Mechanistic insight into the impact of nanomaterials on asthma and allergic airway disease
Meldrum et al. Particle and Fibre Toxicology
Mechanistic insight into the impact of nanomaterials on asthma and allergic airway disease
Kirsty Meldrum 0 1
Chang Guo 0 1
Emma L. Marczylo 0 1
Timothy W. Gant 0 1
Rachel Smith 0 1
Martin O. Leonard 0 1
0 Toxicology Department, Centre for Radiation, Chemical and Environmental Hazards, Public Health England , Chilton, Harwell Campus OX11 0RQ , UK
1 3 O 2 2 2 O e 2 2 2 a a O u -F e
Asthma is a chronic respiratory disease known for its high susceptibility to environmental exposure. Inadvertent inhalation of engineered or incidental nanomaterials is a concern for human health, particularly for those with underlying disease susceptibility. In this review we provide a comprehensive analysis of those studies focussed on safety assessment of different nanomaterials and their unique characteristics on asthma and allergic airway disease. These include in vivo and in vitro approaches as well as human and population studies. The weight of evidence presented supports a modifying role for nanomaterial exposure on established asthma as well as the development of the condition. Due to the variability in modelling approaches, nanomaterial characterisation and endpoints used for assessment in these studies, there is insufficient information for how one may assign relative hazard potential to individual nanoscale properties. New developments including the adoption of standardised models and focussed in vitro and in silico approaches have the potential to more reliably identify properties of concern through comparative analysis across robust and select testing systems. Importantly, key to refinement and choice of the most appropriate testing systems is a more complete understanding of how these materials may influence disease at the cellular and molecular level. Detailed mechanistic insight also brings with it opportunities to build important population and exposure susceptibilities into models. Ultimately, such approaches have the potential to more clearly extrapolate relevant toxicological information, which can be used to improve nanomaterial safety assessment for human disease susceptibility.
Asthma; Allergy; Nanomaterials; Nanoparticles; Ultrafine Pollutant; Inhalation; Lung
Background
Asthma affects over 330 million individuals worldwide
and is associated with significant avoidable mortality,
morbidity and economic burden [
1
]. In pathophysiological
terms, asthma is a chronic inflammatory condition of the
airway and is increasingly considered a term to cover a
broad spectrum disease category with multiple causes and
phenotypes [
2
]. Clinical symptoms include airway
obstruction, bronchospasm, wheezing, coughing, shortness of
breath and airway hyper-responsiveness (AHR) [
3
]. The
most common underlying disease process is chronic
inflammation, which encompasses a wide array of resident
and immune cell types. As a consequence of this
inflammation, airway remodelling occurs with specific changes
manifesting as sub-epithelial fibrosis, smooth muscle
thickening, neo-vascularisation and epithelial barrier
modification. This restructuring together with increased
smooth muscle contractility and enhanced mucus
secretion cause obstructive events and the clinical symptoms of
disease [
4–7
].
There are a number of environmental factors with
strong links to the development of asthma, including early
in utero and childhood exposure to microbes, infection,
diet, obesity, vitamin D levels, allergens, chemicals and
tobacco smoke [
8–17
]. Exposures, particularly to allergens,
particulates and chemicals, and respiratory infections also
account for the majority of known triggers for obstructive
events in asthma [
9, 18–20
]. It has been recognised for
some time that certain chemicals can cause asthma and
allergic airway disease (AAD) [
21, 22
]. Properties intrinsic
to these chemicals and more recently for particulate
exposure, have been suggested to govern their ability to
produce adverse effects. Nanomaterials (NMs), including
ultrafine particulates, defined as having at least one
dimension less than 100nm [
23, 24
], are recognised as
possessing not simply chemical but also physical
characteristics, with the potential to modify disease risk and
outcome.
Concerns have been raised regarding the safety of both
engineered NMs and incidental ultrafine particulates upon
inadvertent exposure in humans [
25
]. Epidemiological
approaches to understanding how nanoscale materials affect
asthma and AAD have been restricted to a handful of
studies with limited power to attribute causal effect. The
study of pollutant mixtures has suggested that nanoscale
components of fossil fuel combustion products such as
diesel exhaust particulates may contribute to asthma
and allergic airway disease [
20, 26, 27
]. As chemicals
including polycyclic aromatic hydrocarbons and quinones
adhered to ultrafine particles are increasingly suggested
as the primary factor responsible for adverse effects
[
26, 28–30
], it is important to properly define the
nanoscale characteristic contribution. Such information also
has the potential to increase our understanding of
how engineered nanoscale materials may impact
asthma. As a focus for this review, we will describe
current knowledge from human and experimental
observations, on how respiratory exposure to
nanomaterials with different compositions and characteristics
modify asthma and AAD. We will also describe current
understanding of the mechanisms through which such
modification can occur, and explore important
knowledge gaps to allow prioritisation of research focus into
the future.
There are different ways in which nanomaterial (NM)
effects on asthma can be considered. In terms of
experimental modelling, there are those approaches that
have examined either naïve or genetically susceptible
animals in the absence of allergen initiated disease.
There are other approaches that have used allergen
induced inflammation as a conditioning step for disease
initiation and used different protocols to examine
effects on the development (adjuvancy) or exacerbation of
established allergic airway disease. Using the search
criteria detailed in Additional file 1 to encompass all aspects
of disease susceptibility, we have performed a
comprehensive analysis of those studies, which examine the effect of
pulmonary exposure to nanoparticles (NPs), including
nano-sized ultrafine pollutants, on humans and
experimental models of asthma and AAD. Those studies
focussed on medical applications of NMs were excluded
from the main analysis and discussed separately.
Experimental models and evidence for modifying effect of nanomaterials
Experimental modelling as a means to identify hazards
and develop risk assessment and management approaches
is fundamental to how materials are assessed for potential
safety concerns. The majority of such testing is carried out
using in vivo exposure systems and occasionally
incorporates models of disease susceptibility. A number of
experimental in vivo approaches have been used to assess the
impact of NMs on asthma and AAD. In order to interpret
these findings as translational information that can be
applied to human exposure, we must first describe the
pathological features of asthma and its endotype
subcategories as measurable endpoints that can be
experimentally modelled and assessed.
Current approaches to the categorisation of asthma
involve subdivision based on immunological
characteristics as well as clinical symptoms and severity of
disease [
2
]. The most common form of asthma found in
children and half of adults is atopic in nature and
correlates with increased IgE antibody levels and/or a
positive skin prick test for allergens such as house dust
mite (HDM). Increased IgE levels in atopic conditions
are a functional consequence of CD4+ T-cell patterning
towards a Th2 phenotype, which switches B-cell antibody
production to IgE. In AAD, IgE coated eosinophils, mast
cells and basophils are activated upon re-exposure to
allergen and an inflammatory response is initiated.
Allergen specific activation of Th2 CD4+ T-cells in lung tissue
increases the production of IL-4, IL-5 and IL-13 cytokines.
This results in an increase in IgE production from B-cells,
eosinophil recruitment and goblet cell metaplasia as well
as increased mucus production (MUC5AC), all of which
contribute to airway restriction [
31
]. Within the category
of a high Th2 asthma endotype, there is a subdivision of
disease, which displays eosinophilia and a Th2 cytokine
environment in the absence of an adaptive immune
response and is often referred to as intrinsic asthma [
32
]. It
has been proposed that the newly discovered innate
lymphoid ILC2 cells drive this type of disease in response
to chronic stimulation from infection, pollution and
irritants. Injury to and activation of the epithelial cell layer in
the airway is a key feature of Th2 mediated responses with
epithelial derived factors including IL-33, TSLP, IL-25 and
GM-CSF all suggested to play a role [
31
].
Another important endotype typically associated with
late onset and more severe forms of asthma is one where
there is a predominant neutrophilic inflammation and the
presence of a mixed Th1 and Th17 immune response,
producing IL-17A, IL-22, IFNγ and TNFα among other
mediators [
33
]. It is also important to consider
overlapping endotypes, where individuals will have varying
degrees of inflammation and disease severity, for example
combined eosinophilic and neutrophilic disease with a
Th2 and Th17 profile [
34
]. Airway obstruction,
hyperresponsiveness, remodelling (cellular proliferation &
extracellular matrix deposition) and excessive mucus
production with goblet cell hyperplasia are common
features across all endotypes of asthma and are directly
measureable in model systems [
35, 36
]. Together with
endotype specific endpoints as described above, they
can all be used to model the impact of material
exposure on disease outcome.
Nanomaterial exposure and the development of allergic airway disease
Models of atopic asthma typically involve sensitisation
to allergens such as ovalbumin (OVA) in rodents and
testing of materials for their ability to modify allergic
inflammation and airway function, and have allowed a
greater understanding of key events in asthmatic
disease progression. NM testing for their impact on the
sensitisation steps in these models aims to examine
how such materials may influence the development and
severity of new cases of the disease, which includes
adjuvant activity and have been summarized in Table 1.
One of the earliest studies investigating adjuvant
activity of NPs demonstrated that carbon nanoparticles
(CNPs) with a smaller size (14nm) and greater surface
area aggravated OVA induced allergic inflammation
and mucus hypersecretion, while larger sized particles
(56nm) of the same material did not [
37
]. Similar
adjuvant effects were observed in other studies from this
group using the same particles [
38
] [
39
] but there was
not always a size dependent distinction for example
when examining AHR [
40
]. The adjuvant effects of
CNPs have also been observed in studies from other
groups [
41
] and display dose dependent effects [
42
] as
well as material composition dependent differences
when compared to titanium dioxide NPs (TiO2NPs) for
example [
43
].
Comparison to non-nano sized particles has also been
carried out within these adjuvant studies and further
supports the hypothesis that smaller size and greater surface
area have a larger impact on biological reactivity and
AAD. This was observed for example, when comparing
nano-sized carbon and TiO2 to sub-micron sized particles
of the same material [
43
]. It has also been observed with
polystyrene particles (PSP), where nano-sized PSP
produced stronger allergic inflammatory responses to OVA
than sub-micron or micron sized PSPs [
44
].
Interestingly this study addressed potential gender response
differences and observed that while PSPs had adjuvant
effects in both male and female mice, there were
significantly higher IgE and eosinophilic responses in
females when compared to males. Examination of silicon
dioxide NPs (SiO2NPs) also demonstrated size and dose
dependent effects, where 30nm sized SiO2NPs increased
OVA specific IgE and Th2 type cytokine production while
larger sized SiO2NPs (70nm) or sub-micron and micron
sized silica did not [
45
]. The adjuvant effects of
SiO2NPs have been well documented with additional
studies revealing dose dependent increases in AHR,
allergic as well as innate airway inflammation (both
Th2 and Th17 related) and mucus hypersecretion [
46,
47
]. A more detailed exploration of how SiO2NPs have
adjuvant effects was recently carried out using three
different types of SiO2NPs (unmodified, mesoporous
and polyethylene glycol (PEG) conjugated). The
strongest adjuvant activity, measured as increased AHR,
inflammatory responses and mucus hypersecretion,
was found for the mesoporous form of SiO2NPs. This
form has a similar size to the unmodified SiO2NPs but
has a much larger surface area. It was argued that
surface area is the key component for adjuvant activity. This
was further supported by the observation that the
PEGSiO2NPs, which had agglomerates four times larger, and
thus a lower surface area per mass dose, failed to produce
any adjuvant activity [48].
