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.