Effect of Malondialdehyde-Acetaldehyde-Protein Adducts on the Protein Kinase C-Dependent Secretion of Urokinase-Type Plasminogen Activator in Hepatic Stellate Cells

Previous studies from our laboratory have shown that malondialdehyde-acetaldehyde-protein adducts (MAA adducts) are formed in hepatocytes of ethanol-fed rats and directly influence the hepatic stellate cells (HSCs) to induce their secretion of chemokines and to up-regulate their expression of adhesion molecules. Since protein kinase C (PKC) is known to play a major role in many diverse intracellular signal transduction processes, we investigated whether MAA adducts influence the function of HSCs via a PKC-dependent pathway. HSCs in culture were exposed to MAA adducts, and PKC activity was determined. We observed a time- and concentration-dependent activation of PKC when cultures were exposed to BSA-MAA as compared with unmodified BSA. Using PKC isoform-specific inhibitors, we also showed that BSA-MAA induces the activation of a specific isoform of PKC, PKC-a, in HSCs. No activation of PKC was observed when HSCs were exposed to other aldehyde adducts such as BSA-acetaldehyde or BSA-malondialdehyde, indicating that the effects of MAA adducts on HSCs were somewhat specific. We further examined whether the observed increase in PKC activation induced by MAA adducts in HSCs, in turn, causes a functional effect. We observed that BSA-MAA induces the increased secretion of urokinase-type plasminogen activator, a key component of the plasmin-generating system, and that PKC activation is necessary for this enhanced urokinase-type plasminogen activator secretion. These results indicate that MAA adducts via a PKC-mediated pathway may regulate plasmin-mediated matrix degradation in the liver, thereby contributing to the progression of hepatic fibrosis

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

The omega-3 fatty acid docosahexaenoic acid attenuates organic dust-induced airway inflammation.

Workers exposed to organic dusts from concentrated animal feeding operations (CAFOs) are at risk for developing airway inflammatory diseases. Available preventative and therapeutic measures for alleviating dust-induced lung disease are inadequate. Because omega-3 fatty acids can mitigate inflammatory processes, we aimed to determine whether nutritional supplementation with the omega-3 fatty acid docosahexaenoic acid (DHA) could reduce the airway inflammatory consequences of exposures to organic dust. Aqueous extracts of organic dusts from swine CAFOs (ODE) were utilized. In DHA-pretreated human bronchial epithelial cells, lung fibroblasts, monocyte cell cultures, and precision-cut murine lung slices, we found that DHA pretreatment dose-dependently decreased ODE-induced inflammatory cytokine production. To determine the in vivo significance of DHA, C57BL/6 mice were orally administered DHA for seven days prior to treatment with intranasal ODE or saline inhalations. Animals treated with 2 mg DHA demonstrated significant reductions in ODE-induced bronchial alveolar lavage neutrophil influx and pro-inflammatory cytokine/chemokine production compared to mice exposed to ODE alone. Collectively, these data demonstrate that DHA affects several lung cells to reduce the airway inflammatory response to organic dust exposures. Dietary supplementation with DHA may be an effective therapeutic strategy to reduce the airway inflammatory consequences in individuals exposed to agriculture dust environments

Maresin-1 reduces the pro-inflammatory response of bronchial epithelial cells to organic dust

BACKGROUND: Exposure to organic dust causes detrimental airway inflammation. Current preventative and therapeutic measures do not adequately treat resulting disease, necessitating novel therapeutic interventions. Recently identified mediators derived from polyunsaturated fatty acids exhibit anti-inflammatory and pro-resolving actions. We tested the potential of one of these mediators, maresin-1 (MaR1), in reducing organic dust-associated airway inflammation. METHODS: As bronchial epithelial cells (BECs) are pivotal in initiating organic dust-induced inflammation, we investigated the in vitro effects of MaR1 on a human BEC cell line (BEAS-2B). Cells were pretreated for 1 hour with 0–200 nM MaR1, followed by 1–24 hour treatment with 5% hog confinement facility-derived organic dust extract (HDE). Alternatively, a mouse lung slice model was utilized in supportive cytokine studies. Supernatants were harvested and cytokine levels determined via enzyme-linked immunosorbent assays. Epithelial cell protein kinase C (PKC) isoforms α and ϵ, and PKA activities were assessed via radioactivity assays, and NFκB and MAPK-related signaling mechanisms were investigated using luciferase vector reporters. RESULTS: MaR1 dose-dependently reduced IL-6 and IL-8 production following HDE treatment of BECs. MaR1 also reduced HDE-stimulated cytokine release including TNF-α in a mouse lung slice model when given before or following HDE treatment. Previous studies have established that HDE sequentially activates epithelial PKCα and PKCϵ at 1 and 6 hours, respectively that regulated TNF-α, IL-6, and IL-8 release. MaR1 pretreatment abrogated these HDE-induced PKC activities. Furthermore, HDE treatment over a 24-hour period revealed temporal increases in NFκB, AP-1, SP-1, and SRE DNA binding activities, using luciferase reporter assays. MaR1 pretreatment did not alter the activation of NFκB, AP-1, or SP-1, but did reduce the activation of DNA binding at SRE. CONCLUSIONS: These observations indicate a role for MaR1 in attenuating the pro-inflammatory responses of BECs to organic dust extract, through a mechanism that does not appear to rely on reduced NFκB, AP-1, or SP-1-related signaling, but may be mediated partly through SRE-related signaling. These data offer insights for a novel mechanistic action of MaR1 in bronchial epithelial cells, and support future in vivo studies to test MaR1’s utility in reducing the deleterious inflammatory effects of environmental dust exposures