Multi-walled carbon nanotubes (MWCNTs) are a
particular concern for human health and have been
observed to worsen the development of AAI with
increased IgE, eosinophils, lymphocytes, IL-4 and mucus
hypersecretion in a mouse model of AAD [
49
]. This
was also accompanied by innate immune responses
including increased neutrophils, IL-1β and IL-33 [
49
].
Similar effects for MWCNTs [
50, 51
] and single walled
carbon nanotubes (SWCNTs) [
52, 53
] were observed
in subsequent studies. Intriguingly, an analysis of
different carbon NMs revealed that both SWCNTs and
MWCNTs produced significantly higher levels of IgE
and pulmonary eosinophils than either carbon
nanofibres (CNFs) or CNPs. CNFs in general had a lesser
impact on inflammatory responses and were correlated
to a lower specific surface area. Different surface areas
however were not sufficient to fully explain allergic
airway responses, and nanoparticle (NP)
characteristics such as thin tubular structure and biopersistance
were suggested as more likely factors contributing to
adverse effects [
54
].
In addition to size, material type and structure,
solubility of NPs has also been proposed as an important
factor for the ability to modulate the development of
AAD. Comparison of different NPs for their potential
to modify IgE responses to OVA in mice, revealed that
zinc oxide nanoparticles (ZnONPs) but not SiO2NPs
or TiO2NPs produced an adjuvant effect [
55
]. It was
suggested that the higher solubility of ZnONPs as a
contributing factor. Similar to effects independent of
allergen, worsening of OVA induced allergic airway
inflammation has also been demonstrated in response to
ZnONPs but not ZnCl2 ion treatment [
56
]. Given the
suggestion that the nanoparticle intracellular release
of Zn ions over a sustained period of time is the key
factor for biological effect [
57
], the use of an ionic
metal bolus may not be entirely appropriate to
replicate all effects.
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Nanomaterial effects on established allergic airway disease
Modification of pre-existing asthma involves alterations
to airway function and inflammatory status. In this
section we will summarise current information on how
nanomaterials can modify established allergic airway
disease in vivo (Table 2).
Early investigations into CNPs and their ability to
modify pre-existing ragweed pollen induced AAI in the
beagle dog, apart from a mild neutrophilic response, did
not show any major effects on airway reactivity or
immune activation [
58
]. More recent studies demonstrated
a neutrophilic response when CNPs were administered
in mouse models of OVA induced inflammatory disease
[
59, 60
]. In addition, earlier studies from these same
authors revealed exacerbation effects of CNPs on AHR,
eosinophils, lymphocytes, IL-13 and mucus hypersecretion,
which was more severe when CNPs were administered
just before, rather than after allergen challenge [
61, 62
].
Gold NPs (AuNPs) and TiO2NPs have been examined
using a model of occupational asthma, where exposure
of toluene diisocyanate sensitised mice to either NP
resulted in an increase in AAD with AuNPs showing
greater effects [
63
]. The doses used in this study were
comparably lower than the majority of other similar in
vivo studies and aimed to reproduce occupational
exposure levels.
Copper oxide NPs (CuONPs) displayed noted potency
for their ability to exacerbate AHR, mucus
hypersecretion and allergic inflammatory markers including IgE,
IL-5 and IL-13 in murine OVA induced AAD, with
dose dependent effects observed from 25 to 100μg/kg
[
64
]. Graphene oxide (GO) is another NP examined for
exacerbation effects in an allergic airway model of
exposure. Repeat administration at the challenge phase of
the protocol did not significantly modulate allergic
endpoints but did cause significant neutrophilia [
65
].
Dose dependent increases in inflammatory cell
infiltrates including macrophages and eosinophils were
induced by SiO2NPs and accompanied by increased Th2
type inflammation and mucin hypersecretion [
66
].
Interestingly, the effects of MWCNTs in their ability
to aggravate pre-existing AAD appear modest.
RymanRasmussen and colleagues demonstrated no effect on
inflammatory cell infiltration at either 1 or 14 days
post challenge exposure. There was however a modest
increase in IL-5 which was suggested as a potential
contributor to fibrotic events observed only with
MWCNTs and OVA treatment at day 14 [
67
]. This
lack of modifying effect was also observed in a rat
model of trimellitic anhydride induced allergy [
68
]
and a murine OVA allergy model [
69
] while
inhibitory effects were observed for eosinophil and
macrophage infiltration in another study [
70
]. Moderate
exacerbation responses were also observed in a HDM
model of AAI [
71
].
Mechanistic insight into how nanomaterials may influence asthma
An understanding of how NMs with defined
characteristics influence models of asthma and AAD has the
potential to identify characteristics or material types that can be
linked to molecular and cellular events critical for disease
initiation and exacerbation in humans. This information
can be used to identify NM “properties of concern” but
importantly can also be used to improve choices
surrounding relevant endpoints of disease, model selection,
testing approaches and prediction strategies towards a
more complete assessment of NM hazard. In this part of
the review we will document those studies, which
have explored key mechanistic events in models of
asthma and AAD suggested as underpinning NM
adverse effects. This will include discussion of not just
in vivo approaches but those attempts at modelling
key events in vitro (Additional file 1: Table S3).
Direct effects on sensitisation in allergic airway disease
The development of an adaptive immune response to
an allergen is central to those with AAD. This process
typically involves dendritic cells, which take up antigen,
become activated, mature and travel to lymph nodes
where they present these antigens to T and B cells to
direct their differentiation to specific functional phenotypes.
The influence of NMs on this process has been
examined in vivo and in vitro, with initial studies focussing on
the role of the dendritic cell and T-cell interaction. Using
adoptively transferred DO11.10 CD4+ T-cells in mice,
which respond specifically to OVA peptides presented
from antigen presenting cells, CNPs when administered to
the lung in combination with OVA induced a proliferative
response [
72
]. This T-cell response indicates increased
dendritic cell (DC) antigen presentation. The number of
myeloid dendritic cells (DCs) as well as the expression of
DC maturation markers CD80 and CD86 in the
peribronchial lymph nodes of these mice were also increased.
Knockout (KO) mice deficient for these dendritic cell
costimulatory molecules failed to produce the adjuvant
effects of CNPs on AAI. A direct maturation effect of
CNPs on dendritic cells in vitro was also demonstrated,
altogether providing strong evidence that the sensitising
effects of CNPs in AAD in this KO model arise from
direct effects on dendritic cell maturation [
72
]. A size
dependent accumulation of antigen presenting cells
induced by CNPs in the mouse lung has also been observed,
where 14nm but not 56nm sized particles increased the
number of cells positive for CD80, CD86 and MHC class
II [
39
]. Moreover, this size dependency was observed in
vitro through the ability of 14nm but not 56nm CNPs to
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enhance T-cell proliferation in an allogeneic mixed
lymphocyte reaction assay [
73
]. Direct effects of CNPs on
T-cell differentiation have also been documented. Splenic
leukocytes from DO11.10 transgenic mice were incubated
with ovalbumin and two different sizes of CNPs (22nm
and 39nm) were examined and shown to have little effect
on OVA induced T-cell proliferation. However, the
smaller CNPs induced the expression of Th2 cytokines
including IL-4 and IL-13 to a greater extent than the larger
[
74
]. Interestingly, cell free BAL from CNPs instilled mice
can induce the maturation of bone marrow derived
dendritic cells (CCR7 expression) pointing towards soluble
factors released within the lung as also having a role in
directing the sensitisation process [
41
].
In addition to CNPs other types of nanomaterials have
been investigated for their ability to modify dendritic and
T-cell responses. Chen and colleagues demonstrated that
PEGylated SiO2NPs have direct effects on T-cell
differentiation signals (IL-2 and IFNγ), but this was only observed
for CD8+, not CD4+ T-cells [
75
]. The ability of SiO2NPs
to enhance cross-presentation of dendritic cell antigens to
CD8+ T-cells has also been observed [
76
]. Both these
studies demonstrate size dependent effects and while no
direct effects on allergy related CD4+ T cell differentiation
were observed, it is further evidence that NPs can
influence the adaptive immune response.
Studies on the mechanisms through which adjuvant
activity of NPs may occur have focussed not only on
how cellular behaviour and differentiation can be
altered but also on what molecular interactions drive
such responses. Early insight stemmed from initial
observations that CNPs enhanced AAD, and that this
correlated with the level of cellular oxidative damage in
lung tissue [
37
]. This oxidative injury has also been
observed with SWCNTs as increased lipid peroxidation
and 8-hydroxy-2′-deoxyguanosine staining within the
lung [
52
]. These changes were paralleled by an increase
in the maturation of bone marrow derived dendritic cells
and antigen-specific syngeneic T-cell-stimulating capacity
in vitro. However, it was not determined which cell type
accounted for oxidative injury or what characteristic of
the SWCNTs may be responsible for these changes [
52
].
A role for oxidative injury in SWCNTs mediated
worsening of AAD development and adjuvant activity was also
suggested from the observations that vitamin E
coadministration attenuated oxidation biomarkers and the
detrimental effects of the NP [
53
]. Again however, there
was no suggestion as to the precise cellular and molecular
events underlying these observations.
Oxidative surface chemistry and increases in reactive
oxidant species (ROS) within living systems are not
necessarily directly related but have been put forward to
explain differential dendritic cell maturation and T-cell
responses. Human primary monocyte derived dendritic
cells were exposed to either TiO2NPs or cerium dioxide
NPs (CeO2NPs) identified as having oxidative and
reductive surface chemistry respectively. TiO2NPs induced
cellular injury was accompanied by DC maturation
and increased expression of CD83, CD80, CD86 and
CCR7 together with the induction of IL-12 and TNF-α.
CeO2NPs on the other hand did not result in any
significant changes in cellular viability or maturation markers,
but did produce IL-10 and IL-6. T-cell responses to these
NP treated dendritic cells (DCs) were also examined, with
TiO2NPs inducing a profile of Th1 cytokine production
(IL-2, IFN-y) and CeO2NPs inducing a Th2 response
(IL4, IL-5, IL-10). The authors suggest that the oxidative
capacity of TiO2NPs was responsible for increased ROS
mediated inflammasome activation (IL-1β), DC maturation
and differentiation of T-cells towards a Th1 profile. They
also suggest, in contrast that the CeO2NPs, which induced
anti-inflammatory IL-10 and Th2 biased responses, could
be attributed to the anti-oxidant surface chemistry of this
NP [
77
]. While this is an interesting set of observations, it
cannot be ruled out that surface chemistry independent
effects played a role.
