CD103-mediated regulation of airway hypersensitivity responses to bioaerosol-associated antigens. Thèse Emilie Bernatchez Doctorat en microbiologie-immunologie Philosophiae doctor (Ph. D.) Québec, Canada © Emilie Bernatchez, 2018
CD103-mediated regulation of airway hypersensitivity responses to bioaerosol-associated antigens.
Thèse
Emilie Bernatchez
Sous la direction de :
Marie-Renée Blanchet, directrice de recherche David Marsolais, codirecteur de recherche
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Résumé
Les mécanismes immunitaires impliqués dans le maintien de l’homéostasie pulmonaire sont finement régulés étant donné l’exposition constante des voies aériennes aux bioaérosols. Plusieurs cellules participent au maintien de l’homéostasie pulmonaire, telles les cellules dendritiques. Un sous-type de cellules dendritiques pulmonaires attire particulièrement l’attention dans l’homéostasie pulmonaire, les cellules dendritiques CD103+, étant donné
qu’il a été démontré qu’elles participent dans la tolérance immune. Toutefois, ce rôle reste controversé, car des études démontrent qu’elles participent plutôt au développement de réponses inflammatoires pulmonaires. De plus, le CD103 (une intégrine exprimée par des sous-types de cellules dendritiques et de lymphocytes T), est surtout utilisé comme marqueur cellulaire et le rôle spécifique joué par l’expression du CD103 sur ces cellules reste inconnu.
L’homéostasie pulmonaire n’est pas toujours maintenue. Chez des individus susceptibles, l’exposition aux bioaérosols peut mener au développement de réponses inflammatoires. C’est le cas pour l’asthme et l’alvéolite allergique extrinsèque, deux réponses d’hypersensibilités pulmonaires, de type I et de type mixte III/IV respectivement. Récemment, des espèces d’archées, Methanosphaera stadtmanae (MSS) et Methanobrevibacter smithii (MBS), ont été retrouvées en grande concentration dans les bioaérosols d’environnements agricoles et il a été démontré que l’exposition pulmonaire à leur extrait mène au développement d’une réponse immune chez la souris. Toutefois, le type de réponse d’hypersensibilité pulmonaire qu’elles induisent reste méconnu, une information cruciale qui permettra la poursuite de la recherche sur leur potentiel d’induire une réponse pulmonaire chez l’humain. De plus, même si plusieurs thérapies contre les maladies d’hypersensibilité pulmonaires existent, ce ne sont pas tous les sous-groupes de patients qui répondent à la médication, menant à des conséquences socio-économiques importantes pour le système de santé et pour les patients. Ainsi, il demeure important de poursuivre la recherche sur de potentielles cibles thérapeutiques, telles les cellules impliquées dans le maintien de l’homéostasie pulmonaire.
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Cette thèse vise donc à évaluer le rôle de l’expression du CD103 dans le maintien de l’homéostasie pulmonaire dans le contexte de maladies d’hypersensibilité pulmonaires induites par des antigènes retrouvés dans les bioaérosols.
Le rôle de l’expression du CD103 dans l’hypersensibilité de type I induite par l’ovalbumine ou l’extrait d’acariens (modèles d’asthme) a d’abord été a évalué via l’utilisation de souris
Cd103-/-. Nous démontrons que l’expression du CD103 est cruciale pour le contrôle de la
sévérité de l’inflammation pulmonaire et qu’elle pourrait être impliquée dans l’initiation de la phase de résolution de la réponse inflammatoire. De plus, l’expression du CD103 sur les cellules dendritiques joue un rôle dans leur migration aux ganglions lymphatiques.
Ensuite, nous avons évalué le rôle de l’expression du CD103 dans la réponse d’hypersensibilité de type mixte III/IV en réponse à Saccharopolyspora rectivirgula (SR; modèle d’alvéolite allergique extrinsèque) en utilisant des souris Cd103-/-. De plus, en utilisant des modèles de transfert de cellules, nous avons évalué le rôle de l’expression du CD103 dans la réponse au SR lorsque seulement exprimé par les cellules dendritiques ou seulement par les lymphocytes T CD4. Nous démontrons que c’est l’expression du CD103 sur les cellules dendritiques spécifiquement qui est impliquée dans la régulation de l’initiation de la réponse inflammatoire.
