, and is included in some processed
foods (E171 on ingredients lists), toothpastes, capsules
and tablets. From these sources, the average daily intake
of pigment-grade TiO2 for an adult in the UK is about
1012 particles/day [
3, 4
], nominally ~0.04 mg/kg/day for a
70 kg adult. These findings are supported by a recent
Dutch study with mean long term intakes of
pigmentgrade TiO2 ranging from 0.06 mg/kg/day in elderly
subjects to 0.17 mg/kg/day for 7–69-year-olds [
5
]. In 2–6 year
old children, however, it was higher at 0.67 mg/kg/day [
5
].
It is well established that particles of TiO2, likely
derived from sources of the Western lifestyle described
above, accumulate in certain cells, such as macrophages
in Peyer’s patches of the human small intestine [
6–10
].
Whether they have any deleterious impact in this
environment remains a matter of speculation, but, if they do,
both cell accumulation and host factors are likely to be
important [
4
]. Indeed, it has been often noted that the
accumulation of these particles occurs where the earliest
signs of Crohn’s disease have been reported [
11
]. With
respect to cell accumulation and stimulation, the pristine
particle is probably of limited relevance. The intestinal
lumen is a ‘soup’ of proteins, bacterial fragments, ions,
small organic molecules etc. and these will modify the
surface of the particles through adsorptive interactions.
Consistent with this, there are several reports of how
TiO2 particles act as an adjuvant for cellular responses
to the bacterial-derived molecule lipopolysaccharide
(LPS), either through formation of ‘conjugates’ or by
coincubation [
12–15
].
More recently it has been shown that pigment-grade
TiO2 is a modest trigger of the NLR family pyrin
domain-containing 3 (NLRP3) inflammasome and that
this activity may contribute to intestinal inflammatory
properties of the particle in murine models [
16
]. The
inflammasome regulates the activation of caspase-1
which, in turn, determines cleavage of inactive
pro-interleukin 1β (pro-IL-1β) to form mature pro-inflammatory
interleukin 1β (IL-1β). If such a pro-inflammatory effect
from oral TiO2 exposure translates significantly from
murine models to humans, it must be occurring in a
small minority of the population because most children
and adults do not have intestinal disease. In this respect
some variants of human genotype could be important.
Indeed, it is well recognised that inflammatory bowel
diseases are complex polygenic disorders [
17
]. Certain
mutations in the nucleotide-binding oligomerisation
domain-containing 2 (NOD2) gene, for example, are
associated with an increased risk of the inflammatory
bowel disease, Crohn’s disease [
18, 19
]. Maeda et al. have
shown that in mice at least one form of Nod2 mutation
potentiates IL-1β processing and enhances risk of
intestinal inflammation [20]. These mice carry a known
Crohn’s disease-associated ‘knockin’ mutation in the
Nod2 locus but also carry a duplication of the 3′ end of
the wild-type (WT) Nod2 locus [
21
], and herein are
designated as Nod2m/m mice. Specifically, development of a
modest pro-inflammatory phenotype in these animals is
reportedly triggered by a bacterial peptidoglycan moiety,
muramyl dipeptide (MDP), in an IL-1β-dependent
fashion [
20
]. Since bacterial peptidoglycan is taken up by
Peyer’s patch phagocytes [
22, 23
] it raises the possibility
that TiO2 could act as an adjuvant for the
proinflammatory effects of peptidoglycan, and especially so
where the genotype potentiates IL-1β processing. Hence,
using bone marrow-derived macrophages (BMDMs)
from WT and Nod2m/m mice, we have tested these
possibilities using an assay of short ‘pulse and chase’ format,
to determine if and how TiO2 could amplify IL-1β
secretion at the cellular level.
Methods
Study design
The macrophage-stimulatory effects of dietary TiO2 were
investigated, either alone or in combination with
microbial-associated molecular patterns (MAMPs), using
cells from WT and Nod2m/m mice. MAMP concentrations
were fixed whereas a range of TiO2 concentrations was
investigated. LPS pre-stimulation of cells was employed as
this MAMP is abundant in the intestinal lumen and can
prime cells for an inflammasome-driven response (IL-1β
secretion), as described in the Introduction. Parameters
assessed were overall cell viability, particle uptake, and
secretion of the pro-inflammatory cytokines IL-1β and
tumour necrosis factor alpha (TNF-α).
TiO2 particles
Food- and pharmaceutical-grade TiO2 particles with
anatase crystal structure and a purity of not less than 99%
were obtained from Sensient Colors (St. Louis, USA).
