DISCUSSION
In this study, we demonstrated associations between high ambient
exposure to PM2.5 and the frequency of specific
circulating monocyte subtypes. These associations were enhanced for
certain monocyte clusters in asthmatic children who are chronically
exposed to high levels of PM2.5. Thus, the frequency of
monocyte subtypes as determined by deep phenotyping could be considered
a novel measure of disease status in highly polluted areas. To model a
global real-life air pollution issue, we focused on the total level of
PM2.5 as averaged from 4 monitoring stations located in
the same area in Fresno, CA. Of key importance, no significant
associations between circulating monocyte subsets and the levels of
ambient pollutants other than PM2.5, including
PM10, CO, and NO, were observed. For example, see data
for PM10 in Supplementary Fig. 6. Our results thus
suggest a novel monocyte association specific to PM2.5.
Association between episodic exposure to PM2.5 and
elevation of total circulating monocytes, but not specific subsets, has
been previously reported (22 ). Classical monocytes are the most
abundant circulating monocytes. Thus, it is not surprising to observe
elevation in the level of total monocytes when the classical subset is
increased. Also, previous studies report that classical monocytes
typically play a pro-inflammatory role, while non-classical monocytes
often act as anti-inflammatory cells and they are activated, for
instance, in viral infections (23, 24 ). Based on our results,
associations between exposure to high levels of PM2.5and increased frequency of classical monocytes may favor a
pro-inflammatory condition. This notion is further supported by a
significant reduction in frequency and number of anti-inflammatory
non-classical monocytes in children highly exposed to
PM2.5. Changes in major monocyte subsets did not show a
specific impact on asthma onset in our pediatric population.
Our computational unsupervised clustering method revealed additional
heterogeneity, in addition to the canonical classical, intermediate and
non-classical monocyte subsets. Among 8 distinct monocyte subtypes, 2
classical clusters (1 and 3) were found to not only be associated with
PM2.5 levels, but also with asthma diagnosis in
children. Maurer et al . have previously reported that the
expression of FcεRI, the high affinity IgE receptor and a key marker of
monocyte cluster 3, is enhanced on circulating monocytes from allergic
patients compared to those of non-allergic individuals (25 ). In
addition, treatment of allergic asthmatic patients with glucocorticoids
lowers IL-4-mediated upregulation of FcεRI on circulating monocytes,
suggesting an anti-type 2 mechanism of action (26 ).
Interestingly, similar to dendritic cells, aggregation of this receptor
on human monocytes has been shown to activate NF-κB signaling
(27 ). Finally, relevant to our findings, PM2.5induces FcεRI-mediated signaling in bone marrow-derived mast cells,
leading to degranulation and cytokine production (28 ). Effects of
PM2.5 on FcεRI expression on monocytes had not been
examined previously; our findings demonstrate the correlation between
exposure to high levels of PM2.5 with significantly
increased frequency of monocytes expressing FcεRI (cluster 3).
Increased frequency of cluster 4 [CD16+CD11chi CD11bdimCD64dim CD14dim/-], a subtype
within intermediate monocytes, was associated with asthma status in
children chronically exposed to elevated levels of
PM2.5. This warrants further investigation because, to
our knowledge, there is no study investigating the potential
contribution of these cells in pollution-mediated pathogenesis of
pediatric asthma. It would be tempting to speculate that clusters 1, 2,
and 4 might undergo trained immunity upon exposure to high levels of
PM2.5 through metabolic and epigenetic reprogramming.
Finally, among non-classical subtypes, cluster 5
[CD16hi CD11b-CCR2- CCR5-CX3CR1+ monocytes] was associated with low levels of
PM2.5 exposure, particularly in asthmatic children.
CX3CR1 is suppressed in circulating non-classical monocytes from severe
asthmatic patients (29 ). Signaling by this chemokine receptor
preserves lung function in fungal-induced allergic airway inflammation,
which is mediated via a novel regulatory mechanism (30 ). Our
results suggest an anti-inflammatory role of this specific subtype,
reversely linked to ambient PM2.5 level. Collectively,
our CyTOF data provides a detailed analytical approach that yielded
novel monocyte clusters which, if confirmed as biomarkers, may allow
improved management of asthma in children living in areas with elevated
levels of PM2.5.
