Combined exposure of diesel exhaust particles and respirable Soufrière Hills volcanic ash causes a (pro-)inflammatory response in an in vitro multicellular epithelial tissue barrier model
Tomašek et al. Particle and Fibre Toxicology
Combined exposure of diesel exhaust particles and respirable Soufrière Hills volcanic ash causes a (pro-)inflammatory response in an in vitro multicellular epithelial tissue barrier model
Ines Tomašek 0 1
Claire J. Horwell 1
David E. Damby 2 3
Hana Barošová 0
Christoph Geers 0
Alke Petri-Fink 0 4
Barbara Rothen-Rutishauser 0
Martin J. D. Clift 0 5
0 BioNanomaterials, Adolphe Merkle Institute, University of Fribourg , Chemin des Verdiers 4, CH-1700 Fribourg , Switzerland
1 Institute of Hazard, Risk and Resilience, Department of Earth Sciences, Durham University , Science Labs, Durham DH1 3LE , UK
2 Department of Earth and Environmental Sciences, Section for Mineralogy, Petrology and Geochemistry, Ludwig-Maximilians-Universität München , Theresienstrasse 41, 80333 Munich , Germany
3 United States Geological Survey , 345 Middlefield Road, Menlo Park, CA 94025 , USA
4 Chemistry Department, University of Fribourg , Chemin des Musee, CH-1700 Fribourg , Switzerland
5 In Vitro Toxicology Group, Institute of Life Sciences, Swansea University Medical School , Singleton Park Campus, Swansea SA2 8PP, Wales , UK
Background: There are justifiable health concerns regarding the potential adverse effects associated with human exposure to volcanic ash (VA) particles, especially when considering communities living in urban areas already exposed to heightened air pollution. The aim of this study was, therefore, to gain an imperative, first understanding of the biological impacts of respirable VA when exposed concomitantly with diesel particles. Results: The combination of respirable VA and DEP, in all scenarios, incited an heightened release of TNF-α and IL8 as well as significant increases in IL-1β, when applied at sub-lethal doses to the co-culture compared to VA exposure alone. Notably, the augmented (pro-)inflammatory responses observed were not mediated by oxidative stress. LSM supported the quantitative assessment of cytotoxicity, with no changes in cell morphology within the barrier model evident. A direct interaction of the VA with all three cell types of the multicellular system was observed by SEM. (Continued on next page) © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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Conclusions: Combined exposure of respirable Soufrière Hills VA with DEP causes a (pro-)inflammatory effect in an
advanced in vitro multicellular model of the epithelial airway barrier. This finding suggests that the combined
exposure to volcanic and urban particulate matter should be further investigated in order to deduce the potential
human health hazard, especially how it may influence the respiratory function of susceptible individuals (i.e. with
pre-existing lung diseases) in the population.
With nearly 10% of the world’s population living near a
historically active volcano , there is long-standing
concern over the capacity of respirable-sized volcanic
ash (VA) to cause acute and chronic respiratory health
effects . Substantial knowledge of the posed
respiratory hazard, alongside extensive characterisation of the
physicochemical properties of respirable VA, has been
obtained in recent years [3, 4], leading to a better
understanding of the structure-toxicity relationship .
However, with many volcanoes situated near large cities, VA is
rarely inhaled in isolation; instead, VA is commonly
exposed to the human population concomitantly with
additional substances, notably anthropogenic pollution. A
prime example of this is Mexico City, which was named
by the United Nations as the world’s most polluted city in
1992  and sits just 70 km from the frequently-erupting
Exposure to anthropogenic pollution is strongly
associated with adverse health effects, predominantly
pulmonary and cardiovascular diseases, as well as reduced
respiratory health [7–12]. The human population
resident in urban areas is particularly affected by high levels
of anthropogenic particulate matter (PM) since vehicles
are primary emitters of PM, with diesel exhaust particles
(DEP) being the main constituent . Yet, currently,
limited understanding surrounds the human health
hazard associated with the combined exposures (i.e.
inhalation) that results from the addition of volcanic PM
to the urban environment. Of particular importance is
the consideration of how respirable VA may interact
with DEP and how this may contribute, or not, to a
heightened, potential respiratory hazard.
The aim of the present study, therefore, was to
investigate the biological impact of a concomitant exposure to
VA (Soufrière Hills volcano, Montserrat) and a
standardised DEP sample (National Institute of Standards and
Technology’s Standard Reference Material (NIST SRM)
2975))  for the first time, using an established,
advanced multicellular in vitro model mimicking the human
epithelial tissue barrier . The basis for this project
stemmed from the British Geological Survey’s report to
the UK Government on characteristics of a future large,
effusive Icelandic eruption, which highlighted the urgent
need to evaluate the role of mixing volcanic emissions
with anthropogenic pollutants and whether this would
affect the individual respiratory hazard of either particle
independently . Thus, the current study provides a
landmark first assessment of these issues, the findings of
which are highly relevant for volcanic health hazard
management on a global scale.
Particle size analysis of an isolated respirable fraction from
the Soufrière Hills ash sample MVO12/7/03 showed that
all particles were <10 μm. The sample consisted of 12.2,
41.5 and 72.5% volume of particles with sizes of <1, <2.5
and <4 μm, respectively (Additional file 1). Specific surface
area, as determined by the Brunauer-Emmett-Teller (BET)
 analysis with nitrogen adsorption, was 3.2 m2/g.
Characteristics for the NIST SRM 2975 used were
previously reported in . Briefly, DEP exhibited a mean
diameter of 1.62 μm (denoted by number distribution).
DEP specific surface area was 91 m2/g, as determined by
BET with nitrogen adsorption.