Après avoir déterminé le type de réponse d’hypersensibilité induite par l’extrait de MSS ou MBS, nous avons étudié le rôle de l’expression du CD103 en réponse à ces archées. Nous démontrons que l’exposition à MSS induit une réponse immune typique d’une hypersensibilité pulmonaire de type IV. Les résultats obtenus après l’exposition à MBS indiquent aussi que la réponse développée est une hypersensibilité de type IV, même si cela reste à confirmer. Finalement, étant donné une grande variabilité entre nos expériences chez les souris Cd103-/-, nous n’avons pu obtenir de conclusion sur le rôle de l’expression du CD103 dans les réponses d’hypersensibilités induites par les archées.
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Ces résultats démontrent que l’expression du CD103 sur les cellules dendritiques joue un rôle dans le contrôle de l’homéostasie pulmonaire en réponse à des bioaérosols spécifiques qui induisent une hypersensibilité pulmonaire. Les mécanismes exacts régulés par le CD103 sur les cellules dendritiques menant au maintien de l’homéostasie pulmonaire restent à être élucidé. De plus, nos résultats confirment que les espèces d’archées MSS et MBS induisent chacune une réponse d’hypersensibilité pulmonaire qui lui est spécifique, des résultats qui contribueront à déterminer si ces microorganismes induisent une pathologie chez l’Homme.
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Abstract
As we breathe, the lungs are constantly exposed to bioaerosols that challenge the maintenance of airway homeostasis. Many cells are involved in the maintenance of lung homeostasis, such as airway dendritic cells (DCs). A subset of airway DCs has gained special interest in the past years for its role in immune tolerance: CD103+ DCs. Yet, this role remains controversial as there are also reports that they induce airway inflammatory responses. Furthermore, CD103 (an integrin expressed by subsets of DCs and T cells) is mostly used as a marker and whether CD103 expression on these cells plays a specific role remains unknown.
Airway homeostasis is not always maintained. Exposure to bioaerosols can elicit an immune response in susceptible individuals, such as in asthma and hypersensitivity pneumonitis, two common airway hypersensitivity diseases of type I and mixed type III/IV hypersensitivity, respectively. Recently, archaea species Methanosphaera stadtmanae (MSS) and
Methanobrevibacter smithii (MBS) were identified in high concentrations in bioaerosols
from agricultural environments and their extracts were shown to induce an immune response in the airways of mice. However, the type of airway hypersensitivity response they induce remains unknown, a key information that is required if research is pursued on whether they elicit an airway hypersensitivity response in humans. Furthermore, although many therapies for airway hypersensitivity diseases exist, not all subsets of patients respond to the current medication, resulting in high social and economic impacts on the health system and patients. Therefore, research on potential therapy targets for airway hypersensitivity diseases, such as those involved in the maintenance of airway homeostasis, remains important.
This thesis focuses on the role of CD103 expression in the maintenance of lung homeostasis in the context of airway hypersensitivity responses induced by antigens found in bioaerosols.
We first assessed the role of CD103 expression in type I hypersensitivity in response to ovalbumin or house dust mite extract (models of experimental asthma) using Cd103-/- mice. We found that CD103 expression is crucial in controlling the severity of airway inflammation
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and could be involved in initiating the resolution of the inflammatory response. Furthermore, CD103 expression on DCs regulates DC trafficking to the draining lymph nodes.
We then assessed the role for CD103 expression in mixed type III/IV hypersensitivity in response to Saccharopolyspora rectivirgula extract (SR; model of experimental hypersensitivity pneumonitis) using Cd103-/- mice. Furthermore, using models of cell transfers, we evaluated the role for CD103 expression in the response to SR when specifically expressed by dendritic cells or specifically by CD4 T cells. We demonstrate that CD103 expression on DCs specifically is involved in regulating the onset of the inflammatory response.
