The cigarette smoke-induced CXCL-8 release from human neutrophils is inhibited by TLR-9 antagonists

Background: Exacerbations of COPD are associated with bacterial infections and neutrophil influx. Cigarettes are contaminated with microbes derived from soil and tobacco plants and due to fermentation processes; bacterial components such as endotoxins remain present in cigarette smoke. Previously we suggested that cigarette smoke-induces CXCL-8 release from human neutrophils partly via activation of Toll like receptor 9 (TLR9). In the previous study we used Chloroquine as a TLR inhibitor. In the current study we investigated the effectiveness of specific TLR9 antagonists to inhibit a cigarette smoke induced immune response. Methods: Human polymorphonuclear cells (PMNs) were isolated from a buffy coat and incubated for 16 h with a TLR9 agonist (ODN 2395) in 10 fold dilution steps (0.01-10 nM) or cigarette smoke extract (CSE) in two fold dilution steps (0.03-0.12 OD). Supernatants were stored at -20°C until analyzed using a human CXCL8 ELISA kit. Next, PMNs were pre-incubated for one hour with TLR9 antagonists ODN 2088 and ODN INH-I in ten fold dilution steps (0.01-10 nM) then stimulated for 16 h with 1 nM ODN 2395 and 0.12 OD CSE. Five donors were used for each experiment. Results: CSE and the TLR-9 agonist dose dependently increased the CXCL-8 production. A submaximal dose of CSE or the TLR-9 agonist was used in further experiments. In four of the donors, INH-1 inhibited CXCL8 production back to baseline levels while the mouse preferred antagonist ODN-2088 was only effective in two of the donors. Both antagonists significantly reduced (>35%) the CSE induced CXCL8 production (P <0.0001). This partial reduction can be explained by the fact that CSE has more than 4500 compounds some of which stimulate CXCL8 production via other pathways. Moreover between person variability can be explained by a positive correlation (R2 = 0.4642) between the CSE induced CXCL8 release and the number of isolated neutrophils. Conclusions: CSE induced CXCL-8 release from human neutrophils is significantly reduced by TLR-9 antagonists. This data provides support for new therapeutic avenues in the control of inflammation in COPD patients.