Alcohol up-regulates TLR2 through a NO/cGMP dependent pathway

Abstract

Background: Heavy alcohol consumption is associated with severe bronchitis. This is likely related to increased inflammation in the airways of alcohol abusers. Toll-like receptor 2 (TLR2) is an important mediator of inflammation in the airway epithelium. TLR2 initiates an inflammatory cascade in response to gram-positive bacteria. We have previously shown that alcohol up-regulates TLR2 in the airway epithelium. However, the mechanism of alcohol-mediated up-regulation of TLR2 has not been identified.

Methods: A human airway epithelial cell line, 16HBE14o-, was exposed to biologically relevant concentrations of alcohol (100 mM) in the presence and absence of N(omega)-Nitro-l-arginine methyl ester hydrochloride, a nitric oxide (NO) synthase inhibitor; and Rp-8-Br-cGMP-S, an antagonist analogue of cGMP. TLR2 was measured using real-time PCR and Western blots. In addition, 16HBE14o- cells were incubated with sodium nitroprusside (SNP), an NO donor, and 8-Br-cGMP, a cGMP analogue. TLR2 was measured using real-time PCR.

Results: N(omega)-Nitro-l-arginine methyl ester hydrochloride blocked the alcohol-mediated up-regulation of TLR2. This indicates that NO plays a key role in alcohol's up-regulation of TLR2. SNP, a NO donor, up-regulated TLR2. Rp-8-Br-CGMP-S attenuated alcohol's up-regulation of TLR2, suggesting that NO was working through cGMP/PKG. 8-Br-cGMP up-regulated TLR2, also demonstrating the importance of cGMP/PKG.

Conclusions: Alcohol up-regulates TLR2 through a NO/cGMP/PKG dependent pathway in the airway epithelium. This is an important observation in the understanding how alcohol modulates airway inflammation. In addition, this is the first time that cyclic nucleotides have been shown to play a role in the regulation of TLR2.

Figure 1

16HBE14o- cells were preincubated with and without L-NAME, an NOS inhibitor, for 1 hour. The indicated cells were then exposed to 100mM alcohol for 6 hours. TLR2 mRNA was measured using real-time PCR. L-NAME blocked alcohol's upregulation of TLR2 (n=4).

Figure 2

16HBE14o- cells were exposed to 0.001-0.1 μM Sodium Nitroprusside (SNP), a nitric oxide donor, for 6 hours. TLR2 mRNA was measured using real-time PCR. SNP upregulated the amount of TLR2 mRNA as much as alcohol (n=5).

Figure 3

16HBE14o- cells were pre-incubated for 1 hour with Rp-8-Br-cGMPS, an antagonist analogue of cGMP. Cells were then stimulated with 100 mM alcohol for 24 hours in the presence of the antagonist. Alcohol caused a 2-fold increase in TLR2 mRNA (p< 0.001) Rp-8-Br-cGMPS inhibited the upregulation of TLR2 mRNA by alcohol (p<0.05). The alcohol + Rp-8-Br-cGMPSgroup is not statistically different from control (p>0.05;n=4).

Figure 4

16HBE14o- were exposed to 0, 1, 10, and 100 μM 8-Br-cGMP (A) and 8-Br-cAMP (B) for 6 hours. TLR2 mRNA increased with 8-Br-cGMP, as with alcohol (n=4).

Figure 5

16HBE14o- were stimulated with 100 mM alcohol in the presence or absence of Rp-8-Br-cGMPS for 48 hours. Cell monolayers were harvested and a Western blot for TLR2 was performed. A. Representative blot of 5 separate experiments. B. Densitometry of the ratio of TLR2: Beta Actin ratio of the 5 Western blots performed. TLR2 protein is upregulated by alcohol, and this effect is blocked by Rp-8-Br-cGMPS.