Nebulisation of VA
A dry powder insufflator (DP-4, Penn Century, USA) was
used to nebulise the respirable fraction of the VA for
direct deposition onto the in vitro lung cell culture at the
air-liquid interface (ALI). As this was the first study to
administer VA at the ALI, an initial dose-dependent
analysis of the VA deposition was conducted to determine
cell-delivered dose, as well as its biological impact at these
different doses. The cell-delivered dose was monitored
using an integrated quartz crystal microbalance (QCM)
and showed a concentration-dependent deposition of the
VA sample (Fig. 1a). The average deposited doses were
0.13 ± 0.03, 0.21 ± 0.06, 0.26 ± 0.09 and 0.89 ± 0.29 μg/cm2
(relative to an administered mass of 4, 6, 8 mg and a
repeated administered mass of 8 mg, respectfully). The
threshold limit for the QCM was 0.09 μg/cm2. Scanning
electron microscopy (SEM) imaging of the nebulised
respirable ash sample (0.89 ± 0.29 μg/cm2) revealed a
heterogeneously dispersed deposition of ash particles (Fig. 1b).
Fig. 1 Deposition of nebulized respirable fraction of volcanic ash. a Average mass deposition (μg/cm2) of volcanic ash (VA) quantified using a
quartz crystal microbalance (QCM), following nebulisation of dry respirable ash (MVO12/7/03) using a dry powder insufflator (DP-4, Penn Century,
USA) under the following conditions: single exposure (SEVA) with 4 mg (n = 14), 6 mg (n = 14) or 8 mg (n = 17), as well as repeated exposure
(REVA) to 8 mg (nebulised 3× within 15 min; n = 9). Data are presented as the mean ± standard error of the mean. Scanning electron
micrographs of nebulized, uncoated ash sample (REVA), show b heterogeneous particle dispersion (WD: 5.53 mm, MAG: 97×) and c an inset of
image (b) (WD: 7 mm, MAG: 3.80 k ×). Images were collected at 10 kV. Scale bars are 1 mm (b) and 20 μm (c)
An initial dose-dependent analysis of cytotoxicity,
oxidative stress potential and (pro-)inflammatory
response using administered VA masses of 4, 6 and 8 mg
(Additional files 2 and 3) indicated all doses to be
sublethal to the co-culture system. Due to the reliability,
as well as a greater and effective deposition of the highest
administered mass, it was subsequently used to assess the
biological impact of VA as either a single exposure (SEVA;
0.26 ± 0.09 μg/cm2) or repeated exposure (REVA; 0.89 ±
0.29 μg/cm2) towards the in vitro triple cell co-culture
model of the epithelial tissue barrier.
As determined via release of the cytosolic enzyme lactate
dehydrogenase (LDH), no significant cytotoxicity (p > 0.05)
was observed after 24 h following exposure to either SEVA
or REVA compared to the negative control (defined as
supplemented cell culture medium with no particle
exposure) (Fig. 2g). The lack of any cytotoxicity associated with
the SEVA and REVA exposures upon the cell cultures was
qualitatively supported by the observation that no
alteration to cellular morphology occurred, as visualised by
laser scanning microscopy (LSM) (Fig. 2b and c). It was
further observed, by LSM, that the epithelial layer was
tightly bound together, forming a monolayer, with cells
undergoing mitosis, suggestive of normal homeostasis
(Fig. 2b and c). Further assessment of the biochemical
impact of SEVA and REVA upon the triple cell co-culture
showed no significant (p > 0.05) loss in total reduced
glutathione (GSH), a key indicator of oxidative stress in vitro
 (Fig. 3a). Similar, negative effects were also observed
for the ability of either SEVA or REVA to elucidate a
(pro)inflammatory response, with no significant (p > 0.05)
production of tumour necrosis factor-α (TNF-α) or
interleukin-8 (IL-8) after 24 h exposure (Fig. 3b and c). It is
important to note that, alongside these negative datasets,
all positive controls used (i.e. tert-Butyl Hydrogen Peroxide
(tBHP; GSH assay) and lipopolysaccharide (LPS; TNF-α
and IL-8)) caused significant increases within the
respective biological marker, indicating that the biological model
used was responsive for all assay endpoints measured.
Diesel exhaust particles
Similar findings were observed following exposure of
the co-culture to DEP alone, with no significant
cytotoxicity (Fig. 2d and g) or changes (p > 0.05) to the
oxidative stress status of cells observed (Fig. 3a).
Importantly, the dose used of DEP was based upon the
findings previously shown by Clift et al. , who
undertook a dose-dependent analysis of the same DEP
Fig. 2 Cell morphology and cytotoxicity of triple cell co-cultures exposed to volcanic ash and diesel exhaust particles. Confocal laser scanning
microscopy (LSM) images show the complete triple cell co-culture (i.e. A549 type-II ‘like’ epithelial cell monolayer with human blood monocyte
macrophages (MDM) and dendritic cells (MDDC) on the apical and basal sides, respectively) stained for F-actin cytoskeleton (red) and the
nuclei (blue). a Control and cultures exposed to b 0.26 ± 0.09 μg/cm2 of single exposure to volcanic ash (SEVA), c 0.89 ± 0.29 μg/cm2, repeated
exposure to volcanic ash (REVA), d diesel exhaust particles (DEP; 0.02 mg/mL), e diesel exhaust particles and 0.26 ± 0.09 μg/cm2 of single exposure
to volcanic ash (DEP + SEVA), and f diesel exhaust particles and 0.89 ± 0.29 μg/cm2 of repeated exposure to volcanic ash (DEP + REVA). Yellow
arrows indicate cells undergoing cell division. Scale bars are 20 μm (a-b) and 15 μm (c-f). Images were collected at magnification 63×.
g Cytotoxicity determined by the release of lactate dehydrogenase (LDH) from the triple cell co-culture following exposure to SEVA, REVA, DEP,
DEP + SEVA and DEP + REVA. Data are presented as fold increase relative to the negative control (supplemented cell culture medium only) ±
standard error of the mean. Triton X-100 at 0.2% in phosphate buffered saline (PBS) acted as the positive assay control. LDH data shown are related to
the following repetitions for each exposure: SEVA n = 4; REVA, DEP, DEP + SEVA and DEP + REVA n = 3; negative and positive controls n = 8
sample upon the same co-culture system. Again, the
positive assay control, tBHP, showed a significant depletion
of GSH in the co-culture system, confirming the
observation that the array of sample exposures incited no oxidative
stress. Despite these findings, the DEP only exposures to
the in vitro multicellular system elicited a significant
(p < 0.05) interleukin-1β (IL-1β) and non-significant
(p > 0.05) TNF-α and IL-8 responses compared to the
negative control (Fig. 3b and c).
