FIGURE 1.

Responses of Calu-3 cells to 10 nM formoterol and 100 nM budesonide (Form/Bud) were examined in the context of cannabis smoke extract (CSE)-conditioned media. Calu-3 cells were exposed to either control, 10% CSE (orange) or 10% tobacco smoke extract (TSE) (blue) with either vehicle or Form/Bud for 24 h. To assess the impact of Form/Bud on inflammatory mediators, cytokine release of a) transforming growth factor (TGF)-α, b) interferon-γ-induced protein (IP)-10/CXCL10 and c) interleukin (IL)-8/CXCL8 was measured. d) The effect of Form/Bud intervention and CSE or TSE exposure on airway epithelial gene expression was assessed by RNA sequencing. The log₂ fold change (FC) of CSE+Form/Bud versus vehicle+Form/Bud was plotted against the log₂FC of TSE+Form/Bud versus vehicle+Form/Bud to reveal significant TSE- and CSE-specific genes under Form/Bud intervention conditions. r=0.771; p<1×10⁻¹⁵. Genes that were significantly differentially expressed (q<0.05) in CSE+Form/Bud and TSE+Form/Bud versus vehicle+Form/Bud are highlighted in purple, where as CSE- and TSE-specific genes are highlighted in blue and red respectively. To examine the similarity of CSE+Form/Bud and TSE+Form/Bud transcriptomic profiles, correlation of differential gene expression was determined by Pearson's correlation. e) Functional enrichment analysis of genes shown to be significantly differentially expressed (q<0.05) in CSE+Form/Bud and TSE+Form/Bud versus vehicle+Form/Bud. Top 10 gene ontology biological processes were ranked by decreasing adjusted p-value, with number of significantly differentially expressed genes (count) contributing to the total number of genes associated with the given pathway (n) denoted by the size of circle. f) Expression of genes involved in NRF2 and AHR responses. g–l) Oxidative stress was assessed using CellROX and m) the fluorescent intensity was quantified n) with complementary quantification using 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA). All experiments were performed using four biological replicates from serial passages. Different letters indicate significantly different group means (Tukey Honest Significant Difference, p<0.05). There are no significant differences between means sharing a common letter. Error bars represent sem. Scale bars=100 μm. RFU: relative fluorescence unit.