Use of TLR2/6 Expressing HEK Cells and Transcriptomic Analysis to Identify Target Genes Induced by Cigarette Smoke

Sarah Mazi1,2, William Wright1, Mark Paul-Clark1, Jane Mitchell1. 1Imperial College London, London, UK, 2King Saud University, Riyadh, Saudi Arabia

Smoking Cigarettes is a risk factor in a number of chronic inflammatory diseases that include chronic obstructive pulmonary disease, atherosclerosis and lung cancer and is responsible for more than 5 million deaths per year worldwide. Our group has previously shown that the oxidant component of cigarette smoke extract (CSE) activates human cells and causes inflammation (Walters, Paul-Clark et al. 2005). We also demonstrated that inflammation caused by acute CSE exposure is partially mediated through the innate immune receptor toll-like receptor (TLR) 2 (Paul-Clark, McMaster et al.). Further, acute CSE exposure also causes significant transcriptomic changes in human monocytes that include the up regulation of oxidant-related, inflammatory and cancer promoting genes (Wright, Parzych et al. 2012). To assess which of these genes changed by acute CSE exposure are TLR2 dependent, HEK293 cells, containing TLR2/6 plasmid or null vector, were treated for 8h with CSE (10% v/v) or H2O2 (1mM) or RPMI alone as a control. Total mRNA was extracted from these cells and converted first to cDNA and then to cRNA. The cRNA was labelled and hybridised to Illumina Human Ref8v3 transcripome chips and analysed for changes in 26,000 genes. Data was loaded into GeneSpring software for analysis where it was first normalised using a quantile algorithm to remove non-biological differences. Gene changes between groups were assessed using a T-test with a Benjamini-Hochberg false discovery rate correction with a p<0.005 considered to be significant. A further filter of 1.5-fold cut off was also applied. After 8h in HEK293 null cells, 556 genes were significantly altered in the H2O2 group and 75 genes changed after CSE treatment when compare with RMPI. In the HEK293 cells transfected with TLR2/6, 271 genes were significantly altered by H2O2 and 49 by CSE. When we looked at the common genes in both H2O2 and CSE we found that only SRGN (serglycin) was TLR2/6 dependent. In contrast, there were 39 TLR2-independent genes that were commonly altered by H2O2 and CSE (Figure 1). When these genes were placed in a pathway analysis program (Ingenuity Pathway Analysis) we saw that oxidants caused a characteristic expression of antioxidant and xenobiotic genes together with a widespread spectrum of inflammation-related genes, and oncogenic genes. We believe that this study may further our understanding of the pathways involved in smoking related pathogenesis and highlight possible novel targets.

Figure 1. TLR2/6-independent gene changes in HEK293 cells treated with H202 or CSE. (A) Venn diagram between TLR2/6-independentgenes altered by CSE or H202 showing the mutual genes that are found in both gene lists. (B) Heat map representation of genes altered by both H202 and CSE in a TLR2/6-independent manner. High gene expression is represented as red and turquoise is represented as low gene expression. Samples for each group are n=3, and each sample is a replicates of 2.

Walters MJ , et al. Mol Pharmacol 2005 Nov;68(5):1343-1353

Paul-Clark MJ, et al. Am J Respir Crit Care Med 2009 Feb 15; 179(4):299-306.

Wright WR, et al. PLoS One 2012; 7(2):e30120