The effects of PSNPs on AAD have also been attributed
to alterations in DC function. These NPs (PS50G; glycine
coated 50nm size) when administered 12 days before
sensitisation to OVA resulted in an inhibition of AAI, mucus
hypersecretion, allergen specific IgE levels and Th2
cytokines in the lung draining lymph nodes [
78
], effects not
observed with larger sub-micron sized particles of the
same material [
79
]. This inhibition was not attributed to
effects on sensitisation but rather DC function at the
challenge phase, explained by a reduction in the numbers of
allergen-containing CD11bhiMHCIIhi DCs in the lung and
CD11c+MHCIIhi DCs in the lung and draining lymph
nodes. An additional mechanism was suggested as PS50G
abolished the ability of CD11b+ DCs to induce OVA
specific CD4+ T-cell proliferation [
78
]. Further investigation
into these effects demonstrated that PS50G induced DC
recruitment and maturation through cytokine induction
in the lung but with a reduced number of stimulatory
DCs translocating to the lymph node [
79
]. Interestingly
the authors have suggested that given the timescale of NP
administration 12 days before the start of sensitisation,
DC refractoriness as a consequence of activation in the
absence of OVA may account for the inability to mount
an adaptive immune response at the challenge phase [
78
].
Furthermore, it was speculated that evolutionary
conserved responses to viral sized particles, which would aim
to limit inflammatory reactions not focussed on anti-viral
immunity (e.g. Th1 T-cell activation) such as Th2 type
adaptive immune responses, may be activated by the
nanoscale properties of NPs and contribute to the
inhibitory effects. This can be supported by observations that
PS50G did not result in inhibition of allergen specific
lymph node production of IFNγ or have any effect on Th1
anti-viral immunity in an acute lung influenza virus
infection model [
78
].
The inhibitory effects of PS50G on AAI also did not
extend to inhibition of AHR [
78
]. This disparity
between NP effects on allergic inflammation and AHR
has been observed for other types of NPs. Graphene
oxide (GO) augments AHR, goblet cell hyperplasia and
smooth muscle hypertrophy in OVA induced allergic
airway disease while at the same time inhibiting Th2
cytokine production, eosinophilia and IgE levels [
65
].
The mechanism put forward to explain this suggested
that increased macrophages within the lung and their
production of chitinases such as CHI3L1 contributes to
airway remodelling/hyperresponsiveness and favours
the development of Th1 over Th2 type responses. These
observations were supported by increased levels of
OVA specific IgG2a [
65
]. Inhibition of adjuvant activity
and AAI has been observed for other nanomaterials,
including Fe2O3 [
80
] and SiO2 coated TiO2 [
81
] where
they were found to have size and dose dependent
effects. While the authors speculate that the mechanism
through which this inhibition occurred involved
dendritic cell modification, modulation of the balance of
Th1/Th2 T-cell responses and direct effects on
regulatory T-cells, no supporting evidence was offered.
Aluminium salts, the most commonly used adjuvant
in humans, have been well characterised for their ability
to promote the development of adaptive immunity to
co-administered allergens, such as OVA, producing a
predominant Th2 type immune response [
82
]. The
classic understanding of how these adjuvants have their
effects has been described in terms of their ability to
aggregate antigens, stabilise protein structure, prevent
degradation and to provide a pool of material for
continuous release to antigen presenting cells [
83
].
However, recent work has suggested that other mechanisms
are involved as a likely consequence of direct cellular
injury, including the activation of innate immune
responses and the recruitment of inflammatory cells.
Activation of the NLRP3 inflammasome as a consequence
of particulate lysosomal overload in resident phagocytes
(including macrophages and dendritic cells), uric acid
release and subsequent caspase-1 activation and release
of inflammatory cytokines, such as IL-1β, IL-18 and
IL33, have also been suggested as important events
underlying adjuvant activity [
84, 85
].
Given the particulate nature of NPs it is perhaps
unsurprising that these same mechanisms have been
suggested to underlie NP modifying effects on adjuvancy
[
86
]. Particular attention has been given to the potential
role of the NLRP3 inflammasome, with nanomaterials
including AgNPs, TiO2NPs, SiO2NPs and CNTs all
inducing markers of this pathway’s activation [
87–89
]. Using
gene KO approaches, a direct role for the NLRP3
inflammasome in mediating the IL-1β response to
TiO2NPs in macrophages and dendritic cells has been
demonstrated. IL-1α but not NLRP3 however, was
identified as a major control point for neutrophilic responses
after instillation in murine lung [
90
]. Nano sized
particulate forms of TiO2 and SiO2 were observed to have a
greater impact than submicron sized particles of the same
material on the maturation of bone marrow derived
dendritic cells. Using KO mice, IL-1β responses to these NPs
in dendritic cells were found to be NLRP3 and caspase-1
dependent [
91
]. Mechanistic insight into how NPs may
preferentially activate the inflammasome to induce release
of IL-1β and IL-18 in dendritic cells stemmed from
observations that production of these mediators was greater
with 30nm SiO2NPs than larger nano or sub-micron sized
particles. The authors went on to demonstrate size
dependent effects on cellular uptake, ATP release and
ROS production and through interventional studies
suggest a sequential mechanism involving NP uptake,
lysosomal injury, ATP mediated activation of purinergic
receptors and NADPH oxidase dependent production of
ROS as a terminal signalling event for inflammasome
activation [
92
].
Other mechanisms of NP effects on the adaptive
immune system include examples where exosomes were
induced in the alveolar region after inhalation of magnetic
iron NPs. These exosomes incorporate antigen for
systemic delivery to antigen presenting cells such as dendritic
cells and modulate T-cell differentiation towards a Th1
Tcell phenotype [
93
]. While Th2 and Th17 immune
responses are more commonly associated with asthma it is
still of considerable interest that modulation of the
immune system in this way by NPs can occur. Furthermore,
it is interesting to speculate as to whether other types of
material can induce exosome formation to direct other
types of hypersensitivity reactions.
Th17 inflammation and the neutrophilic response are
a significant component within the asthma phenotype
particularly in those with severe asthma [
94, 95
]. Through
the use of genetic KO and cell depletion models, evidence
points towards a direct role for neutrophils in controlling
AHR [96]. Whether NP induced neutrophilic responses
contribute to AHR and modify Th17 responses is
relatively unexplored. One study using the chemical
compound ectoine, which preferentially reduced BAL
neutrophils (with no changes in other BAL cells),
suggested that the protective effects of this compound on
CNPs adjuvancy (IgE, Th2 profile) may be attributed to
the reduction in neutrophils [
41
].
Lastly, the airway epithelium has been observed to
play a central role in the development of sensitisation in
asthma, mainly through the ability to detect allergens
and associated injury and respond by producing signals
such as TSLP, GMCSF, IL-25 and IL-33, which target
dendritic cells to initiate sensitisation mechanisms [
97
].
Examples of how NPs can impact on this process
include an in vivo HDM induced allergy model where the
airway epithelium was proposed as the primary source
for signals governing airway sensitisation in response to
MWCNTs [
51
]. Similar suggestions have been made for
IL-33 production in the murine lung by MWCNTs in an
OVA induced AAD model [
49
]. 16HBE14o- airway
epithelial cell exposure to CNPs and TiO2 NPs resulted in an
increase in GMCSF, which was suggested as a
consequence of either NP intrinsic capability or cellular
induction of oxidative stress depending on the material type,
and that this effect was size, surface area and cellular
internalisation dependent [
98
]. SiO2NPs have also been
observed to induce GMCSF from BEAS2B airway epithelial
cells [
99
].
Insight into the impact of nanomaterials on established allergic airway disease
With the shift to Th2 type adaptive immunity in allergy,
B-cell class switching to IgE production and its
subsequent release occurs. This is followed by FCΕRIα
receptor binding in cells and tissues resulting in inflammatory
responses when allergen is reencountered. In atopic
individuals, allergen binding to IgE present on mast cells
and basophils induces the release of pre-existing
pro-inflammatory mediators such as histamine, proteases,
proteoglycans and cytokines [
100, 101
]. This process is a
central mechanism for how allergen induced asthma
exacerbations manifest at a cellular level.
A number of studies have examined how NPs can
impact mast cell function. TiO2NPs have been
demonstrated to induce the degranulation of RBL-2H3 mast
cells with the release of histamine, in an oxidative stress
and calcium flux signalling dependent manner [
102
].
This effect was observed without any IgE mast cell
priming and suggests that these NPs may directly
activate mast cells in vivo independent of allergen. Similar
findings were observed with AgNPs where
degranulation of murine bone marrow derived mast cells was also
examined for effects independent of IgE. The smaller sized
20nm but not 110nm sized particles resulted in
degranulation, which was found to be dependent on scavenger
receptor signalling, indicating cellular uptake as important.
Extracellular Ag+ ions did not have any effect [
103
].
Interestingly, pulmonary instillation of CeO2NPs induced
inflammatory cell and mediator production, which was not
observed in mice deficient in mast cells. This study also
examined bone marrow derived mast cells and found
that CeO2NPs induced inflammatory mediator
production from IgE/allergen primed cells but did not induce
degranulation [
104
]. As observations of the effects of
nano-sized materials on asthma exacerbation also
include inhibitory or protective effects, it is interesting
to note examples where attempts have been made to
interrogate how this may occur. Fullerene derivatives
have been observed to stabilise mast cells, prevent IgE
dependent activation and to attenuate disease
parameters in both exacerbation and sensitisation models of
asthma [
105, 106
]. These fullerene derivatives possess
inherent anti-oxidant capabilities derived from their
ability to catalytically scavenge large numbers of oxygen
free radicals [107] and this has been suggested as a
mechanism through which they elicit their biological
effects. This material has also been reported to increase
production of P-450 derived cis-epoxyeicosatrienoic
acids, eicosanoid metabolites in the lung, which
stabilise and prevent IgE mediated mast cell degranulation
[
106
]. SWCNTs were also observed to inhibit IgE
mediated RBL2H3 degranulation. In this study the authors
pointed to structural similarities to fullerene derivatives
but in the absence of a demonstrated ROS scavenging
capability of SWCNTs the mechanism underlying this
attenuating effect is yet to be determined [
108
].
ZnONPs were found to inhibit OVA induced
degranulation of OVA specific IgE primed RBL2H3 cells, which was
not observed with larger sized ZnO particles. These effects
were correlated with levels of intracellular Zn2+ ions, and
administration of ZnSO4 also produced an inhibitory
effect on mast cell degranulation. ZnONPs have also been
observed to inhibit both basal and IgE mediated
degranulation of RBL2H3 and bone marrow derived mast cells,
which was observed to a much greater extent for the
30nm rather than the 200nm sized particles. In contrast,
TiO2NPs examined in the same study, produced a modest
increase in mast cell degranulation. Observations that
smaller particles produced a greater increase in
intracellular Zn2+ ions and a decrease in Ca2+ concentrations than
larger particles was suggested as a mechanism through
which degranulation was inhibited [
109
].
Similar to investigations of sensitisation and AAD
development, oxidative and electrophilic stress has also been
put forward as a molecular control point for exacerbation
of pre-existing AAD. Inhalation of CNPs, for example just
prior to allergen challenge in vivo resulted in exacerbation
of allergic inflammation and AHR, which was attenuated
by systemic administration of the anti-oxidant compound
N-acetyl cysteine [
110
]. CuONPs have also been reported
to increase disease associated ROS levels in a murine
model of OVA induced AAI, which correlated with the
degree of exacerbation [
64
]. NPs can have pro-oxidant
functional groups, can possess redox cycling ability and
may generate ROS from perturbation of cellular
processes such as the mitochondria. Determining whether
a NP has any or all of these characteristics will be
important for attempts at classification of materials likely
to contribute to asthma exacerbations in humans.