According to the manufacturer, the TiO2 particles had
an average particle size of 300 nm and a maximum
particle size of 1.0 μm, which had been determined using a
sediograph instrument. We undertook further analysis of
the powder, initially with transmission electron
microscopy. A 1 mg/mL suspension of TiO2 powder in distilled
water (Life Technologies, Auckland, New Zealand) with
0.5% bovine serum albumin (BSA; Life Technologies) as
a dispersant was prepared. A drop of the TiO2 particle
suspension was placed on a 200-mesh carbon-coated
copper grid, and excessive liquid was absorbed with filter
paper. The particles were analysed with a Philips CM10
transmission electron microscope at 80 kV. The image
analysis software iTEM (Olympus Soft Imaging
Solutions, Münster, Germany) was used to record the
images digitally and subsequently measure the diameter
of the particles.
In addition, particle size under cell culture conditions
was determined with nanoparticle tracking analysis,
which is a method to analyse dispersed particles based
on their Brownian motion, similar to analysis with
dynamic light scattering [
24
]. A 100 μg/mL TiO2 particle
suspension was prepared in tissue culture medium
(TCM) consisting of RPMI 1640 medium
(Sigma-Aldrich, Gillingham, UK) with 10% foetal bovine serum
(FBS; PAA Laboratories, Yeovil, UK) and 1%
penicillinstreptomycin antibiotics (Sigma-Aldrich). The
suspension was sonicated for 10 min to facilitate distribution of
the TiO2 particles in the medium. The motion of the
particles in suspension was digitally recorded with a
NanoSight NS500 instrument (NanoSight, Amesbury,
UK). Three TiO2 suspensions were analysed
independently. The particle sizes were calculated from the
recorded videos with nanoparticle tracking analysis
software (Nanosight).
Animals
For the cell culture experiments, bone marrow was
obtained from 10 to 18 week old female C57BL/6 WT and
Nod2m/m mice. The original WT breeding pairs were
purchased from the Jackson Laboratory (Bar Harbor,
USA) and bred at the AgResearch Small Animal Colony
(Hamilton, New Zealand). Breeding pairs for Nod2m/m
mice on a C57BL/6 background were kindly provided by
Lars Eckmann [
20
], and backcrossed with WT mice for
10 generations at the AgResearch Small Animal Colony.
The mice were kept under conventional conditions at all
times [
25
].
Harvest of BMDMs and cell culture
For the bone marrow collection, the mice were
euthanised with CO2 asphyxiation and cervical dislocation.
Femurs and tibias were collected, sterilised in 70%
ethanol for 10 s, and the bone marrow flushed out with cold
RPMI 1640 medium (Life Technologies). Single cell
suspensions were prepared by passing the cells repeatedly
through a 19G needle (BD Biosciences, Singapore) and a
70 μm cell strainer (BD Labware, Franklin Lakes, USA).
Bone marrow cells were re-suspended in TCM
consisting of RPMI 1640 medium (Life Technologies) with 10%
FBS (Life Technologies), 1% penicillin-streptomycin
antibiotics (Life Technologies), and 10 μg/mL macrophage
colony-stimulating factor (eBioscience, San Diego, USA).
The cells were transferred to non-tissue culture treated
24-well plates (BD Labware) at a concentration of 1 ×
106 cells/well in 1 mL TCM and cultured at 37 °C in 7%
CO2/93% air. Half of the TCM was replaced every 3 days
with fresh TCM throughout the culture period. Bone
marrow cells were fully differentiated into BMDMs on day 7
and used for experiments between day 8 and day 10.
Stimulation of BMDMs with TiO2 particles +/−
peptidoglycan or MDP
As previously noted, a short ‘pulse (LPS) and chase
(TiO2 +/− peptidoglycan or MDP)’ format was used to
dissect out the point in the pathway that the particles
might act as pro-inflammatory adjuvants of MAMPs. To
that effect, harvested murine BMDMs from each
genotype, +/− LPS pre-stimulation, were exposed to a range
of TiO2 particle concentrations +/− peptidoglycan or
MDP, as detailed below.
To activate the cells, especially for pro-IL-1β
induction, BMDMs were first primed in culture with 1 mL
TCM containing 10 ng/mL LPS from Escherichia coli
O111:B4 (Sigma-Aldrich, Auckland, New Zealand) for
3 h at 37 °C in 7% CO2/93% air. Unprimed BMDMs
were cultured under identical conditions but without
LPS. All cells were then washed in TCM before the TiO2
suspensions were added. A 1 mg/mL TiO2 stock
suspension was first prepared in distilled water and autoclaved.
This stock suspension was used to prepare TiO2
suspensions in the TCM with final concentrations from 5 μg/mL
to 100 μg/mL. Similar concentrations have been used in
previous studies that examined cytokine secretion by
phagocytic cells after TiO2 exposure [
13, 26–28
]. The
TiO2 suspensions were sonicated in a water bath for
10 min before 1 mL of the respective TiO2 suspension
was added to the cells. When the BMDMs were
co-stimulated with MAMPs, either synthetic MDP or
peptidoglycan from Bacillus subtilis (both from
Sigma-Aldrich) was added to the respective TiO2
suspensions in TCM, both at a final concentration of
10 μg/mL. The BMDMs were incubated with TiO2
particles in TCM with or without the co-stimulants
for 3 h at 37 °C in 7% CO2/93% air.