We finally studied the role for CD103 expression in response to the airway exposure of MSS and MBS extracts, after elucidating the type of hypersensitivity response they induce. We demonstrate that exposure to MSS induces a typical type IV hypersensitivity response. The results obtained after exposure to MBS also indicate development of a type IV hypersensitivity response, although it remains to be confirmed. Finally, due to high variability in the results using Cd103-/- mice, we were unable to reach a conclusion on the role for CD103 expression in response to archaea species.
These results demonstrate that CD103 expression by DCs is involved in the control of airway homeostasis to specific airway hypersensitivity-inducing bioaerosols. The exact mechanisms regulated by CD103 on DCs leading to the maintenance of airway homeostasis remain to be elucidated. Furthermore, our results confirm that archaea species MSS and MBS induce a specific type of hypersensitivity response, which will contribute to the elucidation of whether they induce an airway pathology in humans.
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Table of Content
Résumé ... iii
Abstract ... vi
Table of Content ... viii
List of Figures ... xi
List of Abbreviations and Symbols ... xiii
Dedication ... xv
Acknowledgements ... xvi
Foreword ... xix
Chapter 1: Introduction ... 1
1.1 Lung Homeostasis ... 1
1.1.1 Airway Epithelial Cells ... 1
1.1.2 Alveolar macrophages ... 2
1.2 Pulmonary Dendritic Cells ... 3
1.2.1 Development of pulmonary DCs ... 3
1.2.2 pDCs ... 4
1.2.3 moDCs ... 4
1.2.4 CD11b+ cDCs ... 4
1.2.5 CD103+ cDCs ... 5
1.2.6 CD103: solely a cell subset marker? ... 6
1.3 Bioaerosols and Hypersensitivity Airway Diseases ... 9
1.3.1 Bioaerosols ... 9
1.3.2 Hypersensitivity Responses: Definition ... 10
1.3.3 Asthma ... 12
1.3.3.1 Pathology ... 12
1.3.3.2 Immune response ... 13
1.3.3.3 Remodeling and ASM cells contractility ... 15
1.3.4 Hypersensitivity Pneumonitis ... 17
1.3.4.1 Pathology ... 18
1.3.4.2 Immune response ... 19
1.3.5 Methanogenic Archaea Species and Airway Inflammation ... 21
1.3.5.1 Bioaerosols of Methanogens from the GI tract of Animals and Airway Inflammation ... 23
1.3.5.1 Human Endogenous Methanogens and Association to Diseases ... Erreur ! Signet non défini. 1.3.6 Medication for Hypersensitivity Airway Diseases ... 24
Chapter 2: Research questions, hypotheses et aims ... 27
Chapter 3: Pulmonary CD103 expression regulates airway inflammation in asthma. ... 31
3.1 Résumé ... 32
3.2 Abstract ... 32
3.3 Introduction ... 33
3.4 Material and Methods ... 34
3.5 Results ... 36
3.6 Discussion ... 41
3.7 Acknowledgments ... 43
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3.9 Disclosures ... 43
3.10 References ... 43
3.11 Figure Legends ... 47
3.12 Figures ... 51
Chapter 4: Hypersensitivity Pneumonitis Onset and Severity is Regulated by CD103 Dendritic Cell Expression ... 58
4.1 Résumé ... 59
4.2 Abstract ... 59
4.3 Introduction ... 60
4.4 Material and Methods ... 61
4.5 Results ... 65 4.6 Discussion ... 69 4.7 Conclusions ... 72 4.8 Acknowledgements ... 72 4.9 References ... 72 4.10 Figure Legends ... 77 4.11 Figures ... 80
Chapter 5: Methanosphaera stadtmanae induces a type IV hypersensitivity response in a mouse model of airway inflammation ... 87
5.1 Résumé ... 88
5.2 Abstract ... 88
5.3 Introduction ... 89
5.4 Material and Methods ... 91
5.5 Results ... 94 5.6 Discussion ... 98 5.7 Acknowledgements ... 101 5.8 Grants ... 102 5.9 Disclosures ... 102 5.10 References ... 102 5.11 Figure Legends ... 108 5.12 Figures ... 111
