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CF intestinal pathology involves both a disease milieu and absence of CFTR from epithelial cells

Hao, Shuyu
http://rave.ohiolink.edu/etdc/view?acc_num=case1562160689163065

Abstract Details

2019, Doctor of Philosophy, Case Western Reserve University, Genetics.
Cystic fibrosis (CF) is a monogenic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Lung disease is the major cause of CF-related mortality, and pulmonary decline is largely driven by inflammation. Pathogenesis of CF-associated inflammation continues to be debated, with data suggesting that the inflammatory state is due to infection and other studies supporting a model in which CF cells are inherently hyperinflammatory. This thesis demonstrates that both the absence of CFTR function and inflammatory stimuli contribute to the inflammatory profile and uses intestinal model systems to study these processes. We propose that the absence of CFTR results in a different epigenetic profile of the genes, including inflammatory mediators, than in cells with functional CFTR. We hypothesize that chromatin modifications respond to CFTR’s absence and that this remodeling influences the transcriptional status of many genes. In vitro, stimulation by inflammatory mediators reveals a transcriptome difference between cells with and without functional CFTR and appears to reflect stimuli in vivo, where infection evokes the differential expression of genes as a consequence of CFTR status. Caco2 cells were modified to study CF-related inflammation in the intestines. Multiple CFTR-null clonal lines were generated by CRISPR/Cas9 technology. Characterization of the cells lines showed that CFTR protein and activity were completely absent while they maintain other epithelial properties, such as tight junctions and response to inflammatory stimuli. Baseline expression of cytokines was not different between the lines regardless of CFTR genotype. With TNFα stimulation, clonal lines lacking CFTR produce more CXCL8 mRNA compared to lines with functional CFTR. Transcriptome analyses reveal that there are many differentially expressed genes between CFTR+ and CFTR- cells after stimulation, but also a large number of genes that are different under baseline conditions. These data indicate that absence of CFTR itself changes gene expression patterns in the cells, but also the pathways and specific genes that are differentially expressed are context-dependent. For example, many genes that were not differentially expressed under basal conditions were significantly different between CFTR+ and CFTR- cells in response to TNFα stimulation. Our results suggest a model in which CFTR and environment interact in such a way as to make CF cells behave differently in response to stimuli. Further investigation was done on tissues and cells from CF and non-CF animals. Transcriptome analyses showed that simply knocking out Cftr causes immune response changes in CF intestinal tissue and cells. Also, metabolic pathways are directly affected by Cftr’s absence. Defense response to bacteria is decreased in CF cells and it is probably caused by dysregulation of Paneth cells in the crypts. Interestingly, besides Cftr, environmental factors also largely affect transcriptome profiles in CF metabolic tissues. In mouse liver and skeletal muscle tissue, there is no detectable Cftr expression but thousands of differentially expressed genes showed up between CF and non-CF tissues. This study, and the tools developed for it, provide a tractable combination of in vitro and in vivo systems to investigate interactions between gene and environment not feasible to carry out in humans. It also suggests that the epigenome may be a therapeutic target for CF.
Mitch Drumm (Advisor)
Craig Hodges (Committee Chair)
Zhenghe Wang (Committee Member)
Tracy Bonfield (Committee Member)
Yan Li (Committee Member)
Biomedical Research

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Citations

  • Hao, S. (2019). CF intestinal pathology involves both a disease milieu and absence of CFTR from epithelial cells [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1562160689163065

    APA Style (7th edition)

  • Hao, Shuyu. CF intestinal pathology involves both a disease milieu and absence of CFTR from epithelial cells. 2019. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1562160689163065.

    MLA Style (8th edition)

  • Hao, Shuyu. "CF intestinal pathology involves both a disease milieu and absence of CFTR from epithelial cells." Doctoral dissertation, Case Western Reserve University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=case1562160689163065

    Chicago Manual of Style (17th edition)

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This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.