Neutrophil STING and Type I Interferon Activation in Cardiovascular Disease Restricted; Files Only

Abstract

Cardiovascular disease (CVD) is the leading cause of death worldwide and it encompasses a broad spectrum of disorders affecting the heart and vasculature, including coronary artery disease, atherosclerosis stroke, myocardial infarction (MI), and heart failure (HF). Despite advances in preventive and therapeutic strategies, the burden of CVD continues to rise due to aging populations and increasing rates of comorbidities such as obesity, diabetes, and hypertension. Among these conditions HF is a major driver of cardiovascular mortality and morbidity. Emerging evidence implicates neutrophil-driven inflammation in maladaptive cardiac remodeling, fibrosis, and progressive ventricular dysfunction in subtypes of HF, which highlights neutrophils as both mediators of injury and potential therapeutic targets. A deeper understanding of how specific neutrophil functions and inflammatory mediators contribute to cardiac remodeling and dysfunction could lead to more effective and safer therapeutic strategies.

Despite growing recognition of the role of neutrophils in CVD, effective therapies that specifically target neutrophil-mediated inflammation remain limited. This work aims to dissect the contribution of neutrophil-intrinsic Stimulator of Interferon Genes (STING) and type I interferon (IFN) signaling in distinct models of HF. We explore how STING and type I IFN pathways function within the neutrophil response period to drive cardiac inflammation and remodeling in HF resulting in the development of neutrophil-specific STING (neuSTING KO) and IFNAR1 (neuIFNAR1 KO) knockout mouse models. We focus on the impact of neutrophil-specific STING deletion in the setting of myocardial ischemia/reperfusion (MI/R) injury, testing the hypothesis that neutrophil-specific STING promotes early inflammation and its loss confers cardioprotection.

We also investigate the role of neutrophil-intrinsic type I IFN signaling in the development of HF with preserved ejection fraction (HFpEF), hypothesizing that IFNAR1 deletion in neutrophils reduces systemic and myocardial inflammation and prevents diastolic dysfunction. Together, these studies aim to provide new mechanistic insight into neutrophil-driven inflammation and identify novel immune targets for therapeutic intervention in CVD.

Table of Contents

INDEX OF FIGURES: _________________________________________________________ i

INDEX OF TABLES: ________________________________________________________ vii

ABBREVIATIONS: _________________________________________________________ viii

CHAPTER 1: INTRODUCTION _______________________________________________ 1

1.1 Cardiovascular Disease ________________________________________________ 1

1.1.1 Myocardial-Ischemia Reperfusion Injury: __________________________________ 1

1.1.2 Heart Failure with Preserved Ejection Fraction:_____________________________ 5

1.2 Neutrophils in Cardiovascular Disease ____________________________________ 8

1.2.1 Neutrophils: _________________________________________________________ 8

1.2.2 Neutrophils in Myocardial Ischemia/Reperfusion Injury: ______________________ 9

1.2.3 Neutrophils in Heart Failure with Preserved Ejection Fraction: _________________11

1.3 STING and Type I IFNs in Cardiovascular Disease ________________________ 12

1.3.1 STING and Type I Interferons: __________________________________________ 12

1.3.2 STING and Type I Interferons in Neutrophils: ______________________________ 14

1.3.3 STING and Type I Interferons in Cardiovascular Disease: ____________________ 16

1.4 Targeting Neutrophils and Inflammation in Cardiovascular Disease ___________ 17

1.4.1 COLCOT Trial: ______________________________________________________ 17

1.4.2 CANTOS Trial: ______________________________________________________ 19

1.4.3 ENDEAVOR Trial: ___________________________________________________ 20

1.5 Objectives __________________________________________________________ 22

CHAPTER 2: STING AND TYPE I INTERFERON SIGNALING IN CARDIOVASCULAR DISEASE ______________________________________________ 24

2.1 Introduction ___________________________________________________________ 24

2.2 Materials and Methods __________________________________________________ 25

2.3 Results _______________________________________________________________ 37

2.3.1 STING and Type I IFNs in Human Neutrophils _____________________________ 37

2.3.2 Human Neutrophils Respond to Type I Interferons __________________________ 37

2.3.3 Neutrophils Elicit a Type I Interferon Response after MI/R ____________________ 39

2.3.4 Inhibition of STING and IFNAR1 Improves Cardiac Function after MI/R ________ 45

2.3.5 Neutrophils, STING, and Type I Interferons in Heart Failure with Preserved Ejection Fraction ________________________________________________________________ 54

