Browsing by Author "Podell, Brendan, committee member"

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Open Access
A comprehensive approach to modeling musculoskeletal aging and injury: an emphasis on Nrf2-related pathogenesis
(Colorado State University. Libraries, 2021) Andrie, Kendra M., author; Santangelo, Kelly S., advisor; Hamilton, Karyn, committee member; Goodrich, Laurie, committee member; Podell, Brendan, committee member; Muñoz Gutiérrez, Juan, committee member; Miller, Benjamin, committee member
Osteoarthritis (OA) is a degenerative joint disease that affects over 730 million people globally, over 30 million Americans, and is the leading cause of disability in adults. The underlying pathogenesis is multifactorial and largely undetermined, with a variety of cellular pathways and risk factors contributing to disease onset and progression. The crux of this work is that downregulation in nuclear factor erythroid-2 related factor-2 (Nrf2)-signaling in musculoskeletal tissue serves as a central driver for persistent low-grade inflammation, dysregulation of redox homeostasis, mitochondrial dysfunction, and protein dyshomeostasis, all of which contribute to OA progression. To explore the role of this pathway in OA, we utilized the Hartley OA-prone guinea pig model, which develops naturally occurring idiopathic disease with pathology that mimics human disease. My global hypothesis is supported by preliminary data that demonstrates that aging Hartley guinea pig knee joint tissues have decreased expression of Nrf2 mRNA and protein, which coincides with disease onset and remains decreased throughout OA progression. We investigated the utility of a novel nutraceutical and Nrf2-activator in delaying both the onset and progression of idiopathic OA in this model. The ultimate goal of this work is to (1) identify key molecular pathways involved in the etiopathogenesis of OA, with a particular focus on the contribution of the Nrf2 pathway; (2) investigate the utility of a novel nutraceutical and Nrf2-activator in delaying the onset and/or progression of OA in the Hartley guinea pig, and (3) examine the effects of Nrf2-activation on long bone strength. The inclusion of a musculoskeletal condition beyond OA was also pursued; as such, the clinical and histologic manifestations of a novel rectus femoris myotendinous junction injury model was characterized in rats. Ultimately, this work seeks to advance the understanding of musculoskeletal aging and injury through the analysis of key structural and functional outcome measures to further develop appropriate therapeutic targets for disease prevention and treatment.
Open Access
Host directed therapy targeting M. tuberculosis infected macrophages
(Colorado State University. Libraries, 2016) Lakey, Natalie, author; Basaraba, Randall, advisor; Podell, Brendan, committee member; Perera, Rushika, committee member; Chicco, Adam, committee member
With the rise of drug resistant strains of Mycobacterium tuberculosis (Mtb) and lags in antimicrobial drug development, it is imperative to explore alternative methods of treatment through host-directed adjunct therapies. Hallmarks of Mtb infection are altered host cell glucose metabolism and non-diabetic hyperglycemia, which increase disease severity and bacterial burden. This can be targeted using a combination of metformin and 2-deoxyglucose (2DG) to lower systemic blood glucose and increase metabolic stress in infected macrophages to induce apoptotic cell death, enabling Mtb clearance and antigen presentation to activate cell-mediated immune responses. We hypothesized that bacterial survival is aided by glycolysis-dependent macrophages, which can be targeted using a combination of metformin and 2DG to strengthen host immune responses. Using an in vitro model of guinea pig bone marrow derived macrophages under normal and high glucose conditions, we determined that both basal respiration and glycolytic activity increased after infection. When singly treated, metformin inhibited basal respiration while increasing glycolysis while 2DG inhibited both processes. In combination metformin and 2DG treatment inhibited basal respiration more effectively than metformin treatment alone and inhibited glycolysis as effectively as 2DG by itself. Efficacy of metformin-2DG treatment is dependent on high cellular glycolytic activity, a characteristic of granulomatous cells. Metformin-2DG treatment decreased cell survival 48 hours post-treatment by increasing apoptotic cell death and decreased Mtb survival more effectively than metformin or 2DG alone. We conclude that apoptotic induction of macrophages by metformin-2DG treatment may be a viable adjunct treatment to antimicrobial drugs to reduce bacterial burden and increase an effective host adaptive immune response.
Open Access
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.