Chapter 6: Complementary Results on Mechanisms Involved in the Airway Hypersensitivity Response to Archaea Species ... 116
6.1 Abstract ... 116
6.2 Introduction ... 117
6.3 Material and Methods ... 118
6.4 Results ... 120
6.5 Discussion ... 121
6.6 Figure Legends ... 124
6.7 Figures ... 126
Chapter 7: Discussion, conclusions and perspectives ... 129
7.1 Differential Role of CD103 Expression in Airway Hypersensitivities Induced by Bioaerosols ... 129
7.1.1 CD103 Expression in Type I and Mixed Type III/IV Airway Hypersensitivity Responses to OVA/HDM and SR Respectively ... 130
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7.1.1.2 CD103 expression and the control of different phases of the immune
response ... 131
7.1.1.3 Exacerbation phenotype in Cd103-/- mice and airway cDCs ... 131
7.1.1.4 DC transfer experiment ... 132
7.1.1.5 Type I hypersensitivity: contradictory study not discussed in Chapter 3 ... 133
7.1.2 CD103 Expression in Type IV Airway Hypersensitivity in Response to MSS and MBS ... 134
7.2 Potential mechanisms regulated by CD103 expression on DCs to maintain lung homeostasis ... 134 7.2.1 AEC barrier ... 135 7.2.2 Activation of T cells ... 136 7.2.2.1 Cross-Priming of CD8 T cells ... 137 7.2.2.2 Production of IL-12 ... 137 7.2.2.3 Production of IL-2 ... 138
7.2.2.4 Co-stimulatory Proteins and Migration ... 139
7.2.3 Summary ... 139
7.3 Human DCs: CD103+ DCs and CD141+ DCs ... 140
7.4 CD103 Expression by T Cell Subsets: Potential Pitfall to CD103 Targeting for Therapy? ... 141
7.5 Impacts of the identification of MSS and MBS as airway hypersensitivity IBs for agricultural workers and clinicians ... 144
7.6 General conclusion ... 146
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List of Figures
CHAPTER 3
Figure 3. 1 Airway inflammation and lung function in wt and Cd103-/- mice: OVA mouse
model of asthma. ... 51
Figure 3. 2. Histology: OVA mouse model of asthma. ... 52
Figure 3. 3. Recall responses, lung inflammatory cells: OVA mouse model of asthma. ... 53
Figure 3. 4. Airway inflammation in WT and Cd103-/- mice: HDM mouse model of asthma. ... 54
Figure 3. 5. CD103 expression on DC and T cell subsets in lung: HDM mouse model of asthma. ... 55
Figure 3. 6 CD103 in the resolution of inflammation: HDM mouse model of asthma. ... 56
Figure 3. 7 Comparison of migration and expression of markers between WT and Cd103 -/-DCs. ... 57
CHAPTER 4 Figure 4. 1 Characterization of the airway inflammatory response in the chronic model of HP: BAL and IgGs. ... 80
Figure 4. 2 Characterization of the airway inflammatory response in the chronic model of HP: histology and cytokine... 81
Figure 4. 3 Airway inflammatory response in WT and Cd103-/- mice in the acute model of HP. ... 82
Figure 4. 4 Characterization of lung DCs populations in response to SR. ... 83
Figure 4. 5 Modulation of CD103 expression on DCs in response to SR in vitro. ... 84
Figure 4. 6 Characterization of T cells populations in response to SR. ... 85
Figure 4. 7 Transfers of DCs and CD4+ T cells. ... 86
CHAPTER 5 Figure 5. 1 MSS induces a mixed TH2 / TH17 immune lung response. ... 111
Figure 5. 2 MSS mainly induces a TH17 polarized immune response. ... 112
Figure 5. 3 MBS induces a weak TH17 immune lung response. ... 113
Figure 5. 4 IL-17A blockade leads to reduced total inflammation and eosinophil influx in the airways. ... 114
Figure 5. 5 Eosinophils and mast cells are not essential for development of MSS-induced airway inflammation. ... 115
