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

Effect of low-level CO2 on innate inflammatory protein response to organic dust from swine confinement barns.

BACKGROUND: Organic hog barn dust (HDE) exposure induces lung inflammation and long-term decreases in lung function in agricultural workers. While concentrations of common gasses in confined animal facilities are well characterized, few studies have been done addressing if exposure to elevated barn gasses impacts the lung immune response to organic dusts. Given the well documented effects of hypercapnia at much higher levels we hypothesized that CO2 at 8 h exposure limit levels (5000 ppm) could alter innate immune responses to HDE. METHODS: Using a mouse model, C57BL/6 mice were nasally instilled with defined barn dust extracts and then housed in an exposure box maintained at one of several CO2 levels for six hours. Bronchiolar lavage (BAL) was tested for several cytokines while lung tissue was saved for mRNA purification and immunohistochemistry. RESULTS: Exposure to elevated CO2 significantly increased the expression of pro-inflammatory markers, IL-6 and KC, in BAL fluid as compared to dust exposure alone. Expression of other pro-inflammatory markers, such as ICAM-1 and matrix metalloproteinase-9 (MMP-9), were also tested and showed similar increased expression upon HDE + CO2 exposure. A chemokine array analysis of BAL fluid revealed that MIP-1γ (CCL9) shows a similar increased response to HDE + CO2. Further testing showed CCL9 was significantly elevated by barn dust and further enhanced by CO2 co-exposure in a dose-dependent manner that was noticeable at the protein and mRNA levels. In all cases, except for ICAM-1, increases in tested markers in the presence of elevated CO2 were only significant in the presence of HDE as well. CONCLUSIONS: We show that even at mandated safe exposure limits, CO2 is capable of enhancing multiple markers of inflammation in response to HDE

Effect of low-level CO2 on innate inflammatory protein response to organic dust from swine confinement barns.

BACKGROUND: Organic hog barn dust (HDE) exposure induces lung inflammation and long-term decreases in lung function in agricultural workers. While concentrations of common gasses in confined animal facilities are well characterized, few studies have been done addressing if exposure to elevated barn gasses impacts the lung immune response to organic dusts. Given the well documented effects of hypercapnia at much higher levels we hypothesized that CO2 at 8 h exposure limit levels (5000 ppm) could alter innate immune responses to HDE. METHODS: Using a mouse model, C57BL/6 mice were nasally instilled with defined barn dust extracts and then housed in an exposure box maintained at one of several CO2 levels for six hours. Bronchiolar lavage (BAL) was tested for several cytokines while lung tissue was saved for mRNA purification and immunohistochemistry. RESULTS: Exposure to elevated CO2 significantly increased the expression of pro-inflammatory markers, IL-6 and KC, in BAL fluid as compared to dust exposure alone. Expression of other pro-inflammatory markers, such as ICAM-1 and matrix metalloproteinase-9 (MMP-9), were also tested and showed similar increased expression upon HDE + CO2 exposure. A chemokine array analysis of BAL fluid revealed that MIP-1γ (CCL9) shows a similar increased response to HDE + CO2. Further testing showed CCL9 was significantly elevated by barn dust and further enhanced by CO2 co-exposure in a dose-dependent manner that was noticeable at the protein and mRNA levels. In all cases, except for ICAM-1, increases in tested markers in the presence of elevated CO2 were only significant in the presence of HDE as well. CONCLUSIONS: We show that even at mandated safe exposure limits, CO2 is capable of enhancing multiple markers of inflammation in response to HDE

Organic barn dust inhibits surfactant protein D production through protein kinase-c alpha dependent increase of GPR116.