Flow cytometry analysis of BMDMs
Only LPS pre-stimulated BMDMs were used for flow
cytometry analysis. After incubation with particle
suspensions with or without the other MAMPs, the cells were
collected for analysis with flow cytometry. Briefly, cells
were washed with TCM, incubated for 30 min with cold
phosphate-buffered saline (PBS; Life Technologies) on
ice, and collected by vigorous pipetting. The BMDMs
were re-suspended in 150 μL PBS containing 5% FBS,
2% ethylenediaminetetraacetic acid (Life Technologies),
and 1% sodium azide (BDH Laboratory Supplies, Poole,
UK). The cells were first incubated for 15 min on ice
with 1 μg/mL anti-mouse CD16/32 blocking antibody
(clone 93; BioLegend, San Diego, USA) and then stained
for 15 min on ice with 1 μg/mL anti-mouse
phycoerythrin-labelled F4/80 antibody (clone BM8;
BioLegend), a specific marker for murine macrophages.
In addition, 0.8 μg/mL propidium iodide (PI; Life
Technologies) was added to each sample immediately
before analysis for viability assessment. The cells were
analysed with a FACS Calibur flow cytometer (BD
Biosciences, San Jose, USA), and at least 12,000 events
per sample were acquired with the CellQuest Pro
software (BD Biosciences). Data analysis was performed with
FlowJo (Tree Star, Ashland, USA). For details on the
gating strategy see Additional file 1. The percentage of
viable cells in relation to the total number of detected
events was assessed with PI staining. Cells that did not
show PI staining (PI−) were considered to be viable cells.
BMDMs were identified among the PI− cells based on
the expression of F4/80, i.e. viable cells that expressed
F4/80 (PI−F4/80+) were classified as viable BMDMs. The
percentages of PI−F4/80+ cells in relation to the total
number of viable cells are shown in Additional file 2.
Uptake of TiO2 particles by BMDMs was assessed
with the median side scatter (SSC) intensity of the
PI−F4/80+ cell populations. According to previous
studies, an increase in SSC intensity indicated TiO2
particle uptake [
12, 29, 30
].
Validation of SSC analysis by flow cytometry as a
measure of TiO2 cellular uptake
To confirm that increases in SSC intensity did indeed
indicate TiO2 particle uptake, we undertook correlative
studies with conventional flow cytometry and imaging
cytometry which allows visualisation of TiO2 uptake by
individual cells [
31
]. This technique was not available in
the laboratory that undertook the above work and is
impractical for a very large number of samples, so only the
lower concentration range was investigated and
correlated to ensure true discrimination from background.
To quantify TiO2 cellular uptake (i.e. association and
localisation) by peripheral myeloid cell populations, fresh
leukocyte cones were purchased from the National
Blood Service (Cambridge, UK). Peripheral blood
mononuclear cells (PBMCs) were isolated by density
centrifugation using the separating medium
Lymphoprep (Axis Shield Diagnostics, Dundee, UK) and frozen
until use. PBMCs from 3 leucocyte cones were thawed
and rested for 2 h prior to incubation at 1 × 106 cells/mL
with 0 μg/mL, 5 μg/mL, or 10 μg/mL TiO2 and
incubated for 24 h in RPMI 1640 medium
(SigmaAldrich, Gillingham, UK) supplemented with 10%
FBS (Sigma-Aldrich).
After incubation, cells were washed with ice cold PBS
(Sigma-Aldrich) containing 1% BSA (Sigma-Aldrich) and
stained for the human monocyte/myeloid cell markers
CD14 Alexa Fluor 488 or CD11c fluorescein
isothiocyante (both from BD Biosciences), respectively. Single
stain compensation tubes and unstained PBMC tubes,
with and without TiO2, were also prepared at this time
from PBMC samples for the generation of compensation
matrices. After staining, PBMCs were washed with ice
cold PBS containing 1% BSA, re-suspended in a small
volume of PBS containing 2% paraformaldehyde
(SigmaAldrich) solution, and placed on ice in the dark until
acquisition.
Imaging cytometry analysis was undertaken using an
ImageStreamX Mark I platform
(Amnis-Merck-Millipore, Seattle, USA), equipped with 405 nm and 488 nm
lasers for excitation, a 785 nm laser for a scatter signal
with standard filter sets, multi magnification (20×/40×/
60×) and extended depth of field. INSPIRE software
(Amnis) was used for acquisition and IDEAS software
(Amnis) for analysis. The machine passed all tests and
was fully calibrated prior to acquisition of samples.
