INTRODUCTION
Air pollution is a global concern with detrimental effects on health and a significant socioeconomic burden worldwide. Approximately 4.2 million people die every year due to exposure to a specific air pollutant, particulate matter with aerodynamic diameters ≤ 2.5 micrometer (PM2.5) (1 ). Elevation of PM2.5is a risk factor associated with prevalence of inflammatory diseases such as asthma (2, 3 ). However, the mechanisms underlying PM2.5 effects on asthma prevalence are unclear.
Asthma is the most common chronic disease among children, typically associated with inflammation of the airways and massive structural changes collectively called airway remodeling (4 ). Despite various endotypes of asthma, eosinophilic-rich type 2 immunity is a characteristic feature of the disease in nearly half of the patients. Type 2-low asthma is associated with neutrophilic inflammation and refractoriness to corticosteroids (5 ). However, the role of monocytes in asthma has remained to be fully understood. It has been previously reported that monocytes accumulate in the airways of children with fatal asthma attacks (6 ). In addition, our previous studies showed that circulating monocytes are enriched in adolescents highly exposed to PM2.5 (7 ). Deep phenotyping of circulating monocytes in healthy versus asthmatic children exposed to various levels of PM2.5 may uncover a specific endotype of the disease.
Monocytes encompass a heterogeneous population that can be divided into distinct subsets based on expression of immune markers under inflammatory conditions (8 ). We postulated that immune signatures reflecting phenotypic and functional changes of circulating monocytes differ in healthy versus asthmatic children exposed to low versus high levels of PM2.5. These immune signatures may provide novel biomarkers to better stratify disease progression and outcomes, particularly in children. Furthermore, they may enable development of treatments for patients living in highly polluted areas, which is an unmet clinical need.
Monocytes undergo innate training, a cellular process by which they are educated to respond differently to secondary stimulation compared to the initial encounter (9 ). This process is tightly regulated by epigenetic reprogramming. Diverse triggers of epigenetic reprogramming for trained immunity include β-Glucan (10 ), viral infections (11 ), catecholamines (12 ), and Bacillus Calmette–Guérin (BCG) vaccine (13 ). It is not known whether particulate pollutants are able to train innate immune cells to elicit a specific hyper-inflammatory response that could eventually predispose individuals to inflammation via epigenetic mechanisms such as histone modifications (10, 14 ). Acetylation of histone 3 at lysine 27 residue (H3K27ac) is a canonical epigenetic mark that distinguishes active enhancers from inactive/poised enhancer elements and contributes to underlying induction of trained immunity in monocytes (10, 15, 16 ).
In this study, we compared circulating monocytes from healthy versus asthmatic children exposed to varying levels of ambient PM2.5 to determine differences in expression of monocyte-specific markers as detected by mass cytometry time-of-flight (CyTOF). In addition, we postulated that fine particulate pollutants provoke trained immunity characterized by elevation of pro-inflammatory mediators in circulating monocytes after stimulation with house dust mite serving as an asthma-relevant aeroallergen in vitro . Using chromatin immunoprecipitation sequencing (ChIP Seq), we further aimed to understand whether pollutant-induced trained immunity in monocytes is mediated via regulation of histone modifications, particularly H3K27ac.