Combined exposure of SEVA and REVA with DEP
Combined exposures to DEP and VA (DEP + SEVA, DEP +
REVA) also resulted in no significant cytotoxicity (Fig. 2e, f
and g) or changes (p > 0.05) to the oxidative stress status of
cells (Fig. 3a). It was observed, however, that although the
combined exposures did induce an heightened
(pro-)inflammatory response in the co-cultures for TNF-α and IL-8
(p > 0.05), only a significant (p < 0.05) release of IL-1β,
compared to the negative control, was noted (Fig. 3b–d).
Fig. 3 Biochemical response of triple cell co-culture system following exposures to volcanic ash and diesel exhaust particles. a Total reduced
glutathione (GSH), b tumour necrosis factor-α (TNF-α) release, c interleukin-8 (IL-8) release and d interleukin-1β (IL-1β) release of the triple cell
co-culture model after exposure to 0.26 ± 0.09 μg/cm2 of single exposure to volcanic ash (SEVA), 0.89 ± 0.29 μg/cm2 of repeated exposure to
volcanic ash (REVA), diesel exhaust particles (DEP; 0.02 mg/mL), co-exposure to diesel exhaust particles and 0.26 ± 0.09 μg/cm2 of single exposure
to volcanic ash (DEP + SEVA), and co-exposure to diesel exhaust particles and 0.89 ± 0.29 μg/cm2 of repeated exposure to volcanic ash (DEP + REVA).
The respective positive assay controls are tert-Butyl Hydrogen Peroxide (tBHP; 250 μL of 100 mM) and lipopolysaccharide (LPS; 100 μL of 1 μg/mL),
added to the apical and basal compartment of the triple cell co-culture, respectively. The negative control was cell culture medium only. Data are
presented as the mean ± standard error of the mean. Data shown are related to the following repetitions for each exposure: SEVA n = 4; REVA, DEP,
DEP + SEVA and DEP + REVA n = 3; negative and positive controls n = 8. * indicates p < 0.05
Interaction of VA with triple cell co-cultures
SEM images of the upper surface of the triple cell
coculture exposed to dry VA at the ALI showed that VA
was able to directly interact with the macrophage cache
of the co-culture system. Interestingly, VA particles were
also observed on the basal layer of the triple cell
coculture, elucidative of a potential interaction with the
MDDC present in this region (Fig. 4).
For VA, concentrations of 0.26 ± 0.09 μg/cm2 (SEVA)
and 0.89 ± 0.29 μg/cm2 (REVA) were chosen. It is
difficult to state how representative these concentrations are
in relation to ambient air concentrations following a
volcanic eruption, due to the lack of reliable in vivo
dosimetry data available and the fact that airborne
volcanic ash concentrations are highly dependent upon the
distance of a person from the volcano and the dynamics
of the eruption itself. In addition, concentrations of
respirable ash will be raised during ashfall but, also, later,
due to resuspension by wind and human activity in dry
conditions, and will dramatically reduce following rain,
making average and cumulative exposures difficult to
constrain. Searl et al.  measured PM10 on Montserrat
during a period of frequent ashfall (1997–1999) from the
Soufrière Hills volcano and found daily mean
concentrations ranging from 0.05 to 1 mg/m3 when plentiful ash
was in the environment, and 0.02–0.15 mg/m3 when
there was little ash. It is also important to note that
personal exposure to volcanic ash is highly influenced by
activities undertaken by individuals as well as the general
dustiness of the environment; hence concentrations
Fig. 4 Interaction of volcanic ash with the triple cell co-culture. Scanning electron micrographs of the triple cell co-culture membrane exposed to
0.26 ± 0.09 μg/cm2 of single exposure to volcanic ash (SEVA), showing direct interaction of VA particles with the different cell types. a and b (inset
of (a)) show a representative interaction of the human blood monocyte-derived macrophages (MDM) with VA, which appears to be engulfed by
the MDM (b). Images (c) and d (inset of (c)) show a representative interaction of the human monocyte-derived dendritic cells (MDDC) with the
VA particles, which are interacting with the pseudopodia of the MDDC (d; indicated with white arrows). Images were collected at 3 kV and
10 mm working distance. Scale bars are 20 μm (a), 2 μm (b), 50 μm (c) and 20 μm (d)
associated with activities such as cleaning, or clearing
the roads, may be higher than background
concentrations, especially considering children [21, 22]. However,
the general community will have lower personal
exposures than the ambient levels as people will protect
themselves (e.g. staying indoors) during ashfall,
overnight and at times of high resuspension.
Assuming a daily inhaled air volume for humans to be
25 m3 and an alveolar lung surface area of ca. 100 m2,
which correspond to a healthy, moderately-active adult
, and an alveolar deposition efficiency of about 10%
, it can be estimated  that the doses used in this
study correspond to airborne concentrations which
would not be encountered over a 24 h exposure relative
to dry conditions of the highest ash concentration areas
during the most active phase of the Soufrière Hills
eruption . Thus, from a hazard assessment approach,
the doses used in the present study can be considered as
a ‘worst-case’, or a particle overload scenario relative to
such an exposure period to humans.