Before acquisition, cells were filtered through 35 μm cell
strainers (BD Labware). A minimum of 10,000 events
per sample were acquired. Compensation matrices were
generated by running single stained cells (i.e. single cell
surface marker) and analysed using IDEAS software. For
analysis, TiO2 positive cells were identified and
quantified using a spot count analysis of dark spots appearing
within the cells based on bright-field images of CD14
positive (CD14+) cells. Briefly, cells were first plotted as
area versus aspect ratio of the bright-field images and
a single cell gate drawn, followed by a focused gate.
CD14+ cells were then gated based on fluorescence
intensity. A custom dark spot count mask was
generated to quantify CD14+ cells, with cells positive for 2
or more darks pots gated as dark spot positive.
Conventional flow cytometry analysis was performed
using a CyAn ADP 9 colour analyser (Beckman Coulter,
Brea, USA) equipped with 405 nm, 488 nm and 642 nm
solid-state lasers and 11 detectors in standard
configuration. Summit software was used for acquisition and
analysis (Beckman Coulter, USA). At least 500,000
events were acquired on the flow cytometer using a
lowered SSC setting on a logarithmic scale. Samples
were filtered through 35 μm cell strainers (BD Labware)
directly prior to acquisition. For data analysis, events
were first plotted as forward versus side scatter using
SSC on a logarithmic scale, and a large gate was drawn
excluding debris. Cells were then further gated for
CD11c positivity based on fluorescence intensity for the
mean fluorescence intensity (MFI) of the SSC signal of
CD11c+ myeloid cells.
Stimulation of PBMCs with monosodium urate crystals or silica nanoparticles
We confirmed that other exemplar
inflammasomeactivating particles to which humans are exposed,
namely monosodium urate (MSU) crystals and silica
nanoparticles (SNPs) [
32, 33
], promote IL-1β processing
in our short ‘pulse and chase’ format. Isolated PBMCs
(n = 4) were thawed and rested overnight. Cells (1.106
cells/mL) were then subjected to LPS pre-stimulation
(10 ng/mL, Escherichia coli O111:B4; Sigma-Aldrich) to
induce the production of pro-IL-1β or with TCM as a
negative control. Following 3 h, cells were washed and
then challenged with 100 μg/mL MSU crystals (Caltag
Medsystems, Buckingham, UK) or 100 μg/mL SNPs
(InvivoGen, San Diego, USA) for a further 3 h.
Following this, cells were washed and replenished with
fresh TCM for a further 21 h (3 + 21 h). Supernatants
were collected at the 3 h and 3 + 21 h time points for
IL-1β analyses.
Cytokine detection in cell supernatants
Cell supernatants were collected at the time points
indicated and stored at −20 °C until required for cytokine
analysis. IL-1β (TiO2 and exemplar
inflammasomeactivating particles) and TNF-α (TiO2 only) were
investigated with enzyme-linked immunosorbent assay (ELISA)
using DuoSet ELISA kits (R&D Systems, Minneapolis,
USA) according to the manufacturer’s instructions. The
cytokine concentrations were generally determined with
a FlexStation 3 microplate scanner (Molecular Devices,
Sunnyvale, USA) and Soft Max Pro software (Molecular
Devices).
Statistical analysis
All statistical comparisons were carried out using R (R
Development Core Team, Vienna, Austria). For analysis
of the flow cytometry results, the groups according to
genotype (WT or Nod2m/m BMDMs) were compared
with two-way analysis of variance (ANOVA) using
co-stimulation condition and TiO2 exposure as the two
factors. For analysis of the cytokine secretion results
without co-stimulation, the groups according to
genotype (WT or Nod2m/m BMDMs) were compared with
one-way ANOVA using TiO2 exposure as the single
factor. For analysis of the cytokine secretion results with
co-stimulation, the groups according to co-stimulation
condition (MDP or peptidoglycan) were compared with
two-way ANOVA using genotype and TiO2 exposure as
the two factors. In instances where two-way ANOVA
results showed a significant interaction effect or the
one-way ANOVA results indicated a significant
difference between groups, pairwise group comparisons were
performed with Tukey’s post-hoc test. Figures depict
group means ± standard deviation (SD). Finally, paired
T tests were used to compare supernatant levels of
IL1β for cells exposed to MSU crystals or SNPs versus
non-particle-exposed control cells. Group means ±
standard error of the mean (SEM) are depicted in the
corresponding figure.
Results
TiO2 particle characterisation
Several images of TiO2 particles were obtained with
transmission electron microscopy and a representative
image is shown in Fig. 1. The diameters of individual
particles were measured with image analysis software.
The average primary particle size was 119 nm with a SD
of 45 nm, and the observed particle sizes ranged from
50 nm to 350 nm with a maximum frequency at 100 nm
(Fig. 2a). Approximately 54% of the particles had a
diameter between 125 nm and 200 nm, and about 40% had a
diameter of 100 nm or less.
TiO2 particles were suspended in TCM for the
subsequent cell culture experiments, so the particle sizes
in TCM were also investigated, using nanoparticle
tracking analysis. According to this method, the
average particle size was 160 nm, and the sizes
ranged between 20 nm and 450 nm (Fig. 2b).
