Frequency of classical versus non-classical circulating monocytes
is inversely altered in children highly exposed to
PM2.5
A subset of fifty children (mean age of 8.02±0.60 years) from a
previously characterized air pollution cohort (17 ) were selected
based on exposure to the lowest (7.47±1.45 µg/m3,
N=25) versus highest (21.33±4.61 µg/m3, N=25) ambient
levels of PM2.5 (P <0.001) (Fig. 1A).
Subjects were excluded if they had taken oral immune suppressants within
5 days of the blood draw, had a history of allergen immunotherapy within
1 year of the clinical visit, had a chronic disease other than asthma,
or had an acute infection. We utilized CyTOF analysis of a 35-marker
panel focused on identifying circulating monocyte changes (Supplementary
Table 1). Cell populations other than monocytes were manually excluded
(Supplementary Fig. 1 and Fig. 1B). Major monocyte subsets from children
exposed to low versus high levels of PM2.5 were
computationally visualized (Fig. 1C). Environmental exposure to high
levels of PM2.5 was associated with significantly
increased frequency of total (Fig. 1D, P =0.008) and classical
(CD14+/CD16-; Fig. 1E,P =0.004) but not intermediate
(CD14+/CD16+; Fig. 1F,P =0.071) monocytes. Conversely, the frequency of non-classical
monocytes (CD14lo/-/CD16+) was
significantly diminished in those highly exposed to
PM2.5 (Fig. 1G, P =0.015). Elevation of classical
and reduction of non-classical circulating monocytes in children highly
exposed to PM2.5 was further confirmed by comparison of
absolute number (Supplementary Fig. 2). Monocyte subsets did not differ
between healthy and asthmatic children exposed to high levels of
PM2.5. Therefore, these results suggest that the level
of total and major monocyte subsets, classical and non-classical, could
reflect the impact of exposure to the fine particulate pollutants.
Importantly, monocytes have been found to be more heterogeneous than the
major subsets indicate (18 ), leading us to take an unsupervised
approach to identify additional subsets for our investigation of the
impact of exposure to PM2.5 on circulating monocytes
from healthy versus asthmatic children.
Distinct monocyte clusters are associated with exposure toPM2.5 levels in healthy versus asthmatic children
Using an unsupervised FlowSOM approach (19 ), we identified 8
monocyte clusters that were projected onto a two-dimensional Uniform
Manifold Approximation and Projection (UMAP) plot (Fig. 2A-B). Also, we
determined the proportion of each monocyte cluster per subject and found
considerable interpersonal variation (Supplementary Fig. 3). UMAPs in
Supplementary Fig. 4 represent the expression levels of individual
markers within the predicted monocyte clusters.
We next addressed whether specific monocyte clusters display an
association with ambient exposure to PM2.5. In
accordance with our initial gating approach (Fig. 1), the frequencies of
classical monocytes from clusters 1, 2, and 3 were significantly
increased in subjects highly exposed to PM2.5. In these
subjects, the frequency of cluster 1
[FcεR1a- CCR5-CD163dim CD123dimCLEC4D+ CD11b+CD14+], the major monocyte population, as well as
cluster 3 [FcεR1ahi CCR5+CD163+ CD123+CLEC4D- CD11bdimCD14+] were particularly enhanced in children
diagnosed with asthma compared to the healthy control group. The
frequency of these monocytes was not associated with the disease status
in subjects exposed to low levels of PM2.5 (Fig. 2C).
Similarly, frequency of cluster 4 [CD16+CD11chi CD11bdimCD64dim CD14dim/-], i.e.,
intermediate monocytes, was associated with asthma in subjects highly
exposed to PM2.5. In contrast, no significant
association was observed in those exposed to low levels of
PM2.5 regardless of disease status (Fig. 2C). Among
non-classical monocytes, the frequency of cluster 5
[CD16+ CX3CR1dimCD11b- CD14-] was negatively
associated with PM2.5 exposure. However, decreased
frequency of these monocytes in asthmatic individuals was not
significant compared to a healthy control group with exposure to high
levels of PM2.5. Interestingly, a higher frequency of
cluster 5 was associated with asthma diagnosis in subjects exposed to
low levels of PM2.5. Other clusters did not show a
significant difference between individuals exposed to low versus high
PM2.5 or to those with or without asthma (Fig. 2C).