Open Access
Metabolic interventions in the treatment of Mycobacterium tuberculosis infection
(Colorado State University. Libraries, 2023) Ackart, David, author; Basaraba, Randall, advisor; Podell, Brendan, committee member; Chicco, Adam, committee member
Tuberculosis remains a global threat. For the first time in over a decade, there was an increase in deaths in 2021. Current antimicrobial treatments are lengthy and costly, which leads to non-compliance. Host-directed therapies have emerged as a possible adjuvant to antimicrobial treatment. It has become clear that immune cell function and metabolic pathways are intertwined and a target for therapy. Previously, metformin, a partial inhibitor of complex I, was associated with decreased disease burden in the guinea pig model. Based on these findings, we hypothesized that limiting glycolysis would reduce lesion burden and bacterial viability. To test our hypothesis, we infected guinea pigs for thirty days and then administered metformin or 2-deoxy-glucose alone, and in combination. After 90 days of infection, histological analysis revealed increased healing in the combination treatment. To determine the effect of treatment on cellular metabolism, we evaluated treatments in guinea pig bone marrow-derived macrophages. We found that the combination treatment reduced bacterial viability, prevented mitochondrial damage, and increased apoptosis, which helps support in vivo findings. These findings suggest that the metabolic switch of immune cells to glycolysis alone is insufficient to control Mycobacterium tuberculosis infection; it is essential, as is oxidative phosphorylation.
Open Access
Mycobacterium tuberculosis – mediated modulation of host macrophage metabolism in the granuloma microenvironment
(Colorado State University. Libraries, 2020) Kiran, Dilara, author; Basaraba, Randall, advisor; Podell, Brendan, committee member; Obregon-Henao, Andres, committee member; Olver, Christine, committee member; Chicco, Adam, committee member
Mycobacterium tuberculosis (Mtb) is the leading cause of death by an infectious agent, and tuberculosis (TB) disease continues to be a prominent global health concern. Infection with Mtb incites granulomatous inflammation, chronic antigen stimulation, and the development of granuloma lesions. These lesions compress tissue architecture in a way that reduces blood supply and creates central regions of hypoxia. Complex lesion pathology, multi-drug resistance of Mtb, co-morbidities with other endemic diseases, the lack of an effective vaccine, and slow drug development pipelines have hindered progress in the field. Researchers have worked to combat these difficulties through their exploration of host-directed therapeutic strategies, which aim to better equip the host immune system to respond to Mtb infection, with a focus on immunometabolism as a target pathway. The metabolism of host macrophages plays a role in modulating disease pathogenesis, with a metabolic switch from oxidative phosphorylation to glycolysis characterizing Mtb infected macrophages. This metabolic switch is primarily regulated at the transcriptional level by hypoxia inducible factor-1α (HIF-1α), which regulates the cellular response to hypoxic stressors encountered within the chronic granuloma lesion microenvironment. Downstream impacts of HIF-1α activation include increased glycolysis, increased lactate production, and increased lactate transport. HIF-1α becomes stable and undergoes its transcriptional activity in conditions of low oxygen, as a result of prolyl hydroxylase (PHD) inhibition. Additionally, hypoxia-independent factors interfere with PHD leading to HIF-1α stabilization, including iron chelation. Bacteria, such as Mtb, have developed iron chelating siderophores to sequester iron from host cells, and knocking-out these iron chelators has been demonstrated to reduce stable HIF-1α activation. As a result, we hypothesized that the Mtb siderophore, mycobactin, plays a role in driving stabilization of HIF-1α during early infection, prior to the development of hypoxic lesion microenvironments. This would serve as a pathogen-driven mechanism that would support macrophage adaptation to hypoxia later during disease progression, and thus develop an Mtb survival niche. Using purified iron chelators deferoxamine (DFO) and mycobactin J (MbtJ), we demonstrated that treated CD1 mouse bone marrow derived macrophages (BMDMs) increase HIF-1α via Western Blot and potently increase glycolytic metabolism as demonstrated by Seahorse Extracellular Flux Analysis. Additionally, the use of mycobactin synthase K (mbtK) knock-out, complement, or wild-type H37Rv strains of Mtb demonstrated the role that mycobactin plays in the metabolic response of macrophages in vitro, having a significant impact on oxidative metabolism. Hypoxia-independent mechanisms of HIF-1α activation by mycobactin may be a critical pathway through which Mtb drives macrophages toward a phenotype conducive for bacterial survival. Lactate produced as a result of increased glycolytic metabolism during infection may also play an important role as a metabolic intermediate and as a signaling molecule during Mtb infection. Metabolic symbioses exist in multiple systems between highly