Approximately 20% of the particles had a diameter of
less than 100 nm. The slight increase in particle sizes
versus electron microscopy measures probably results
from the differing environments as, in solution,
particles have a hydration shell and are liable to adsorb
TCM molecules. However, the possibility of a small
degree of agglomeration in this environment cannot
be precluded.
Cellular effects of TiO2 particles
As intended with our short ‘pulse and chase’ style assay,
BMDMs of both genotypes that were not primed with
LPS (i.e. sham-pulsed) did not secrete meaningful
amounts of IL-1β when chased for 3 h with TiO2 from
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
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0 μg/mL to 100 μg/mL +/− MDP or peptidoglycan
(IL1β secretion always <5 pg/mL; data not shown). All
subsequent data therefore refer to results with
LPSprimed cells.
Cell viability
The viability of LPS-pulsed cells, from WT (Fig. 3a) and
Nod2m/m mice (Fig. 3b), was significantly reduced by
chasing with TiO2 particles, in a dose-responsive fashion
(p < 0.001 for trend, Fig. 3a and b). Addition of
peptidoglycan or MDP during the chase phase marginally, but
significantly, decreased cell viability further (p < 0.001 for
trend), although there was no interaction effect with
TiO2 exposure (Fig. 3a and b).
Particle uptake
Particle uptake was assessed by flow cytometric SSC
intensities for LPS-pulsed viable (PI−) F4/80+ WT (Fig. 4a)
and Nod2m/m BMDMs (Fig. 4b). During the chase phase,
SSC intensities of WT and Nod2m/m BMDMs increased
with increasing TiO2 concentrations (p < 0.001 for trend)
but were unaffected by the presence of peptidoglycan or
MDP (Fig. 4a and b). To confirm that such increases in
SSC intensities did result from TiO2 uptake, as
anticipated and as previously reported [
12, 29, 30
], we
compared this form of analysis with imaging cytometry
which allows visualisation of particle uptake [
31
]. Using
PBMCs, and the lower end of the exposure range (where
error would be greatest), increases in SSC intensity of
myeloid-gated cells correlated positively and closely with
observed TiO2 uptake (r = 0.84, p < 0.01; Fig. 5).
IL-1β secretion
In LPS-pulsed BMDMs chased with TCM alone (i.e. zero
dose TiO2 in Fig. 6a), there was no secretion of mature
IL-1β, consistent with the role of LPS in stimulating
pro-IL-1β but not triggering the inflammasome [
34, 35
].
5 10 25 50
Dose of TiO2 ( g/mL)
100
WT TCM
WT MDP
WT PGN
Nod2 m/m TCM
Nod2 m/m MDP
Nod2 m/m PGN
a
1000
Again as anticipated, chasing LPS-primed BMDMs
with TiO2 led to mature IL-1β secretion in a
dosedependent fashion (p < 0.001; Fig. 6a) as these
particles are a modest activator of the inflammasome
[
16, 28
]. Pairwise group comparison with Tukey’s
post-hoc test indicated significant IL-1β stimulation
with TiO2 doses in TCM of ≥50 μg/mL (p between
<0.01 and <0.001; Fig. 6a).
Similarly, chasing LPS-primed BMDMs with TiO2 +
peptidoglycan or MDP increased IL-1β secretion in a
dose-dependent fashion, for cells of both genotypes (p <
0.001, Fig. 6b). However, genotype significantly
influenced the extent of the IL-1β response (p < 0.01
for + MDP and p < 0.001 for + peptidoglycan).
Furthermore, an interaction effect between genotype and
TiO2 exposure was observed for peptidoglycan (p < 0.001),
but not for MDP. Pairwise comparisons between groups
with Tukey’s post-hoc test, when chasing with TiO2 +
peptidoglycan, showed that the amount of IL-1β released
by WT and Nod2m/m BMDMs differed significantly when
the cells were similarly exposed to ≥10 μg/mL TiO2 (p
between <0.05 and <0.001; Fig. 6b).
TNF-α secretion
LPS priming led to marked secretion of TNF-α even
when chased with TCM alone (Fig. 7a) because, unlike
IL-1β [
36
], there is no requirement for a second signal
to enable protein formation and secretion of this
cytokine. Chasing LPS-primed BMDMs with TiO2 led to
further TNF-α secretion in a dose-dependent fashion (p <
0.001; Fig. 7a) and, again, Tukey’s post-hoc test indicated
significant TNF-α stimulation with TiO2 doses in TCM of
≥50 μg/mL (p between <0.05 and <0.001; Fig. 7a).
