Impaired Ciliogenesis in differentiating human bronchial epithelia exposed to non-Cytotoxic doses of multi-walled carbon Nanotubes
Snyder et al. Particle and Fibre Toxicology
Impaired Ciliogenesis in differentiating human bronchial epithelia exposed to non-Cytotoxic doses of multi-walled carbon Nanotubes
Ryan J. Snyder 0
Salik Hussain 0
Charles J. Tucker 0
Scott H. Randell
Stavros Garantziotis 0
0 National Institutes of Health (NIH), National Institute of Environmental Health Sciences (NIEHS) , Research Triangle Park, Durham, NC 27709 , USA
Background: Multi-walled carbon nanotubes (MWCNTs) are engineered nanomaterials used for a variety of industrial and consumer products. Their high tensile strength, hydrophobicity, and semi-conductive properties have enabled many novel applications, increasing the possibility of accidental nanotube inhalation by either consumers or factory workers. While MWCNT inhalation has been previously shown to cause inflammation and pulmonary fibrosis at high doses, the susceptibility of differentiating bronchial epithelia to MWCNT exposure remains unexplored. In this study, we investigate the effect of MWCNT exposure on cilia development in a differentiating air-liquid interface (ALI) model. Primary bronchial epithelial cells (BECs) were isolated from human donors via bronchoscopy and treated with non-cytotoxic doses of MWCNTs in submerged culture for 24 h. Cultures were then allowed to differentiate in ALI for 28 days in the absence of further MWCNT exposure. At 28 days, mucociliary differentiation endpoints were assessed, including whole-mount immunofluorescent staining, histological, immunohistochemical and ultrastructural analysis, gene expression, and cilia beating analysis. Results: We found a reduction in the prevalence and beating of ciliated cells in MWCNT-treated cultures, which appeared to be caused by a disruption of cellular microtubules and cytoskeleton during ciliogenesis and basal body docking. Expression of gene markers of mucociliary differentiation, such as FOXJ1 and MUC5AC/B, were not affected by treatment. Colocalization of basal body marker CEP164 with γ-tubulin during days 1-3 of ciliogenesis, as well as abundance of basal bodies up to day 14, were attenuated by treatment with MWCNTs. Conclusions: Our results suggest that a single exposure of bronchial cells to MWCNT during a vulnerable period before differentiation may impair their ability to develop into fully functional ciliated cells.
Carbon Nanotubes; Cilia; Ciliogenesis; Epithelial cells
Nanomaterial science has made significant
advancements over the past decade, taking advantage of the
unique properties of nanoscale substances to enable a
staggering variety of novel applications and products.
Multi-walled carbon nanotubes (MWCNTs) are among
the most commonly used engineered nanomaterials, and
their commercial production has increased significantly
in recent years [
]. MWCNTs are nanoscale overlapping
cylinders of graphene, manufactured for their high
tensile strength and hydrophobic properties. These
properties have made them especially useful in industrial
applications and consumer products such as reinforced
], sporting equipment [
], textiles [
sprayon coatings [
], and other uses [
However, the stiff and fiber-like nature of MWCNTs
has raised concerns about their safety. Similarities to
asbestos fibers [
], as well as cytotoxic and
proinflammatory effects [
] have been demonstrated in a
number of studies. MWCNTs enter epithelial cells and
have been found in the cytoplasm [
], and endosomes [
]. Inhalation of MWCNTs
induces pulmonary inflammation and fibrosis [
retained fibers have been found in the lungs of exposed
rodent models months after MWCNT exposure [
These adverse health effects were demonstrated at high
doses; however, even lower, more
occupationallyrelevant doses can have significant effects on pulmonary
]. We have previously demonstrated effects
of MWCNTs on the barrier function of primary human
BECs and cytoskeletal disruptions resulting from
noncytotoxic exposures  in submerged cultures.
Impaired barrier function in the pulmonary epithelium of
MWCNT-exposed individuals may further increase their
susceptibility to environmental insult. These effects may
be particularly important in susceptible individuals with
pre-existing lung disease, or in cases of long-term
chronic exposure. Denuded epithelium resulting from
tobacco smoke [
] or viral infection [
] could expose
undifferentiated basal cells to nanoparticles, potentiating
further injury or attenuating epithelial regeneration.