CHAPTER 6 Figure 6. 1 Effects of the lack of CD103 expression on the severity of the inflammatory response after exposure to MSS or MBS. ... 126
Figure 6. 2 CD103+ cDCs in the lung are not increased at 3µg MSS or 6.25µg MBS. ... 127
Figure 6. 3 Lack of TLR4 expression leads to a reduced inflammatory response after exposure to MSS. ... 128
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Figure 7. 1 Comparison of cDC subsets found in the airways of WT and Cd103-/- mice. . 132 Figure 7. 2 CD103 expression on splenic-DCs. ... 133 Figure 7. 3 Populations of CD103+ T cells in WT mice compared to Rag-/- mice reconstituted with CD3 lymphocytes isolated from WT mice. ... 143 Figure 7. 4 Relative quantification of MSS-specific IgG1 in plasma of workers from swine
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List of Abbreviations and Symbols
ATP: adenosine triphosphate AJ: adherens junctions AECs: airway epithelial cells AHR: airway hyperresponsiveness ASM: airway smooth muscle AMs: alveolar macrophages
BATF3: basic leucine zipper ATF-like transcription factor 3 BF: bird fancier’s
BM: bone marrow
BAL: bronchoalveolar lavages CCL: C-C motif ligand CCR: C-C motif receptor CD: cluster of differentiation CDPs: common DC progenitors cDCs: conventional DCs CXCL : C-X-C motif ligand Tc: cytotoxic T cells
DAMPs: danger-associated molecular patterns DCs: dendritic cells
DTR: diphtheria toxin receptor ECMs: extracellular matrices FL: farmer’s lung
GI: gastro-intestinal
GATA3: GATA binding protein 3
G-CSF: granulocyte-colony stimulating factor
GM-CSF: Granulocyte-macrophage colony-stimulating factor TH: helper T cells
FCεRI: high-affinity receptor for the Fc region of IgE HDM: house dust mites
HP: hypersensitivity pneumonitis Ig: immunoglobulin
ICOS-L: inducible T-cell co-stimulator ligand IB: inducing bioaerosol
IBD: inflammatory bowel disease ICS: Inhaled corticosteroids ID2: inhibitor of DNA binding 2 ILC2s: innate lymphoid cells type 2 IFN-γ: interferon-gamma
IRF: interferon regulatory factor IL: interleukin
IL-R: IL-receptor
IDL: interstitial lung disease ITGAE: αE integrin
xiv Flt3L: ligand for the receptor tyrosine kinase Flt3 LPS: lipopolysaccharide
LABAs: Long-acting beta-agonists Ly: lymphocyte antigen
LN: lymph nodes
MDPs: macrophage DC progenitors
MIP-2: macrophage inflammatory protein 2 MBS: Methanobrevibacter smithii
MSS: Methanosphaera stadtmanae moDCs: monocyte-derived DCs MUC5AC: mucin 5AC
MUC5B: mucin 5B
MCC: mucociliary clearance
Myd88: myeloid differentiation primary response gene 88 NKT: natural killer T
NETs: neutrophil extracellular traps NLRs: Nod-like receptors
PAMPs: pathogen-associated molecular patterns PRRs: pattern recognition receptors
PMBCs: peripheral mononuclear blood cells pDCs: plasmacytoid DCs
PARs: protease-activated receptors ROR: RAR-related orphan receptor Tregs: regulatory T cells
RA: retinoic acid
SR: Saccharopolyspora rectivirgula
STAT: signal transducer and activator of transcription TJ: tight junctions
TSLP: thymic stromal lymphopoietin TGF-β: Transforming growth factor-beta TLRs: toll-like receptors
TNF-α: tumor necrosis factor-alpha TH1: type 1 TH cells
TH2: type 2 TH cells
TH17: type 17 TH cells
VEGF: vascular endothelial growth factor Wnt: Wingless/int
XCR: X-C Motif Chemokine Receptor XCL: X-C Motif Chemokine Ligand
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Dedication
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Acknowledgements
There are many people without whom the accomplishment of this thesis would not have been possible.