For DEP, a pseudo-ALI approach was adopted as it
was not possible to aerosolize the hydrophobic DEP with
the dry powder insufflator device, due to the
electrostatic nature of DEP in dry powder form. For this
reason, VA and DEP could not be applied concomitantly to
the cell cultures as a dry powder mixture. Instead, as
previously described in , a volume of 100 μl of DEP
suspension in supplemented medium at 0.02 mg/mL
was added to the cells grown on a 4.2 cm2 surface insert.
Although these particles highly agglomerate, it was
assumed that the majority of the DEP deposited on the cell
surface at a dose of 0.5 μg/cm .
Notably, the present study is a first screening of
potential adverse effects of combined exposure to VA and DEP,
therefore a simple approach of comparing the effect of a
combined exposure with the effects of individual particles
at comparable concentrations and exposure duration was
used . Importantly, the effects following exposures to
individual particle types were considered, and single
exposure data for DEP or VA are now compared to previous
investigations using both in vitro and in vivo experiments.
Single particle exposure biological effects
In the present study, DEP, alone, caused no significant
(p > 0.05) cytotoxicity or oxidative stress to the
coculture. Previous studies also conducted with the same
multicellular model have shown that DEP do not
enhance the release of LDH, although it has been found
that they induce oxidative stress [20, 28, 29]. Variation
among studies can be attributed to differences in the
applied exposure method [30, 31] as previously, the same
co-culture as used in the present study was exposed to
the same type of DEP (NIST 2975), albeit applied via
suspension (in supplemented cell culture medium) to
the apical chamber of the insert [20, 29], thus initiating
different particle-cell interaction kinetics. In the present
study, as previously mentioned, a pseudo-ALI approach
was used, as previously described . Notably, the lack
of cytotoxicity and oxidative stress observed in the
present study is contrary to previous findings using
monocultures of these cell types [32–36]. This difference
can be associated with the cellular interplay exhibited by
this multicellular model, where two immune cell types
(macrophages and dendritic cells) can directly interact
with each other at the epithelium during reactions to
particulate antigens , thus further highlighting the
advantages of using multicellular models. Overall, though,
it should be taken into consideration that different cell
cultures, DEP compositions, the preparation of particle
suspension and doses used in different studies vary, which
makes a direct comparison amongst these studies
challenging. Nonetheless, in the current study, DEP did cause an
increased release of measured (pro-)inflammatory markers
(TNF-α, IL-8 and IL-1β) compared to the negative control
(supplemented cell culture medium). These findings
concur with previous observations of monoculture in vitro
studies, which reported DEP to be highly
(pro)-inflammatory [38, 39], as well as with studies using the same triple
cell co-culture system .
Ash from the Soufrière Hills volcano has been
extensively studied over the past two decades  and is
well characterised for its physical and chemical
properties, including the sample used here [3, 41, 42]. The
biological impact of Soufrière Hills ash has also received
increased attention during this time [43–46], particularly
due to the substantial crystalline silica present in ash
derived from collapses of the Soufrière Hills lava dome
(a pile of extruded, viscous lava sitting within the crater)
. Previous results from monoculture in vitro studies
performed with Soufrière Hills ash are variable, due to
the various and numerous different experimental designs
employed and endpoints considered as well as the large
amount of natural variability amongst ash samples
[2, 46]. Despite this variability, Soufrière Hills ash is
generally considered to be non-cytotoxic and have a
low oxidative potential, but has the capacity to incite
a limited (pro-)inflammatory response. Previous
cellspecific studies on macrophages (PMA-differentiated
THP-1 monocytes) and A549 epithelial 'like' cells
indicated minimal cytotoxicity (measured by LDH release)
and GSH depletion following exposures , however the
response of other antigen presenting cells to VA
particles has been largely, to date, uncharacterised. Similar
findings have also been noted from in vivo studies [44,
47–49]. In this study, assessment of the biological
response from the triple cell co-cultures following VA
exposures alone resulted in no significant (p > 0.05)
cytotoxicity, changes in cellular morphology, oxidative
stress or release of (pro-)inflammatory mediators
(TNF-α and IL-8). Therefore, the findings of the initial
dose-respose analysis of VA exposures alone are largely
in congruence with previous research with Soufrière
Hills ash [43, 46, 50].
As evident from the discussion above, all previous in
vitro studies performed on VA have used monoculture cell
models, where the cell cultures have been immersed in
cell medium and VA particles added, already suspended
i.e. a pre-mixed sample, in liquid medium. Whilst this
approach is commonplace, it does not adequately reflect the
physiological condition of a respiratory exposure to VA as
particles do not settle in the lung already immersed in
liquid (which may affect the surface reactivity of the VA
sample). The nebulisation method of VA using the dry
powder insufflator (DP-4, Penn Century, USA) has
enabled, for the first time for in vitro studies, application of
VA to cells in its pristine, dry state. This method
represents a more realistic scenario in comparison to all
previous studies which used suspended ash in cell medium.
The method has also shown good reproducibility, and
can, therefore, be considered as a suitable method for
conducting realistic in vitro respiratory hazard assessment of
VA in future research activities. Furthermore, it has
recently been shown that multicellular models can be useful
tools in determining the specific (pro-)inflammatory and
oxidative stress effects of particles compared to
monocultures; as these models additionally take into account
intercellular signalling among cells as it would occur in vivo .