In addition to fullerene derivatives [
105, 106
],
antioxidant capabilities have also been suggested as a
mechanism of inhibition of OVA induced AAD by AgNPs [111].
The authors in this study did not identify cellular targets
for the attenuating effects on ROS levels and disease
parameters, or whether inherent anti-oxidant properties of
AgNPs or the prevention of cellular events leading to ROS
generation were responsible for the observed inhibitory
effects. Using the same model in a follow on study the
authors suggest signalling molecules including HIF-1α and
RTK signalling as possible contributors [
112
]. In contrast,
AgNPs have been observed to induce ROS in cell free
[
113
] as well as upon interaction with cellular [
114, 115
]
systems, and have been demonstrated to induce oxidative
stress in other investigations of OVA induced murine
AAD [116]. The reasons for these differential effects with
AgNPs could be attributed to particle differences such as
size, as well as model specific parameters including dose
effects. The inhibitory effects of AuNPs on AAD have
been reported in a study by Barreto and colleagues, using
two different mouse strains, an effect that was associated
with a decrease in ROS generation in BAL cells [
117
].
One similarity between these studies on AuNPs and
AgNPs was that the smaller primary-sized particles (6nm)
of both materials were associated with inhibitory effects,
while the larger-sized was associated with exacerbation
effects.
TiO2NPs have been observed to reduce AAI in rats with
pre-existing disease. Here it was argued that a direct effect
on Th2 type inflammation was the mechanism through
which this effect manifested. It was also suggested that the
lack of inhibitory effects in other studies, could be
attributable to different routes of administration (aerosol
versus instillation) causing different agglomeration,
deposition patterns and biological responses within the lung
[
118
]. Inhibitory effects of TiO2NPs on AAI were also
observed in different rat strains [
119
]. This was paralleled by
TiO2NPs induced increases in neutrophil and lymphocyte
pulmonary responses in dark agouti rats, which are
predisposed to chronic inflammatory disorders, but not in
Brown Norway rats, which are pre-disposed to allergic
responses. [
119
]. In addition to these effects, another study
demonstrated that a single dose of TiO2NPs given at the
challenge phase resulted in an exacerbation of AAI and
AHR. Repeat dose exposure in the same model, while
resulting in reduced allergic inflammatory markers and
AHR, resulted in a marked neutrophilic response and
body weight loss pointing towards effects associated with
general health decline rather than any protective response
attributable to the particles [
120
]. The authors also suggest
that activation of the inflammasome via ROS generation
may contribute to the responses observed in this study.
Surface chemistry has been proposed as an important
determinant of NM effects on biological systems.
Uncoated SiO2NPs were observed to exacerbate AAD in
an OVA-induced mouse model. When the NPs were
coated with either amino or phosphate groups this
exacerbation effect was attenuated. Macrophage activation
markers were not induced by the modified particles and it
was suggested that recognition and uptake of the NPs by
macrophages were attenuated potentially as a result of
protein corona differences in the NPs. Other mechanisms
suggested for the lack of effect with coated materials
include a modified ability to affect the thermodynamic
characteristics of lipid monolayers, including lung surfactant
and cell membranes [
66
].
Additional mechanisms for how NM may affect those
with pre-existing AAD include the ability to enhance the
presentation of allergens. The HDM allergen DERP1 when
coated onto AuNPs produced stronger basophil activation
from allergic patients compared to exposure to the same
amount of free allergen and was correlated with protease
activity of the allergen [
121
]. In addition to activation and
degranulation of mast cells and basophils as part of
exacerbation events, direct effects of NPs on neutrophil and
eosinophil responses have also been investigated. Primary
human peripheral blood derived neutrophil degranulation
was significantly induced by TiO2NPs and CeO2NPs with
ZnONPs producing a more modest effect. Interestingly
these effects were more potent than the classical bacterial
FMLP agonist [
122
]. A number of other studies have
demonstrated activation of neutrophils by NPs, with
intracellular events such as calcium signalling proposed as key
in the induction of a respiratory burst and inflammatory
mediator transcription and release. One such study
investigated the sub-lethal impact of different NPs on HL-60
neutrophil like cells and found that AgNPs but not
TiO2NPs, ZnONPs, CNPs or MWCNTs induced increases
in intracellular Ca2+ levels [
123
]. Direct activation of
eosinophils was observed for ZnONPs and 20nm AgNPs but
not CeO2NPs, TiO2NPs and 70nm AgNPs [
124
]. ZnONPs
were also observed to have similar effects in primary
human eosinophils in another study from the same group
[
125
]. Whether such effects are relevant for modulation of
exacerbation in AAD has yet to be determined.
Regulation, recruitment and control of immune and
inflammatory signalling is becoming more of a focus
for how NMs elicit biological responses and contribute
to exacerbation of airway disease. Interventional
strategies including gene KO approaches have been used to
interrogate how particular inflammatory signalling events
and pathways contribute to NM exacerbation effects.
Using such knockout approaches, MWCNTs were shown
to exacerbate OVA induced AAI through a mechanism
involving cyclooxygenase 2 (PTGS2) activity, an enzyme
that controls levels of prostaglandins, key regulators of
immune system responses [
70
]. In addition, the ability
of MWCNTs to cause exacerbation of AAI in vivo was
further enhanced through KO of the STAT1 gene [
69
],
a transcription factor important for mediating
interferon and Th1 signals, immune cell signals suggested as
having protective effects in allergic asthma. Similar to
effects on sensitisation, exosome formation in the
alveolar region after inhalation of magnetic iron oxide
(Fe2O3) NPs, has also been suggested as a mechanism
through which regulation of Th1 T-cell responses are
guided in exacerbation of AAI [
93
]. Lastly, MWCNTs
administered to mice were found to produce a strong
neutrophilic response, which was diminished in animals
with pre-existing HDM induced AAI [
71
]. MWCNTs
did not alter the level of eosinophils or other Th2
inflammatory markers but did result in a more severe
airway fibrosis. Decreased IL-1β levels together with in
vitro investigations in this study suggest that an allergic
inflammatory environment inhibits inflammasome
activation through a STAT6 mechanism and that this may
be responsible for the exacerbated fibrotic response
observed with MWCNTs exposure [
71
].
Mechanisms of asthma development and exacerbation independent of allergic sensitisation
Modelling of asthma using rodent allergic airway
disease models as the prototypical testing paradigm does
not reflect all types of asthmatic disease in humans.
Mechanistic insight into how environmental exposures
may influence disease is therefore, potentially
overshadowed by predominant discussion of how materials
affect allergic responses and Th2 type inflammation. A
summary of those studies with observations
independent of allergy driven disease identified within our search
parameters are documented in Additional file 1: Tables
S1 and S2. Those studies primarily focussed on allergen
independent models are summarized in Additional file
1: Table S1, while those observations independent of
allergen effects but documented as part of larger allergen
driven model studies are summarized in Additional file
1: Table S2.
Some of the earliest studies to investigate the pulmonary
effects of nanomaterial exposure did not specifically set
out to model the potential impact on disease conditions
such as asthma. However observations including
neutrophil and inflammatory mediator alterations can be viewed
not just in terms of broad pulmonary toxicity effects but
as having the potential to influence disease initiation and
progression in conditions such as asthma and AAD. One
of these early studies set out to compare similar sized
metallic NPs after pulmonary instillation in rats. It was
demonstrated that the level of toxicity and neutrophil
response was material specific with nickel (Ni) and cobalt
(Co) having the greatest effects. It was suggested that the
surface chemistry of these materials as expressed in terms
of their ability to generate reactive oxygen species,
governed specific potency [
126, 127
]. Other factors
including NP solubility and surface area have been put forward
as the most significant characteristics influencing NP
toxicity after pulmonary exposure and have been reviewed
extensively elsewhere [
128, 129
].
In a clinical setting, treatment strategies targeting Th2
type inflammation reduce allergic exacerbation rates but
have little effect on baseline measures of asthma activity
such as AHR [
130
]. These and other observations support
the suggestion that asthma should be considered as a
condition with a fundamental abnormality in smooth muscle
function that underlies diminished lung function and
AHR [
130, 131
]. The concept that AHR and allergic
inflammation are not exclusively linked is consistent with
observations in this review and has been discussed
previously. Indeed, direct effects on AHR have been observed
in the absence of allergic airway modelling for AgNPs,
TiO2NPs, SWCNTs and MWCNTs exposures [
132–136
].
While an understanding of how such effects on AHR
manifest is unknown at this time, it is clear that smooth
muscle cells are likely to have a central role [137]. The
direct impact of nanomaterials on smooth muscle has been
explored to a limited extent. TiO2NPs instillation in mice
resulted in changes in lung gene expression consistent
with effects on ion homeostasis and smooth muscle
contractibility [
138
]. Ex vivo examination of AgNP applied
directly to rat tracheal rings found that the nanoparticles
caused a non-reversible contractile response to
acetylcholine [
139
]. It was suggested that these NPs may interact
with and modify muscarinic receptors on smooth muscle
and induce nitric oxide as a mechanism to directly
influence contractility. Additional observations using ex vivo
smooth muscle preparations have also revealed increased
contractility on exposure to SnO2NPs and CoFe2O4NPs
[
140, 141
]. In vitro modelling of smooth muscle cells and
exposure to NPs has also been attempted to a limited
extent. AuNPs were observed to directly alter plasma
membrane potential and intracellular calcium signalling in
human airway smooth muscle cells in a charge dependent
manner [142]. Intracellular calcium signalling is intimately
involved in muscle contraction and suggests such direct
modifications by AuNPs could impact contractility and
ultimately AHR. An examination of different NP effects on
human airway smooth muscle cell mechanical function
after direct exposure, using optical magnetic twisting
cytometry revealed material specific, dose and size
dependent effects [
143
]. These included CuONPs, which
inhibited smooth muscle cell stiffness, histamine
contractility and isoproterenol relaxation, while larger micron
sized CuO particles did not.
