Ethanol suppresses the pulmonary response to Streptococcus pneumoniae: Role of NOD2 and NF-kB signaling in alveolar macrophages exposed to muramyl dipeptide.

Abstract

Abstract Alcohol use decreases host immune defense and increases susceptibility to pathogens such as Streptococcus pneumoniae. NOD2 is an intracellular pattern recognition receptor that binds muramyl dipeptide (MDP), a cell wall component of S. pneumoniae, and activates NF-kB signaling. To limit inflammation, NF-kB signaling is negatively regulated in part by TNFAIP3 (TNF Alpha Induced Protein 3), or A20. We hypothesize that ethanol impairs NOD2 signaling and A20 expression in alveolar macrophages, leading to excessive inflammation and reduced bacterial clearance after infection. For in vivo experiments, we gavaged mice once daily for 3 consecutive days with water or ethanol (blood alcohol concentration ~ 80 mg/dL at 30 min), and infected with 104 cfu S. pneumoniae. In parallel experiments, MH-S cells were treated with 25 mM ethanol and 10 μg/ml MDP. We found that ethanol treated animals, compared to vehicle treated, had impaired respiratory function (17% lowered respiration rate [p < 0.01] and 33% increased penh [p = 0.09]) and increased lung bacteria (log10 increase of 0.8 [p = 0.11]) at 24 hours. RNA sequencing of alveolar macrophages from vehicle- or ethanol-gavaged mice revealed that ethanol intake led to 25% less Nod2 and 60% less A20 (p < 0.05) in the absence of infection. These results were confirmed by qPCR in ethanol-treated MH-S cells (40% decrease in Nod2 and 50% reduction in A20 [p < 0.01]), along with 15% lower A20 protein levels, which was sustained after MDP stimulation (29% less A20 compared to vehicle-treated, stimulated cells). Future experiments aim to further investigate ethanol’s effect on A20 regulation of NF-kB signaling, and will provide insight into immune dysregulation seen in alcohol-consuming patients with pneumonia.