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Browsing by Author "Nordgren, Tara, advisor"

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    Mediators of mucosal integrity in the context of agriculture dust exposure
    (Colorado State University. Libraries, 2023) Chesterman, Carly Sue, author; Nordgren, Tara, advisor; Moreno, Julie, committee member; Podell, Brendan, committee member
    Inhalation of particulate matter, such as agricultural dust, can lead to lung inflammation and increased risk for chronic respiratory diseases. Agricultural workers, such as hog farmers, are constantly exposed to organic dusts from the workplace. Chronic pulmonary obstructive disease (COPD) is a type of lower respiratory disease, which results in an excessive inflammation cycle that leads to disease progression. Currently, available treatments merely treat the patient's symptoms with no effect on the prevention of disease progression. Metabolites of omega-3 fatty acids, called specialized pro-resolving mediators (SPM), can aid in inflammation resolution and promote immunity in the context of respiratory infection. IL-22 promotes mucosal immunity by regulating inflammation, inducing the production of antimicrobial peptides (AMP), and promoting epithelial repair. Mucosal surfaces in the airways are lined with epithelial cells, a mucus layer, and immune cells that act as the first line of defense against inhaled pathogens. The respiratory epithelium cells express antimicrobial peptides like beta-defensin-2 and Regenerating islet-derived protein 3 gamma (Reg3g); this expression can be stimulated by IL-22. In this study, we explored the effects of omega-3 fatty acids after organic dust exposure using a fat-1 transgenic mouse model, which represents the ideal ratio between omega-3 and omega-6 fatty acids. To further explore the impacts of IL-22 on AMP expression after organic dust exposure, a whole-body IL-22 knockout mouse model also was used in this study. Since omega-3 fatty acids and IL-22 promote inflammation resolution, we studied their impact on mucosal immunity and epithelial repair following repetitive challenges with extracts of organic dust in vivo. Wildtype, IL-22 knockout, and fat-1 mice were exposed by intranasal installations five times a week for 3 weeks. Lung tissue from mice exposed to either organic dust or saline were obtained and evaluated for AMP and wound repair markers. To evaluate the impact of IL-22 on AMP expression in the context of organic dust exposure, immunofluorescence (IF) staining, enzyme-linked immunosorbent assays (ELISAs), and RT-qPCR arrays were used. IF staining was done to assess beta-defensin-2 expression within the bronchial epithelial cells in IL-22 knockout and wildtype mice. While trends of positive staining for beta-defensin-2 were observed, no statistical significance was found. ELISAs were performed to assess for Reg3g expression in mice lung tissue; concentrations were found to be present in both the wildtype and IL-22 knockout models after saline and dust exposure. To assess for markers of wound repair in IL-22 knockout and wildtype mice models, a custom RT2 profiler PCR array was ordered to detect gene expression of different antimicrobial peptides, anti- and pro-inflammatory markers, and wound repair markers. The findings were as follows. Expression of AMPs—specifically S100A8 and S100A9—indicates a decrease among organic dust-exposed groups. The expression of wound repair markers CCL7 and ITGA3 exhibited decreases in the context of organic dust exposure. Expression of the anti-inflammatory marker MIF also exhibited a decrease among dust-exposed groups. To evaluate the impact of omega-3 fatty acids on AMP expression in the context of organic dust exposure, lung tissue from fat-1 and wildtype mice were stained for Reg3g expression. While trends of positive staining for Reg3g were observed, no statistical significance was found. IL-22 signaling exhibited trends of increased expression of AMP and pro-resolution mediators of mucosal integrity in the context of chronic dust exposure. Further studies should be conducted to determine the effects of omega-3 fatty acids on AMP expression in the context of organic dust exposure. These findings can be utilized to develop new treatment strategies for lung disease that focus on pro-resolution rather than solely anti-inflammatory methods.
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    Resolvin D1 modulates the pulmonary immune response to agriculture dust exposure
    (Colorado State University. Libraries, 2023) Threatt, Alissa Nicole, author; Nordgren, Tara, advisor; Schaffer, Joshua, committee member; Schenkel, Alan, committee member
    Occupational exposure to agriculture dust causes a variety of acute and chronic pulmonary diseases including allergies, asthma, chronic obstructive pulmonary disease (COPD) and organic dust toxic syndrome (ODTS). These diseases have high impact on the healthcare system and limited treatments with variable efficacy. In addition, workers often display low compliance with required workplace personal protective equipment (PPE), increasing their risk for developing these diseases. Therefore, the development of new pharmacological interventions is critical to alleviate the burden on the healthcare system and improve the quality of life for patients who will inevitably develop occupational-related pulmonary diseases. Interleukin-22 (IL-22) is a cytokine in the anti-inflammatory interleukin-10 (IL-10) family of cytokines that has demonstrated a protective role in murine models of acute and chronic lung injury. It has been described as being exclusively produced by lymphocytes, however methodological limitations of the primary cited study restricted the exploration of other cell types as producers of IL-22. Upregulation of this cytokine by pharmacological means could prove beneficial for delaying the progression of occupational chronic pulmonary diseases. Omega-3 fatty acids and their metabolites have well-documented anti-inflammatory and pro-resolution functions in chronic pulmonary diseases and have been implicated in the induction of IL-22. Omega-3 fatty acids have shown overwhelming evidence in being anti-inflammatory by their function as substrates for the production of specialized pro-resolving mediators (SPMs), lipid metabolites that signal immune cells to transition to a resolution and repair state following inflammation. Resolvin D1 (RvD1), a metabolite of the omega-3 fatty acid docosahexaenoic acid (DHA), has shown to have anti-inflammatory and protective functions in a murine acute lung injury model. To evaluate the source of IL-22 in the pulmonary response to agricultural dust, mouse alveolar macrophages were co-exposed to 1% hog dust extract (DE) collected from swine confinement facilities in the Midwest US and treated with either 10 nM or 100 nM RvD1. Cells were incubated for up to 24 hours, supernate was collected at the desired timepoint, and enzyme-linked immunosorbent assays (ELISAs) were performed to assess protein expression. Cells were also lysed to determine intracellular IL-22 protein concentrations. Cells exposed to DE exhibited increased pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) as well as increased IL-10 and IL-22 production, demonstrating macrophages as a source of IL-22 in the immune response to organic dust. Cells exposed to DE and treated with RvD1 demonstrated significant decreases in IL-6 and TNF-α and increases in IL-10. To determine the efficacy of RvD1 as an inducer of IL-22, and as a potential treatment for organic dust-induced lung injury, C57BL/6 (WT) and full-body IL-22 knock-out (KO) mice were intranasally instilled (IN) with 12.5% DE 5 days/week for 3 weeks and injected intraperitoneally (IP) with 250 ng RvD1 once per week. Animals were allowed to recover for 5 hours or 3 days before sacrifice where bronchoalveolar lavage fluid (BALF) was collected for cytokine and cellular infiltrate evaluation to determine the role of RvD1 in the reduction of the immune response to organic dust exposure. BALF cytokines exhibited significant increases in the production of IL-10 in KO mice exposed to DE and treated with RvD1 with a 3-day recovery. Cellular infiltrates demonstrated decreased neutrophil infiltration and increased lymphocyte recruitment in KO mice exposed to DE after a 3-day recovery and further significant decreases in mice treated with RvD1 with a 3 day recovery. The data support the production of IL-22 by alveolar macrophages and its induction by RvD1. They also demonstrate the effects of RvD1 on the pulmonary immune response to agriculture dust and as a potential therapeutic for organic dust-induced chronic pulmonary diseases.