In addition to smooth muscle cells, airway remodelling
and AHR in asthma are associated with peribronchial
fibrosis and fibroblast expansion [
144
]. Nanomaterial
influence on this aspect of airway disease has revealed direct
effects, including the ability of graphene oxide, NiNPs and
MWCNTs to increase extracellular matrix deposition after
pulmonary instillation [
145–148
]. This profibrotic effect
within the lung has also been observed for CeO2NPs,
ZnONPs and SWCNTs [
148–151
]. The involvement of
the innate immune system may also play a role here. An
investigation into MWCNTs effects suggests that airway
epithelial signals initiated by inflammasome activation
induce fibroblast proliferation and pro-fibrotic gene
expression [
152
]. IL-33 is an epithelial cell derived innate
immune signal, which can influence type 2 inflammatory
events. Knockout (KO) of IL-33 in mice causes an
inhibition of the inflammation, peribronchial fibrosis and AHR
induced by MWCNTs [
153
]. The STAT1 transcription
factor, which is involved in intracellular inflammatory
signalling, also contributes to MWCNTs induced airway
profibrotic exacerbation in mice [
69
]. Further examination of
NP effects in pre-existing allergic airway disease revealed
that SWCNTs and MWCNTs also induce airway fibrosis
[
53, 67
]. Whether allergic inflammation was necessary for
these later effects was not fully investigated. Airway
epithelial cells have been argued as central players not only
in directing inflammation but also in the control of
fibrotic signalling in response to inhaled material. In
addition to fibroblast to myofibroblast transition, epithelial
to mesenchymal transition (EMT) in response to
MWCNTs exposure has been proposed to contribute to
pulmonary fibrosis. [154]. Instillation of MWCNTs in the
mouse lung also resulted in peribronchial as well as
alveolar fibrosis in another study and a role for epithelial
mesenchymal transition was also suggested [
155
]. While a
significant body of work has focussed on MWCNTs, there
is a distinct lack of interrogation of other types of particles
for their effects on airway remodelling and fibrosis.
Airway hyper-responsiveness and excessive mucin
production are the principal mechanisms contributing
to airway obstruction in asthma. The airway epithelium
is the primary site for mucin release and is therefore an
essential focus for the examination of nanomaterial
effects. A recent study investigating the effects of NPs on
mucin rheology found that positively charged
polystyrene nanoparticles impaired mucin gel swelling causing
mucin aggregation [
156
]. It was suggested that NPs
with these properties would impinge mucociliary
clearance and contribute to adverse disease outcomes in
conditions such as asthma. Studies to date have shown
induction of mucus secretion accompanied by AHR
independent of allergic sensitisation for CuONPs [
64
]
and MWCNTs [
134
]. It is unclear how nanomaterials
cause these effects. Whether it is a result of direct
action on epithelial cells, secondary signalling events from
inflammatory and other resident cells or a combination of
all is still unknown. Some insight however has emerged
from observations that cytokines including IL-13, Il-17A
and IL-1β can induce mucin production from airway
epithelial cells. For example, in a rat pulmonary exposure
study, it could be argued that TiO2NPs induced goblet cell
hyperplasia and MUC5AC expression may be attributable
to IL-13 produced from mast cells [
157
]. Direct effects on
airway epithelial cells through ROS and calcium signalling
was also suggested to control TiO2NPs induced mucin
secretion [
158
]. Additional signalling events that may
be important in the control of mucin production
include airway epithelial MAPK signalling, an observation
documented for CuONPs induced MUC5AC in H292
epithelial cells [
159
].
Airway epithelial cells are a primary target for inhaled
material deposition and a control point for the
recruitment of inflammatory cells. Chemokines such as IL-8,
MCP1 and CCL28 act to recruit cells including
neutrophils, monocytes, lymphocytes, eosinophils and
dendritic cells. Production of these mediators from airway
epithelial cells has been demonstrated in response to
NPs including CNPs, TiO2NPs, MWCNTs, CeO2NPs,
SiO2NPs, CoNPs and ZnONPs [
160–167
]. A greater
understanding of these primary events in nanomaterial
exposure and airway response is likely to lead to a more
complete mechanistic understanding of the potential to
contribute to asthma and airway disease.
Mast cells are an important part not only for atopic
but also non-atopic asthma [
168
]. Exposure of mice to
MWCNTs resulted in inflammatory responses and
pulmonary functional changes similar to those observed in
asthma, which were dependent on the presence of mast
cells and their ability to respond to IL-33 [
169
].
Additional investigations demonstrated a role for the IL-33/
ST2 axis together with IL-13 signalling in MWCNTs
induced AHR and inflammatory events, which were
independent of T and B cell involvement [
135
]. The
authors suggest type 2 innate lymphoid cells (ILC2)
recruited to the airways as a consequence of epithelial
injury and IL-33 release as the primary events
governing adverse effects. As these cells have the ability to
produce type 2 cytokines such as IL-4, IL-5 and IL-13
in the absence of any adaptive immune response, they
also represent an important target for how
nanomaterials may impact asthma and obstructive airway disease
[
170
]. Other innate lymphoid cells such as ILC3 cells,
which produce IL-22 and IL-17, through inflammasome
activation, may also be important as they have been
directly implicated in the mechanisms of AHR induction
in the absence of adaptive immunity [
171
].
It is recognised that respiratory tract viral infection,
mainly from rhinovirus (RV), is associated with the
majority of asthma exacerbations in children and more than
half of adults [
172
]. The presence of Th2 type
inflammation and asthma has also been associated with reduced
antiviral defence in plasmacytoid dendritic cells and the
airway epithelium, indicating increased susceptibility to
viral mediated exacerbations in those with allergic
inflammation [
173, 174
]. Despite this being a primary
mechanism for asthma exacerbation, only a handful of
investigations have been directed towards understanding
how nanomaterial exposure, including ultrafine
components of air pollution, may impact respiratory viral
infections relevant for asthma. This is also true for the
development of asthma. One of the earliest studies that
did investigate such interactions focussed on mice
exposed to CNPs prior to respiratory syncytial virus (RSV)
infection [175]. This virus causes lower respiratory tract
infection and bronchiolitis and the severity of infection
has been associated with the development of asthma.
Despite alterations in the inflammatory response, including
induction of Th2 markers such as IL-13, there were no
significant effects of CNPs on RSV replication or viral
clearance [
175
]. In a similar study from the same group,
CNPs were administered to mice after RSV infection and
despite there being no difference in viral titre and
clearance, there was an increase in pulmonary inflammation
with CNPs, which was correlated to an increase in AHR
[
176
]. Similar to CNPs, TiO2NPs when administered prior
to RSV infection in mice did not alter viral titres but did
exacerbate inflammation and pneumonia [
177
]. Gold
nanorods interestingly have been observed to inhibit RSV
infection levels in mice and airway epithelial cells, which
correlated with the induction of antiviral gene expression
and modulation of pattern recognition receptors [
178
].
Other nanomaterials including SWCNTs, AgNPs and
polystyrene NPs have all demonstrated modifying effects
on viral infectivity or inflammatory consequences of
respiratory viral infection [
179–181
]. These later studies
however did not focus on viruses typically associated with
asthma.
Insight from nanomedicine approaches to vaccines and allergy
NPs intended for medical use have also been investigated
for modulation of sensitisation. PVA coated
superparamagnetic iron oxide nanoparticles (PVA-SPIONs)
caused an inhibition of monocyte derived dendritic cells’
ability to process antigen and stimulate CD4+ T cells,
without affecting antigen uptake or markers of
maturation. It was suggested that PVA-SPIONs induced
reversion to a more immature dendritic cell phenotype
involving the regulation of lysosomal function as a
potential mechanism of action. PVA-SPIONs have a slightly
positive charge and therefore bind proteins with an
isoelectric point (pI) of > 5.5. While not tested, it was
suggested that NPs may bind proteins with a higher pI
involved in antigen processing, such as Cathepsin B, H, L1
and DMB, and subsequently sequester their function, as a
mechanism for how they attenuate dendritic cell function
[
182
]. The use of NMs in the development of novel
vaccines has contributed significantly to our understanding of
how NMs direct dendritic cell function and sensitisation
to antigen. Supporting a role for particle charge in this
process, a number of studies have observed that cationic
NPs produce superior adjuvant responses following
pulmonary delivery, when compared to anionic particles of
the same material. Pulmonary immunization using OVA
conjugated cationic rod shaped hydrogel NPs led to
increased antibody titres, B-cell expansion and increased
activation of CD4+ T-cell populations in the draining lymph
nodes. These effects were not observed using anionic
particles of the same material. Ex vivo treatment of dendritic
cells with these cationic conjugated particles induced
dendritic cell maturation markers and robust antigen specific
T-cell proliferation, effects not observed with anionic
particles or with OVA alone [
183
]. In a follow on study from
the same group, it was demonstrated that these positively
charged NPs are preferentially taken up by dendritic cells
(DCs) in contrast to negatively charged NPs, and were
associated with an increased expression of the chemokines
Ccl2 and Cxcl10, which possibly contribute to recruitment
of Th2 promoting CD11b DCs to the lung [
184
]. Using
the same approach as the previous hydrogel studies to
modify NP surface by the addition of chemical species for
negative carboxyl (−COOH) and positive amino (−NH2)
charge, a study examining the effect of PVA coated AuNPs
found that after instillation into the lung, cationic particles
were preferentially taken up by all antigen presenting cells
including macrophages and DCs. This was accompanied
by an increase in CD4+ T-cell OVA proliferative responses
in lung draining lymph nodes, an effect not observed with
the anionic NPs [
185
].
Interestingly, rapid translocation of NPs to the lymph
nodes after pulmonary instillation, suggested as
independent of cell mediated transport, in addition to being
influenced by charge is highly size dependent [
186
].
Size dependent preferential uptake of NPs by dendritic
cells and translocation to lymph nodes was
accompanied by enhanced T-cell responses when compared to
larger sized particles [
187
]. These studies have
suggested an optimal NP uptake size of between 20 and
50nm for lymph node homing migratory DCs and other
antigen presenting cells after pulmonary
administration. In contrast, other studies have suggested roles for
larger particles in adaptive immunity modulation. As
part of strategic approaches to improve vaccine
development, one study examined how antigen conjugated
to particles in the viral size range influenced induction
of adaptive immune responses. Intranasal
administration of larger 200nm pluronic-stabilized poly(propylene
sulphide) NPs (PSPNPs) were observed to deliver OVA
antigen more efficiently into both MHC I and II
presentation pathways and increase the number of poly
functional CD+ T-cells when compared to the smaller
30nm size [
188
]. Moreover, immunisation approaches
using different size OVA conjugated PS beads
administered intradermally found that larger sized beads
(93nm, 101nm & 123nm) induced IL-4 release from
spleen derived CD4+ T-cells ex vivo, while smaller sized
(40nm, 49nm) induced the release of IFN-γ from CD8+
T-cells. This size dependent effect on adaptive
immunity patterning was suggested to be a direct result of
modification of dendritic cells [
189
].
Immunotherapy is an established treatment for allergies.
New approaches using viral like particles (VLPs) loaded
with TLR ligands such as unmethylated CpG, targeting
antiviral innate immune responses have the potential for
more effective treatment in the absence of side effects
associated with other strategies [
190
]. It is proposed that the
advantages of this approach stem from the nanosized
structure of VLPs, allowing transport to lymph nodes
where they can be taken up by resident DCs. Processing
then allows endosomal TLR9 activation by
unmethylated CpG and the subsequent patterning of T-cells
towards a Th1 phenotype, which in turn competes and/or
inhibits Th2 type allergic responses [
190
]. It has been
suggested that improvements to the nanocarrier approach to
delivering unmethylated CpG within the lung may further
enhance the ability of this TLR ligand to modulate and
inhibit allergy and to overcome aspects of efficacy of VLPs
observed in preclinical and clinical trials. One approach
involved conjugation of unmethylated CpG to the surface
of PSPNPs, where intranasal administration prior to
sensitisation significantly reduced AAI and IgE levels in a
murine model of HDM induced disease [
191
]. It was
suggested that surface associated, rather than
encapsulated CpG (observed with VLP approaches) may allow a
more robust approach to modifying adaptive immunity
towards Th1 responses. This mechanism of delivery for
these nanomaterial associated pathogen associated
molecular patterns (PAMPs), towards antigen processing,
presentation and adaptive immune response influence,
is one which should be considered carefully as a
significant component to allergy development and
exacerbation events. Indeed, the bacterial cell component and
TLR4 agonist lipopolysaccharide (LPS) is necessary for
the induction of metal allergy to Ag when administered
as AgNPs [
192
]. Ag+ ions and metal NPs of low
solubility failed to induce allergy under similar conditions.