MWCNT-induced effects on the cytoskeleton are of
particular interest in the context of pulmonary exposure,
because of the importance of actin and tubulin networks
in the organization, orientation, docking, and structure of
airway cilia. Multiciliated cells differentiate from epithelial
precursors in the conducting airways and are critical to
the removal of debris and pathogens from the lung [
While an intact, healthy, functioning mucociliary
epithelium constitutes a significant barrier to inhaled
], exposed epithelial precursors in an injured lung
may be more susceptible to MWCNT effects [
nanotubes can damage the cytoskeletal network [
have steric interactions [
] with microtubules and may
therefore have detrimental effects on cilia formation and/
or function even after exposure has ceased.
This study investigated whether undifferentiated cell
exposure to MWCNT affects the cells’ differentiation
potential. We utilized a brief, 24-h exposure to
noncytotoxic doses of MWCNTs during submerged,
undifferentiated culture and evaluated subsequent mucociliary
differentiation. Primary BECs from healthy human
donors were exposed to MWCNT in confluent submerged
culture, then converted to an air-liquid interface (ALI)
model and allowed to differentiate for 4 weeks in the
absence of further MWCNT exposure. Mucociliary
differentiation in MWCNT-treated cultures was compared to
dispersion vehicle-treated and graphitized
carbontreated controls. We found that a single 24-h exposure
of undifferentiated cells to MWCNTs decreased
formation and function of cilia in differentiated cells analyzed
28 days later. To our knowledge, this is the first
demonstration that MWCNT exposure during the vulnerable
undifferentiated state may perturb cell development, and
suggests that windows of susceptibility may exist for
specific MWCNT effects.
Attenuated cilia staining in differentiated ALI culture 28 days after single MWCNT exposure
We first analyzed the effect of a single MWCNT
exposure on cell differentiation. Characterization of the
particles used in this study can be found in the Methods and
in Additional file 1. We did not find any cytotoxicity
from our MWCNT at any used dose, based on LDH
release (Additional file 2). Ciliated cell area in 28-day
differentiated ALI cultures was attenuated in
MWCNTtreated BECs compared to vehicle controls (Fig. 1). The
area covered by fluorescently-labeled ciliary α-tubulin
was reduced from 25.46 ± 6.25% (Mean ± SD) in control
cultures to 7.83 ± 1.84% in the MWCNT-treated
cultures (p < 0.0001). Analysis of F-actin staining coverage
also revealed a significant decrease in tight junction
actin from 22.43 ± 4.56% to 14.36 ± 3.93% (p < 0.006).
By contrast, cells which were treated with MWCNTs
after they had differentiated in ALI for 21 days had
ciliary α-tubulin and tight junction actin staining of
32.07 ± 7.32% and 23.83 ± 7.86% (respectively), and were
not significantly different from control cultures.
Reduced prevalence of ciliated cells compared to goblet cells in differentiated air-liquid interface culture 28 days after MWCNT exposure
To determine whether MWCNT exposure specifically
affected the formation of ciliated cells or could affect
multiple cell lineages, we also studied the differentiation of
mucin-secreting goblet cells. Cross-sections of
paraffinembedded membranes were used to count both ciliated
and goblet cells. Sections from differentiated 28-day
cultures revealed a dose-dependent decrease in ciliated
cell counts with increasing MWCNT dose (Fig. 2). Pooled
counts from NG-treated cells had an average of
27.75 ± 4.93 (mean ± SD) ciliated cells per 500 μm
section, while the sections from cultures treated with
MWCNT 1 μg/cm2 contained an average of 13.65 ± 3.26
ciliated cells per 500 μm (p < 0.003). Goblet cells, by
contrast, were not significantly affected by MWCNT
exposure and were observed in equivalent abundance
between control (8.6 ± 2.62 goblet cells per 500 μm section)
and 1 μg/cm2 MWCNT-treated (6.3 ± 2.31) ALI cultures.
MWCNT treatment induced heterogeneity in cilia beating frequency
Having shown that MWCNT exposure affects cilia
development, we wanted to investigate MWCNT effects
on cilia function. We therefore analyzed the effect of
MWCNT exposure on cilia beating by measuring the
frequency of intensity changes in motion capture video
taken at the center of each insert. Control inserts
developed cilia with a relatively uniform beating frequency
and produced a dominant peak at approximately 20 Hz
accounting for an average of 51.6 ± 1.76% (mean ± SD)
of the total signal (Fig. 3). Cultures which had been
pretreated with MWCNT also had a dominant peak at
20 Hz; however, the percentage of the total cilia beating
signal which fell beneath this dominant peak was
decreased in a dose-dependent manner. Ciliated cells
that formed in the inserts previously treated with 1 μg/
cm2 MWCNTs possessed a much wider range of beating
frequencies, with the dominant 20 Hz peak accounting
for only 16.07 ± 9.07% of the total signal. Additionally,
the amplitude of cilia beating intensity was greatly
elevated in MWCNT-treated cells (Fig. 3c), at
45.7 ± 30.1 dB (standard deviation of peak amplitude)
compared to 5.14 ± 3.97 dB in control wells.