The observation, by SEM, that the macrophages
directly interacted with VA was expected, due to their role
in the clearance of foreign material, and previous studies
have shown the capacity of macrophages to internalise
VA [46, 51]. The interaction with epithelial cells is not
completely unexpected either, due to the surface area
that they cover in the alveolar epithelial airway barrier in
vitro system (insert membrane is 4.2 cm2 with 1 × 106
epithelial cells seeded compared to 5 × 104 macrophages
seeded in the co-culture) [15, 37]. It is worth noting that,
to the best of the authors’ knowledge, this was the first
time that dendritic cells have been considered in terms of
the biological impact of VA in vitro. The observed
interaction of the VA with the MDDC can be hypothesized to
occur either through (I) translocation of the VA particles
via cell-cell interactions, as previously described for other
particle types , (II) direct translocation through the
pores of the micro-porous membrane insert (3 μm), or
(III) deposition between the micro-porous membrane
outer ridge and the side of the well of the six-well plate
during the nebulisation process. Further research is
therefore necessary to determine how VA becomes potentially
available to interact, or not, with the dendritic cells of the
co-culture system, such as through translocation studies
previously performed with this 3D in vitro lung model
[28, 52, 53], as well as to deduce what biological impact
this interaction may potentially elucidate.
Co-exposure biological effects
At sub-lethal concentrations, as with the SEVA, REVA
and DEP alone, the combined exposures (DEP + SEVA
and DEP + REVA) showed no significant cytotoxicity in
the triple cell co-culture. By working in this
concentration range, it was therefore possible to study further,
mechanistic effects. The impact of any particle type
upon the respiratory system is commonly associated
with an increased level of oxidative stress . Yet, in
the current study, it was observed that no significant
differences in oxidative stress levels were evident in any of
the combined particle exposures compared to the
negative control. In light of these observations, DEP showed
no deviation from the negative control but VA treatment
increased the relative abundance of GSH, an observation
previously attributed to increased production by
macrophages (in monoculture) to cope with volcanic ash .
Therefore, comparatively, the effect of DEP on GSH
levels is greater than the effect seen with VA alone, and
the effects noted with the combined exposure scenario
could be attributed to the DEP driving an oxidative
stress environment in the cell cultures rather than the
absence of any oxidative stress. However, to elucidate
the underlying mechanisms controlling oxidative stress
levels in this combined exposure, further research is
needed. Despite the lack of any cytotoxic and limited
oxidative stress response following VA and DEP
combined exposures, it was found that a heightened
(pro)inflammatory response occurred following exposure to
respirable VA and DEP when applied as a combined
exposure. Focussing firstly on TNF-α release, although
there was a greater effect with the DEP + SEVA exposure
than that of the individual response for both SEVA and
DEP, the greatest response was observed following the
combined exposure of DEP + REVA. It is difficult to state
though whether this effect can be described as
synergistic in comparison to the two particle treatments alone
. Furthermore, the impact of the DEP can also be
seen in the combined exposure IL-8 response, as the
DEP, DEP + SEVA and DEP + REVA responses are all
raised, albeit not significantly (p > 0.05), in comparison
to the negative control. Given that the apparent driver of
the (pro-)inflammatory signal is DEP present in the
exposure, this was further assessed by analysing the
(pro)inflammatory cytokine IL-1β. Release of this marker
was significantly higher (p < 0.05) in the DEP + REVA
scenario compared to the negative control and to DEP
alone. The observed higher response of
(pro-)inflammatory markers following exposure to the DEP + REVA
compared to the DEP + SEVA scenario can be attributed
to the effect of the greater combined dose of particles
delivered to the cell surface.
In summary, the current study provides a first insight
into the biological effects of combined exposure to VA
and an urban pollutant (DEP) and implies a potentially
greater hazard of simultaneously inhaling both particle
types. The observations indicate that combined exposure
to VA and DEP induces a (pro-)inflammatory response
in cells at the respiratory epithelial tissue barrier, but it
is not yet clear whether this effect is directly driven by
the individual particle-cell interactions, secondary
toxicology mechanisms incited via the particles’
physicochemical characteristics, or through particle-particle
interactions leading to the combined effect noted. It is
known that increased release of (pro-)inflammatory
mediators may augment, as well as prolong,
inflammatory reactions and, if the exposure persists, can result in
chronic inflammation . Airway inflammation not
only promotes the development of lung diseases, but it
may increase the susceptibility to acute cardiovascular
disease . Thus, the significance of these findings lies in
the potential effects on respiratory health that this
combined exposure may elucidate. These initial results are
being used to inform future, planned work investigating
chemical interactions between particles and the particle/
gas/volatile mixtures of complete vehicle exhaust [56, 57]
as well as volcanic emissions.
The findings in the present study show that exposure to
sub-lethal concentrations of VA with an urban pollutant
(i.e. DEP) can promote a heightened and significantly
increased (pro-)inflammatory response in vitro, absent of
mediation by oxidative stress. The observed effects of
the combined exposures are of further significance as, in
some circumstances, they are greater than the response
noted for DEP or VA, independently. It is envisaged that,
in the event of future eruptions, the findings of this
study will serve for a better understanding of the
potential respiratory risk posed by combined exposure to
urban pollution and VA towards human health. These
findings will provide the basis for further investigations
into the mechanisms driving the heightened
(pro-)inflammatory response, in order to deduce the specific human
health hazard, as well as how it may influence the
respiratory function of susceptible individuals (i.e. with
preexisting lung diseases) in the population.
Source Ash from a dome-collapse ash-fall deposit of
Soufrière Hills volcano, Montserrat (erupted and ash
sample collected on 12 July 2003) was used (MVO12/7/
03). The ash on Montserrat erupts into a very clean
atmosphere (occasionally polluted by transfer of dust
from the Sahara), so was chosen as a pristine example of
ash which had no prior interaction with anthropogenic
pollution. The bulk ash has previously been extensively
characterised [3, 4, 41], and contains substantial
quantities of respirable particles (cumulative volume % is 22.5
and 11.5 for <10 μm and <4 μm, respectively)  and is
rich in crystalline silica (13.5 weight %) .