These observations again highlight the importance of
nanostructure and co-exposure to pattern recognition
receptor (PRR) ligands in allergy development.
Interestingly, in addition to being suggested as a central
mechanism for how adjuvants can enhance and direct
adaptive immune responses [
83, 85
], PRR activation has
been observed directly by NPs. TLR4 binding and
activation by TiO2NPs for example has been observed in
pulmonary epithelial cells [193] and using a TLR4 KO
murine inhalation model system was also demonstrated to
mediate ZnONPs pulmonary inflammatory effects [
194
].
Ultrafine pollutant nanomaterial exposure and asthma
Air pollution is one of the greatest non communicable
disease related public health concerns. An estimated
3.3 million premature deaths worldwide per year have
been attributed to outdoor air pollution with the
particulate fraction playing a major role [
195
]. Respiratory
health effects are among the primary concerns, with
asthma accounting for a large proportion [
196
].
Evidence for an association between air pollution and in
particular traffic derived material, and asthma has
accumulated over the last decades, with systematic
reviews continuing to build a weight of evidence for
direct causal effects [
20, 196–199
]. The precise nature of
the exposure material(s) and the mechanisms
underlying these associations is not complete and further
understanding may allow more targeted and efficient
strategies to reduce adverse health effects. The
ultrafine particulate (UFP) component of pollutant
exposure has been highlighted as a particular focus for
investigation due to unique physicochemical
properties and interactions with biological systems. It is the
aim of the discussion in this section, to document
evidence for the role ultrafine particulates (UFPs) may
play in asthmatic disease and the potential
mechanisms involved.
Human ultrafine and nanomaterial exposure effects
Most of the information on human exposure to
nanoscale materials and their influence on asthma has
originated from the study of pollutant ultrafine material.
The majority of particles in terms of numbers within
urban pollutant particulate matter (PM) are found
within the UFP fraction (typically described as <100nm
diameter), while most of the mass is found in particles
>100nm in size [
200
]. One of the earliest
epidemiological studies in asthmatic adults found that
associations between the UFP fraction and asthma outcome,
assessed using lung function parameters (Peak Expiratory
Flow (PEF)), were greater than those of the fraction
between 0.1-10μm (PM10) [
200
]. A follow on study from
the same research group in an independent adult
asthmatic population, found associations between cumulative
(up to 14 days) exposure to both fine (<2.5μm (PM2.5))
and UFP fractions and asthma steroid medication use. It
was however, difficult to separate out the effect of UFP
material on asthma symptoms from other pollutant
components, including NO2 and larger particulates [
201
].
An association between UFP levels and asthma
incidence has also been observed in an urban environment
but again confounding factors including other
pollutants cannot be ruled out [
202
]. A separate series of
studies examining air pollutant exposure in a Finnish
adult asthmatic population found an association
between a larger particle number and decreased lung
function (PEF), which they suggested could be
attributed to the level of UFPs. Yet, they did stress that the
associations could not be entirely separated from the
potential effects of other pollutants such as SO2, NO2
and CO [
203, 204
]. Separation of the UFP effects of air
pollutant material on asthma has been observed to
some extent, however, in a study examining lung
function (forced expiratory volume-one second (FEV1) and
forced volume capacity (FVC)) in adult asthma
sufferers after a 2hr walk in either high or low air
pollutant environments. In this study significant associations
between higher levels of atmospheric pollutants and
reduced lung function were observed for UFP and carbon
content, which were not observed to the same extent
for PM2.5 or NO2 levels. These effects were more
pronounced in those volunteers with moderate versus mild
asthma [205]. Severe asthma sufferers were not
recruited in this study. A similar 2hr walking adult
volunteer study found associations between lung function
decline and higher levels of pollutant material, the
effects being greater in those with severe asthma. In this
study however it was not possible to separate out the
effect of UFPs from other pollutants [
206
].
Studies in paediatric asthmatic populations have also
been carried out. One early examination failed to
distinguish any association of UFPs with diminished lung
function as distinct from other pollutant material [
207
].
Indeed, more recent observations have found that
nitrogen oxide pollutants, coarse and fine particles but not
UFPs in urban air correlated with hospital admissions for
asthma in children [
208
]. There are however other studies
that do suggest associations between UFPs and reduced
lung function/respiratory problems/asthma in children.
Paediatric visits to health care facilities for acute asthmatic
events requiring prednisolone were significantly associated
with UFP levels in the previous 7 days. This association
was also found for CO levels but not for larger particles,
carbon black, ozone or SO2 [
209
]. Similarly, a separate
study demonstrated paediatric asthma hospital admissions
were associated with ambient urban background levels of
total particle numbers, UFPs (mass) and NO2 but not
PM10 (mass) [
210
]. When examining wheezing in infants,
researchers found a positive correlation with increased
UFPs in those below 1 year. Caution was expressed,
however, over this observation due to a protective effect
observed in over 3 year olds [
211
]. A more recent analysis of
asthmatic children, referred to a clinic for respiratory
symptoms found that levels of UFPs in breath condensate
correlated with wheezing, eosinophilia and breath
symptom score [
212
]. Other studies in children have also
indicated some correlation with increased levels of UFPs
and functional asthma outcomes but again additional
confounding factors cannot be ruled out [
213–216
].
Additional investigations to identify the effects of NMs
on asthma have centred mainly on experimentally
controlled exposure studies in human volunteers. These
include a study examining the effect of isolated UFPs
collected from a motor vehicle polluted area in the US,
which found decreases in lung function (FEV1). There
was, however, no difference between asthmatic and
control volunteers [
217
]. Other studies have investigated the
effects of inhaled CNPs as model UFPs. Allergic
asthmatic subjects exposed to CNPs 28 days before allergen
challenge demonstrated increased markers of disease
including eosinophilia. There was however no alteration in
lung function (FEV1) [
218
]. Another study found that
inhalation of CNPs caused mild small airway dysfunction
together with impaired alveolar gas exchange in normal
subjects. When examined in asthmatic subjects there were
no differences in responses to those observed for normal
volunteers [
219
]. An additional study from the same
group found that there were some small changes in
vasculature function after CNPs exposure but there were no
changes in asthmatic symptoms, pulmonary function, or
markers of airway inflammation in both asthmatic and
control volunteers [
220
]. This study did, however, identify
that asthmatic subjects have an increased deposition of
NMs after inhalation as compared to control volunteers,
an observation also found in other studies [
221, 222
]. This
effect raises the possibility of increased potentially
hazardous effects of these materials in asthmatic populations due
to higher exposures over the long term.
Despite there being a weight of evidence for adverse
effects of UFPs on conditions such as cardiovascular
disease in humans [
223–225
], the picture is less clear for
asthmatic disease. Much of the epidemiology suffers
from a lack of ability to separate the effects of UFPs
from other pollutant material, while human volunteer
exposure studies are limited and do not offer clear cut
evidence for a direct impact. The studies described in
this section focus almost completely on the potential for
acute exacerbation effects of these nanosized materials.
Apart from a study indicating long term and repeated
exposure to NP abrasion products from diamond
polishing [
226
] and a case report on long term exposure to
Nickel NPs [
227
], there are virtually no long term
epidemiological analysis of the effects of NPs and their
potential to influence the development of asthma.
Experimental models of ultrafine air pollutant exposure effects in asthma
Concern for how particulate pollutants and the nanoscale
constituents therein, contribute to adverse health effects
were raised by early in vivo observations that pulmonary
exposure of rats to urban PM10 produces an
inflammatory response. In one such study the authors
demonstrated a greater inflammatory response with CNPs and
proposed that the UFP component of PM10 was the main
contributor to the effects seen [
228
]. In this section
we will summarise those studies that have attempted
to examine the effect of the UFP fraction of air
pollution on models of asthma exacerbation and
development (Table 3).
The UFP fraction of urban PM produced a greater lung
inflammatory response than the larger sub-micron-sized
fraction, when administered during the sensitisation phase
of a HDM murine model of AAD. The effects were
correlated with an oxidative stress cellular response [
229
].
Labile chemicals were suggested as responsible for the
increases in antigen presentation marker expression on
monocytes induced by coarse, fine and ultrafine fractions
of freshly collected urban PM [
230
]. Indeed, in a murine
OVA model of AAD, the higher level of redox cycling
organic chemicals, PAHs and intrinsic oxidative potential
within the ultrafine fraction (<150nm) of ambient PM,
was put forward as the mechanism for increased
adjuvancy observed with these particulates when compared to
the larger PM2.5 sized fraction [
231
]. The idea that
oxidative potential of ultrafine material is a determining factor
for adjuvant behaviour was also supported by another
study from the same group, which demonstrated an
increase in Th2 inflammation and augmentation of UFP
directed adjuvant activity in KO mice lacking the
antioxidant transcription factor Nrf2 in dendritic cells [
232
].
Moreover, maternal exposure of mice to free radical
chemical containing UFPs (engineered flame combustion
particulates) resulted in enhanced OVA induced AAD in
offspring, including increased AHR, eosinophils and Th2
responses, which was attributable to an imbalance in
Tcell development and inhibition of Th1 maturation [
233
].
Using similar particles also containing free radical
chemicals, the same group demonstrated increased oxidative
injury and AHR in mice. These effects were not observed
however when UFPs had the pro-oxidant chemicals
removed [
234
].
In another study, these oxidant chemical containing
UFPs were observed to induce EMT in infant mouse
lungs. The authors suggest that these changes could
represent a mechanism through which pollutant material
affects airway development and predisposition to conditions
such as asthma [
235
]. In a similar study, UFPs high in
PAH chemicals were observed to have increased toxicity
in neonatal as compared to adult mice. The authors
suggest that neonates have an impaired response to
environmental exposures that make them susceptible to pollutant
oxidant toxicity and that this may impact airway
development and predispose to pulmonary disease development
[
236
]. The effect of nanoscale properties as opposed to
adhered chemicals effects in these studies were not
investigated, but given that these materials have carbonaceous
properties which also have the potential to induce
oxidative stress [
237
] it is possible that the NPs themselves also
contribute. Indeed, CNPs as well as OVA and NO2 have
all been observed to reduce the rate of end expiratory lung
volume increase in maturing rats, indicating a direct
impact on lung development [
238
]. As reduced lung function
has been put forward as a contributor for the development
of asthma [
239
] and given the observations that air
pollution exposure is associated with compromised lung
development [
240, 241
], the potential for both nanoscale effects
and chemical components within pollutant materials to
adversely affect lung development, and thus ultimately to
influence the development asthma is an important
consideration that warrants further investigation.
