Matrisome alterations in lung inflammatory disease

229 Pages·2017·7.4 MB·English

by  Lauren Cholewa

Matrisome alterations in
lung inflammatory disease

A thesis submitted to the University of Manchester for the
degree of Doctor of Philosophy in the Faculty of Biology,
Medicine and Health

2017

Lauren Cholewa

School of Biological Sciences
Division of Infection, Immunity and Respiratory Medicine
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Contents
Table of Figures and Tables ............................................................................................................... 7
Table of Abbreviations ...................................................................................................................... 10
Abstract ............................................................................................................................................. 13
Declaration ........................................................................................................................................ 14
Copyright ........................................................................................................................................... 14
Acknowledgements ........................................................................................................................... 15
1. Introduction ................................................................................................................................ 16
1.1 The structure of the lung ................................................................................................... 17
1.1.1 Lung Structure ............................................................................................................... 17
1.1.2 Macrophages in the lung ............................................................................................... 17
1.1.3 Surfactant proteins ........................................................................................................ 18
1.1.4 Microbiota ...................................................................................................................... 18
1.2 The extracellular matrix of the lung ................................................................................... 19
1.2.1 Degradation of the ECM ................................................................................................ 20
1.2.2 Basement membrane .................................................................................................... 21
1.2.3 ECM components .......................................................................................................... 23
1.2.4 Airway macrophages and ECM ..................................................................................... 27
1.2.5 Effects of influenza virus on the ECM ........................................................................... 29
1.3 Long term changes to the lung microenvironment ............................................................ 31
1.3.1 The lung microenvironment in health ............................................................................ 31
1.3.2 Long term changes to airway macrophages ................................................................. 31
1.3.3 Other long-term cellular changes .................................................................................. 32
1.3.4 Long-term alterations to the ECM ................................................................................. 32
1.4 Immune regulation by non-coding RNA ............................................................................ 33
1.4.1 miRNA ........................................................................................................................... 33
1.4.2 miRNA and Influenza virus infection ............................................................................. 35
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1.4.3 miRNA and pulmonary fibrosis ...................................................................................... 37
1.5 Thesis hypothesis and aims .............................................................................................. 38
2. Materials and Methods............................................................................................................... 40
2.1 Laboratory Animals and in vivo models ............................................................................ 41
2.1.1 Animals .......................................................................................................................... 41
2.1.2 Sample Collection ......................................................................................................... 41
2.1.3 Influenza model ............................................................................................................. 41
2.1.4 Carbon nanotube model of pulmonary inflammation with and without infection ........... 41
2.1.5 Quantification of bacteria .............................................................................................. 42
2.1.6 In vivo treatment with hyaluronan ................................................................................. 42
2.2 In vitro cell treatments ....................................................................................................... 42
2.2.1 In vitro treatment of murine airway macrophages with ECM components.................... 42
2.3 Histology ............................................................................................................................ 43
2.3.1 Haematoxylin and Eosin ............................................................................................... 43
2.3.2 Masson’s trichrome ....................................................................................................... 43
2.3.3 Fluorescent immunohistochemistry staining for hyaluronan ......................................... 43
2.3.4 Image acquisition .......................................................................................................... 44
2.3.5 Quantification of hyaluronan staining ............................................................................ 44
2.4 RNA extraction and qPCR ................................................................................................ 44
2.4.1 RNA extraction .............................................................................................................. 44
2.4.2 Reverse Transcription and RT-qPCR ........................................................................... 44
2.5 Mass Spectrometry ........................................................................................................... 45
2.5.1 Mass spectrometry reagents ......................................................................................... 45
2.5.2 Protein extraction from whole lung ................................................................................ 46
2.5.3 In-gel proteolytic digestion ............................................................................................ 46
2.5.4 Offline peptide desalting ................................................................................................ 47
2.5.5 MS data acquisition ....................................................................................................... 47
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2.5.6 MS data analysis ........................................................................................................... 47
2.5.7 MS data quantification ................................................................................................... 48
2.5.8 Hierarchical clustering analysis ..................................................................................... 48
