Synergistic Amplification of Lipopolysaccharide‐ and Histamine‐induced TLR4 and COX2 Expression by Cigarette Smoke Extract and Nicotine in Endothelial Cells
Authors: Barua Rajat S , Sharma Mukut , De Alok , Zhou Jianping , Parashara Deepak , Dileepan Kottarappat N 
Published: November 2023

BackgroundIncreasing evidence suggests that infection and persistent inflammation are key players in the pathogenesis of cardiovascular disease (CVD). Cigarette smoke (CS) and innate immune upregulation are important risk factors for CVD, and infections significantly contribute to the morbidity and mortality caused by cigarette smoking. Although it is well‐established that CS promotes atherosclerotic CVD, very little is known about potential impact of the collective effects of CS and over‐active innate immune system on vascular inflammation and CVD. Mast cells (MC) and Toll‐like receptors (TLRs) are constituents of the innate immune system. Our work has shown that MC‐derived histamine and lipopolysaccharide (LPS) synergistically enhance pro‐atherogenic inflammatory response via bidirectional upregulation of histamine‐1 receptor (H1R) and TLR4 in human endothelial cells. These results suggest that the combined and persistent effects of MC mediators and bacterial agents on the vasculature are risk factors of CVD. Here, we tested the hypothesis that CS and nicotine would amplify histamine‐ and/or LPS‐mediated pro‐atherogenic inflammatory responses in endothelial cells via innate immune upregulation.MethodsHuman umbilical vein endothelial cells (HUVECs) were incubated with medium (control), histamine (10 μM), LPS (100 ng/mL), CS extract (CSE, 11μg/mL equivalent to 100 nM nicotine) or nicotine (100 nM) for 24 h. To assess the synergistic effects, HUVECs were also incubated with histamine+LPS, histamine+LPS+nicotine or histamine+LPS+CSE for 24 h. The expression of TLR4, COX2, and nicotinic acetylcholine receptor‐1α (NAchRα1) mRNA were determined by RT‐qPCR. Immunocytochemical analyses were performed to determine changes in the protein expression.ResultsCSE and nicotine significantly increased the basal as well as histamine‐ and LPS‐induced COX2 gene expression with varying magnitudes of synergy. While nicotine alone did not increase COX2 expression, histamine+LPS+nicotine caused a >100‐fold increase in COX2 mRNA expression. Effect of CSE was more pronounced than that of nicotine in all settings. CSE significantly increased the expression of TLR4 in quiescent and in histamine‐ and LPS‐treated cells. Histamine alone as well as histamine+LPS induced significant increases in NAchα1R mRNA expression (2.3 and 3.5 fold) over the baseline suggesting their role in this synergetic effect.ConclusionsThese results suggest that CS and nicotine have synergistic amplifying effects on histamine‐ and LPS‐mediated pro‐atherogenic inflammatory response and innate immune upregulation. We propose that CS and its constituents acting in concert with bacterial toxins and MC products may amplify vascular inflammation via H1R‐TLR4‐COX2 axis.Support or Funding InformationSupported by Kansas City VA Medical Center, The Midwest Biomedical Research Foundation, Joseph & Carey Arthritis Foundation, and Kansas University Endowment Foundadtion