Gene expression in ALI cultures is unaltered by early
We then investigated the mechanism of MWCNT
inhibition of cilia development and function. We analyzed
expression of genes that are known to be associated with
differentiation of ciliary or goblet cells. FOXJ1, MUC5AC
and MUC5B genes were analyzed in ALI converted
cultures 1 day after MWCNT exposure, as well as in ALI
day 28 cultures (Fig. 4). FOXJ1, MUC5AC and MUC5B
expression was not affected by any of the MWCNT
treatments at any time point, suggesting that the
attenuation of cilia by MWCNTs was not mediated via changes
in gene expression. We also examined the expression of
retinoid signaling marker genes RARRES1 and RDH12,
as well as keratinocyte marker CRNN, and found no
MWCNT-induced changes in these genes at either day 1
or day 28 following exposure.
MWCNT is associated with modest ciliary axoneme abnormalities
Elongation of motile cilia depends on an intact
microtubule axoneme structure and unobstructed dynein arm
]. While previous research has effectively
demonstrated physical interaction and biomimicry [
between carbon nanotubes and intracellular microtubules,
interactions with ciliary microtubules has not yet been
shown. To investigate whether MWCNT exposure alters
axoneme structure and, by this mechanism, impairs
development of normal cilia, cross sections of cilia from day 28
BECs treated with MWCNT 1 μg/cm2 were compared to
those from control cells by TEM (Fig. 5). Axoneme
abnormalities from the typical “9 + 2” arrangement appeared
elevated with MWCNT treatment, occurring in 14 out of
234 cross sections examined (6.0%), compared to 4 out of
224 control cilia (1.9%). Dynein arm abnormalities were
not observed in either treatment.
exposure could affect cilia docking. Confocal images
of actin cytoskeleton in ALI days 3, 5, and 14 show
disorganized formation of the apical cytoskeletal web
by day 3 in cultures treated with MWCNT 1 μg/cm2
compared to vehicle controls (Fig. 6). Basal bodies,
stained via γ-tubulin (magenta), were apparent and
frequent in the day 5 control cultures, but tubulin
staining remained mostly in intracellular centrioles by
the same time point in cultures treated with
MWCNTs. By day 14, MWCNT-treated cultures had
also developed basal bodies, but with greatly reduced
occurrence compared to control cultures. Pixel area
analysis (using Image J) of maximum intensity
projections from these confocal images was used to quantify
the docking of basal bodies (Fig. 6b). By day 14, basal
body docking was significantly reduced in
MWCNTexposed BECs compared to control cultures (p < 0.05,
Multiple t-tests, Holm-Sidak comparison correction).
Disruption of actin web formation and apical translocation of basal bodies by MWCNTs
We evaluated apical actin web morphology and
appearance of γ-tubulin-staining basal bodies during
early ciliogenesis to determine whether MWCNT
Attenuated colocalization of CEP164 and γ-tubulin in
basal bodies during early ciliogenesis
We examined the apical surfaces, to a depth of 6 μm, of
individual cells at post-ALI days 1 and 3 for CEP164
staining and colocalization with γ-tubulin in the ciliary
necklace/transition zone. In Fig. 7, we show that BECs
treated with 1 μg/cm2 MWCNTs had noticeably less
CEP164 staining, and that its colocalization with
γ-tubulin was patchy and inconsistent compared to the
vehicle-treated BECs. This effect was apparent with
MWCNT treatment at post-ALI day 1 and by day 3
resulted in defined “clusters” of ciliary precursors on the
apical surface despite having wider coverage of CEP164.
Trans-epithelial electrical resistance (TEER) is unaffected by MWCNT exposure
As the actin translocation to cell junctions appeared
attenuated by MWCNT exposure, we measured TEER in
exposed ALI cultures to determine whether the barrier
function of the differentiated cells could also be
impaired. In Fig. 8, TEER measured in ALI cultures treated
pre-differentiation with MWCNTs or controls is
recorded every 2 days after treatment until day 12,
averaging the measurements from 3 donors. TEER from all
ALI cultures increases rapidly during differentiation.
However, no significant effect from any treatment was
observed on the TEER of differentiating cultures,
suggesting that epithelial barrier function is retained at the
study doses despite the MWCNTs’ effect on developing
cilia. Staining for ZO-1, a tight junction-specific marker,
showed no significant treatment effect on junction
staining, supporting the TEER results (Additional file 3).