glycolytic, hypoxic cells and more oxidative, normoxic cells, wherein glycolytic cells uptake glucose, convert glucose to lactate via lactate dehydrogenase (LDHA) and export lactate in large amounts via monocarboxylate transporter 4 (MCT4). Normoxic cells import lactate via monocarboxylate transporter 1 (MCT1) and convert it back to pyruvate via lactate dehydrogenase B (LDHB) and utilize lactate-derived pyruvate to fuel mitochondrial respiration. This preserves glucose for hypoxic cells which rely heavily on glycolysis for metabolic survival. While this type of lactate shuttle has been demonstrated to regulate the tumor microenvironment, it has yet to be explored within the context of the similar TB granuloma microenvironment. As a result, we explored the role of a lactate shuttle within Mtb infection by detecting lactate in guinea pig plasma, detecting lactate shuttle components within guinea pig granuloma lesions, and by using the LDHA inhibitor sodium oxamate and the MCT1 inhibitor α-Cyano-4-hydroxycinnamic acid (α-CHC), both of which are commercially available. We showed that Mtb infection significantly increases lactate on both a systemic and cellular level. We successfully demonstrated that LDH and MCT inhibition augments metabolism in macrophages by blocking glycolysis and decreasing mitochondrial spare capacity. Through in vitro Mtb infection models, we were able to show that inhibitor treatment can reduce the amount of lactate accumulated. These studies demonstrated proof of concept for the role of a lactate shuttle in modulating macrophage metabolism during Mtb infection and maintaining infection dynamics within the granuloma microenvironment. Overall, the research presented herein seeks to understand the ways in which Mtb infection drives host macrophages to alter their metabolic phenotype in a way that promotes Mtb survival and contributes to disease pathogenesis. A better understanding of the interactions which occur at the host-pathogen interface will provide important insight for the development of host-directed therapeutic strategies which will better equip host cells to combat Mtb infection.
Open Access
The role of prion protein glycosylation in prion propagation
(Colorado State University. Libraries, 2018) Walker, Deandra Leigh, author; Telling, Glenn, advisor; Bamburg, James, committee member; Podell, Brendan, committee member; Zabel, Mark, committee member
Transmissible Spongiform Encephalopathies (TSEs) are a group of neurodegenerative diseases that affect humans and animals alike. TSEs are caused by the accumulation of a disease producing isoform referred to as PrPSc that results from the misfolding of the normal cellular prion protein PrPC. The pathological outcomes of TSEs include amyloid plaque build-up and spongiform degeneration in the brain of infected hosts. Clinical signs of prion disease can vary between TSEs, but often include neurologic impairment that is subtle in onset and tends to progress slowly. Prion diseases are relatively recently discovered and have sparked much controversy due to the scientific findings that directly challenge some of the most well established scientific dogmas. Among these is that the infectious agent responsible for the transmission of TSEs is proteinacious in nature and devoid of the nucleic acids present in pathogens like viruses and bacteria. As a result of this hypothesis, both PrPC and PrPSc share the same amino acid sequence in the host. Therefore, central to our understanding of the prion hypothesis is to recognize the structural differences between PrPC and PrPSc. PrPC has been proven to include three α-helices and two, short β-pleated sheets whereas PrPSc consists of high β-sheet content, aggregates in the presence of detergents, and is resistant to protease treatment. These characteristics of PrPSc have inhibited researchers to successfully examine the abnormal isoform in high-resolution structural studies. Therefore, an alternative means of distinguishing PrPC and PrPSc is necessary. Since then, several groups have created monoclonal antibodies (mAbs) that differentiate between infectious prion protein (PrP) aggregates. Two such mAbs, PRC5 and PRC7 were the first mAbs discovered in which the involvement of individual residues in functional, discontinuous, and conformationaly dependent epitopes was studied. Of these antibodies, PRC7, is dependent on N-linked glycosylation at mono-1 of the prion protein and specifically binds to the infected isoform of PrP. Therefore, we hypothesized that an underglycosylated form of PrP is preferentially generated during prion replication in the infected host. In this body of work, we have systemically ablated mono-1, one of the two N-linked glycan attachment sites on the murine prion protein to address the role of underglycosylation in prion propagation at N180 and at S/T182 of the consensus sequence by mutating N or S/T to each of the other 19 amino acids individually. Here we present novel evidence showing the effects of underglycosylation in prion propagation of prion isolates RML, 22L, 139A, and mCWD. These preliminary data demonstrate the importance of post-translational differences between PrPC and PrPSc which represent a fundamental, unresolved aspect of the prion hypothesis.