Determining the effects of hyperglycemia on WT and CF bronchial epithelial barrier function Open Access

Abstract

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Dysfunction of the CFTR protein, which primarily functions as anion channel, leads to multiorgan disease. However, the leading cause of CF mortality is respiratory failure. CFTR dysfunction in the lungs leads to inflammation, bacterial infections, as well as neutrophil recruitment and activation of neutrophils in the airways. All these factors lead to progressive lung tissue damage in CF. Highly effective modulator therapies (HEMTs) have drastically improved the life quality and increased the life expectancy of people with CF (pwCF) who are able to take them. As a result, CF co-morbidities might soon become the life-limiting factor for pwCF. The most common CF co-morbidity is cystic fibrosis related-diabetes (CFRD), affecting around half of pwCF by adulthood. CFRD is a devastating co-morbidity since it leads to more frequent pulmonary exacerbations and accelerates the rate of lung function decline. Despite the negative impact on patient health, the mechanisms driving CFRD pathophysiology are unknown. It is also unclear whether current HEMTs will have an impact on CFRD onset and disease progression. We hypothesize that hyperglycemia in the context of CF induces severe alterations in airway epithelial monolayers that prevent the formation of proper tight junctions (TJs) between cells. To investigate this hypothesis, we took several distinct yet complementary approaches. We studied the physiological changes experienced by 16HBE cells expressing either wildtype (WT) or ΔF508 CFTR (CF) in response to hyperglycemia. Further, we developed a programmable automated cell culture system (PACCS) capable of mimicking blood glucose fluctuations experienced by CFRD patients to better study CFRD in vitro. We also tested the effects of chronic hyperglycemia in a Scnn1b-Tg murine model, an in vivo model that develops similar lung pathology as seen in CF. Finally, we created a novel in vitro model to study the mechanisms driving neutrophil transmigration across airway epithelial monolayers. Using 16HBE cells, gene expression analyses identified claudin-4 (CLDN4) as a key tight junction protein dysregulated in CF cells in response to hyperglycemia. Further investigation into CLDN4 protein localization using a novel confocal microscopy technique revealed increased CLDN4 abundance at TJs in CF cells, which was further increased under hyperglycemic conditions. Treatment with HEMT reversed this trend, normalizing CLDN4 expression levels at TJs in CF cells toward WT levels. Bulk RNA sequencing showed differing transcriptional responses to hyperglycemia between WT and CF cells, thereby highlighting a few promising targets for further investigation. Culturing immortalized and primary airway epithelial cell monolayers using PACCS showed that our system could be successfully used to culture airway epithelial cells with meal-like glucose fluctuations, better mimicking CFRD-like conditions. Our studies using Scnn1b-Tg mice showed that chronic hyperglycemia aggravated the lung pathology of this mouse model, mimicking lung pathology seen in pwCF. Our novel in vitro assay to study neutrophil transmigration across epithelial monolayers showed that transmigrated PMNs differ from naïve PMNs, and that they are differentially impacted by the cell monolayer they transmigrate through. Further, pretreatment of the epithelial monolayers with high glucose media impacted PMN transmigration, with lower PMN transmigration efficiency in CF 16HBE cells. Overall, our research aims to identify mechanisms driving CFRD pathophysiology with the goal of informing the development of future therapeutics to prevent its deleterious effects.

Table of Contents

Chapter 1 – Introduction

1.1. Cystic Fibrosis: an overview

1.1.1. Discovery of the disease and the CFTR gene

1.1.2. CFTR protein structure and function

1.1.3. CFTR variants

1.2. CF pathology

1.2.1. Organs affected by CF

1.2.2. CF and respiratory bacterial pathogens

1.2.3. CF and the immune system

1.2.4. CF and metabolism

1.3. CF therapies

1.3.1. Airway clearance

1.3.2. Highly effective modulator therapies

1.3.3. New CF therapies under development

1.4. Cystic Fibrosis Related Diabetes: the main CF co-morbidity

1.4.1. What is CFRD?

1.4.2. How is CFRD different from type 1 and type 2 diabetes?

1.4.3. Potential causes of CFRD

1.4.4. CFRD diagnosis

1.4.5. CFRD treatments

1.4.6. CFRD challenges and unmet needs

1.5. In vitro models to study CFRD

1.5.1. Immortalized airway epithelial cells

1.5.2. Primary airway epithelial cells

1.6. In vivo models to study CFRD

Chapter 2 - Effects of hyperglycemia on airway epithelial barrier function in WT and CF 16HBE cells