Taken together, our results suggest that a brief, 24-h
exposure of undifferentiated BECs to low, non-cytotoxic
doses of MWCNTs is sufficient to specifically impair
ciliogenesis during air-liquid interface differentiation.
The abundance of ciliated cells in exposed cultures is
attenuated and cilia beating frequencies remain
heterogeneous and disrupted at 28 days following the end of
MWCNT exposure. This implies that exposure to
MWCNTs during a vulnerable period can permanently
affect differentiation of precursor epithelial cells in the
Whole-mount staining reveals that total cilia coverage
in differentiating BECs is reduced by a brief 24-h
MWCNT exposure during submerged culture 28 days
prior. As the MWCNTs were no longer present in the
culture medium by the time the cultures were converted to
ALI, intracellular changes induced by the MWCNT
exposure influenced later ciliogenesis and differentiation.
Additionally, F-actin staining was also reduced in the
MWCNT-treated cultures. While this did not have an
impact on the tight junctions or barrier function (see Fig. 8
and Additional file 3), altered intracellular distribution of
F-actin is consistent with our previous research [
submerged cultures, and also found by other researchers
] investigating carbon nanotubes. Interactions between
carbon nanotubes and cytoskeletal components have been
described previously, both for actin fibers [
]. Our results suggest that cytoskeletal
involvement in ciliogenesis may be particularly susceptible
to sustained effects of MWCNT exposure.
In addition to a reduction in cilia, BECs which
differentiated after MWCNT treatment had altered cilia
beating behavior. A significant decrease in the
percentage of normal cilia beating frequencies was observed in
treated cultures, as well as a significant increase in
amplitude. These results indicate a loss of uniformity in
ciliary beating in cells which developed cilia after
MWCNT exposure, compared to controls. The
concerted motion of beating cilia is critical to efficient
clearance of mucus by the mucociliary escalator in vivo
]. Therefore, this finding could have implications on
the buildup of mucus and particulates in the
Counts of ciliated and goblet cells in PAS and
H&E-stained cross sections also reveal that the effect of
MWCNT treatment appears to be confined to ciliated
cells alone. The finding that goblet cells are not affected
suggests that general differentiation signaling in BECs is
not impaired by MWCNTs, and the effect is specific to
ciliogenesis. This is further supported by the lack of any
MWCNT-induced effects on the expression of various
differentiation gene markers, such as FOXJ1, MUC5AC/
B, RDH12, and RARRES1. These results, combined with
negative results from a previous microarray analysis (not
shown), suggest that the mechanism by which
MWCNTs impair ciliogenesis is not transcriptionally
mediated and is independent of differentiation signaling
cascades. We therefore investigated interactions between
MWCNTs and the synthesis and docking of basal bodies
during early ciliogenesis.
Ultrastructural imaging of the cilia which developed in
treated and control BECs revealed a modest increase in
axoneme abnormalities, from 1.9% of counted cross
sections in controls to 6.0% after MWCNT treatment.
This suggests that MWCNTs may interfere with ciliary
axoneme formation, which could contribute to the
observed ciliary dysfunction. However, normal ciliary
function is thought to be retained when there are less
than 10% abnormal/dysfunctional cilia [
we investigated other effects of MWCNT exposure on
early cilia formation.
The docking of basal bodies with the apical cell
membrane is necessary for the development of motile cilia
] and depends on interaction between the actin
cytoskeletal web and basal body precursor components
(procentriole and ciliary vesicle). Disruption of the
cytoskeletal web by carbon nanotubes has been described
], and our findings in Fig. 6 confirm that a
similar effect can be replicated in our cell cultures using
the multi-walled nanotubes implemented in this study.
As cytoskeletal disruption was observed prior to the
appearance of γ-tubulin-staining basal bodies in treated
cultures, MWCNTs may be disrupting early ciliogenesis
events leading to docking. Further, as γ-tubulin staining
of ciliary basal bodies is attenuated in MWCNT-exposed
cells as early as ALI day 5, at least a week prior to the
appearance of mature cilia in culture, our results support
that the intracellular mechanism mediating impaired
ciliogenesis occurs during or prior to basal body docking.
