DNA hypomethylation of Th17 cells by ambient particulate pollutants enhances neutrophil recruitment in murine models of allergic airway inflammation

Despite the deleterious effects of particulate matter (PM) exposure on asthma, the underlying mechanism remains unclear currently.1 DNA methylation of peripheral blood mononuclear cells (PBMCs) is a potential mechanism that bridges the gap between exogenous exposure and genome, which rapidly influences incidence and progression of diseases.2 In this study, human PBMCs (hPBMCs) isolated from individuals pre- and post-PM exposure were subjected to the methylated DNA immunoprecipitation sequencing. Among 1, 443 differentially methylated genes following PM exposure, we identified 49 overlapping genes related to PM-induced diseases based on the comparative toxicogenomics database (CTD, https://ctdbase.org/)3 (Figure 1A). KEGG pathway analysis upon overlapping genes presented enrichment of the retinoic acid receptor-related orphan receptor C (RORC) in the T helper 17 (Th17) cell differentiation pathway (Figure 1B). Therefore, we hypothesized that PM exposure-induced hypomethylation of RORC may initiate Th17 cell differentiation. Using DNA bisulfite assay and sequencing, hypomethylation in the promoter region of Rorc was confirmed in PM-exposed mouse PBMCs (mPBMCs) (Figure 1C), as well as in CD4+ T cells (Figure S1A,B). This may be attributed to the evaluated expression levels of 10–11 translocation methylcytosine dioxygenase 2 (TET2) (Figure S1C,D), a pivotal protein to promote DNA demethylation in T cells.4 Treated with TET2 inhibitor, Bobcat339, PM exposure-induced activation of RORC and IL17A was blunted in T cells (Figure S1E–H). To clarify the upstream driver of Rorc hypomethylation, mPBMCs isolated from PM-exposed mice and control mice were subjected to RNA sequencing and bioinformatics analysis displayed significant alterations in transcription factor expression levels (Figure 1D). The expression of E-26 variant gene 5 (ETV5) was significantly up-regulated following PM exposure in a TET2-independent manner (Figure 1E,F). Expression of Rorc were transcriptionally driven by ETV5 following PM exposure; however, inhibition of demethylation in binding motif of ETV5 to Rorc promoter effectively counteracted Rorc expression (Figure 1G–J). Accordingly, PM exposure-induced secretion of IL17A was significantly decreased with Etv5 knockdown (KD) in Th17 cells (Figure 1K,L). The binding of ETV5 to Rorc and activation of Th17 cells were observed in lungs from PM-exposed mice (Figure S2). As the neutrophilic asthma was characterized by type-17 inflammation,5, 6 we assumed that PM-activated Th17 cells might aggravate symptom and pathology in asthma. To elucidate that, ovalbumin (OVA)-induced allergic airway inflammation model was established and challenged with PM or PBS (Figure 2A). PM exposure caused massive infiltration of immune cells and more severe airway embolism than those of PBS-challenged mice (Figure 2B). Significantly increased proportions of Th17 cells and neutrophils in both lung tissue and bronchoalveolar lavage fluid (BALF), rather than eosinophils or other cells, were observed in OVA-PM-challenged mice, comparing with OVA-PBS control (Figure 2C; Figure S3A,B). Meanwhile, the eosinophils-to-neutrophils ratio in BALF significantly declined in OVA-PM group, comparing with OVA-PBS control (Figure S3C,D). These results suggest that along with the accumulation of neutrophils recruited by PM-primed Th17 cells, type 2 inflammation has been converted to a mixed inflammation following PM exposure. Mechanically, activation of ETV5 and demethylation of ETV5 binding motif in Rorc promoter region were confirmed in RORC+ cells from PM-challenged murine lungs (Figure 2D,E). Next, PM-primed WT or Etv5-KD Th17 cells (labeled with CFSE) were intravenously transferred to recipient asthmatic mice (Figure S4A). Blockade of ETV5 significantly reduced the recruitments of Th17 cells to lung tissue and attenuated infiltration of neutrophils in the murine circulation and lungs (Figure S4B–D). hPBMCs from 14 asthma patients residing in Daxing District, Beijing, China were collected pre- and post-exposure to PM pollution (Figure 2F). Significantly elevated counts of Th17 cells and neutrophils, rather than eosinophils, were observed in participants after exposure to air pollution (Figure 2G and Figure 2H). Accordingly, mRNA expression levels of ETV5 and RORC in hPBMCs isolated from asthma patients after exposure to air pollutants were also significantly increased (Figure 2I). The percentage of Th17 cells (Figure 2J), expression levels of ETV5 or RORC (Figure 2K) in asthma patients were significantly correlated with concentration of ambient PM2.5 or PM10 in Daxing District, Beijing, China. Besides, the demethylation of RORC promoter in ETV5 binding sites in asthma patients post-exposure to air pollution was raised (Figure 2L). In summary, PM exposure aggravates endotype of neutrophilic asthma through activation of ETV5/RORC/Th17 signaling (Figure 2M). Our findings shed light on the precise pathophysiological processes of PM exposure-induced Th 17 inflammatory response in asthma. Bin Li was involved in investigation, methodology, and writing— original draft. Yuan Zhang was involved in patient recruiting. Nannan Huang, Jing Wang, Xianan Zhang, Qingtao Meng, Chengshuo Wang, and Jiajia Wang were involved in methodology and validation. Luo Zhang was involved in project administration and supervision. Xiaobo Li was involved in writing—original draft, methodology, validation and funding acquisition. Rui Chen was involved in supervision, writing—review & editing and funding acquisition. All authors contributed to manuscript revision and approved the final version of the manuscript. Not applicable. This work was financially supported by National Key R&D Program of China (2022YFC2504100 and 2022YFC2504101, 2023YFC3708300 and 2023YFC3708303); the National Science Fund for Distinguished Young Scholars (82025031); the Beijing Outstanding Young Scientist Program (JWZQ20240101024); the Youth Beijing Scholar Program; the Chinese Institutes for Medical Research (CX23YZ01), Beijing; High level talent in Public Health of Beijing (Discipline leaders-03-29). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. All authors approved the manuscript. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The data that support the findings of this study are available from the corresponding author upon reasonable request. Data S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.