Analysis of inflammatory mediators after induction of trained immunity
by particulate pollutants indicates that the pattern is a relatively
specific innate immunity response. The levels of certain mediators, such
as MCP-1 and MDC, did not significantly change upon pollution-induced
trained immunity. Interestingly, the type of secondary stimulation, HDM
versus LPS, was a determining factor in the case of EGF secretion after
primary training with the particulate pollutant. To our knowledge, the
impact of trained immunity has not been tested after re-exposure to the
primary stimulant. In our model, a trained immunity phenotype was
sustained upon pollutant re-exposure for IL-8 and TNF, but not IL-6.
These results imply that the nature of secondary stimulus affects the
final functional outcome.
A major strength of our study was that we specifically linked innate
training of monocytes with a fine particulate pollutant to the
activation of certain inflammatory pathways. We report not only
increased secretion of pro-inflammatory mediators, but also differential
H3K27 acetylation peaks in proximity to the transcription start sites of
the genes encoding these mediators. Our evidence supports the dynamic
role of H3K27ac, a mark of active transcription, in trained immunity at
both promoter and enhancer sites (10 ) and its differential
landscape in subjects highly exposed to PM2.5,
particularly for genes involved in immune cell activation (14 ).
Besides cytokines and chemokines, pollutant-induced H3K27ac marks at the
transcription factors IRF 8 and AHR reflect the impact of
air pollution on monocyte development and xenobiotic detoxification via
a previously unknown trained immunity mechanism (31, 32 ). In line
with H3K27ac mark at AHR , the pathway analysis revealed that
xenobiotic metabolism is also highly enriched, suggesting a novel
immuno-detoxification pathway, as previously reported in the liver
(33 ) and kidney (34 ). Furthermore, identification of the
strongest H3K27Ac signal among transcription factors upstream ofTCF3 was unexpected as this transcription factor is known as a
master regulator in B cell fate (35 ). The H3K27ac mark at
promoter-TSS region of SETD1A , a dedicated H3K4
methyltransferase, suggests an active methylation axis under H3K27
acetylation (36 ). Identification of MAPK signaling pathway with a
high H3K27ac mark indicates pollutant-induced training could be mediated
by metabolic reprogramming in monocytes (37 ).
Lastly, our analysis of biological pathways on ChIP Seq data highlighted
the inflammatory pathways relevant to asthma such as chemokines, TNF,
and MAPK signaling, as well as autophagy (38 ) and axon guidance
(39, 40 ) which were epigenetically activated upon pollution
training in monocytes. In addition, other pathways that contribute to
pathogenesis of cardiovascular, infectious, metabolic diseases, and
cancers were found to be activated upon induction of innate trained
immunity by air pollution in monocytes. Although these pathways are
beyond the focus of current study, these results suggest the importance
of pollution-induced training of monocytes as a potential risk factor
underlying several diseases. Finally, similar to β-glucan-trained
control group, changes in cell culture media from pollutant-trained
monocytes support a plausible metabolic reprograming towards glycolysis
to be measured the lactate levels in the future.
Our study had some limitations and challenges to be considered for
interpretation of results and development of future directions. First,
PM2.5 contains several organic and inorganic
constituents, making it difficult to dissect which compound(s)
specifically induce phenotypic and functional changes in circulating
monocytes. Variations in chemical composition and the source of ambient
PM2.5 should be considered when designing sample
collection strategies and monitoring environmental pollution levels
across different populations or across monitoring seasons for the same
population. Another limitation is that our study was cross-sectional.
Continuous exposure to high levels of PM2.5 over time
might be associated with disease progression or exacerbation. Therefore,
monitoring of monocytes in blood samples longitudinally collected from
the same subjects could determine the pattern of alterations in monocyte
subtypes as a potential biomarker of asthma status. Also, sample
collection was prioritized based on pollution exposure levels and the
asthma diagnosis was a secondary co-morbidity. Therefore, including
several samples from children diagnosed with asthma exposed to low
versus high levels of PM2.5 and their corresponding
healthy controls was not possible. In our trained immunity in
vitro studies, we selected the subjects with lowest versus highest
levels of exposure to PM2.5 regardless of asthma status
due to small sample size. Our hypothesis to understand the epigenetic
mechanism underlying trained immunity by pollutants was centered on
H3K27ac. However, various histone marks might be modified, and these
could be comprehensively deciphered using the mass cytometry technology,
EpiTOF (37, 41 ). Additionally, treatment of monocytes with
specific pharmacologic inhibitors against H3K27ac in future studies
would be important to corroborate our proposed mechanism.
Uncovering the full epigenetic mechanism is a high priority for future
studies. Targeting epigenetic alterations of monocytes induced by
pollution exposure may provide novel therapeutic strategies for
individuals living in areas with increased ambient levels of particulate
pollutants.