Abstract
Single-cell genomics, particularly single-cell RNA sequencing (scRNA-seq), enables the comprehensive profiling of gene expression at the resolution of individual cells, uncovering cellular heterogeneity, identifying rare populations, and reconstructing developmental trajectories that are obscured in bulk analyses. My studies focused on the combination of complementary single-cell analyses and experimental approaches to dissect immune cell dynamics in the gut and brain. We first examined Crohn’s like disease of the pouch (CDP), a clinically challenging condition that exclusively affects patients who underwent ileal pouch anal anastomosis (IPAA) for ulcerative colitis (UC), by performing scRNA-seq/TCR/BCR-seq of paired ileoanal pouch and pre-pouch ileum tissues of individuals with CDP and healthy controls. We identified myeloid cell as the pathogenic signaling hub for chronic inflammation of the ileoanal pouch, and that the myeloid cells in CDP represent a more terminally differentiated state compared to pouchitis. We found that Th17 cells are clonally expanded in the pouch, but not the inflamed pre-pouch ileum, suggesting that pre-pouch ileitis in CDP occurs following acute-to-chronic transition of pouch inflammation. We identified shared immune and stromal inflammatory mediators between CDP, pouchitis, Crohn’s disease, and UC. Lastly, we identified endoplasmic reticulum stress as a potential biomarker for CDP diagnosis and treatment. We followed with studying perianal fistulizing Crohn’s disease (PCD) using scRNA-seq and spatial transcriptomics. We found extensive upregulation of type II interferon responses in PCD, and further show that type II interferon and TNF signal on rectal epithelial cells and potentiate their epithelial to mesenchymal transition. We identified pathogenic Th17 cells as the chief source of IFN-g in PCD, and demonstrate their co-localization with elevated interferon signaling in the fistula tract. These findings suggest the use of systemic anti-IFNg therapy in the treatment of this highly morbid complication. We then characterized macrophage populations in the choroid plexus, a critical interface between the central nervous system and systemic circulation facing continuous immunological challenges. Through high-throughput scRNA-seq, flow cytometry, lineage tracing, and imaging techniques, we identified three phenotypically and functionally distinct macrophage populations in the homeostatic choroid plexus, characterized by differential expression of CD163, MHCII, or CD9. These subsets originate from distinct combinations of primitive and definitive hematopoietic waves, occupy discrete spatial niches, and rely on different survival factors. During neuroinflammation, these macrophages orchestrated IFN responses and chemokine production, targeting effector CD8 T cells, while microglia predominantly expressed chemokines for B cells. We also identified both conserved and unique macrophage subsets within the human choroid plexus. Finally, we developed and validated novel anti-amyloid chimeric antigen receptor astrocytes (CAR-A) targeting Alzheimer’s disease, characterized by a pathological cascade initiated by amyloid accumulation and progressing to tau-mediated neurodegeneration. We engineered CAR-A constructs and validated their in vitro functionality. In vivo studies demonstrated the efficacy of two CAR-A designs in reducing amyloid plaque burden and ameliorating related pathologies in a murine amyloidosis model. Single-nucleus analysis indicated that CAR-A treatment induced a distinctive glial response towards amyloid pathology, implicating both astrocytes and microglia in amyloid clearance. These findings provide in vivo evidence supporting CAR-A-based cell or gene therapies as viable strategies for treating Alzheimer’s disease.
Committee Chair
Marco Colonna
Committee Members
Benjamin Humphreys; Gwendalyn Randolph; Sidharth Puram; Siyan Cao
Degree
Doctor of Philosophy (PhD)
Author's Department
Biology & Biomedical Sciences (Immunology)
Document Type
Dissertation
Date of Award
8-6-2025
Language
English (en)
DOI
https://doi.org/10.7936/djaf-ae64
Recommended Citation
Nguyen, Khai Mac, "Single-cell and spatial immunogenomics to dissect immune mechanisms in inflammatory bowel diseases and the central nervous system" (2025). Arts & Sciences Theses and Dissertations. 3605.
The definitive version is available at https://doi.org/10.7936/djaf-ae64