During very early ciliogenesis, CEP164 bound to
procentrioles forms basal bodies and assists their docking to
the apical membrane [
]. This allows CEP164 to be
used as a biomarker for basal bodies prior to the
development of the γ-tubulin-rich transition zone. In this
study, we found that γ-tubulin staining and CEP164
staining at the apical surface of treated cells were poorly
colocalized, defining a more specific molecular target for
MWCNT-induced disruption of basal body synthesis in
multiciliated cells. While in control BECs most CEP164
staining is colocalized with γ-tubulin and permits the
formation of cilia, much of the CEP164 staining found
in MWCNT-treated BECs is not associated with
γ-tubulin at the transition zone. Therefore, it appears that
MWCNTs may be interfering with the development of
the ciliary transition zone during basal body synthesis.
In combination with the disruption of the apical
cytoskeleton, these molecular events could have a significant
impact on early ciliogenesis given a brief exposure to
In contrast to the nanotubes, mesoporous graphitized
nano-scale carbon (NG) induced no significant effects
on cell differentiation or ciliary function. The NG
controls were applied to BECs at four-fold higher
concentration than the highest MWCNT treatments, but
ciliated cell abundance and beating frequencies remained
unchanged from vehicle control values. This result
strongly suggests that the mechanism for
MWCNTdriven impairment of ciliogenesis is dependent on the
unique tube structure and/or aspect ratio of the
nanotubes. Biomimicry between carbon nanotubes and
microtubules, on account of their similar shape and size,
has been reported previously [
] and physical
interactions between nanotubes and cytoskeletal components
remain a subject of active research [
Our cilia-specific findings contrast with those of
Boublil et al. [
] who found mucociliary differentiation,
as a whole, to be modulated by ultrafine particulate
exposure. We believe that our methodology differs from
theirs in several important ways, which may account for
the discrepancy. The materials used in the Boublil study
(while nano-scale and carbon-based) were taken from
exhaust and ambient particulates and contain residual
organic compounds. These particles also lack the tubular
shape and high aspect ratio of the carbon nanotubes
used for this study, and would therefore not be expected
to physically interact with cytoskeletal components as
nanotubes are known to do. Finally, while our study
dosage was restricted to a single 1 μg/cm2 exposure
prior to conversion to air-liquid interface, the Boublil
study applied doses up to 10 μg/cm2 every week until
differentiation. Consequently, we do not believe there is
any conflict or overlap between these studies or their
The results of our study are consistent with an attenuation
of ciliogenesis and ciliary beating in BECs previously
exposed to non-cytotoxic doses of MWCNTs during their
undifferentiated phase. Since we do not find prospective
loss of cilia in fully-differentiated cells which were exposed
to MWCNTs, our results suggest that undifferentiated
cells are especially vulnerable to MWCNTs, in particular
with regard to ciliogenesis. Undifferentiated basal cells can
become exposed to exogenous substances following
airway injury, when the differentiated columnar cells are
]. Our findings therefore suggest that
preexisting lung injury (e.g. by noxious or infectious agents)
could compound the adverse effects of MWCNT at
concentrations which are not normally harmful;
furthermore, chronic, low-dose MWCNT exposure may have
long-lasting effects on ciliary differentiation and
mucociliary clearance. Additional studies to investigate this
previously-unreported mechanism of carbon
nanotubeinduced lung injury are needed to assess their long-term
impact on ciliary development and lung health in vivo.
Characterization of the multi-walled carbon nanotubes
(MWCNTs) used for this study has been previously
]. In brief, MWCNTs were purchased
from Helix Nanomaterial Solutions and synthesized by
chemical vapor deposition. Elemental carbon content
was >95%, with catalytic metal impurities of nickel and
lanthanum at <0.2 and <0.1% respectively. Nanotubes
measured 10-30 nm in diameter and 500-4000 nm in
length, as determined by TEM.
Mesoporous graphitized nanocarbon (NG) was used at
4 μg/cm2 as a control particle, as it possesses a similar
graphitized surface chemistry without the tubular shape
and aspect ratio of MWCNTs. NG was purchased from
Sigma Aldrich (St. Louis, MO) and was of high purity
Further information on the characterization of the
nanomaterials used in this study can be found in
Additional file 1.
Cell culture and treatment
Primary human bronchial epithelial cells (BECs) were
taken from bronchoscopy brushings of healthy donors
(Additional file 1). Cells were grown in submerged
culture using BEGM medium (Lonza) and frozen after 1
passage. Thawed BECs were seeded onto 12 mm
Millicell standing porous inserts (EMD Millipore,
Darmstadt, Germany) at ~105 cells/cm2 in ALI
differentiation medium [
] (University of North Carolina,
Cystic Fibrosis Laboratory). Human collagen IV was
used to coat insert membranes 24 h prior to seeding.
