Neutrophil Plasticity Enables the Development of Pathological Microenvironments: Implications for Cystic Fibrosis Airway Disease Público

Margaroli, Camilla (Summer 2019)

Permanent URL: https://etd.library.emory.edu/concern/etds/3j333345d?locale=pt-BR
Published

Abstract

Neutrophils constitute 60% of blood leukocytes and act as one of the first lines of defense against sterile and pathogen-induced inflammation. The peculiarity of their nuclear shape, showing as hypercondensed chromatin with three to four nuclear lobes instead of the canonical nuclear round shape, together with low abundance of cytoplasmic RNA, has led to the paradigm holding these immune cells as short-lived, terminally differentiated and with little opportunity for plasticity. Over the course of the last decade, research has shown that tissue neutrophils can alter their fate to remain metabolically active and avoid rapid cell death. In cystic fibrosis (CF) specifically, airway neutrophils undergo profound functional and phenotypical changes (resulting in the “GRIM” fate) and play a dominant pathogenic role. The work presented in this thesis addresses three key questions related to clinical and basic biology of CF airway disease. First, we show the presence of GRIM neutrophils in the airways of CF infants, at very early stages of disease, prior to chronic microbial infection. We also show a potential role for GRIM neutrophils in modulating the immune response of resident airway macrophages in CF infants. Second, the work presented here demonstrates a strict transcriptional dependency of GRIM reprogramming. This novel mechanism by which neutrophils actively adapt to the CF airway microenvironment contradicts the conventional paradigm holding neutrophils as pre-programmed. Third, we illustrate the use of a new drug delivery system that can be customized to match proteolytic microenvironments and efficiently deliver hydrophobic drugs to diseased airways, including but not limited to CF. In conclusion, this dissertation challenges prevailing paradigms in clinical development of CF lung disease and basic neutrophil biology, and provides insights into mechanisms of neutrophilic inflammation, thus opening opportunities for immunotherapies targeting this key component of airway pathology in CF and other diseases.

Table of Contents

Chapter 1................................................................................................ 1

1.1. Introduction.................................................................................. 1

1.2 Neutrophil plasticity in CF lung disease: emergent mechanisms...... 2

Lifespan and aging................................................................................................................. 2

Overview of effector functions.............................................................................................. 5

Focus on neutrophil elastase (NE)........................................................................................ 7

Figure 1.2.1................................................................................................................... 8

Table 1.2.1................................................................................................................... 10

Table 1.2.2................................................................................................................... 11

Impact of CFTR on neutrophil function............................................................................. 13

Immunomodulatory role of neutrophils............................................................................ 16

Metabolic licensing of neutrophils...................................................................................... 18

The CF airway microenvironment..................................................................................... 21

Treatment opportunities..................................................................................................... 22

Figure 1.2.2................................................................................................................ 23

Figure 1.2.3................................................................................................................ 25

1.3 Conclusions................................................................................. 26

Box 1.3.1. Open.......................................................................................................... 27

Chapter 2............................................................................................. 57

Inflammatory mechanisms in early CF lung disease: interplay between neutrophils and macrophages

2.1 Elastase exocytosis by airway neutrophils associates with early lung damage in cystic fibrosis children         58

2.1.1 At-a-glance commentary............................................................ 58

2.1.2 Abstract.................................................................................... 59

2.1.3 Introduction............................................................................. 60

2.1.4 Methods................................................................................... 62

Table 2.1.4.1............................................................................................................... 64

Table 2.1.4.2.............................................................................................................. 66

2.1.5 Results...................................................................................... 70

Macrophages and neutrophils coexist in BAL from CF and disease control children 70

Table 2.1.5.1................................................................................................................ 71

Airway neutrophils in CF children show distinct changes consistent with hyperexocytosis           71

Figure 2.1.5.1.............................................................................................................. 73

Figure 2.1.5.2............................................................................................................. 74

Cell-based measure of NE exocytosis by airway neutrophils correlates cross-sectionally with structural lung damage in CF children.......................................................................................................................................... 75

Figure 2.1.5.3............................................................................................................. 75

Table 2.1.5.2............................................................................................................... 76

Cell-based measure of NE exocytosis by airway neutrophils and free extracellular NE activity are not impacted by infection status in CF children...................................................................................................................... 77

Figure 2.1.5.4............................................................................................................. 77

Extracellular NE is counteracted by antiproteases and compartmentalizes in airway leukocytes in BALF of CF children        78

Figure 2.1.5.5............................................................................................................. 79

Figure 2.1.5.6............................................................................................................. 80

2.1.6 Discussion................................................................................. 81

2.2 Macrophage exhaustion signals neutrophil takeover in early cystic fibrosis airway disease         85

2.2.1 At-a-glance commentary........................................................... 85

2.2.2 Abstract................................................................................... 86

2.2.3 Introduction............................................................................. 87

2.2.4 Methods................................................................................... 88

Table 2.2.4.1.............................................................................................................. 89