Sample preparation A respirable fraction of VA was
isolated using a Sioutas Cascade Impactor (SKC Inc.,
USA) and Leland Legacy sample pump (SKC Inc., USA)
attached to a gravitational separation chamber. VA was
introduced into an airstream established by operating
the pump at a constant flow rate of 5 L/min. Aerosolised
particles then entered the separation chamber where
particles above a theoretical spherical aerodynamic diameter
of 5 μm sedimented in accordance with Stoke’s law,
calculated for a particle density of 1.0 g/cm3 in the system.
These parameters have been empirically observed to
produce an appropriate respirable-sized fraction in this set
up. Remaining airborne particles were then sampled by
the Impactor, which was assembled without impaction
stage filters to enable sample recovery. Size-fractionated
samples were then collated for use in characterisation and
toxicity assays. Separation of the respirable fraction from
the sedimented ash was conducted upon the same
subsample on three different occasions, and then combined
in order to maximise the recovery of respirable material
needed for the study.
Characterisation of VA respirable fraction
Particle size distribution analyses of isolated respirable
samples were performed using a Coulter LS230 (Coulter
Corporation, USA) in water without sonication. Data
were analysed according to the Mie theory of light
scattering . Results are the mean of three consecutive
runs of the sample. Runs were 60 s each.
Surface area was determined using samples dried
overnight at 105 °C according to the BET method , and
analysed by nitrogen adsorption measurements at
-196.15 °C using a Gemini III 2375 surface area analyser
(Micromeritics Instrument Corporation, USA). Results
are the mean of three independent measurements of the
Nebulisation of VA respirable fraction
Respirable VA was nebulised over the cells using a dry
powder insufflator (Model DP-4; PennCentury Inc.,
Philadelphia, USA). The use of the dry powder
insufflator was based upon the method previously described by
Bihari et al. . Briefly, the ash was loaded into a
sample chamber and then pushed through the device by
small pulses of air administered to the device using a
10 mL commercial air syringe. The ash was discharged
as a cloud from the end of a delivery tube and, in this
way, nebulised over the cell culture plate located below
the delivery tube within a closed nebulisation chamber.
The quantification of deposited material was monitored
by a QCM (with a detection limit 90 ng/cm2, AT-cut
quartz, 5 MHz resonance frequency; Stanford Research
Systems, USA) also located within the nebulisation
chamber. Specifically, as material settles onto the QCM,
the frequency of the crystal changes (ΔF). Calculated
from the recorded frequency values before and after
deposition of material, this ΔF value (Hz) is converted to
deposited mass per area (μg/cm2) as described in .
In addition, as previously highlighted in , the
deposition pattern can possibly change across each well
of the six-well culture plate used. Analysis of the data
showed that there was no difference in the deposition
pattern across each well of the six-well culture plates
used (data not shown).
Scanning electron microscopy Nebulised respirable ash
was imaged, uncoated, by a Mira3 LM (Tescan, Czech
Republic) FE-SEM, using a secondary electron (SE)
detector in order to visualise particle deposition and
Diesel exhaust particles
Source Standard diesel exhaust particulate (DEP; NIST
SRM 2975) was used. The key characteristics of this
standard sample have previously been reported [14, 59].
Sample preparation To produce a suspension of DEP,
1 mg of dry DEP was suspended in 1 mL cell culture
medium RPMI 1640 (supplemented with 1% L-Glutamine,
1% Penicillin/Streptomycin and 10% fetal bovine serum).
The pre-mixed solution was subsequently sonicated for
90 min at 37 kHz at 37 °C. This stock suspension of DEP
was diluted with supplemented RPMI 1640 medium to a
working concentration of 0.02 mg/mL.
Lung cell cultures
All in vitro exposure experiments in this study were
conducted with an established 3D triple cell co-culture model
of the human epithelial tissue barrier cultured at the ALI
[15, 52, 60]. This system has previously been described in
detail . Briefly, the model consists of a layer of human
alveolar type II-like epithelial cells (A549) with human
monocyte-derived macrophages (MDM) on the apical side
(upper chamber) and monocyte-derived dendritic cells
(MDDC) on the basal side (lower chamber). A549
epithelial cells were cultured at a density of 1 × 106 cells per
insert on BD Falcon cell culture inserts (high pore density
PET membranes, 4.2 cm2 growth area, 3.0 μm pore size;
Beckton Dickinson AG, Switzerland).
Human blood monocytes were isolated from
different, individual buffy coats received from the Swiss
blood donation service (Bern, Switzerland) (i.e.
different donor for each exposure), using CD14+
MicroBeads as described previously . Due to this,
variations in the background among different sets of
cell cultures were expected to occur. The cell culture
densities of MDM and MDDC were 5 × 104 cells/insert
and 25 × 104 cells/insert, respectively. Quantification of
the cell-cell ratio for this co-culture system has previously
been analysed and reported . Co-cultures were
incubated for 24 h under suspension conditions in order to
allow cell-cell habituation. Subsequently cell culture
medium was extracted from the apical layer to allow
formation of the ALI over an extra 24 h period at 37 °C,
5% CO2 prior to particle exposures.
Lung cell exposures
VA exposures In the approach used in this study, VA
was administered as dry powder onto the upper surface
of the co-culture at the ALI [37, 60] using a dry powder
insufflator (Model DP-4; PennCentury Inc., Philadelphia,
USA). Compared to the conventional particle suspension
exposure, exposure at the ALI has been found to be a
more sensitive in vitro exposure method, as it exhibits
similar cellular responses at lower doses . In
addition, changes to the surface chemistry, morphology
and size of the particles, which might affect the
toxicological response of the system, are minimised.