Cultures were allowed to reach confluence while
submerged in ALI medium prior to treatment with
nanomaterials. Cultures were then treated in the apical
chamber only with 0, 0.25, or 1 μg/cm2 (0, 0.75, and
3 μg/ml respectively) MWCNTs or 4 μg/cm2 (12 μg/ml)
NG dispersed in ALI medium supplemented with 10 μg/
ml 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)
and 600 μg/ml sterile bovine serum albumin (BSA). Both
nanomaterial suspensions and control media were
dispersed via sonication in a cup horn (Misonix,
Farmingdale, NY) at amplitude 100 in 5 pulses of 3 min
each, replacing the cup horn with cold water between
pulses. Dynamic light scattering (DLS) measurements of
nanotube agglomerates in this vehicle solution had
hydrodynamic diameters between 10 and 200 nm, and
graphitized carbon agglomerates formed between 20 and
150 nm, as previously shown [
suspensions were dynamic, and nano-scale aggregates
would settle into larger 1+ μm agglomerates within
hours (see Additional file 1), so the μg/ml concentration
is more accurate in the first few hours while the μg/cm2
concentration is more appropriate when the suspension
has fully settled. Following a 24-h incubation with the
nanomaterials, treatment medium was removed from
the apical chamber to convert the inserts into air-liquid
interface (ALI) cultures. Cultures were washed with ALI
medium immediately following removal of the apical
treatment and during replacement of basal chamber
medium every 2 days until fixation/harvest at 28 days
post-exposure. A fixed timepoint of 28 days was used in
this study to allow for cultures from multiple donors to
fully differentiate and control for variable rates of
differentiation in each donor, though all had
fullydifferentiated before day 20. A post-differentiation
treatment group was also examined, in which the untreated
cultures were allowed to differentiate in ALI for 20 days
and were then treated with 1 μg/cm2 MWNCTs for
24 h. These cells were fixed 7 days following treatment,
at day 28 of differentiation.
Lactate dehydrogenase assay
Cytotoxicity resulting from nanomaterial exposure was
measured by lactate dehydrogenase (LDH) release into
the apical chamber. Apical chambers were washed with
phosphate buffered saline (PBS) 24 h prior to collection
and accumulated LDH collected in a second apical wash
was quantified using the CytoTox 98 colorimetric assay
(Promega, Madison, WI). Absorbance at 495 nm by the
conversion of the formazan dye product indicated the
elevation of LDH concentrations and increased
cytotoxicity. Total 24-h accumulated LDH release was
normalized to vehicle control LDH (Additional file 2). While
MWCNTs have been shown to interfere with
colorimetric assays such as this [
], we have demonstrated in
previous work [
] that the relatively low concentrations
we utilize do not significantly alter assay results. Results
of acellular assays containing only MWCNT or NG were
subtracted from the treatment results to account for
direct 495 nm absorbance by these materials, though
these wells were not significantly different from media
blanks (not shown).
Whole mount immunocytochemistry
Culture inserts were fixed at day 28 following treatment
and removal of apical medium (ALI day 28). Inserts were
washed with PBS to remove mucus and fixed with 4%
EM-grade paraformaldehyde (PFA) for 30 min. Cells
were permeabilized with 0.2% TritonX-100 in PBS for
30 min, washed again with PBS, and blocked (with a
solution of 1% BSA, 1% fish gelatin, 0.1% Triton-100, and
5% goat serum in PBS) for 1-h at room temperature.
Primary incubation with rat anti-tubulin mAb, clone
YL1/2 (EMD Millipore, Darmstadt, Germany, diluted to
5 μg/ml in blocking solution) was applied overnight at 4
°C. Wells were washed 3× for 30 min in 25% blocking
solution in PBS before adding Alexa488-conjugated
phalloidin (Thermo Fisher, Waltham, MA, 1:200) and
Alexa647 anti-rat secondary antibody (Thermo Fisher,
Waltham, MA, 1:200). Secondary incubation was
performed for 2-h at room temperature, after which the
membranes were washed with PBS 3× at 5 min each,
excised from their plastic sprues, and mounted onto slides
with Prolong gold plus DAPI (Thermo Fisher, Waltham,
MA). Z-stacks taken from 10 μm of the apical surface of
each membrane were imaged using a Zeiss 710 confocal
microscope. Parameters such as gain, pinhole, laser
intensity, z-planes per stack, and image post-processing
were kept consistent between treatments. Eight z-stacks
were taken from randomly-selected locations in the
center of each membrane and used to produce maximum
intensity projections. Pixel area analyses of these
projections (using Image J software) were used to quantify
ciliary tubulin staining and F-actin at tight junctions. Pixel
areas were normalized to DAPI nuclear staining to
account for reduced cellularity; however, cellularity was
not altered by any treatment (see Additional file 4).