WT
Nod2m/m
WT MDP
Nod2m/m MDP
WT PGN
Nod2m/m PGN
0 5 10 25 50100 0 5 10 25 50100
Dose of TiO2 ( g/mL)
Fig. 6 IL-1β secretion by LPS-primed BMDMs after chasing with TiO2
+/− peptidoglycan or MDP. BMDMs from WT and Nod2m/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 Nod2m/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 Nod2m/m cells compared to WT cells cultured
with the same TiO2 concentration, †††p < 0.001 for WT and Nod2m/m
cells compared to respective WT or Nod2m/m cells incubated without
TiO2, ‡p < 0.05 for Nod2m/m cells compared to Nod2m/m cells
incubated without TiO2
In contrast to IL-1β, the secretion of TNF-α by
LPS-primed BMDMs that were chased with MDP or
peptidoglycan was not affected by additional TiO2
exposure regardless of dose (i.e. the MAMPs rather
than the particles dominated the scene for TNF-α
secretion; Fig. 7b).
Although in all cases the genotype had a significant
influence (p < 0.001) on TNF-α secretion, being
greater for cells from Nod2m/m than WT mice, there
was no interaction effect between genotype and TiO2
exposure (Fig. 7a and b).
Specificity of TiO2 effect
Activation of the inflammasome is by no means specific
to TiO2 particles although Pele et al. have shown that
correct design of in vitro experiments is critical. Notably,
cell gorging of particles through extended particle
0 5 10 25 50100 0 5 10 25 50100
Dose of TiO2 ( g/mL)
Fig. 7 TNF-α secretion by LPS-primed BMDMs after chasing with
TiO2 +/− peptidoglycan or MDP. BMDMs from WT and Nod2m/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 Nod2m/m cells
incubated without TiO2
exposure (e.g. over 24 h) can lead to false positives [
35
].
SNPs and MSU crystals are considered exemplar
particulate stimulants of the inflammasome, and we
confirmed that, with similar short exposures as for our TiO2
particles (3 h) and LPS priming, IL-1β secretion was
enhanced compared to non-particle-exposed cells (Fig. 8).
Discussion
Relevance and context of our findings
The distal intestinal tract is bathed in high
concentrations of MAMPs such as LPS and peptidoglycan (and
their fragments) due to the continuous turnover of the
microbiome. Since ingested particles, such as
pigmentgrade TiO2, are taken up by intestinal cells from this
distal environment it is important to consider
interactions of these components (i.e. MAMPs + particles)
when looking at potential cellular effects. In this work
we have further considered the impact of genotype,
namely one that imparts greater potential for an
inflammatory phenotype (Nod2m/m) than the WT version. We
confirm that (a) primed cells from Nod2m/m mice secrete
higher concentrations of pro-inflammatory cytokines,
namely IL-1β and TNF-α, in response to
MDPcontaining MAMPs than cells from WT mice [
20
] and
(b) TiO2 particles are mediators of inflammasome
activation [
12, 16, 28, 33
]. Additionally, we show for the first
time that, in primed cells exposed to peptidoglycan, the
concentration of TiO2 that is required to trigger the
inflammasome and induce IL-1β secretion is lower for
cells from Nod2m/m mice than it is from WT mice. This
may have important implications as discussed below.
It is established that at least some ingested TiO2
particles are taken up by intestinal cells, especially by
macrophages of large lymphoid follicles of the ileum termed
Peyer’s patches [
6–10
]. Recent data suggest that cells of
the large bowel can also scavenge particles of
pigmentgrade TiO2 and that oral administration of this pigment
can lead to pre-cancerous lesions of the colon, termed
aberrant crypt foci, in about a third of WT animals but
not in controls without TiO2 exposure [
37
]. In that
work, intestinal mucosal levels of TNF-α and IL-1β were
modestly increased for animals fed TiO2 versus controls
[
37
]. Whilst our data support these findings from a cell
culture perspective they also show that particle dose is
critical as a determinant of the cytokine response. The
precise pathway of TiO2 uptake by intestinal cells is still
not understood, but it is likely that particles in the
lumen have their surfaces ‘decorated’ by soluble
molecules of the intestinal lumen so that conjugates (with
MAMPs for example), rather than pristine particles, are
seen by intestinal cells. Moreover, it is not clear how
basal macrophages of the human Peyer’s patches become
loaded with particles such as TiO2 as, following M cell
uptake, particles should be scavenged by phagocytes that
are more apical than the observed basal tissue-fixed
macrophages [
38
]. However, despite the pathway not
being fully elucidated, the important point is that
macrophages of the Peyer’s patches do accumulate TiO2
particles in humans [
9, 39
]. If, as we show here, certain
genotypes require a lesser cell dose of particles to
respond in a pro-inflammatory fashion compared to other
genotypes then, in vivo, the initiation of a cascade of
inflammation may be host-dependent as well as
dosedependent.
Specificity of the IL-1β adjuvant effect to TiO2
nanoparticles
The ‘role’ of the TiO2 particles in the work presented
here involves boosting the pro-inflammatory effects of
MAMPs via particle-activation of the inflammasome.