Asymmetric dimethylarginine blocks nitric oxide-mediated alcohol-stimulated cilia beating.

The airway epithelium is exposed to alcohol during drinking through direct exhalation of volatized ethanol from the bronchial circulation. Alcohol exposure leads to a rapid increase in the cilia beat frequency (CBF) of bronchial epithelial cells followed by a chronic desensitization of cilia stimulatory responses. This effect is governed in part by the nitric oxide regulation of cyclic guanosine and adenosine monophosphate-dependent protein kinases (PKG and PKA) and is not fully understood. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is implicated in the pathogenesis of several pulmonary disorders. We hypothesized that the inhibition of nitric oxide synthase by ADMA blocks alcohol-stimulated increases in CBF. To test this hypothesis, ciliated primary bovine bronchial epithelial cells (BBEC) were preincubated with ADMA (100  µM) and stimulated with 100 mM ethanol. CBF was measured and PKA assayed. By 1 hr, ethanol activated PKA, resulting in elevated CBF. Both alcohol-induced PKA activation and CBF were inhibited in the presence of ADMA. ADMA alone had no effect on PKA activity or CBF. Using a mouse model overexpressing the ADMA-degrading enzyme, dimethylarginine dimethylaminohydrolase (DDAH), we examined PKA and CBF in precision-cut mouse lung slices. Alcohol-stimulated increases in lung slice PKA and CBF were temporally enhanced in the DDAH mice versus control mice

MyD88 in lung resident cells governs airway inflammatory and pulmonary function responses to organic dust treatment.

Inhalation of organic dusts within agriculture environments contributes to the development and/or severity of airway diseases, including asthma and chronic bronchitis. MyD88 KO (knockout) mice are nearly completely protected against the inflammatory and bronchoconstriction effects induced by acute organic dust extract (ODE) treatments. However, the contribution of MyD88 in lung epithelial cell responses remains unclear. In the present study, we first addressed whether ODE-induced changes in epithelial cell responses were MyD88-dependent by quantitating ciliary beat frequency and cell migration following wounding by electric cell-substrate impedance sensing. We demonstrate that the normative ciliary beat slowing response to ODE is delayed in MyD88 KO tracheal epithelial cells as compared to wild type (WT) control. Similarly, the normative ODE-induced slowing of cell migration in response to wound repair was aberrant in MyD88 KO cells. Next, we created MyD88 bone marrow chimera mice to investigate the relative contribution of MyD88-dependent signaling in lung resident (predominately epithelial cells) versus hematopoietic cells. Importantly, we demonstrate that ODE-induced airway hyperresponsiveness is MyD88-dependent in lung resident cells, whereas MyD88 action in hematopoietic cells is mainly responsible for ODE-induced TNF-α release. MyD88 signaling in lung resident and hematopoietic cells are necessary for ODE-induced IL-6 and neutrophil chemoattractant (CXCL1 and CXCL2) release and neutrophil influx. Collectively, these findings underscore an important role for MyD88 in lung resident cells for regulating ciliary motility, wound repair and inflammatory responses to ODE, and moreover, show that airway hyperresponsiveness appears uncoupled from airway inflammatory consequences to organic dust challenge in terms of MyD88 involvement

Race Inclusion Diversity & Equity: The RIDE Report

The Race, Inclusion, Diversity & Equity (RIDE) Report is presented as a resource to augment, strengthen and support existing and future diversity, inclusion & equity efforts at the University of Nebraska Medical Center (UNMC). It is organized into 7 sections and highlights the voices, perspectives and ideas of over 150 UNMC administrators, students, faculty, staff and community partners (key stakeholders).https://digitalcommons.unmc.edu/coph_disp_reports/1001/thumbnail.jp

Alcohol and Cigarette Smoke Decrease Lung Epithelial Cell Proliferation

https://digitalcommons.unmc.edu/surp2021/1019/thumbnail.jp