2.1. Introduction

2.1. Methods

2.1.1. Airway epithelial cell culture

2.1.1. Short-circuit current measurements

2.1.2. Paracellular dye flux experiment

2.1.3. Quantitative reverse transcription PCR (qRT-PCR)

2.1.4. ETI and insulin conditioning

2.1.5. Immunostaining

2.1.6. Image Analysis

2.1.7. RNA Sequencing

2.1.8. Gene Set Enrichment Analysis

2.1.9. Statistical analysis

2.1.10.      Data Accessibility

2.2. Results

2.2.1. Hyperglycemia increases CFTR current and decreases trans-epithelial electrical resistance in WT cells but leads to no change in CF cells.

2.2.2. Increased paracellular flux in CF cells under hyperglycemia upon insulin treatment

2.2.3. Rescue of CFTR by ETI in CF cells is not compromised by hyperglycemia or insulin treatment.

2.2.4. Key proteins of the airway glucose barrier are dysregulated in CF cells.

2.2.5. Transcriptional responses are different in CF versus WT cells under normal or high glucose.

2.2.6. Gene set enrichment analysis shows that hallmark gene sets are dysregulated in CF versus WT cells under normal and high glucose culture conditions.

2.3. Discussion

2.4. Conclusion and Future Directions

Chapter 3 – Development of a Programmable Automated Cell Culture System to Study the Lung Pathophysiology of Cystic Fibrosis Related Diabetes

3.1. Introduction

3.2. Materials and Methods

3.2.1. Cell culture of CFBE immortalized cell line

3.2.2. Culture of primary cells

3.2.3. Programmable and automated control of PACCS

3.2.4. Design iterations of PACCS cell culture plate components

3.2.5. 3D-Printing

3.2.6. PACCS quality control

3.2.7. Running PACCS

3.2.8. Ussing chamber analysis of cells cultured with PACCS and controls

3.2.9. Data Analysis

3.3. Results

3.3.1. The PACCS design incorporates several unique components to create a reliable automated cell culture platform for mammalian cells plated on permeable Transwell supports

3.3.2. PACCS plate design iterations to reduce media consumption and improve fluid exchange

3.3.3. Quality control of the final PACCS plate design showed good media exchange from fasting to meal-like conditions

3.3.4. Immortalized cells and primary airway epithelial cells can be successfully cultured with PACCS

3.4. Discussion

3.5. Conclusions and Future Directions

Chapter 4 – Chronic hyperglycemia aggravates lung function in a Scnn1b-Tg murine model

4.1. Introduction

4.2. Materials and Methods

4.2.1. Experimental Animals

4.2.2. Electrogenic Ion Transport Measurements

4.2.3. CFRD animal model

4.2.4. BALF collection and BALF cell counting

4.2.5. Glucose Determination using Mass Spectrometry

4.2.6. Lung tissue RNA sequencing and analysis

4.2.7. PAO1 inoculum preparation and PAO1 infection

4.2.8. Histopathological evaluation

4.2.9. Source of Reagents

4.2.10.      Statistical Analysis

4.3. Results

4.3.1. Bioelectric studies on mouse tracheas

4.3.2. STZ-mediated induction of CF-related diabetes both murine models

4.3.3. Scnn1b-Tg-D mice exhibit increased pulmonary inflammation and infection

4.3.4. Bulk RNA sequencing reveals distinct gene expression in WT-D and Scnn1b-Tg-D mice

4.3.5. Scnn1b-Tg-con and Scnn1b-Tg-D mice exhibited histopathological lung features

4.3.6. Pulmonary infection with PAO1 in mouse models

4.4. Discussion

4.5. Conclusion and Future Directions

Chapter 5 – Neutrophil transepithelial migration through monolayers of human bronchial epithelial cells is altered by chronic hyperglycemic conditioning