In vitro models investigating the effects of ultrafine
pollutant material on aspects of asthma pathophysiology have
focussed mainly on airway epithelial responses. An early
study demonstrated that UFPs (<0.18μm), but not the
larger-sized fractions of ambient urban PM, induced
GMCSF production from primary human bronchial
epithelial cells. This effect on GMCSF, which is a cytokine
involved in activation of dendritic cells and Th2 type allergic
inflammatory responses was not replicated by exposure of
cells to similar sized CNPs, with the suggestion that
chemicals adhered to the UFPs were responsible for the
observed effects [
242
]. Fine and UFP fractions from urban
Paris were also found to induce GMCSF from the human
bronchial 16HBE14o- epithelial cell line [
243
]. A
subsequent study from the same group correlated GMCSF
production from airway epithelium with the organic fraction,
also suggesting that chemical composition in PM is a
major determinant of this response [
244
]. In a separate
study also examining Paris urban as well as rural PM,
induction of GMCSF was only induced by the smaller UFP
and fine fractions and was correlated to the induction of
CYP1A1, NQO1 and HO-1, pointing towards chemical
induced aryl hydrocarbon receptor activation (indicative
of PAH content) and oxidative stress inducing activity as
contributory agents [
245
]. A more recent study of
Lebanese fine and UFPs also indicated the chemical organic
fraction as inducing significant effects in vitro [
246
].
Diesel exhaust particulates (DEPs) are estimated to
account for a significant proportion of the UFP fraction
found in ambient and traffic related air pollution. There
is evidence that DEPs levels are associated with
exacerbation of pre-existing asthma and more recently evidence
also points towards a direct impact on sensitisation and
respiratory effects in early life [
247, 248
]. Indeed, DEPs
have been observed to produce adjuvant effects in humans
[249]. Experimental modelling in mice further supports
the hypothesis that respiratory exposure to DEPs can act
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as an adjuvant [
250
]. Similar to studies of ambient PM in
mice, it has been proposed that the chemical content, in
particular the PAH content of DEPs, are the major
contributor to adjuvant activity [
251
]. In contrast, work
carried out by Tanaka and colleagues revealed that exposure
of mice to NP rich DEPs, optimised to maintain relevant
organic chemical content, resulted in an adjuvant effect
on OVA induced AAD. Interestingly, this adjuvant effect
remained when particulates were filtered from the exhaust
material, implicating gaseous pollutants as the main
effector of adjuvancy [
252
]. The examination of gaseous
pollutants, such as NO2 and ozone, for adjuvant effects in
experimental AAD has revealed mixed responses [
253,
254
]. Clarity is still needed regarding the relative
contribution of individual pollutant components including the
UFP fraction, in complex mixtures.
Exacerbation of AAD, including effects on AHR has
been documented in experimental models of ambient
PM exposure [
255
]. The extent to which individual size
fractions within such material contribute to exacerbation
outcomes has also been examined. Exposure of OVA
sensitised mice to the PM2.5 fraction collected close to a
motorway resulted in an increase in inflammatory
responses, which were not observed to the same extent
with UFP fraction exposures [
256
]. Another study
examining near road collected PM and exacerbation of
inflammatory events in OVA sensitised mice found that
the PM10 and PM2.5 fractions resulted in a greater
inflammatory cell response than the UFP fraction, with
responses more pronounced in samples with a higher level
of traffic related material [
257
]. The UFP fraction of near
motorway PM has been observed to exacerbate OVA
induced AAD in mice but this study did not examine
other fractions of PM to allow size comparisons to be
made [
258
]. Both the fine (PM2.5) and UFP fractions of
traffic associated PM were found to exacerbate AAD in
OVA sensitised mice through a mechanisms involving
JAG1 and the NOTCH signalling pathway, events which
were not observed with carbon black fine particle
exposure [
259
]. This study also examined antigen presentation
aspects and T-cell responses in vitro and in vivo and
through interventional and knockout strategies
demonstrated that UFP induced exacerbation of AAD,
including IgE responses was mediated through the aryl
hydrocarbon receptor. The authors proposed that since
UFPs have chemicals associated with them including
PAHs, which are potent ligands for the aryl hydrocarbon
receptor, that such receptor activation drives JAG1
expression in dendritic cells and consequently allows
enhancement of T-cell responses to aggravate AAD [
259
].
Additional aspects of exacerbation impacts have been
discussed as a consequence of PM and UFP exposure,
including inhibition of macrophage phagocytosis and
clearance of microorganisms [
260
]. Interestingly, a
recent study demonstrated that neonates exposed to
model ultrafine particulates with high oxidant potential
enhanced the severity of influenza virus infection [
261
].
Whether similar effects occur for other respiratory viral
infections, including asthma associated rhinovirus and
RSV exacerbations, is currently unknown.
Knowledge gaps and future directions
Broadly speaking from the studies summarised in this
review, there appears to be evidence for an impact of
NMs on asthma and AAD. There are however many
significant knowledge gaps that need to be addressed in
order to build any weight of evidence towards a
confident assignment of hazard potential relevant for
human exposure. Specifically there is an urgent need for
much more detailed and mechanistic information
surrounding the vast collection of different types of NM
and their properties and how they may each affect the
range of different manifestations of asthma and AAD
observed in humans. There is also a need for study
designs to be tailored towards more human relevant
exposure scenarios, in order to increase confidence in testing
being relevant for human disease. Only a handful of
nanomaterial inhalation studies have been carried out to
date. These studies capture relevant particle dynamics,
properties and deposition patterns, not properly
accounted for when the nanomaterial is administered in
solution. Future efforts should encourage inhalation
exposures coupled with real world dose equivalents to
minimise translational uncertainties. Furthermore, there
is a lack of information on long term exposures to NMs.
New studies together with the development of new
approaches, including in vitro and mechanistic insight to
address these knowledge gaps are to be encouraged.
Nanomaterial characteristics as a predictor for adverse effects
The application of appropriate NM characterisation
approaches is at the forefront of addressing knowledge gaps.
This is fundamental to accurately attribute nanoscale
properties to biological interaction and toxicological
outcome. The extent to which NM characterisation was
carried out across the studies in this review can be
considered substantially inadequate in the vast majority of
cases. Adoption of guidance proposals for minimum sets
of NM characterisation requirements previously proposed
in regulatory and other broader contexts [
262, 263
] would
greatly enhance the power to assign hazard potential in
future work.
How one may confidently assign particular
characteristics of NMs to specific adverse outcome endpoints, is a
substantial challenge and has only been attempted on a
limited scale. Initial attempts were made to compare
nanoparticle composition and characteristics across
studies in this review. However, it was ultimately judged
unworkable as the variabilities in models used; including,
exposure protocol, material dose and route, time points
and endpoints of disease, among many other parameters,
including the extent of NM characterisation, made it
impossible to reliably compare. Therefore the only realistic
comparisons that can be made to assign NM
characteristics to adverse effects with any degree of confidence are
from those studies, which focussed on addressing NM
characteristics within a specific study. These studies are
summarised in Table 4.
Size dependent effects have been observed for
sensitisation, inflammation and airway disease parameters.
This cannot however be said to be a universal effect for
all materials and disease situations (due to lack of ability
to compare across studies). Interestingly, smaller-sized
NMs such as AgNPs and ZnONPs were observed to
have more of an impact on mast cells and eosinophils in
vitro when compared to larger sized particles of the
same material. In addition, smaller primary-sized
particles of both AuNPs and AgNPs materials were
associated with inhibitory effects on AAD, while larger-sized
particles were associated with exacerbation effects.
While it is interesting to speculate as to whether there is
a common size dependent mechanism involved here, the
observations are too few and the models used too
variable to reliably say. Importantly, no in vivo studies have
reported the role of nanomaterial size on exacerbation
effects in AAD. This is a significant knowledge gap in
our understanding.
Agglomeration size whether in aerosol or instillation
fluid was not documented to the same extent as primary
NP size. Agglomeration size and its potential to influence
disease is an often overlooked and little discussed
characteristic that may influence tissue distribution and cellular
interaction. In addition, how the transition to and from
agglomerate state(s) affects biological interaction and
localisation (including subcellular) and whether this
behaviour is material and/or nano property dependent is
unknown.
Solubility, surface charge and surface coating of NPs
have been a focus for a number of studies and have
demonstrated potential mechanisms and concerns.
However these too would be considered of insufficient weight
to attribute hazard with any great confidence to any
specific property or material. The nature of the biological
corona surrounding NPs as they interact with living
tissue has been put forward as an important factor
determining toxicological impact [
264, 265
]. To what
degree this is true and to what extent material type and
associated properties influences this, remains to be
determined.
In terms of NP composition, attempts can be made at
some general observations, albeit with obvious caveats.
CNTs would appear to have a strong adjuvant activity
and a detrimental impact on AAD when exposure
occurs during sensitisation. In contrast, the effect of CNTs
on exacerbation is absent or present to a lesser extent.
In some studies, CNTs have been associated with an
increase in Th2 type inflammation in the absence of
adaptive immune responses, indicating a possible
concern for exacerbation of intrinsic type asthma. Whether
such categorisation based on asthma endotype can be
made confidently, is possibly premature but is something
that should be considered in the future. In addition,
whether these observations can be linked to the high
aspect ratio of CNTs is unknown but also worth further
investigation. Some materials like titanium and
fullerenes are associated more with inhibitory effects on AAD
than others. In addition, there are a number of materials
that have not been tested in different types of exposure
settings. For example AuNPs, AgNPs and CuONPs have
all been tested for their ability to affect pre-existing
AAD but not for any effect on sensitisation and the
development of AAD. SWCNTs on the other hand have
been examined for adjuvant effects but not investigated
for any impact on pre-existing disease.
Surface area is a critical factor for pulmonary toxicity of
NMs, particularly soluble metal NPs [
266
]. Much more
work is needed to investigate this NM characteristic as a
determinant of adverse effects of NMs in asthma and
AAD. Concerted efforts to categorise NMs according
to their adverse effects by reference to their characteristics
such as size, solubility, surface reactivity, shape (e.g. high
aspect ratio, spherical, fibres, platelet-like) and metal
content among other characteristics is a particular focus for
nanotoxicology. While the lack of NM characterisation
data, together with model variability, in the studies in this
review currently limits attempts to link adverse effects to
these groupings, it is imperative to prioritise such
approaches for future work.
Model selection, comparability across studies and protocol standardisation
There is a large variability in the types of protocol and
disease-associated endpoints used to assess adverse effects
relevant for asthma and AAD, which make it extremely
difficult to directly compare results across studies.
Adoption of a standardised set of protocols, addressing these
concerns would allow more confident comparisons to be
made across studies. We have summarised those
approaches used to date in terms of model set-up and
characteristics, target cells and tissues, key events in disease
manifestation as well as molecular endpoints of adverse
effects (Fig. 1) in order to highlight the immense set of
challenges, which need to be addressed if one is to bring
forward NM testing approaches to become more
informative and relevant for human disease.
