Chronic Alcohol Consumption Induces Alveolar Epithelial Barrier Dysfunction in vivo Pubblico
Jeffers, Lauren (Spring 2022)
Published
Abstract
Chronic alcohol abuse is a major risk factor for developing Acute Respiratory Distress Syndrome, a serious lung condition and a form of respiratory failure characterized by widespread airspace flooding and inflammation. One mechanism by which alcohol use weakens the integrity of the alveolar epithelial barrier is by changing the composition of tight junction proteins that are critically necessary to maintain a functional air-liquid interface. Previously, we have shown that alcohol exposure increases the paracellular permeability and decreases the transepithelial resistance across alveolar epithelial cells in vitro. Here, we first establish a novel model using Evans Blue dye to assess paracellular permeability across both epithelial and endothelial barriers in vivo using a murine animal model. We then tested the effects of alcohol and an endotoxemia injury on pulmonary function by comparing direct (intratracheal, IT) to indirect (intraperitoneal, IP) administration of lipopolysaccharide (LPS). Chronic alcohol ingestion primes the lung for increased alveolar macromolecular leak when coupled with either route of endotoxin injury and further compromises the vascular endothelial barrier when the LPS is administered directly (IT). Moreover, we show that nebulized treatment of a mimetic peptide of a tight junction-associated protein rescues the alcohol-mediated alveolar leak in mice treated with IT-administered LPS. Additionally, we begin to test Klebsiella pneumoniae as a model for infectious pneumonia and its effect on alcohol-primed lungs. We found a disease burden-dependent increase on alveolar permeability and that alcohol-fed mice had decreased bacterial clearance, providing a potential new infectious injury model against which barrier-enhancing therapeutics can be tested.
Table of Contents
Table of Contents
DISTRIBUTION AGREEMENT ................................................................................................ 1
CHAPTER 1: INTRODUCTION ................................................................................................ 1
Lung anatomy ........................................................................................................................... 1
The epithelial barrier and tight junctions .............................................................................. 2
Alcohol and acute respiratory distress syndrome ................................................................. 5
Scope of Dissertation ................................................................................................................ 7
Literature Cited ........................................................................................................................ 9
CHAPTER 2: MEASUREMENT OF LUNG VESSEL AND EPITHELIAL PERMEABILITY IN VIVO WITH EVANS BLUE .................................................... 15
Abstract ................................................................................................................................... 15
Introduction ............................................................................................................................ 15
Materials ................................................................................................................................. 16
Evans Blue Injection .......................................................................................................... 16
Tissue Harvest .................................................................................................................... 17
Tissue Analysis ................................................................................................................... 18
Methods ................................................................................................................................... 18
Tail Vein Injection of Evans Blue ..................................................................................... 18
Serum Collection ................................................................................................................ 20
BAL Fluid Collection ......................................................................................................... 20
Lung Tissue Collection ...................................................................................................... 21
Evans Blue Measurement and Analysis ............................................................................ 22
Notes 23
Figure 2.1. Diagram depicting a cross-section though a normal lung alveolus comprised of an epithelial sac surrounded by blood vessels. .................................................. 27
Figure 2.2. Tail vein injection. ........................................................................................... 28
Figure 2.3. Appearance of Evans Blue in mice and murine lungs. ................................. 29
Figure 2.4. Effect of alcohol consumption and lipopolysaccharide on Evans Blue accumulation in lung airspaces and tissue. .......................................................... 30
Literature Cited ...................................................................................................................... 31
CHAPTER 3: EFFECTS OF DIFFERENT ROUTES OF ENDOTOXIN INJURY ON BARRIER FUNCTION IN ALCOHOLIC LUNG SYNDROME.............................. 33
Abstract ................................................................................................................................... 33
Introduction ............................................................................................................................ 34
Materials and Methods .......................................................................................................... 35
Results ..................................................................................................................................... 38
LPS insult and chronic alcohol ingestion altered body weight ........................................ 38
Alcohol alone promoted the appearance of airspace protein that is further exacerbated by direct LPS injury ................................................................................................ 39
Alcohol and LPS independently induced interstitial edema ............................................. 40
Direct LPS challenge in alcohol-fed mice increased lung permeability .......................... 40
Discussion ................................................................................................................................ 41
Figure 3.1. Effect of alcohol consumption and lipopolysaccharide on mouse weight. ... 44
Figure 3.2. Alcohol increased lung airspace protein content. .......................................... 45
Figure 3.3. Alcohol and intratracheal lipopolysaccharide induced an inflammatory response in lung tissue. .......................................................................................... 46
Figure 3.4. Alcohol consumption and lipopolysaccharide independently increased alveolar septal thickness. ........................................................................................ 47
Figure 3.5. Effect of alcohol consumption and lipopolysaccharide on Evans Blue accumulation in lung tissue. .................................................................................. 48
Figure 3.6. Effect of alcohol consumption and LPS on Evans Blue in airspaces. ......... 49
Figure 3.7. Model of lung permeability after alcohol and intratracheal lipopolysaccharide. ................................................................................................. 50
Literature Cited ...................................................................................................................... 51
CHAPTER 4: EFFECTS OF INTERSTITIAL FLUID PRESSURE ON ALVEOLAR BARRIER FUNCTION .................................................................................................. 55
Introduction ............................................................................................................................ 55
Materials and Methods .......................................................................................................... 56
