OS03-05 Differential toxicology of air pollution: co-exposure of particulate matter and NO2 induces transcriptomic alterations at the epithelial airway barrier in vitro

Due to the heterogeneous nature of air pollution, humans are exposed to a variety of different pollutants simultaneously. Particulate matter (PM) and nitrogen dioxide (NO 2 ) have both shown to be associated with morbidities, though, the ambient levels of each these pollutants are closely correlated. This makes it difficult to distinguish pollutant specific effects within epidemiological studies. This therefore warrants further investigation into these pollutants within a controlled environment using a model system. Herein, an advanced in vitro triple cell co-culture of the epithelial airway barrier, comprising ATI (hAELVi), ATII (NCI-H441) and alveolar macrophages (differentiated THP-1) cultured at an anatomically relevant ratio at the air-liquid interface (ALI) were exposed to PM and/or NO 2 . The PM samples used within this work were indoor dust (ID) (NIST SRM 2583) (780ng/cm 2 ) and diesel emission particles (DEPs) (32.8 μg/mL) from a controlled combustion engine in a modern heavy-duty truck using ultralow sulphur MK1 diesel fuel at high (17%) oxygen intake. In vitro cultures were first exposed to ID using a VITROCELL® Cloud aerosol exposure system or equivalent DEP concentration through quasi-ALI, before incubation in a controlled NO 2 exposure chamber at 2.5 ppm for 24 hr. These exposure parameters had been defined in previous work investigating the biological impact ( e.g., viability, pro-inflammatory, and oxidative stress response) of each single pollutant at the protein level. Cells were then lysed, and RNA was isolated. Transcriptomic changes were analysed using DNBSEQ PE150 sequencing. All exposures resulted in differential gene expression compared to respective controls, though NO 2 and DEP exposures resulted in more differentially expressed genes (DEGs) than ID at 94, 468 and 6 DEGs, respectively. Gene ontology pathway analysis showed that NO 2 exposure altered the expression of genes associated with neutrophil activation and negative regulation of autophagic cell death. DEPs altered expression of genes associated with IL-27 signalling, chemotaxis and extracellular matrix constituent secretion. Co-exposure of DEP and NO 2 also increased expression of genes associated with IL-27 signalling, genes associated with a chronic inflammatory response, as well as other pro-inflammatory processes. Comparing DEP and NO 2 co-exposure against DEP single pollutant exposures, a cluster of genes associated with a pro-inflammatory response were upregulated, such as Il1a , Il1b and Tnf . Single gene analysis shows a potential novel mechanism by which NO 2 attenuates IL10 signalling, augmenting PM induced pro-inflammatory response. It therefore appeared that independently NO 2 and PM can exert specific effects, which may then combine to drive a greater response during co-exposure. The findings highlight the importance of deducing the differential toxicity of the various constituents of air pollution which can aid in the better understanding of human health effects.