First, I thank Drs Marie-Renée Blanchet and David Marsolais for accepting me in their teams during my undergraduate studies and for their constant supervision during the years of graduate studies with them. I enjoyed working under your supervision and got to learn and grow a lot from your advices, both personally and professionally. A special mention to David "the intern", when you would still go out with us to have a drink (or more than one drink…we miss you Dave).
I also want to thank all the supervisors who gave me advice during my studies. Dr Nicolas Flamand, for advising me more than once and for allowing me to elaborate collaborations with your team. Dr Mathieu Morissette, for your input on my project and for your career advice. Dr Caroline Duchaine, for allowing a collaboration crucial to my thesis, for the constant support on the project and for the support career-wise. Dr Élyse Bissonnette, for your input on my project and for you career advice. Dr Ynuk Bossé, for your input on my project. Dr Yohan Bossé, for your support during the final straight of my thesis, by allowing me to hide in your team’s office to write.
A special thanks to Dr Kelly McNagny that allowed me to complete a chapter of my thesis under his care, in Vancouver. I learned a lot and I had a blast. I also want to thank Drs Fabio Rossi and Michael Underhill, for all the interesting conversations during Friday@5.
I want to thank all the research professionals that helped me. First and foremost, thank you Anick Langlois for everything. I would not be the research candidate I am without you. Marc Veillette, for all the time spent at the flow cytometer, and for all the beer. Marie-Josée Beaulieu, for all the advice since the beginning, always with a smile. Anne-Marie Lemay, Pascale Blais-Lecours, Dany Patoine, Sophie Plante, Véronique Provost, Cindy Henri, Valérie Létourneau, Sylvie Pilote, Cyril Martin, Nathalie Turgeon, Nathalie Gaudreault,
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Michel Taillefer and Sophie Aubin, thank you for the technical and moral, as well as for all the career advice. Finally, I want to thank all the team from the animal unit, especially Justin Robillard, Audrey Chalifoux, Nicolas Thiboutôt-Gagnon and Sébastien Poulin.
Matt Gold, thank you for your supervision during the 4 months you had to endure me (although I am not sure who endured who ahah!). Thank you for being my beer partner in Vancouver. I shall come soon to Toronto, be a bit more patient. I hope your baldness is not getting worse...
I want to thank all the students with whom I worked, had fun, and shared my frustrations. Julyanne, sorry that you had to get stuck with me. Thank you for all your hard work and for working with me on this project. I enjoyed (and still do) working with you. Jean-Francois Lauzon-Joset, I miss you, your constant smile and all your comments to irritate Anick. David Gendron, remember when we were the super-team? Caroline Turcotte and Marie-Chantale Larose, the courses were less boring with you (you know the ones I am talking about)… Katherine, little ball of stress. Magali, for all the good laughs (mostly the one at the grocery store). Potus and all your craziness and weird music on FIP. Morgan, I should have never invited you to move in the office. To Sam: GOOD MORNING SAM! Carole-Ann, for all your energy. Alisson, for making me laugh and for the good poney times. JC for setting an example on how cool powerpoint presentations can be. Ariane, thank god I am not the only one to have a crazy mom that believes in shamans. Mélissa, I know on who to count for an ice-cream. Thanks to all the others that also shared my good and bad moments.
Bernard Clement (I know you hate me right now) and Regan, without you two, my time in Vancouver wouldn’t have been as enjoyable. You two better not change.
Thank you Guillaume for your support during these four years.
TY, Issan, and Jerome. We went through a lot together, and I don’t think I could thank you enough for all the support you gave me and will continue to give me. SEMUSO4ever.