2.5.9 Protein interaction network analysis.............................................................................. 49
2.6 Electron microscopy .......................................................................................................... 49
2.6.1 Serial block-face scanning electron microscopy ........................................................... 49
3. Changes to the lung matrisome after influenza infection resolution .......................................... 50
3.1 Introduction ........................................................................................................................ 51
3.1.1 The extracellular matrix and influenza infection ............................................................ 51
3.1.2 Whole tissue proteomics by mass spectrometry ........................................................... 52
3.1.3 Objectives ...................................................................................................................... 53
3.2 Results .............................................................................................................................. 54
3.2.1 Extraction and detection of proteins in murine lung at day 14 post-influenza infection 54
3.2.2 Identification of matrisome proteins within the mass spectrometry data ...................... 57
3.2.3 Overlap of proteins identified by mass spectrometry in the different fractions ............. 61
3.2.4 Alteration of matrisome protein solubility after influenza infection ................................ 62
3.2.5 Network analysis of matrisome proteins in the matrisome-enriched fraction ............... 63
3.2.6 mRNA and miRNA expression after influenza infection................................................ 65
3.2.7 Electron microscopy images after flu ............................................................................ 67
3.2.8 HA expression during an influenza time course ............................................................ 68
3.3 Discussion ......................................................................................................................... 69
3.3.1 Extraction and detection of proteins in murine lung at day 14 post-influenza infection 69
3.3.2 Alteration of basement membrane matrix ..................................................................... 70
3.3.3 Interstitial matrix protein alteration after flu ................................................................... 71
3.3.4 Other matrix changes .................................................................................................... 73
3.3.5 mRNA and miRNA expression after influenza infection................................................ 73
4. Influences of commensal bacteria, age and matrix proteins on the lung .................................. 75
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4.1 Introduction ........................................................................................................................ 76
4.1.1 The lung microbiome ..................................................................................................... 76
4.1.2 The effect of the gut microbiome on lung immunity ...................................................... 77
4.1.3 Differences in neonate and adult lung structure ........................................................... 77
4.1.4 Immune differences between neonate and adult .......................................................... 78
4.1.5 ECM effects on macrophages ....................................................................................... 78
4.1.6 Objectives ...................................................................................................................... 79
4.2 Results .............................................................................................................................. 80
4.2.1 Airway macrophages do not have an altered miRNA profile following fibronectin
treatment .................................................................................................................................... 80
4.2.2 Airway macrophages have an altered miRNA profile following hyaluronan treatment . 83
4.2.3 The effects of age and the microbiome on lung architecture and matrix ...................... 85
4.2.4 The effect of the microbiome and age on lung RNA expression in mice ...................... 89
4.3 Discussion ......................................................................................................................... 92
4.3.1 ECM stimulation of airway macrophages ...................................................................... 92
4.3.2 Differences between GF and SPF mouse lung mRNA and miRNA expression ........... 93
4.3.3 Microbiome effects on the ECM .................................................................................... 94
4.3.4 Differences between neonate and adult ....................................................................... 95
5. Carbon Nanotube model of pulmonary inflammation in the lung .............................................. 97
5.1 Introduction ........................................................................................................................ 98
5.1.1 Carbon nanotube exposure and health concerns ......................................................... 98
5.1.2 Respiratory models using CNTs ................................................................................... 99
5.1.3 CNT and macrophages ............................................................................................... 100
5.1.4 Objectives .................................................................................................................... 100
5.2 Results ............................................................................................................................ 102
5.2.1 Development of a carbon nanotube model of pulmonary granulomatous inflammation
102
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5.2.2 RNA expression after CNT administration in whole lung ............................................ 109
5.2.3 miRNA expression after CNT administration in airway macrophages ........................ 111
5.2.4 Effect of pre-administration of CNT on infection with influenza or S. pneumoniae ..... 112
5.3 Discussion ....................................................................................................................... 117
5.3.1 Effect of CNT on macrophages ................................................................................... 117
5.3.2 Induction of miRNA by CNT ........................................................................................ 118
5.3.3 Effects of CNT on the lung ECM ................................................................................. 118
5.3.4 CNT and pulmonary infection ...................................................................................... 119
6. Discussion ................................................................................................................................ 122
6.1 Effects of alteration of the interstitial ECM ...................................................................... 123
6.2 Alteration of the basement membrane ............................................................................ 124
6.3 Therapeutic implications ................................................................................................. 126
6.4 Future Directions ............................................................................................................. 127
6.5 Conclusions ..................................................................................................................... 128
7. Bibliography ............................................................................................................................. 130