As part of the initial dose dependent analysis to
determine the optimal dose to use for VA in the combined
exposure scenario, feed masses to the dry powder
insufflator of 4, 6 and 8 mg of VA were used in a single dose
exposure scenario. The corresponding dose that was
deposited onto the cells was 0.13 ± 0.03, 0.21 ± 0.06 and
0.26 ± 0.09 μg/cm2 for 4, 6, and 8 mg starting (feed)
mass, respectively. Additionally, repeated exposure to
the highest dose (8 mg) was used in order to increase
particle mass deposition onto the cells, as 8 mg was the
feed maximum per nebulisation. A mass of 8 mg was
loaded into the dry powder insufflator and nebulised over
the cells three times within a time period of ~15 min. The
average mass deposited in the repeated exposure scenario
was 0.89 ± 0.29 μg/cm .
DEP exposures DEP were used in a pseudo-ALI
exposure experiment, as previously described . Briefly, a
total volume of 100 μl of DEP at 0.02 mg/mL suspended
in supplemented medium was added to the apical
compartment of the triple cell co-culture model at the ALI,
grown on a 4.2 cm2 surface trans-membrane insert. Upon
assumption that the majority of the DEP would deposit
homogeneously on the cell surface, the applied
concentration would equate to a deposited concentration of 0.5 μg/
cm2. It is important to note that this methodology was
used due to the fact that dry DEP were found to be
unsuitable for nebulisation using the dry powder insufflator due
to their electrostatic nature as a dry powder (data not
VA and DEP combined exposures Directly after the
exposure to DEP, the highest dose (chosen based upon
the biological impact noted from the dose–response
analysis and the efficiency of each dose deposited on the
coculture from the dry powder insufflator) of VA was
applied, either as SEVA or REVA exposure, using the dry
powder insufflator as previously described above in the
section entitled ‘Lung Cell Exposures; VA Exposures’.
Post-exposure and sampling Each exposure was
followed by a 24 h incubation period at 37 °C and 5% CO2.
After this, supernatants were collected and stored at either
4 °C or −80 °C until biochemical assays could be
performed. In addition, insert membranes were fixed and
prepared for immunofluorescent labelling or SEM
microscopy, as described below.
LDH Release Cytotoxicity, indicated by cell membrane
damage, was determined by measuring the release of the
intracellular enzyme lactate dehydrogenase (LDH) into
the co-culture supernatant, assessed using an LDH
cytotoxicity detection kit (Roche Applied Science,
Mannheim, Germany) according to the manufacturer’s
guidelines. The test was conducted in triplicate for each
replication. The following repetitions for each exposure
were conducted: SEVA n = 4; REVA, DEP, DEP + SEVA and
DEP + REVA n = 3; negative and positive controls n = 8.
Absorbance was determined at 490 nm after 10 min using
a microplate reader (Bio-Rad, Switzerland), with a reference
wavelength set at 630 nm. As a positive control, co-cultures
were treated with 100 μl of 0.2% Triton X-100 in
phosphate buffered saline (PBS) on the apical side and incubated
for 24 h at 37 °C, 5% CO2.
Cell morphology After the post-incubation period of
24 h, triple cell co-cultures were prepared for imaging
via laser scanning microscopy (LSM). Cell membranes
were fixed with 3% paraformaldehyde for 15 min at
room temperature and then transferred to 0.1 M glycine
in phosphate buffered saline (PBS) for 10 min. Samples
were then washed x3 with PBS, and treated with 0.2%
Triton X-100 for 15 min at room temperature to
permeabilise the cell membrane for immunofluorescent
staining. Subsequently, samples were stained with
phalloidinrhodamine (R-415; Molecular Probes, Life Technologies
Europe B.V., Zug, Switzerland) in a 1:100 dilution to label
the F-actin cytoskeleton, and with 1:100 dilution of
4′,6diamidin-2-phenylindol (DAPI) at 1 μg/mL in 0.2% Triton
X-100 + 1% BSA in PBS to highlight the cell nuclei.
Visualisation of the samples was conducted with an inverted
confocal LSM 710 (Axio Observer.Z1, Carl Zeiss, Switzerland)
at a magnification of 63×. Representative images (z-stacks)
were recorded at three independent fields of view for each
sample (three independent samples were analysed (n = 3))
and were further processed using the 3D reconstruction
software IMARIS (Bitplane AG, Zurich, Switzerland).
The total amount of reduced glutathione (GSH) in
samples was quantified using a glutathione assay kit
(Cayman Chemical Company, Ann Arbor, Michigan,
USA) according to the manufacturer’s instructions. The
detected concentrations of GSH are reported relative to
the concentrations of total protein of each corresponding
sample (determined by the Pierce BCA Protein Assay kit
(Pierce Protein Research Products, Thermo Scientific,
Rockford, Illionis, USA)), according to the manufacturer’s
instructions. The negative control was cell culture medium
only. As a positive control, co-cultures were treated with
250 μl of 100 mM tert-Butyl Hydrogen Peroxide (tBHP)
on the apical side and incubated for 24 h at 37 °C, 5%
CO2. For each replication, analysis was conducted in
triplicate. The following repetitions for each exposure were
conducted: SEVA n = 4; REVA, DEP, DEP + SEVA and
DEP + REVA n = 3; negative and positive controls n = 8.
The (pro-)inflammatory response was investigated by
quantifying tumour necrosis factor-α (TNF-α),
interleukin8 (IL-8) and interleukin-1β (IL-1β) release from the
coculture system into the basal cell culture well via
enzymelinked immunosorbent assays (ELISA DuoSet
Development Kit, R&D Systems, Minneapolis, Minnesota, USA)
according to the manufacturer’s protocol. The
concentrations were determined spectrophotometrically at
450 nm using a microplate reader (Bio-Rad, Switzerland).
Lipopolysaccharide (LPS, from E-coli at 1 μg/mL) was
applied at a volume of 1.2 mL in the bottom compartment of
the co-cultures and served as the positive control for
TNFα, IL-8 and IL-1β induction. The negative control was cell
culture medium only. Analyses were conducted in
triplicate for each replicate. The following repetitions for each
exposure were conducted: SEVA n = 4; REVA, DEP, DEP +
SEVA and DEP + REVA n = 3; negative and positive
controls n = 8.