Many materials activate the inflammasome, including
other (nano)particles, and some of these will be more
potent than pigment-grade TiO2 given the modest
efficacy of the latter. For example, MSU crystals and silica
particles are activators of the inflammasome (as
exemplified here (Fig. 8) and [
28, 32, 33
]) and have direct
relevance in terms of human exposure. MSU crystals
may precipitate ectopically and are the cause of joint
inflammation in patients with gout, whilst silica exposure
to the lungs is well established as an occupational hazard
that leads to silicosis in miners. However, in terms of an
adjuvant effect on MAMP-primed cells, TiO2 deserves
particular scrutiny because (a) humans are widely
exposed to it orally [
3, 5
], (b) MAMPs are ubiquitous at
high concentrations in the intestinal lumen which is
unlike anywhere else in the body, and (c) pigment-grade
TiO2 is one of two major particle types that accumulates
in intestinal (Peyer’s patch) macrophages [
7, 9, 39
]. The
second major particle type, namely aluminosilicate
which is mostly in the kaolinite form [7], has not been
obviously linked to inflammasome activation although
this merits further careful assessment as prolonged
macrophage exposure to kaolinite leads to modest IL-1β
secretion even in the absence of MAMPS [
40
].
Interestingly, Winkler et al. have shown that
foodgrade silica induced production of pro-IL-1β and
secretion of mature IL-1β when dendritic cells were exposed
to these particles [
41
]. In other words, silica particles
have the capacity to both prime IL-1β formation in the
precursor (pro-) form and to induce cleavage to a
mature form via inflammasome activation. Although, unlike
TiO2, this silica has not been demonstrated to
accumulate in human intestinal immune cells [
7
], further studies
are merited as there is significant oral exposure and
perhaps intestinal cells other than those that have been
so far characterised for particle accumulation in the
intestine are impacted.
In summary for this section, pigment-grade TiO2 is
especially relevant as a potential inflammasome adjuvant
in intestinal tissue because of human exposure,
accumulation, and activity. However, other particles,
such as aluminosilicates and silica, should not be
ignored as there is certainly exposure and accumulation
for the former and exposure and potential for activity
for the latter.
inflammation in response to certain MAMPs, precisely as
has been proposed for Crohn’s disease [
44
]. Further work
with patient samples is therefore merited to scrutinise the
potential for a TiO2 adjuvant effect on MAMPs in terms
of IL-1β secretion.
IL-1β secretion is not a simple consequence of TiO2-induced
cell death
Non-biological particles in a size range that enables
phagocytosis, which includes pigment-grade TiO2, are
readily engulfed by macrophages and accumulate in
lysosomes [
7, 9, 42
]. This in turn leads to lysosomal
membrane disruption which is a trigger for two
concomitant events. The first is cathepsin-dependent IL-1β
release which requires inflammasome activation, and the
second is cell death which again is cathepsin-dependent
but is independent of the inflammasome [
42
]. Hence, as
expected, both events were observed in this study in a
dose-dependent fashion when cells were exposed to
TiO2. In vivo, cell death can lead to pro-IL-1β leakage
into the extracellular environment and its activation
through ‘alternative’ pathways, such as cathepsin
Cneutrophil proteases. However, this does not occur in
‘clean’ cell culture media in vitro [
42
]. Moreover, a short
‘pulse and chase’ routine protects against such longer
term complications. It is therefore anticipated that our
observed IL-1β-inducing effect of TiO2 in LPS-primed
macrophages is independent of the concomitantly
observed cell death. Regardless of mechanism, it does not
alter the potential relevance of these findings to the in
vivo situation where, as noted, pigment-grade TiO2
accumulates in selective intestinal cells of humans.
In vivo relevance for health and disease
Notwithstanding the above, and as discussed earlier,
TiO2 is only a modest activator of the inflammasome, so
whether realistic oral exposure to TiO2 leads to
interactions with MAMPs and whether intestinal cell loading of
both materials is sufficient to trigger inflammation
merits closer attention in a relevant genetically
susceptible model. In particular, such work should focus on (a)
the Peyer’s patches as sites of cellular TiO2 accumulation
with the potential for early inflammatory processes [
11
]
and (b) the colon, given the association of large bowel
cancer with early inflammation and potential
exacerbation of disease by TiO2 [
43
].
In addition, our specific interest concerns
inflammatory bowel disease, especially Crohn’s disease, and the
potential for TiO2 as an adjuvant for pro-inflammatory
responses in recipient Peyer’s patch cells [
7, 38, 39
].
Although the murine model used here does not accurately
mimic Crohn’s type mutations for NOD2 because of the
duplication of the 3′-end of the WT Nod2 locus [
21
], it
does, nonetheless, have a heightened susceptibility to
Conclusions
In summary, in this study we have shown that dietary
TiO2 particles have an impact on the production of the
pro-inflammatory cytokines IL-1β and TNF-α by LPS
pre-stimulated murine macrophages in vitro, and that
TiO2 particles can act as IL-1β-inducing adjuvants for
bacterial MAMPs that contain MDP moieties. We also
demonstrated that the impact of this adjuvant effect is
genotype-dependent. Primed macrophages from Nod2m/m
mice showed an elevated IL-1β response to incubation
with TiO2 particles and peptidoglycan compared to cells
from WT mice. Further work will need to consider if any
human genotypes (sub-populations) are at greater
inflammatory risk than the background population from TiO2
exposure.
