Intoxicating Interactions: The Impacts of Chronic Alcohol Use and SARS-CoV-2 Infection on Airway Epithelial Cells Público
Easley, Kristen Fowler (Summer 2023)
Abstract
The airway epithelial barrier is the first line of defense against environmental and biological insults to the lung. To accurately study how these insults, in addition to airway diseases, affect epithelial cell function, we must develop in vitro cell culture systems that best mimic the in vivo microenvironment. We created a differentiation medium with physiologic glucose levels (150mg/dL) called Emory-ALI (E-ALI) that encouraged differentiation of non-diseased and CF respiratory epithelial cells and allowed for insulin-stimulated glucose uptake and metabolic analysis. We used this improved system to examine whether Alcohol Use Disorder (AUD) was a risk factor for a more severe outcome in response to SARS-CoV-2 infection. We examined early responses to infection using cultured differentiated bronchial epithelial cells derived from brushings obtained from people with AUD or without AUD. We found that AUD cells had a significant decrease in barrier function up to 72 h after infection, while non-AUD cells increased their barrier function. AUD cells secreted more pro-inflammatory cytokines during the 72 h infection time course and displayed increased basolateral secretion compared to non-AUD cells. RNA-seq analysis revealed 164 differentially expressed genes between AUD and non-AUD cells, and suggested that AUD cells adapted an inflammatory, epidermal gene expression profile. To further determine how chronic alcohol use impacts the airway epithelial barrier, I utilized a variety of bronchial epithelial cell sources. I found that ethanol-treated airway cells had a decrease in barrier function during differentiation but had similar barrier function to untreated cells once differentiation into a mucociliary monolayer was complete. Interestingly, differentiation of AUD and non-AUD cells revealed a correlation between patient age and unjamming, a phenomenon where cells undergo aberrant collective cell migration. Together, these data underscore the sensitive yet resilient nature of airway epithelial cells in response to environmental and biological insults and highlight the need to further study the impacts of glucose levels, chronic alcohol use and SARS-CoV-2 infection on the airway epithelium.
Table of Contents
Abstract ............................................................................................................................... iv
Acknowledgements .............................................................................................................. vi
List of Figures ....................................................................................................................... xi
List of Tables ...................................................................................................................... xiii
Abbreviations ..................................................................................................................... xiv
Chapter 1: Introduction .......................................................................................................... 1
1.1 Lung Anatomy and Physiology ..................................................................................................1
1.1.1 The Airway Epithelium................................................................................................................................. 2
1.2 The Apical Junctional Complex ..................................................................................................5
1.2.1 Tight Junctions ............................................................................................................................................. 5
1.2.2 Adherens Junctions ..................................................................................................................................... 6
1.3 Chronic Alcohol Use and the Lung .............................................................................................7
1.3.1 Susceptibility to infections .......................................................................................................................... 7
1.3.2 Oxidative Stress ......................................................................................................................................... 10
1.3.3 Barrier Dysfunction .................................................................................................................................... 11
1.4 Scope of Dissertation .............................................................................................................. 13
References ................................................................................................................................... 16
Chapter 2: A medium composition containing normal resting glucose that supports differentiation of primary human airway cells ...................................................................... 32
2.1 Abstract ................................................................................................................................. 32
2.2 Introduction ........................................................................................................................... 33
2.3 Material and Methods ............................................................................................................ 36
2.3.1 Donor consent ........................................................................................................................................... 36
2.3.2 Tracheal epithelial cell isolation ................................................................................................................ 36
2.3.3 Bronchial epithelial cell isolation............................................................................................................... 37
2.3.4 3T3 Fibroblast feeder cell preparation ...................................................................................................... 38
2.3.5 Airway epithelial cell expansion ................................................................................................................ 38
2.3.6 Airway epithelial cell differentiation ......................................................................................................... 39
2.3.7 Cell stock cryopreservation and use .......................................................................................................... 41
2.3.8 nasal epithelial cell isolation and expansion ............................................................................................. 41
2.3.9 Immunofluorescence and imaging ............................................................................................................ 42
2.3.10 Transepithelial resistance and electrophysiology ................................................................................... 43
2.3.11 Insulin stimulation and glucose uptake ................................................................................................... 44
2.3.12 Statistics ................................................................................................................................................... 44