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Given the diversity of asthma in terms of atopic,
nonatopic and mixed forms of the disease together with the
different stages of susceptibility (e.g. sensitisation and
exacerbation), the ability to compare a defined set of
disease indicators specific for the type of disease stage,
across studies would aid greatly in further clarifying
NM effects within a standardised protocol framework.
Importantly, reversible airway obstruction is the
primary observation used to diagnose those with asthma,
and AHR assessment is used to model this in vivo.
There is a significant knowledge gap surrounding the
effects of NMs on AHR as well as a lack of examination
of smooth muscle effects and airway remodelling as
underlying causes and targets. This can also be said for
mucin production and goblet cell hyperplasia modelling
as another overlooked and underreported aspect to
airway disease in asthma and NM exposure. Both of these
features should be included as a basic requirement for
any NM studies examining the potential impact on
asthma and AAD.
There is also an overrepresentation of in vivo
modelling using ovalbumin sensitisation protocols. The
inclusion of more human relevant antigen exposures
such as house dust mite may allow for broader capture
of effects that may otherwise be protocol type specific.
Aerosolised delivery of nanomaterials in test systems is
also significantly lacking in studies to date. This is
particularly important given that this is the route by which
humans are exposed to NM. The consequences that
instillation of NM dispersions have on deposition patterns,
nanomaterial characteristics and biological response is
rarely discussed. Moreover, the range of doses applied
across all studies is highly variable and how it relates to
human exposure levels is not often referred to.
Translational relevance for human nanomaterial and pollutant exposure
Information on direct effects of engineered
nanomaterials on asthma and AAD in humans is limited and
evidence for the potential for adverse effects comes
mainly from in vivo and in vitro experimental
modelling. However, inadvertent exposure to airborne PM
mainly from traffic related pollution brings with it the
concern that nanosized particles contained within this
inhaled mixture have particular detrimental health
effects including an influence on asthma.
Epidemiological studies have suggested a role for the UFP
fraction of pollutant material in asthma exacerbation,
although the role for confounding factors including
other pollutants cannot be entirely excluded. Evidence
from experimental modelling also suggests that the
UFP fraction from ambient and traffic related
pollutant material may play a role in the sensitisation and
development of AAD. However, experimental models
in vivo suggest that the UFP fraction may be less
important for exacerbation effects than the larger
fractions, particularly the fine (typically PM1 and PM2.5)
fractions. This apparent discordance with
epidemiological observations necessitates clarification.
Such discussion of ultrafine effects in Asthma and AAD
must also be considered in the context of other
constituents of air pollutant materials. Across all experimental
studies including in vitro work, there is strong evidence
that the particle associated chemical component,
containing PAHs among other organics, rather than the particles
themselves are the critical determinant responsible for
pollutant particulate mediated biological impact in asthma
and AAD. However, given that nanomaterials including
carbon nanoparticles can directly impact these same
model systems, further investigation is needed to address
direct versus indirect particulate effects within a pollutant
mixture. It should not be overlooked that more labile and
gaseous components of airborne pollution also are likely
to have a significant role [
252
].
Population studies have pointed towards genetic
susceptibility for air pollutant exposures and asthma
outcome. These include exposures such as PM10, PM2.5,
ozone and NO2 and effects associated with altered
asthma outcomes in those with particular single
nucleotide polymorphisms in genes such as GSTP1, NQO1
and GSTT1 [
267–269
]. These genes are part of the
antioxidant defence pathway within the cell, altered function
of which may impact susceptible individuals through an
inability to mount an appropriate anti-oxidant response.
However, gene-environment interactions have not been
investigated for associations with the smaller UFP
fraction of airborne pollution and represent a significant
knowledge gap. This is also true to a degree for
engineered NP exposure, although some initial studies
comparing different animal strains suggest genetic
background is a strong determinant of pulmonary
response to inhaled or instilled NMs [
117, 119, 132
].
Indeed, a wider analysis of up to 20 different strains of
mice for responses to inhaled ZnONPs found that there
were up to 10 fold differences in PMN accumulation in
the lung [
270
]. Similarly, in a study of 8 different mouse
strains, lung inflammation and injury in response to
inhaled AgNPs was also found to be strain dependent
[
271
]. These later studies did not focus on models of
asthma or AAD but do highlight pulmonary response
susceptible populations. Interestingly, no studies to date
have been reported examining how genetic background
affects NM impact on sensitisation and development of
AAD. Other types of susceptibilities that exist and may
play a role in how pollutant UFPs and engineered NMs
impact asthma include in utero exposure, age, gender,
diet and co-morbidities, including obesity, and have not
been examined to any great extent.
Mechanistic understanding and focus for future testing
The development of new and more refined approaches
to NM testing for effects on asthma and AAD have
begun to incorporate concepts of how mechanistic
information can lead to more detailed understanding of the
most important molecular targets responsible for
pathophysiological change. Identification of molecular
initiating events that are coupled to detailed mechanistic
understanding and NM characterisation, furthers the
potential to more clearly define NM properties of concern.
A more complete understanding of events that are
responsible for NM effects at a tissue, cellular and
molecular level through the use of standardised in vivo
protocols can also lead to the development of more
targeted testing approaches such as those using in vitro
modelling. While in vitro modelling to date has been
used to identify adverse effects as they may affect
sensitisation and other approaches, these remain abstracted
from other cellular and tissue compartments that may
have important contribution to the overall NM effect at
an organism level. Through greater understanding of in
vivo targets we can begin to prioritise and standardise in
vitro tests, which may ultimately replace animal testing.
The adoption of in vitro systems has many advantages
including the ability to greatly increase the number of
different NMs that can be tested. Given the many
combinations of different material types and characteristics,
this is crucial. In vitro testing will also allow for testing
to be carried in a human rather than an animal system,
removing particular concerns regarding species specific
effects (Fig. 2). In addition, technologies including the
use of induced pluripotent stem cells (iPSC) to develop
in vitro testing models will allow for the integration of
population diverse as well as human disease specific
genetic backgrounds, further optimising towards human
relevant testing. Of particular note is the recently funded
Marie Curie - Innovative Training Network “in3”, which
aims to incorporate iPSC models into an overall strategy
to develop better models for chemical and NM safety
assessment.
The recent momentum behind understanding modes
of toxicity and development of adverse outcome
pathways for xenobiotic effects has the potential to help
translate in vitro findings to a whole organism level.
Additionally, it is the hope that through accrual of data
from standardised and higher throughput systems, that
in silico modelling and read across strategies may
develop to a point where experimental testing for new
materials can be minimised (Fig. 2). Through a greater
and more detailed understanding of how NMs may
impact respiratory disease outcome and the identification
of adverse effect linked molecular signatures, including
epigenetic marks, it is hoped that more informative
biomarkers of NM exposure and effect may be developed.
These may also be useful for refining epidemiological
studies of asthma and NM or air pollutant exposure.
Microbial exposure
Lastly, it is becoming ever clearer that microbial
exposure is a critical factor influencing both the development
and exacerbation of asthma and AAD. Exposure to
bacterial and fungal diversity within the microbiome has
been strongly associated with a reduced development of
atopy and asthma [
8
]. Viral infection is also considered
a major factor for both the development and
exacerbation of disease. These interactions and their biological
effects should not be examined in isolation from other
environmental exposures such as air pollution. Whether
NMs can influence these microbial interactions remains
to be fully explored.
Conclusions
Within this review we have produced a detailed analysis of
studies directed to investigate how nanomaterials
including nano-sized pollutant particulates may impact asthma
and allergic airway disease. We have identified significant
knowledge gaps including a lack of investigations targeted
towards understanding how different nanomaterial
properties influence disease outcome in vivo. Attempts at
standardisation for testing may overcome the diverse
nature of different disease models and allow meaningful
comparison of different nanomaterials and their effects
across studies. The development of in vitro models as
representative of in vivo exposure and disease progression
is immature, particularly in terms of how nanomaterial
effects on airway remodelling may be captured. This is also
true for how multiple cell types may interact to produce
adverse effects. In terms of human exposure,
epidemiological studies are rare for engineered materials and
represent another important knowledge gap. Investigations into
how pollutant nanomaterials affect asthma and AAD in
human populations are far from clear due to the inability
to separate co-pollutant effects.
In order to address these knowledge gaps,
mechanistic understanding at a cellular and molecular level, of
disease pathology as well as NM specific outcomes is
required. Some attempts have been made in studies
covered in this review. Apart from some direct effects on the
process of sensitisation involving dendritic cells and
studies examing specific gene function through KO
approaches, mechanistic insight is generally lacking. As
knowledge progresses in our understanding of human
asthma development and exacerbation events, it is
important that cellular and molecular discoveries
underlying adverse effects and outcomes are incorporated
into model development. This allows for a more
targeted and translational approach to testing. With the
inclusion of more detailed NM characterisation, this
approach also allows for increased power to identify
properties of concern that can be linked more readily
to relevant endpoints of disease. Specifically, in terms
of adjuvant activity and the development of
sensitisation to antigen, there is a need to determine how the
innate immune response, including airway epithelial,
macrophage, mast and dendritic cells handle and
respond to different NMs as they are first encountered
within the lung. How NM properties including size and
surface area impact how antigens attached to their
surface are handled by these cells is something to consider
as a priority research area. For established AAD, there
is also a need to tie down how NMs propagate an
allergic response. There is also a need for models to be able
to capture information relevant for other asthma
endotypes such as intrinsic asthma or severe asthma, where
allergy does not play a role. Mechanistic insight should
be prominent in our attempts to achieve this. Finally,
an understanding of airway remodelling and
hyperreactivity is urgently needed; given these events
underlie airway obstruction, arguably the pathological feature
with the greatest impact on patient morbidity and
mortality. Mechanistic understanding of physical airway
responses and in particular how smooth muscle cells,
fibroblasts and other airway resident cells react to NM
exposure is lacking and should also be encouraged as a
priority research area.
Additional file
Additional file 1: Table S1. Effects of nanomaterials in non-allergic in
vivo models of experimental pulmonary exposure. Table S2. Allergen
independent effects of nanomaterials in in vivo models of experimental
pulmonary exposure documented as part of larger allergy driven models
of asthma and allergic airway disease. Table S3. In vitro models and
nanomaterial exposure. (DOCX 295 kb)
Acknowledgements
Not Applicable
Funding
This work was part funded through the Public Health England PhD
Studentship Project Scheme. This work was also part funded by the National
Institute for Health Research Health Protection Research Unit (NIHR HPRU) in
Health Impact of Environmental Hazards at King’s College London in
partnership with Public Health England (PHE). The views expressed are those
of the authors and not necessarily those of the NHS, the NIHR, the
Department of Health or Public Health England.
Availability of data and materials
Not Applicable.
Authors' contributions
All authors have made a significant contribution to the review of published
material and writing of this manuscript. All authors read and approved the
final manuscript.
Ethics approval and consent to participate
Not Applicable.
Consent for publication
All authors consent to publication of this review on acceptance by the journal.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
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