Results ..................................................................................................................................... 58
Intraperitoneal fluid in anesthetized and conscious mice has a minimal impact on alveolar leak ............................................................................................................ 58
Intravascular fluid does not affect alveolar leak of protein ............................................. 59
Increasing vascular pressure with fluid does not increase alveolar leak or cause pulmonary edema ................................................................................................... 59
Figure 4.1 Anesthesia is essential for Evans Blue assays of lung permeability. ............. 61
Figure 4.2. No effect of increased intravascular fluid on total protein levels in bronchoalveolar lavage fluid. ................................................................................ 62
Figure 4.3. Intravascular fluid dilutes Evans Blue in mouse serum in volume-dependent manner. ................................................................................................................... 63
Figure 4.4. No effect of increased intravascular fluid on alveolar leak at baseline. ....... 64
Figure 4.5. No pulmonary edema observed in mice given intravascular bolus of fluid. . 65
Literature Cited ...................................................................................................................... 66
CHAPTER 5: THERAPEUTIC DRUG CANDIDATES TO IMPROVE ALCOHOL-MEDIATED BARRIER DYSFUNCTION ................................................................... 69
Introduction ............................................................................................................................ 69
Interaction between Connexin 43 and ZO-1 ..................................................................... 69
Alpha Carboxyl-Terminal Mimetic Peptide (ɑCT1) .......................................................... 71
Previous therapeutic uses of ɑCT1 .................................................................................... 72
Therapeutic potential for ɑCT1 to treat alcoholic lung syndrome ................................... 73
Role of Claudin 5 in alcoholic lung disease ...................................................................... 73
Claudin 5 peptide mimetic (C5) ......................................................................................... 74
Materials and Methods .......................................................................................................... 75
Results ..................................................................................................................................... 77
Claudin 5 expression is increased after chronic exposure to alcohol .............................. 77
C5 peptide did not impact total blood volume ................................................................... 78
C5 peptide did not improve alcohol-induced alveolar leak ............................................... 78
ɑCT1 does not impact weight loss or total blood volume .................................................. 79
ɑCT1 improved alcohol-induced alveolar leak .................................................................. 80
Discussion ................................................................................................................................ 82
Figure 5.1. Dietary alcohol increases the expression of claudin 5. ................................. 84
Figure 5.2. No difference in serum concentration of Evans Blue dye with C5 treatment. ................................................................................................................................. 85
Figure 5.3. C5 (5 mg/kg) did not reverse alveolar leak of Evans Blue dye in alcohol-fed mice. ........................................................................................................................ 86
Figure 5.4. C5 (50 mg/kg) did not reverse alveolar leak of Evans Blue dye in alcohol-fed mice. ........................................................................................................................ 87
Figure 5.5. ɑCT1 has no significant impact on intratracheal lipopolysaccharide-mediated weight loss. .............................................................................................. 88
Figure 5.6. No difference in serum concentration of Evans Blue dye with ɑCT1 treatment. ................................................................................................................ 89
Figure 5.7. ɑCT1 reverses alveolar leak of Evans Blue dye in alcohol-fed mice. ........... 90
Literature Cited ...................................................................................................................... 91
CHAPTER 6: FUTURE THERAPEUTIC TARGETS AND INJURY MODELS ............. 100
Introduction .......................................................................................................................... 100
Claudin 4 ........................................................................................................................... 100
Klebsiella pneumoniae ..................................................................................................... 101
Materials and Methods ........................................................................................................ 102
Results ................................................................................................................................... 104
Claudin 4 KO mice have similar alveolar inflammation response to LPS .................... 104
Claudin 4 KO mice are more susceptible to intratracheal injury ................................... 105
Alcohol increases levels of IL-6 in lavage fluid after LPS treatment ............................ 105
Klebsiella pneumoniae increases alveolar leak in a dose-dependent manner ............... 106
Alcohol decreases bacterial clearance of Klebsiella pneumoniae .................................. 106
Figure 6.1. Claudin 4 KO mice have similar levels of inflammation in bronchoalveolar lavage to Wild-type mice. ...................................................................................... 107
Figure 6.2. Claudin 4 KO mice are more susceptible to saline-induced intratracheal injury than Wild-type mice. .................................................................................. 108
Figure 6.3. LPS-treated, alcohol-fed mice have higher levels of IL-6. .......................... 109
Figure 6.4. No difference in weight changes with increased Klebsiella inoculation concentration. ....................................................................................................... 110
Figure 6.5. Mice have increased alveolar leak with increased Klebsiella inoculation concentration. ....................................................................................................... 111
Figure 6.6. Alcohol-fed mice have lower lung clearance of Klebsiella infection. ......... 112
Literature Cited .................................................................................................................... 113
CHAPTER 7: DISCUSSION – CONCLUSION AND FUTURE DIRECTIONS ............... 117
Overview of Findings ........................................................................................................... 117
In Vivo Measurement of Lung Vessel and Epithelial Permeability ............................... 117
‘Second-hit’ Injury Models .............................................................................................. 118
Potential Therapeutic for Alcoholic Lung Disease ......................................................... 120
Future Directions ................................................................................................................. 121
Fluid pressure as a novel injury model ........................................................................... 121
aCT1 therapeutic potential with additional injury models ............................................. 123
Therapeutic potential of claudin 5 ................................................................................... 124
Literature Cited .................................................................................................................... 125
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