2.4 Results and Discussion ............................................................................................................ 45
2.4.1 Isolation and differentiation of airway epithelial cells .............................................................................. 45
2.4.2 Cells cultured in E-ALI medium show insulin-stimulated glucose uptake ................................................. 47
2.4.3 Expansion and maturation of CF nasal epithelial cells using E-ALI ........................................................... 49
2.5 Acknowledgements ................................................................................................................ 65
2.6 Author Contributions .............................................................................................................. 65 ix
References ................................................................................................................................... 66
Chapter 3: Chronic alcohol use primes bronchial cells for barrier dysfunction and altered inflammatory response during SARS-CoV-2 infection ............................................................ 73
3.1 Abstract ................................................................................................................................. 73
3.2 Introduction ........................................................................................................................... 74
3.3 Material and Methods ............................................................................................................ 76
3.3.1 Donor Consent ........................................................................................................................................... 76
3.3.2 Airway epithelial cell culture and infection ............................................................................................... 77
3.3.3 RNA-seq Analysis ....................................................................................................................................... 78
3.3.4 Transepithelial Resistance (TER)................................................................................................................ 78
3.3.5 Immunofluorescence Microscopy ............................................................................................................. 79
3.3.6 Cytokine Analysis ....................................................................................................................................... 80
3.3.7 Statistics ..................................................................................................................................................... 80
3.4 Results ................................................................................................................................... 80
3.4.1 RNA-seq reveals differentially expressed genes between non-AUD cells and AUD cells ......................... 80
3.4.2 SARS-CoV-2 infection impairs barrier function of AUD cells ..................................................................... 82
3.4.3 Differential secretion of cytokines by infected AUD and non-AUD cells .................................................. 83
3.5 Discussion .............................................................................................................................. 85
3.6 Acknowledgements .............................................................................................................. 121
3.7 Author Contributions ............................................................................................................ 121
References ................................................................................................................................. 122
Chapter 4: Characterization of alcohol-exposed airway cells ............................................... 138
4.1 Abstract ............................................................................................................................... 138
4.2 Introduction ......................................................................................................................... 139
4.3 Material and Methods .......................................................................................................... 140
4.3.1 Donor Consent ......................................................................................................................................... 140
4.3.2 Airway epithelial cell culture and EtOH treatment ................................................................................. 141
4.3.3 Rat airway epithelial cell isolation and culture ....................................................................................... 142
4.3.4 Transepithelial Resistance (TER).............................................................................................................. 143
4.3.5 Immunofluorescence Microscopy ........................................................................................................... 143
4.3.6 Statistics ................................................................................................................................................... 144
4.4 Results ................................................................................................................................. 144
4.4.1 Ethanol treatment in vitro decreases barrier function only during differentiation ................................ 144
4.4.2 Most AUD patient samples are unjammed ............................................................................................. 146
4.4.3 Some AUD patient samples show diminished JAM-A ............................................................................. 147
4.4.4 Ethanol treatment in vitro does not decrease JAM-A expression and may cause unjamming. ............. 147
4.4.5 Unjamming correlates with patient age .................................................................................................. 148
4.5 Discussion ............................................................................................................................ 148
References ................................................................................................................................. 159
Chapter 5: Conclusions and Future Directions ..................................................................... 163
5.1 Overview of findings and significance ................................................................................... 163 x
5.2 Future Directions .................................................................................................................. 165
5.2.1 Implications of hyperglycemia on airway cell function ........................................................................... 165
5.2.2 Investigating alcohol-induced modifications to the epigenome and transcriptome.............................. 166
5.2.3 Improvements to our in vitro ethanol exposure model .......................................................................... 167
5.2.4 Linking aging-related effects to unjamming in airway epithelial cells .................................................... 168
References ................................................................................................................................. 170
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