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Elevated O-GlcNAcylation promotes an enhanced immune response: When the SARs-CoV-2 virus emerged in late 2019, the world was faced with a challenge of simultaneously treating, fighting and understanding a novel pathogen. While little was initially known about the biology of this novel virus, it became immediately clear that patients with certain underlying diseases, like cancer, cardiovascular disease, and diabetes were at increased risk of developing severe COVID-19. This observation underscored the role a patient’s immune response played in disease risk. One common feature of these underlying diseases is that elevated O-GlcNAcylation has been described in all. Thus, I hypothesized that chronic hyper-O-GlcNAcylation, as seen in diseases like diabetes, predisposes patients to an altered immune response to pathogens.
a. Elevated O-GlcNAcylation promotes inflammatory responses in macrophages: Upon stimulation, inflammatory macrophages are characterized by their glycolytic metabolism. Studies have found that glucose availability impacts pro-inflammatory polarization, regulation of key inflammatory modulators like inducible Nitric Oxide Synthase (iNOS), and expression of inflammatory cytokines. For this reason, understanding how changes in the nutrient-sensing posttranslational modification, O-GlcNAcylation, impacts the inflammatory response of macrophages is of great interest with clinical significance. To model chronically elevated O-GlcNAc, I analyzed classically polarized bone marrow derived macrophages (BMDMs) from mice in which Oga had been deleted within the hematopoietic lineage (OgaVav-Cre). OGA deficient macrophages had significantly elevated gene expression of key pro-inflammatory cytokines like Il-6. Focusing on iNOS, we found that nitric oxide (NO) release was diminished in Oga knockout cells. Further, elevated O-GlcNAc impacted Il-6 expression through the iNOS pathway, as inhibition of iNOS in wildtype cells using the inhibitor L-NIL increased Il-6 expression similar to OGA deficiency alone, but had no further effect on the Oga knockout macrophages. WGA pull-down analysis and in vitro OGT assays provided evidence that iNOS was an OGT target. Together, these data indicated that through interaction with iNOS, elevated O-GlcNAc dampens NO production and contributes to deregulation of specific pro-inflammatory cytokines. Thus, balanced O-GlcNAc cycling is a key component for maintaining macrophage homeostasis. This study was published in Frontiers in Immunology in 2022.
b. Elevated O-GlcNAcylation enhances T cell activation: To further understand the impact of chronically elevated O-GlcNAc on immunity, I have been assessing CD3/CD28 mediated in vitro T cell activation using splenocytes from OgaVav-Cre mice, as well as mice in which Oga has been deleted only in B and T cell lineages (OgaCD2-Cre). Preliminary analysis indicated that Oga knockout T cells respond with higher levels of late stage activation markers than their wildtype counterparts, suggesting an enhanced T cell response. This work is currently ongoing, and I have recently recruited a post baccalaureate fellow, Kaylee Philbrick, to contribute to this project.
Significance/impact: These studies continue to define the impact of excess O-GlcNAc on an enhanced immune response to a pathogen. Defining elevated O-GlcNAc as a biomarker for immune deregulation will help to better define at risk populations for severe disease after infection.
Current project: Development of next generation artificial bioorthogonal precursors to study levels and interaction partners of GlcNAc-containing glycoconjugates.
Description: Changes in GlcNAc-containing glycoconjugates are associated with a variety of human diseases, such as cancer, diabetes, and neurodegenerative diseases, but the mechanistic details linking altered glycosylation to disease pathology remain poorly understood. Recent efforts in the development of methods to study biomolecules in their native environment have unlocked the door of bioorthogonal chemistry. Functional group modifications around the sugar hydroxyl groups are tolerated by the biosynthetic pathways and transform them into the corresponding sugar-nucleotide donors. My work is on the development of accessible and effective methods to monitor the levels and interaction partners of GlcNAc-containing glycoconjugates. N-acetylglucosamine (GlcNAc) represents a critical link between cellular metabolism and glycoconjugates, such as O-GlcNAc and N-linked glycans, which regulate an important and ubiquitous cell signaling paradigm, as well as substrate function, localization, and stability. My goal is to design and synthesis of easy-to-use bioorthogonal tools that can be used by any biomedical researcher to track levels and interaction partners of GlcNAc-containing glycoconjugates, and then to use them to understand their roles in human health and disease. My work prioritizes approaches that are simple to implement and makes use of “off-the shelf” reagents and procedures to develop and engineer bioorthogonal next-generation artificial metabolic reporters capable of specifically labeling GlcNAc-containing glycoconjugates and then applying them to pathogenic disease (metabolic dysfunction, neurodegeneration, and cancer) treatment.