Table 2.2.4.2.............................................................................................................. 92

2.2.5 Results..................................................................................... 95

PD-1 expression is increased on airway macrophages, independently of lipidation.. 95

Figure 2.2.5.1............................................................................................................. 96

Figure 2.2.5.2............................................................................................................ 97

Figure 2.2.5.3............................................................................................................ 98

PD-1 expression on CF airway macrophages is associated with age, and infection status  99

PD-1 expression on CF airway macrophages correlates with structural airway damage and airway neutrophil burden           99

PD-1 expression on CF airway macrophages increases uniformly upon infection... 100

Figure 2.2.5.4........................................................................................................... 101

Figure 2.2.5.5........................................................................................................... 102

Increased PD1 expression on CF airway macrophages coincides with neutrophil takeover           103

Figure 2.2.5.6.......................................................................................................... 103

Figure 2.2.5.7........................................................................................................... 105

Figure 2.2.5.8.......................................................................................................... 106

Airway immune cells express PD-1 ligands.................................................................... 106

Figure 2.2.5.9........................................................................................................... 107

PD-1 blockade in CF BALF short-term cultures increases bacterial killing................ 108

Figure 2.2.5.10......................................................................................................... 108

2.2.6 Discussion.............................................................................. 109

Figure 2.2.6.1............................................................................................................ 111

2.3 References................................................................................. 112

Chapter 3............................................................................................ 131

Transcriptional reprogramming drives neutrophil plasticity in the cystic fibrosis airways

3.1 At-a-glance commentary............................................................. 132

3.2 Abstract...................................................................................... 133

3.3 Introduction............................................................................... 134

3.4 Methods..................................................................................... 135

3.5 Results....................................................................................... 139

CF airway GRIM neutrophils display profound transcriptional changes in vivo...... 139

In vitro-produced CF airway GRIM neutrophils recapitulate transcriptional changes observed in vivo 140

Figure 3.5.1............................................................................................................... 141

Figure 3.5.2.............................................................................................................. 142

The CF airway microenvironment imprints a unique transcriptional and proteomic profile upon recruited neutrophils       143

Figure 3.5.3.............................................................................................................. 144

Figure 3.5.4.............................................................................................................. 145

Figure 3.5.5.............................................................................................................. 147

CF airway GRIM neutrophils lack de novo expression of effector granule proteins in vivo and in vitro  148

Table 3.5.1................................................................................................................. 156

Figure 3.5.6.............................................................................................................. 157

Figure 3.5.7.............................................................................................................. 158

Acquisition of the CF airway GRIM phenotype depends on time-dependent transcriptional reprogramming of neutrophils 159

Figure 3.5.8.............................................................................................................. 160

Figure 3.5.9.............................................................................................................. 161

Transcriptional blockade of CF airway GRIM neutrophils modulates production of pathological extracellular vesicles 162

Figure 3.5.10............................................................................................................ 163

3.5 Discussion.................................................................................. 163

3.6 References................................................................................. 165

Chapter 4............................................................................................ 172

Targeting airway neutrophilic inflammation: exploiting a novel protease-activated drug delivery system

4.1 At-a-glance commentary............................................................. 173

4.2 Abstract..................................................................................... 174

4.3 Introduction............................................................................... 174

4.4 Methods..................................................................................... 176

Figure 4.4.1.............................................................................................................. 179

4.5 Results....................................................................................... 181

N-in-M degrade in presence of neutrophil elastase (CF ASN) resulting in uptake   of nanoparticles by activated PMNs.           181

Figure 4.5.1.............................................................................................................. 183

N-in-M delivered to acute LPS-induced lung injury model degrade, delivering fluorescent nanoparticles to airway PMNs.   184

Figure 4.5.2.............................................................................................................. 185

N-in-M delivering Nexinhib20 attenuates neutrophilic inflammation in vivo........ 186

Figure 4.5.3.............................................................................................................. 188

Figure 4.5.4.............................................................................................................. 189

Figure 4.5.5.............................................................................................................. 190

4.6 Discussion.................................................................................. 191

4.7 References................................................................................. 193

Chapter 5............................................................................................ 197

5.1 Summary of thesis findings......................................................... 198

5.2 Clinical implications and perspectives........................................ 198

Introduction of CFTR modulators and correctors.......................................................... 201

5.3 Basic scientific implications and perspectives.............................. 201

Inflammatory mechanisms of early CF........................................................................... 201

Acquisition of the GRIM phenotype............................................................................... 203

Role of EVs.......................................................................................................................... 204

5.5 Conclusion................................................................................. 205

Figure 5.4.1.............................................................................................................. 206

5.6 References................................................................................. 207

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