ALI day 28 transwells were fixed with 4% PFA and
dehydrated in plastic cassettes using 15 min submersions
in ethanol (70%, 70, 90, 95, 100, 100%, and 100%) followed
by three 15 min submersions in NeoClear xylene
substitute (EMD Millipore, Darmstadt, Germany) prior to
overnight paraffin embedding. Cross sections of
differentiated membranes were stained with hematoxylin and eosin
(H&E) and periodic acid-Schiff (PAS) to visualize cilia and
mucins. Ciliated cells and goblet cells were counted using
a 20× objective and results were expressed as average cell
count per 500 μm length of membrane.
Cilia beating analysis
Cultures at ALI day 28 were washed with ALI medium
and 10 s of cilia beating was recorded with a Hamamatsu
ORCA-Flash motion-capture camera using MetaMorph
software (Molecular Devices, Sunnyvale, CA).
Autoregressive spectral analysis was performed on fast Fourier
transforms of image intensity which provided a spectrum
of beating frequencies in each motion capture [
The percentage of the total frequency spectrum which fell
under the dominant (highest intensity) peak was
calculated for each of 5 regions per membrane. These regions
were selected based on the lack of free-floating debris
during the 10 s recording and were otherwise selected
“blindly” as beating differences could not be visually
determined. Dominant amplitudes of cilia beating within
regions of interest were also calculated by parametric
reconstruction analysis. Analyses were performed using
AutoSignal software, v1.7 (Systat Software, San Jose, CA).
RNA was collected from ALI day 1 and day 28 inserts and
extracted using an RNeasy Plus Mini kit and columns by
the manufacturer’s protocol (Qiagen, Venlo, Netherlands).
Total RNA was converted to cDNA using the iScript
reverse transcriptase kit (BioRad, Hercules, CA). QPCR
was carried out using SYBR Green in an ABI (Waltham,
MA) StepOne sequencer and primers for FOXJ1,
MUC5AC, MUC5B, RARRES1, RDH12, and CRNN. 18S
was used as an endogenous control and fold change in
gene transcription was calculated by ddCt analysis. Primer
sequences (QStar, Origene Technologies, Rockville, MD)
can be found in Additional file 5.
Ultrastructural imaging of cilia axonemes
Cell cultures were fixed in 3% glutaraldehyde at ALI day
28. Membranes were rinsed with PBS prior to post-fix in
1% osmium tetroxide. Membranes were then stained
with uranyl acetate and dehydrated in the previously
described ethanol series, followed by submersion in
acetone. The samples were embedded in Polybed 812
epoxide resin. Membrane blocks were cut into thin sections
of 80–90 nm, placed onto 200 mesh copper grids, and
then stained again with uranyl acetate and lead citrate.
Digital images were captured with a Orius SC1000/
SC600 camera (Gatan, Pleasanton, CA) attached to a
Tecnai T120 (FEI/Thermo Fisher, Wlatham, MA)
transmission electron microscope (TEM). Images with clear,
non-oblique, cross-sections of cilia were used to count
abnormalities in axoneme 9 + 2 microtubule
arrangement and/or dynein arm abnormalities. A total of 224
vehicle control-treated and 234 MWCNT-treated cilia
cross-sections were counted for this purpose.
Confocal microscopy of early ciliogenesis
Actin and γ-tubulin structure were examined in inserts
fixed at ALI days 3, 5, and 14. Membranes were fixed
with 4% EM-grade PFA for 15 min and excess aldehydes
were quenched with 0.1 M glycine in PBS for 5 min.
Cells were permeabilized with 0.1% TritonX-100 in PBS
for 15 min and rinsed 3× with PBS. Membranes were
blocked with 1% BSA and 5% goat serum in PBS for 1 h
at room temperature. Rabbit anti-γ-tubulin mAb
(Sigma-Aldrich, St. Louis, MO, diluted 1:800 in blocking
solution) was applied overnight at 4 °C. Following three
5 min PBS washes, membranes were incubated with
Alexa594-conjugated goat anti-rabbit and
Alexa488conjugated phalloidin (Thermo Fisher, Waltham, MA,
diluted 1:1000 and 1:200 in 25% blocking solution,
respectively) for 1-h at room temperature. Membranes
were again washed with PBS (3× at 5 min each) before
being excised from their plastic sprues and mounted
onto slides with Prolong Gold. Z-stack images of the
apical 10 μm of each membrane were taken with a Zeiss
(Oberkochen, Germany) 880 confocal microscope using
Airyscan imaging on Zen software (63× oil immersion
objective). Parameters such as gain, pinhole, laser
intensity, z-planes per stack, and image post-processing were
kept consistent between treatments.
