Effects of Smoldering Eucalyptus Wood Smoke Extract on Primary Human Brain Microvascular Endothelial Cells (HBMEC) and Immortalized Human Cerebral Microvascular Endothelial Cell Line (hCMEC/D3)

Increase in frequency and size of wildfire raises a significant public health concern due to rapid climate change. Epidemiological studies have shown that wildfire smoke exposure can increase the risk of neurological disorders. Our study aims to understand how wildfire smoke particles affect brain endothelial cells using both primary human brain microvascular endothelial cells (HBMEC) and an immortalized human cerebral microvascular endothelial cell line (hCMEC/D3). HBMEC and hCMEC/D3 were exposed to different levels of smoldering eucalyptus smoke particles (10, 30, 50 ug/mL) for 24 hr. Supernatants were collected for LDH and ELISA array, cell lysates were collected for RNAseq, and cells were fixed and stained for tight junction marker (Zonula Occludens-1, ZO-1) via ICC. Our study found that treatment to smoldering eucalyptus particles can increase inflammatory cytokine production dose-dependently in HBMECs and hCMEC/D3. Moreover, RNAseq analyses resulted in dose-dependent changes in HBMEC and hCMEC/D3 from wood smoke treatment that could be mediated via AhR and Nrf2 pathways. Both HBMEC and hCMEC/D3 were plated in 96 well plates at 5x103 cells/well. Cells were cultured in coated 96 well plates to confluency before the treatment with eucalyptus wood smoke extract (10,30,50 mg/mL or 20,70, 100 mg/mL), lipopolysaccharide (LPS) (1 mg/mL), or LiCl (10 mM) for 24 hours. At the end of treatment, cells were fixed with 4% paraformaldehyde (PFA) for ICC, lysed to collect mRNA for bulk-RNA sequencing, or supernatants were collected for ELISA and cytotoxicity assays. All experiments were repeated with at least two different seedings with n=2 or 3.