Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular, Cellular, and Developmental Biology

First Advisor

Horsley, Valerie

Abstract

Introduction:Delayed wound healing is a major healthcare challenge, creating a significant financial burden and impacting patient quality of life by increasing risks of infections, chronic wounds, and impaired tissue regeneration. Macrophages are key mediators in wound healing, where they transition from a pro-inflammatory to an anti-inflammatory state, balancing inflammation resolution and tissue repair. Dysregulated macrophage function, as seen in chronic wounds, leads to persistent inflammation and poor healing outcomes. Recent studies indicate that glutamine metabolism in macrophages may be crucial for this phenotypic transition by influencing gene expression and epigenetic regulation. However, the specific role of glutamine metabolism in modulating macrophage-driven wound healing remains underexplored. Methods: We used an excisional wound model on dorsal skin to create full-thickness wounds in mice. To investigate the changes in macrophage metabolism in wound healing, we utilized integrative metabolomics and transcriptomics analysis. To further assess the role of glutamine metabolism in wound healing, we utilized dietary and genetic approaches. Mice were fed either a glutamine/glutamate-deficient diet or an isocaloric control diet, and wound healing was assessed. Additionally, we generated LysMCre-expressing glutaminase knockout (Gls cKO) mice to specifically ablate glutamine metabolism in macrophages. Bulk RNA sequencing was used to examine gene expression changes, and DOGMA-seq combined with CUT&Tag assays was employed to assess chromatin accessibility and histone modifications. Results: Metabolomic and transcriptomics analyses revealed metabolic and gene expression shifts in macrophages from glycolysis to TCA cycle intermediates, particularly glutamine/glutamate pathways, at later healing stages. Mice on the Glu/Gln-deficient diet and Gls cKO mice both displayed delayed wound healing, characterized by prolonged inflammation and reduced re-epithelialization. Flow cytometry confirmed increased pro-inflammatory macrophages (CD45+CD11b+F4/80+Ly6C+) and neutrophil recruitment in Gls cKO wounds. Transcriptomic data showed upregulation of genes involved in neutrophil chemotaxis and inflammation in Gls cKO macrophages. Epigenetic profiling demonstrated hypermethylation near pro-inflammatory gene loci and increased chromatin accessibility at inflammatory markers, indicating glutamine’s role in modulating macrophage gene expression during healing. Conclusions: Our findings highlight that glutamine metabolism is essential for macrophage function during wound healing, supporting the transition from inflammation to tissue repair. Disrupted glutamine metabolism leads to sustained inflammation, impaired angiogenesis, and compromised tissue remodeling. This study underscores the potential therapeutic value of targeting macrophage glutamine metabolism to enhance wound healing, particularly in chronic, non-healing wounds. Future research should investigate the molecular pathways linking glutamine metabolism with epigenetic regulation in macrophages and explore its clinical applications in wound management.

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