Prolonged exposure to organic barn dusts can lead to chronic inflammation and a broad range of lung problems over time, mediated by innate immune mechanisms. The immune surfactant or collectin surfactant protein D (SP-D) is a crucial multifunctional innate immune receptor. Little work to date has examined the effect of such collectins in response to organic dusts. We provide evidence here that agricultural organic dusts can inhibit mRNA and protein expression of SP-D in a human alveolar epithelial cell line, and an in vivo mouse model. This inhibition was not a result of lipopolysaccharide (LPS) or peptidoglycans, the two most commonly cited immune active components of these dusts. We further show that inhibition of the signaling molecule protein kinase C alpha (PKCα) can reverse this inhibition implicating it as a mechanism of SP-D inhibition. Examination of the SP-D regulatory receptor GPR116 showed that its mRNA expression was increased in response to dust and inhibited by blocking PKCα, implicating it as a means of inhibiting SP-D in the lungs in response to organic dusts. This reduction shows that organic barn dust can reduce lung SP-D, thus leaving workers potentially at risk for a host of pathogens

Adhesion Molecules in Lung Inflammation from Repeated Glyphosate Exposures

Glyphosate is an active ingredient in herbicides. Exposure to glyphosate-based herbicides has been associated with respiratory dysfunctions in agricultural workers. The ability of inhaled glyphosate to induce lung inflammation is not well understood. Further, the role of adhesion molecules in glyphosate-induced lung inflammation has not been studied. We evaluated lung inflammatory responses from single and repeated glyphosate exposures. Male C57BL/6 mice were intranasally exposed to glyphosate (1 μg/40 μL) for 1 day or once daily for 5 days or 10 days. Lung tissue and bronchoalveolar lavage (BAL) fluid were collected and analyzed. Repeated exposure to glyphosate for 5 days and 10 days resulted in an increase in neutrophils in BAL fluid and higher eosinophil peroxidase levels in lungs, with leukocyte infiltration further confirmed through lung histology. Repetitive exposure to glyphosate increased IL-33 and Th2 cytokines IL-5 and IL-13. A single glyphosate treatment revealed expression for ICAM-1, VCAM-1, and vWF adhesion molecules in the perivascular region of lung sections; with repeated treatment (5 and 10 days), adhesion molecule expression was found in the perivascular, peribronchiolar, and alveolar regions of the lungs. Repetitive exposure to glyphosate induced cellular inflammation in which adhesion molecules may be important to the lung inflammatory process

Effect of low-level CO2 on innate inflammatory protein response to organic dust from swine confinement barns

Background: Organic hog barn dust (HDE) exposure induces lung inflammation and long-term decreases in lung function in agricultural workers. While concentrations of common gasses in confined animal facilities are well characterized, few studies have been done addressing if exposure to elevated barn gasses impacts the lung immune response to organic dusts. Given the well documented effects of hypercapnia at much higher levels we hypothesized that CO2 at 8 h exposure limit levels (5000 ppm) could alter innate immune responses to HDE. Methods: Using a mouse model, C57BL/6 mice were nasally instilled with defined barn dust extracts and then housed in an exposure box maintained at one of several CO2 levels for six hours. Bronchiolar lavage (BAL) was tested for several cytokines while lung tissue was saved for mRNA purification and immunohistochemistry. Results: Exposure to elevated CO2 significantly increased the expression of pro-inflammatory markers, IL-6 and KC, in BAL fluid as compared to dust exposure alone. Expression of other pro-inflammatory markers, such as ICAM-1 and matrix metalloproteinase-9 (MMP-9), were also tested and showed similar increased expression upon HDE + CO2 exposure. A chemokine array analysis of BAL fluid revealed that MIP-1γ (CCL9) shows a similar increased response to HDE + CO2. Further testing showed CCL9 was significantly elevated by barn dust and further enhanced by CO2 co-exposure in a dose-dependent manner that was noticeable at the protein and mRNA levels. In all cases, except for ICAM-1, increases in tested markers in the presence of elevated CO2 were only significant in the presence of HDE as well. Conclusions: We show that even at mandated safe exposure limits, CO2 is capable of enhancing multiple markers of inflammation in response to HDE