CEP164 and γ-tubulin were stained under similar
conditions, using mouse anti-CEP164 (Sigma Aldrich, St.
Louis, MO, diluted 1:800) and a blocking solution
containing 10% goat serum and 5% BSA.
Alexa488conjugated goat anti-mouse and the previously mentioned
Alexa594 anti-rabbit secondary antibodies (Thermo
Fisher, Waltham, MA, diluted 1:1000 in blocking solution)
were used to image CEP164 and γ-tubulin, respectively.
Hoechst stain (diluted 1:1000) was used to identify cells
(not shown in images). Isotype control primary antibodies
were used to control for non-specific staining (found in
Additional file 4). Confocal Z-stacks were taken of the
apical 6 μm of each membrane, also using Airyscan
imaging with a 4× software zoom (same microscope/
TEER measurement of differentiating ALI cultures
ALI cultures from one donor were used for TEER
measurements during differentiation following nanomaterial
treatment. Every 2 days after conversion to ALI, the
apical chamber of each well was briefly filled with 300ul of
ALI medium taken from the basal chamber (as to avoid
introducing any new nutrients which would alter the
cellular biochemistry during measurement) to permit
TEER measurement. A single TEER measurement was
recorded from each well using an ERS-2 Voltohmmeter
(EMD Millipore, Darmstadt, Germany) with an STX01
probe, as per manufacturer’s instructions. Following each
measurement, the apical medium was removed and fresh
medium added to the basal chamber. Measurements
were averaged from 3 wells for each treatment.
Additional file 1: Bronchoscopy procedure and the characterization of
the nanomaterials. (DOCX 680 kb)
Additional file 2: Graph of cytotoxicity in ALI cultures, measured by LDH
release, on days 1, 4, and 7 following MWCNT exposure. (DOCX 40 kb)
Additional file 3: ZO-1 tight junction staining in pre- vs post-differentiation
exposed BECs. (DOCX 328 kb)
Additional file 4: Raw images of confocal Z-stacks used for Fig. 1, and
images of isotype control antibodies used for Fig. 7 (DOCX 2950 kb)
Additional file 5: List of all primers and sequences used in the QPCR
experiments (DOCX 13 kb)
ALI: Air-liquid interface; ANOVA: Analysis of variance; BECs: Bronchial epithelial
cells; BSA: Bovine serum albumin; CoV: Control vehicle / dispersion medium
alone; DPPC: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; H&E: Hematoxylin
and eosin; LDH: Lactate dehydrogenase; MWCNTs: Multi-walled carbon
nanotubes; NG: Mesoporous nanoscale graphitized carbon; PAS: Periodic
Acid-Schiff; PBS: Phosphate buffered saline; TEER: Trans-epithelial
electrical resistance; TEM: Transmission electron microscopy
We would like to thank the staff of the NIEHS Clinical Research Unit for their
support and recruitment of human donors. We would also like to thank Erica
Scappini, Dr. Michael Dykstra, and Deloris Sutton for their technical support and
assistance with confocal microscopy, TEM imaging and sample preparation.
This research was supported by the Intramural Research Program of the NIH,
National Institute of Environmental Health Sciences.
RS is the primary author, performed the experiments, and analyzed the
data. SH assisted with data analysis and co-developed protocols related to
cell culture and treatment. CT assisted with confocal and cilia beating
analysis, as well as co-developing the protocol for cilia beating measurements.
SR developed the protocols for air-liquid interface culture and differentiation of
BECs, and produced the differentiation medium used in these studies. SG is the
senior investigator responsible for this study. All authors contributed to the
writing of the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Bronchoscopy of healthy human donors and the use of cells derived from this
procedure was performed after review and approval by the National Institute of
Environmental Health Sciences Intramural Review Board (NIEHS-IRB,
clinicaltrials.gov identifier: NCT01224691). Bronchoscopies were performed
with the donors’ written consent by a trained pulmonologist at the
NIEHS Clinical Research Unit (CRU).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published
maps and institutional affiliations.
1National Institutes of Health (NIH), National Institute of Environmental
Health Sciences (NIEHS), Research Triangle Park, Durham, NC 27709, USA.
2University of North Carolina Chapel Hill, Chapel Hill, NC 27599-7248, USA.
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