Role of Toll-like receptor 9 in mouse lung inflammation in response to chicken barn air

Lung dysfunction due to exposure to air in high intensity livestock barn operations is a common problem for workers in these facilities. Exposure to this air has been linked to disorders such as chronic bronchitis, occupational asthma, organic dust toxic syndrome, and chronic cough and phlegm. These symptoms have been linked to higher levels of endotoxins in air in chicken and swine barns. However, there are many other toxic molecules such as bacterial DNA and gases capable of inducing respiratory inflammation. Bacterial molecules are recognized through highly conserved pattern recognition molecules called Toll-like receptors (TLR). While lipopolysaccharides are recognized by TLR4, bacterial unmethylated DNA binds to and signals through TLR9. As a prelude to understanding the biology of TLR9 in lung inflammation, it is important to precisely clarify their in situ expression in the lung. I determined expression of TLR9 in intact lungs from cattle, pigs, dogs, horses, mice, and humans. Two samples from normal lungs of cattle, pigs, dogs, three from horses, and two from inflamed calf lungs were tested. Five normal mouse and three normal human lungs were similarly tested as well as 5 human lungs with diagnosis of asthma. The expression was determined with multiple methods such as Western blots, immunohistology, immunogold electron microscopy and in situ hybridization. Lungs from all the species showed TLR9 expression in the bronchial epithelium, vascular endothelium, alveolar septa, alveolar macrophages, and type-II alveolar epithelial cells. Immuno-electron microscopy detected TLR9 on the plasma membrane, cytoplasm and the nucleus of various cells including macrophages. In situ hybridization demonstrated TLR9 mRNA in the bronchial epithelium, vascular endothelium, alveolar septa, alveolar macrophages, and type-II alveolar epithelial cells of mouse and human. Asthmatic human lungs showed many more inflammatory cells expressing TLR9 compared to healthy lungs. In cattle and horses, pulmonary intravascular macrophages showed robust expression of TLR9. Depletion of pulmonary intravascular macrophages in horses resulted in significant reduction in total TLR9 mRNA in the lungs. Having determined that TLR9 expression is similarly expressed on many lung cell types in mice and humans, I determined the role of TLR9 in barn air induced lung inflammation by exposing TLR9-/- and wild-type mice (6 per group) to single or multiple days (5 and 20) in a chicken barn. Each exposure was of 8 hours/day duration. The TLR9-/- mice exposed 5 and 20 times showed significant reductions in TNF-alpha and IFN-gamma expression in lung lavages as well as cellular changes consistent with reduced lung inflammation such as reductions in the number of lung neutrophils. This suggests that barn dust DNA, acting through TLR9, contributes to lung inflammation seen in response to exposure to chicken barn air. These fundamental data advance our knowledge on the cell-specific expression of TLR9 across a range of species including the humans and demonstrate that TLR9-/- partially regulates lung inflammation induced following exposure to chicken barn air