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I also want to thank my precious friends that allowed me to take breaks. Sabrina and Anthony, good food, good wines, good beers, good times. Phelou <3, gaming and music. Alexandra Vien-Nolet, for allowing me to clear my head on Chances Are. Bev&Colin, for all the good laughs and drinks. Sophie&Lloyd, Barbara&John, for all the relaxing times I spent at your houses during my studies, see you soon.
Bab(e), thank you.
Finally, I want to thank my family for all their support throughout my graduate studies. I also thank you for all the practice at vulgarizing my project and for the encouragement to pursue what I like to do, even though no one knows where I will end up!
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Foreword
The results presented in this thesis were obtained by me, under the supervision of Dr Marie-Renée Blanchet (supervisor) and Dr David Marsolais (co-supervisor). An introduction on mechanisms involved in airway homeostasis and on airway hypersensitivity diseases is first presented. Then are presented two chapters (Chapter 3 and 4) constituted of published results covering the role of CD103 expression in a type I airway hypersensitivity disease (asthma) and a mixed type III/IV airway hypersensitivity disease (hypersensitivity pneumonitis). Chapters 5 (published results) and 6 (unpublished results) cover the mechanisms involved in the airway immune response induced by archaea species, including the role of CD103 expression.
The scientific article presented in Chapter 3, Pulmonary CD103 expression regulates airway
inflammation in asthma, was published in The American Journal of Physiology-Lung Cellular and Molecular Physiology in 2015 (PMID: 25681437). This study was designed
under the supervision of Dr Marie-Renée Blanchet. I participated in the writing of this article in collaboration with Dr Blanchet. Drs Matthew J Gold, Anick Langlois, Anne-Marie Lemay, Nicolas Flamand, David Marsolais, Kelly M McNagny and Blanchet revised the manuscript. The results from this paper were obtained and analyzed in collaboration with Dr Gold, Dr Lemay, and Julyanne Brassard. I interpreted the results under the supervision of Dr Blanchet. Dr Cormier was acknowledged for his mentorship and Marc Veillette for his technical assistance in flow cytometry.
The scientific article presented in Chapter 4, Hypersensitivity Pneumonitis Onset and
Severity is Regulated by CD103 Dendritic Cell Expression, was published in PLOS One in
2017 (PMID: 28628641). This study was designed under the supervision of Dr Blanchet. I am the principal author of this article in collaboration with Julyanne Brassard and Dr Blanchet. Juyanne Brassard, Drs Langlois, Flamand, Marsolais, and Blanchet revised the manuscript. The results from this paper were obtained and analyzed in collaboration with Julyanne Brassard. I interpreted the results under the supervision of Dr Blanchet. Marc Veillette was acknowledged for his technical assistance in flow cytometry, the team from the
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animal unit for their technical assistance, Dr Caroline Duchaine for her assistance with the production of SR, and Drs Yvon Cormier, McNagny and Gold for their advices.
The scientific article presented in Chapter 5, Methanosphaera stadtmanae induces a type IV
hypersensitivity response in a mouse model of airway inflammation, was published in Physiological Reports in 2017 (PMID: 28364028). This study was designed under the
supervision of Drs McNagny and Blanchet. I am the principal author of this article in collaboration with Dr Blanchet. Drs Gold, Langlois, Pascale Blais-Lecours, Duchaine, Marsolais, McNagny and Blanchet revised the manuscript. The results from this paper were obtained and analyzed in collaboration with Dr Gold and Magali Boucher. I interpreted the results under the supervision of Drs McNagny and Blanchet. Marc Veillette was acknowledged for his technical assistance in flow cytometry and the team from the animal unit for their technical assistance.
The results presented in Chapter 6 are not part of a manuscript and will not be subject to a future article. The studies were designed under the supervision of Drs McNagny and Blanchet. The results were obtained and analyzed in collaboration with Dr Gold. I interpreted the results under the supervision of Dr Blanchet.