VA:lung cell interactions in vitro
Scanning electron microscopy
Co-cultures exposed to SEVA were fixed with 3%
paraformaldehyde for 15 min at room temperature and then
sequentially washed with 20, 40 and 60% methanol for
5 min, 80% methanol for 3 min and washed 5 times with
100% methanol for 30 s. Samples were then dried in a
vacuum desiccator over a 48 h period. Samples were
then carbon coated and subsequently imaged with a
Mira3 LM (Tescan, Czech Republic) FE-SEM, using an
InBeam detector on a rotated stage (60°).
Data and statistical analysis
All data are presented as the mean ± standard error of the
mean, deriving from three individual experiments (n = 3)
unless otherwise stated. All statistical analyses were
performed using SPSS statistical software (IBM SPSS
Statistics for Windows, Version 22.0, Armonk, NY, USA).
Statistical significance was deduced through the use of a
one-way analysis of variance (ANOVA), based upon
normal distribution of the datasets. Subsequent Tukey’s
post hoc tests were conducted to determine the specific
statistical significance between the VA, DEP, DEP + SEVA
and DEP + REVA exposures to the negative control
(supplemented cell culture medium only). The alpha value
was set at 0.05.
Additional file 1: Particle size distribution of the isolated respirable
fraction of Soufrière Hills volcanic ash. Determined by a Beckman Coulter
LS230 PSD analyser (Coulter Corporation, USA). Data are the mean of n = 3.
(TIF 30 kb)
Additional file 2: Cell morphology and cytotoxicity of the triple cell
co-culture exposed to different single exposure doses of volcanic ash.
Confocal laser scanning microscopy (LSM) images show the F-actin
cytoskeleton (red) and the nuclei (blue) of (a) control and cultures
exposed to (b) 0.13 μg/cm2, (c) 0.21 μg/cm2, and (d) 0.26 μg/cm2 of
respirable volcanic ash. Yellow arrows indicate cells undergoing cell
division. Scale bars are 20 μm. Images were collected at magnification 63×.
(e) Cytotoxicity as determined by the release of lactate dehydrogenase
(LDH) from the triple cell co-culture following single exposure to 0.13 μg/
cm2, 0.21 μg/cm2, and 0.26 μg/cm2 of respirable volcanic ash. Data are
presented as fold increase relative to the negative control (cell culture
medium only) ± standard error of the mean. Triton X-100 at 0.2% in
phosphate buffered saline (PBS) acted as the positive assay control.
LDH data shown are related to the following repetitions for each
exposure: SEVA n = 4; negative and positive controls n = 8. (TIF 524 kb)
Additional file 3: Biochemical response of the triple cell co-culture
following exposures to different doses of volcanic ash. (a) Total reduced
glutathione (GSH), (b) tumour necrosis factor-α (TNF-α) release, and (c)
interleukin-8 (IL-8) release of the triple cell co-culture model after single
exposure to 0.13 μg/cm2, 0.21 μg/cm2, and 0.26 μg/cm2 of respirable
volcanic ash. The respective positive assay controls are tert-Butyl Hydrogen
Peroxide (tBHP; 250 μL of 100 mM) and lipopolysaccharide (LPS; 100 μL of
1 μg/mL), added to the apical and bottom compartment of the triple cell
co-culture, respectively. The negative control was cell culture medium only.
Data are presented as the mean ± standard error of the mean. Data shown
are related to the following repetitions for each exposure: SEVA n = 4;
negative and positive controls n = 8. (TIF 51 kb)
ALI: Air-liquid interface; BET: Brunauer-Emmett-Teller; DEP: Diesel exhaust
particles; ELISA: Enzyme-linked immunosorbent assay; GSH: Total reduced
glutathione; IL-1β: Interleukin-1 beta; IL-8: Interleukin-8; LDH: Lactate
dehydrogenase; LPS: Lipopolysaccharide; LSM: Laser scanning microscopy;
MDDC: Monocyte derived dendritic cells; MDM: Monocyte derived
macrophages; NIST SRM: National Institute of Standards and Technology’s
Standard Reference Material; PBS: Phosphate buffered saline; PM: Particulate
matter; QCM: Quartz crystal microbalance; REVA: Repeated exposure volcanic
ash; SE: Secondary electron; SEM: Scanning electron microscopy; SEVA: Single
exposure volcanic ash; tBHP: tert-Butyl Hydrogen Peroxide; TNF-α: Tumour
necrosis factor - alpha; VA: Volcanic ash
IT is financially supported by the VERTIGO Marie Curie Initial Training
Network (ITN), funded through the European Seventh Framework
Programme (FP7) under Grant Agreement number 607905. The authors
would also like to thank the Swiss National Science Foundation (Grant No.
310030_159847/1) as well as the Adolphe Merkle Foundation for additional
financial support. DED was supported by the AXA Research Grant “Risk from
volcanic ash in the Earth system” and ERC Advanced Investigator Grant No.
IT participated in the design of the study, carried out all biological - based
experimentation and drafted the manuscript. CJH devised the project and
was, as well as DED, AP-F, BR-R and MJDC, involved in the planning and
technical advisory of the study. DED isolated the respirable fraction of VA
and performed PSD and BET characterisation analysis. HB was involved in
performing LSM imaging. CG was involved in SEM imaging. MJDC was the
project leader; he was involved in planning the design of the study, has
intellectually accompanied all experimental work, made substantial
contributions to the analysis and interpretation of the data. CJH, DED, BR-R and
MDJC have been involved in critically revising the manuscript for important
intellectual content. All authors have confirmed approval of the final version
of the submitted manuscript.
Ethics approval and consent to participate
Ethics regarding the use of human blood was considered under Swiss
Federal Law. There were no requirements to obtain ‘consent to participate’,
as it was not applicable for this study.
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