2.3.6 Generation of Neutrophil-Specific STING and IFNAR1 Knockout Mice __________ 54

2.4 Conclusions ___________________________________________________________ 68

CHAPTER 3: NEUTROPHIL-SPECIFIC STING PROMOTES INFLAMMATION RESOLUTION AND CARDIOPROTECTION AFTER MYOCARDIAL ISCHEMIA/REPERFUSION _________________________________________________ 74

3.1 Introduction ___________________________________________________________ 74

3.2 Materials and Methods __________________________________________________ 76

3.3 Results _______________________________________________________________ 94

3.3.1 STING is Activated in Human and Murine Neutrophils after Myocardial Ischemia/Reperfusion Injury ________________________________________________ 94

3.3.2 Loss of STING in Neutrophils Worsens Cardiac Injury After MI/R ______________ 96

3.3.3 Loss of STING in Neutrophils Increases Cardiac Type I Interferon Levels after MI/R ______________________________________________________________________ 102

3.3.4 Single-Cell RNA Sequencing of neuSTING KO Neutrophils Reveals Reduced Inflammatory Signaling after MI/R __________________________________________ 102

3.3.5 Loss of STING in Neutrophils Dampens Recruitment of Neutrophils to the Heart after MI/R ___________________________________________________________________114

3.3.6 Loss of STING in Neutrophils Prevents Polarization of Macrophages to Anti-Inflammatory Phenotype after MI/R __________________________________________117

3.3.7 Loss of STING in Neutrophils Decreases Apoptosis and Efferocytosis of Cardiac Neutrophils after MI/R ____________________________________________________ 126

3.3.8 Loss of STING in Neutrophils Impairs Cardiac Neutrophil Effector Functions after MI/R __________________________________________________________________ 130

3.4 Conclusions __________________________________________________________ 140

CHAPTER 4: NEUTROPHIL-SPECIFIC TYPE I INTERFERON SIGNALING IN HEART FAILURE WITH PRESERVED EJECTION FRACTION _________________ 145

4.1 Introduction __________________________________________________________ 145

4.2 Materials and Methods _________________________________________________ 146

4.3 Results ______________________________________________________________ 152

4.3.1 Cardiac Neutrophil Numbers Significantly Increase in Wildtype Mice after One Week of HFpEF Diet __________________________________________________________ 152

4.3.2 neuIFNAR KO Mice Fed HFpEF-Inducing Diet Weigh Significantly More Than Mice Fed Control Diet ________________________________________________________ 153

4.3.3 neuIFNAR KO Mice Show Significant Increases in Blood Pressure at Early Time Points After Beginning HFpEF- Inducing Diet _________________________________ 156

4.3.4 Wildtype and neuIFNAR KO Mice Fed HFpEF-Inducing Diet Show Significant Glucose Intolerance Compared to Mice Fed Control Diet ________________________ 156

4.3.5 Echocardiography Reveals Diastolic Dysfunction in Mice Fed HFpEF-Inducing Diet Independent of Genotype __________________________________________________ 158

4.4 Conclusions __________________________________________________________ 162

CHAPTER 5: DISCUSSION _________________________________________________ 166

5.1 Summary of Results ___________________________________________________ 166

5.1.1 STING and Type I Interferon Signaling in Cardiovascular Disease ____________ 166

5.1.2 Neutrophil-Specific STING Promotes Inflammation Resolution and Cardioprotection after Myocardial Ischemia/Reperfusion ______________________________________ 168

5.1.3 Neutrophil-Specific Type I Interferon Signaling in Heart Failure with Preserved Ejection Fraction ________________________________________________________ 169

5.2 Contributions to the Field _______________________________________________ 170

5.3 Limitations ___________________________________________________________ 172

5.4 Future Directions _____________________________________________________ 173

5.4.1 STING and Type I Interferon Signaling in Cardiovascular Disease ____________ 173

5.4.2 Neutrophil-Specific STING Promotes Inflammation Resolution and Cardioprotection after Myocardial Ischemia/Reperfusion ______________________________________ 174

5.4.3 Neutrophil-Specific Type I Interferon Signaling in Heart Failure with Preserved Ejection Fraction ________________________________________________________ 179

5.5 Concluding Remarks ___________________________________________________ 181

REFERENCES ____________________________________________________________ 182

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Physiology Health Sciences, Immunology Biology, Systematic
Keyword	STING Neutrophil Interferon Cardiovascular Disease Inflammation
Committee Chair / Thesis Advisor	Rebecca Levit, Emory University
Committee Members	Samantha Yeligar, Emory University Rabindra Tirouvanziam, Emory University John Calvert, Emory University

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