5.1. Introduction

5.2. Materials and methods

5.2.1. 16HBE cell culture on the undersurface of Transwells

5.2.2. Conditionally reprogrammed Human Bronchial Epithelial cells (NhBE/CFhBE) cultured on the undersurface of Transwells

5.2.3. HL-60 cell culture and differentiation

5.2.4. Isolation of human neutrophils

5.2.5. Ussing chamber analysis

5.2.6. dHL-60 cells and PMN transmigration

5.2.7. Immunostaining and confocal microscopy

5.2.8. Source of Reagents

5.2.9. Statistical analysis

5.3. Results

5.3.1. 16HBE cells formed monolayer cultured on the inside and undersurface of Transwells with 3 µm pore size

5.3.2. Differentiated HL-60 cells transmigrated across 16HBE monolayer

5.3.3. Transmigration of healthy human neutrophils across 16HBE cell monolayers

5.3.4. The neutrophils transmigrated across epithelial monolayer exhibited altered morphology

5.3.5. Healthy human neutrophil transmigration across primary human bronchial epithelial monolayers

5.4. Discussion

5.5. Conclusion and Future Directions

Chapter 6 – Understanding CFTR function and CFRD pathophysiology through additional projects

6.1. CFTR immunoprecipitation and immunoblotting

6.1.1. Background

6.1.2. Methods

6.1.3. Results and discussion

6.2. CFTR mutagenesis

6.2.1. Background

6.2.2. Protocol

6.2.3. Results and discussion

6.3. Airway epithelial and bacterial co-culture experiments

6.3.1. Background

6.3.2. Methods

6.3.3. Results and discussion

6.4. Development of the Neutrafluor assay to study live neutrophil transmigration in vitro

6.4.1. Introduction

6.4.2. Methods

6.4.3. Results

6.4.4. Conclusions and Future Directions

6.5. Comparing ATPase activity of ATP-binding cassette subfamily C member 4, lamprey CFTR, and human CFTR using Antimony-phosphomolybdate assay

6.5.1. Abstract

6.6. Discussion and Conclusions

Chapter 7 – Conclusions and Future Directions

Chapter 8 – Protocols

8.1. Cell culture of 16HBE cells

8.1.1. Media composition

8.1.2. Pulling from the freezer

8.1.3. Changing Media

8.1.4. Splitting Cells from a T25 (when 80-90% confluent)

8.1.5. Splitting Cells from a T75 (when 80-90% confluent)

8.1.6. Freezing cells

8.2. Ussing chamber protocol

8.2.1. Warm up

8.2.2. Blanking

8.2.3. Loading Samples

8.2.4. Setting up the software

8.2.5. Other notes

8.3. Dye flux protocol to measure paracellular permeation

8.3.1. Cell Culture

8.3.2. Plate maps

8.3.3. Dye Flux Test

8.3.4. Testing with insulin

8.4. Running qRT-PCR to test for changes in gene expression

8.4.1. Background

8.4.2. RNA isolation

8.4.3. Reverse Transcription

8.4.4. Real Time PCR

8.5. Immunostaining Protocol

8.5.1. Background

8.5.2. Protocol

8.6. Analyzing tight junction protein localization with confocal microscopy

8.7. Running meal-like patters using PACCS

8.7.1. Initial set-up

8.7.2. Starting PACCS

8.7.3. While PACCS is running

About this Dissertation

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Biology, General Engineering, Biomedical
Keyword	CFRD Cystic fibrosis Tight junctions CF Cystic fibrosis-related diabetes Claudin-4
Committee Chair / Thesis Advisor	Joshua Chandler, Emory University Samantha Yeligar, Emory University Nael McCarty, Emory University Michael Koval, Emory University

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