Additional files
Additional file 1: Flow cytometry gating strategy. (DOCX 321 kb)
Additional file 2: F4/80 expression of LPS-primed BMDMs after chasing
with TiO2 +/−peptidoglycan or MDP. (PDF 129 kb)
Abbreviations
ANOVA: Analysis of variance; BMDM: Bone marrow-derived macrophage;
BSA: Bovine serum albumin; FBS: Foetal bovine serum; IL-1β: Interleukin 1β;
LPS: Lipopolysaccharide; MAMP: Microbial-associated molecular pattern;
MDP: Muramyl dipeptide; MFI: Mean fluorescence intensity; MSU: Monosodium
urate; NLRP3: NLR family pyrin domain-containing 3; NOD2: Nucleotide-binding
oligomerisation domain-containing 2; Nod2m/m: Homozygous Nod2 gene
mutation (as described); PBMC: Peripheral blood mononuclear cell;
PBS: Phosphate-buffered saline; PGN: Peptidoglycan; PI: Propidium iodide;
pro-IL-1β: pro-interleukin 1β; SD: Standard deviation; SEM: Standard error of the
mean; SNP: Silica nanoparticle; SSC: Side scatter; TCM: Tissue culture medium;
TiO2: Titanium dioxide; TNF: Tumour necrosis factor; WT: Wild-type
Acknowledgements
The authors would like to thank Doug Hopcroft from the Manawatu Microscopy
and Imaging Centre (Massey University, Palmerston North, New Zealand) for
assistance with electron microscopy. The authors gratefully acknowledge the
technical support provided by Drs Nuno Faria and Carolin Haas (Biomineral
Research Group, MRC Human Nutrition Research, Cambridge, UK) for nanoparticle
tracking analysis and the support for animal-related work and BMDMs preparation
from Genevieve Sheriff (nee Baildon) and Ric Broadhurst (Campus Services,
AgResearch, Hamilton, New Zealand), and Leigh Ryan and Dr. Wayne Young
(Food Nutrition & Health Team, Food & Bio-based Products Group, AgResearch,
Palmerston North, New Zealand). The authors are grateful for advice on statistical
analysis from Dr. John Koolaard and Catherine Lloyd-West (Campus Services,
AgResearch, Palmerston North, New Zealand) and comments on the manuscript
from Drs Matthew Barnett and Wayne Young (Food Nutrition & Health Team,
Food & Bio-based Products Group, AgResearch, Palmerston North, New
Zealand). The authors thank Dr. Sabine Kuhn (Institut für Klinische Chemie und
Pathobiochemie, Klinikum rechts der Isar, Technische Universität München,
Munich, Germany) for her kind assistance with designing the figures and three
anonymous reviewers for their constructive feedback which helped to improve
the manuscript.
Funding
The research was mainly supported by the Riddet Institute through its
Centre of Research Excellence funding which has been awarded to the
Riddet Institute by the New Zealand government. Additional funding was
provided by AgResearch, MRC Elsie Widdowson Laboratory (formerly MRC
Human Nutrition Research, Grant number U105960399) and Nutrigenomics
New Zealand, a collaboration between AgResearch, Plant & Food Research,
and The University of Auckland (primarily supported by funding from the
Ministry for Science & Innovation contract C11X1009). SR was supported by
doctoral scholarships from Massey University and AgResearch. The funding
bodies had no influence on the research or preparation of this manuscript.
Availability of data and materials
The datasets generated during and/or analysed during the current study are
available from the corresponding author on reasonable request.
Authors’ contributions
LCP and JJP developed the research hypothesis. LCP and SR designed the
study with contributions from DEO, HS, NCR, and JJP. SR performed the
experiments, analysed the results, and, together with LCP, designed the figures
(except Figs. 5 and 8). REH and LCP provided the data and associated analyses
for Figs. 5 and 8, respectively, and wrote the corresponding methods and
results sections. All authors contributed to the interpretation of the results. SR
and JJP wrote the manuscript with contributions from all authors. All authors
read and approved the final manuscript.
Ethics approval
Collection of bone marrow from mice for this research was approved by the
Grasslands Ethics Committee (Palmerston North, New Zealand), AgResearch
Animal Ethics Committee, applications AE Tissue Collection 54 and 68 in
compliance with the New Zealand Animal Welfare Act 1999.
Use of human blood for this research was approved by the ethics
committee of the University of Cambridge (Cambridge, UK), Human Biology
Research Ethics Committee, application HBREC.2015.10.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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