Word Count: 53,232
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Table of Figures and Tables
Figure 1.1 The structure of the lung.. ................................................................................................ 19
Figure 1.2 Basement membrane assembly. ..................................................................................... 22
Figure 1.3 Hyaluronan distribution in naïve mouse lung.. ................................................................. 24
Figure 1.4 Influenza virus replication in epithelial cells. .................................................................... 30
Figure 1.5 The miRNA processing pathway. .................................................................................... 34
Table 1.1 Examples of miRNA involvement in immunity .................................................................. 36
Figure 1.6 Thesis objectives. ............................................................................................................ 39
Figure 3.1 Weight loss chart of influenza virus infection in mice.. .................................................... 54
Figure 3.2 Coomassie stained protein gels of fractions 1-3.. ............................................................ 55
Figure 3.3 Flow diagram of murine lung protein extraction for mass spectrometry. ......................... 55
Figure 3.4 Heat map of proteins detected in mouse lungs post-influenza virus infection.. .............. 56
Figure 3.5 Heat map of significantly altered matrisome proteins in the matrisome-enriched fraction 3
from mouse lung post-influenza virus infection. ................................................................................ 57
Figure 3.6 Heat map of significantly altered matrisome proteins in protein fractions 1 and 2 from
mouse lung post-influenza virus infection.. ....................................................................................... 58
Table 3.1 Most highly decreased proteins in the matrisome enriched fraction 3 in mouse lung at day
14 after influenza virus infection. ...................................................................................................... 59
Table 3.2 Most highly increased proteins in the matrisome enriched fraction 3 in mouse lung at day
14 after influenza virus infection. ...................................................................................................... 60
Figure 3.7 Venn diagrams showing the overlap between proteins identified in the different fractions
in both control mice and 14 days post-influenza virus infection in mice. .......................................... 61
Figure 3.8 Solubility plots of proteins identified post influenza virus infection in mouse lung. ......... 62
Figure 3.9 Network map of known interactions between matrisome proteins identified in the
matrisome-enriched fraction 3 post-influenza virus infection in mice. .............................................. 63
Figure 3.10 Network maps of increased and decreased proteins after influenza virus infection in mice
in the matrisome-enriched protein fraction 3..................................................................................... 64
Figure 3.11 mRNA expression of significantly altered matrisome proteins at day 14 post-influenza
virus infection in mouse lung. ............................................................................................................ 65
Figure 3.12 miRNA expression at day 14 post-influenza virus infection in mouse lung.. ................. 66
Figure 3.13 mRNA and miRNA expression at 6 weeks and 10 weeks post-influenza virus infection in
mouse lung. ....................................................................................................................................... 66
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Figure 3.14 EM images of mouse lung at day 14-post influenza virus infection. .............................. 67
Figure 3.15 Hyaluronan fluorescent staining in mouse lung during an influenza virus time course..
.......................................................................................................................................................... 68
Figure 4.1 Treatment of mouse alveolar macrophages with fibronectin in vitro. .............................. 81
Figure 4.2 Fibronectin treatment of mouse airway macrophages via different delivery methods. ... 82
Figure 4.3 Treatment of mouse alveolar macrophages with hyaluronan ex vivo and in vivo.. ......... 84
Figure 4.4 H&E staining of SPF and GF mouse lungs.. ................................................................... 86
Figure 4.5 Masson’s Trichrome staining of SPF and GF mouse lung. ............................................. 87
Figure 4.6 HA in germ free mouse lungs. ......................................................................................... 88
Figure 4.7 Matrix mRNA expression in germ free mice. ................................................................... 89
Figure 4.8 miRNA expression in whole lung of adult GF mice. ........................................................ 90
Figure 4.9 miRNA expression in whole lung of SPF and GF mice over time.. ................................. 91
Figure 5.1 Imaging of carbon nanotubes within the mouse lung.. .................................................. 103
Figure 5.2 Imaging of carbon nanotubes within the mouse lung after 2 different doses.. .............. 104
Figure 5.3 H&E imaging of CNT in mouse lung after a single dose or two doses. ......................... 105
Figure 5.4 Distribution of carbon nanotubes within the mouse lung. .............................................. 106
Figure 5.5 Collagen in mouse lungs treated with CNT. .................................................................. 107
Figure 5.6 Hyaluronan in mouse lungs treated with CNT. .............................................................. 108
Figure 5.7 RNA expression in whole lung after CNT treatment. ..................................................... 109
Figure 5.8 Difference in whole lung miRNA expression in mice treated with two doses of CNT
compared to a single dose. ............................................................................................................. 110
Figure 5.9 miRNA expression in airway macrophages of mice treated with carbon nanotubes. ... 111
Figure 5.10 Difference in airway macrophage miRNA expression in mice treated with two doses of
CNT compared to a single dose. .................................................................................................... 111
Figure 5.11 Weight chart of mice treated with CNT and then infected. .......................................... 112
Figure 5.12 C.F.U. after S. pneumoniae infection with and without pre-treatment with CNT.
administration, followed by intranasal infection with 1x105 C.F.U. S. pneumoniae in 50μL PBS on day
7. ..................................................................................................................................................... 113
Figure 5.13 H&E staining of mouse lung after treatment with carbon nanotubes and infection.. ... 114
Figure 5.14 Hyaluronan staining in mouse lung in the lungs of mice treated with CNT and infected
with influenza or S. pneumoniae.. ................................................................................................... 115
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Figure 5.15 miRNA expression in whole lung after influenza or pneumonia infection in mice pre-
treated with CNT. ............................................................................................................................ 116

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Table of Abbreviations
ADAMTS A disintegrin and metalloproteinase with thrombospondin motif
AF488 Alexa Fluor 488
BAL Bronchoalveolar lavage
C.F.U. Colony forming units
CD Cluster of Differentiation
CNT Carbon nanotubes
DAMP Damage-associated molecular pattern
DAPI 4',6-diamidino-2-phenylindole
DDR Discoidin domain receptor
DTT Dithiothreitol
ECM Extracellular matrix
EDTA Ethylenediaminetetraacetic acid
FBS Foetal bovine serum
FN Fibronectin
GF Germ free
H&E Haematoxylin and Eosin
HA Hyaluronan
HBSS Hank’s balanced salt solution
i.p. Intraperitoneal
IA Iodoacetamide
IHC Immunohistochemistry
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