Neutrophil Plasticity Enables the Development of Pathological Microenvironments: Implications for Cystic Fibrosis Airway Disease Open Access
Margaroli, Camilla (Summer 2019)
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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