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Chapter 1: Introduction
1.1 Lung Homeostasis
The lungs are constantly exposed to pathogenic and non-pathogenic air particles (aerosols) that enter the airways as we breathe. Immune strategies have therefore evolved to defend the lung tissues against these aerosols and to maintain lung homeostasis so that the lungs are not constantly in a state of inflammation. Airway epithelial cells (AECs), alveolar macrophages (AMs), and dendritic cells (DCs) are key players in lung homeostasis that eliminate the aerosols without eliciting a complete inflammatory response when unnecessary (1–3). These cells form the first line of defense against aerosols that we breathe by forming a barrier, eliminating the aerosols by mucociliary clearance (MCC), producing diverse defense molecules, and, importantly, by inducing immune tolerance to non-pathogenic aerosols. The importance of these cells and mechanisms in airway homeostasis will be briefly described in the following sections.
1.1.1 Airway Epithelial Cells
The barrier function of the epithelium consists of separating the air environment, thus the aerosols, from the parenchymal environment of the lungs. This function is maintained by adherens and tight junctions (AJ and TJ respectively), which confer the epithelium a selective permeability (allowing water and ions while excluding larger particles) (3,4). AJs are composed of E-cadherin and catenin proteins and TJs are composed of claudins, occludins, and zonula occluden proteins (5–8). Disruption of these junctions is involved in the development of airway inflammatory responses, as it allows leakage of high-molecular weight proteins and water in the airways, causing pulmonary oedema (3,9,10), and the passage of the aerosols into the tissues, where they induce an inflammatory response.
MCC is an important function in airway homeostasis as it eliminates aerosols trapped in the mucus layer found on the apical side of AECs (11–13). It involves the production of mucus by mucous cells (goblet and Clara cells) and its elimination to the pharynx by ciliated cells (14–16). Mucus is a gel substance composed of water, mucins, ions, lipids and molecules involved in host defense (11,17–20). Mucins are glycoproteins produced by mucous cells,
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such as goblet and Clara cells, and the two main mucus-forming mucins in the lung are mucin 5AC (MUC5AC) and mucin 5B (MUC5B) (21–23). The secretion of these mucins from their secretory granules is tightly regulated to maintain mucus hygroscopicity, viscosity and viscoelasticity for adequate elimination (21). Ciliated cells are also key players in MCC as they are responsible for the movement of mucus and its trapped antigens/pathogens to the pharynx, where they are eliminated (24). An alteration in MCC, either from a change in mucus composition or defective cilia movement, is a factor in airway diseases that can lead to death because of infections or clogging of the airways (12,21).
AECs express a variety of molecules aimed at the maintenance of immune tolerance or at the defense against an insult. For example, they express the tolerogenic protein cluster of differentiation (CD) 200 that binds the receptor CD200R expressed on AMs, which inhibits their activation (25). In case of encounter with aerosols, AECs express pattern recognition receptors (PRRs), such as toll-like receptors (TLRs), that allow for the rapid secretion of defensins or inflammatory molecules, and activation of DCs (26–29). AECs also express protease-activated receptors (PARs), that contribute to the initiation and resolution of the immune response (30). Surfactant proteins, produced by type II alveolar cells, are crucial for the maintenance of a low surface tension in the alveoli and act both as tolerogenic and pro-inflammatory molecules (15,24); depending on which receptors they bind, surfactant proteins can either inhibit the phagocytic function of AMs or facilitate opsonization (24,31–33).
1.1.2 Alveolar macrophages
AMs represent 90-95% of the immune cells in the alveoli under steady-state and they are present next to alveolar AECs (1,34). They participate in lung homeostasis through their phagocytic function and through induction of immune tolerance. Altered surfactant (oxidized) present in the alveoli needs to be cleared and replaced to maintain the required surface tension for gas exchange (35,36). About 20% of surfactant protein clearance is mediated by AMs that catabolize it, the rest is recycled by alveolar type II cells or eliminated by MCC (37,38). AMs also support maintenance of homeostasis by phagocytosis of apoptotic cells, also called efferocytosis (39). Efficient efferocytosis of AECs prevents them to enter
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necrosis, which leads to the release of danger-associated molecular patterns (DAMPs) and initiation of an inflammatory response, thus damaging the tissue (39–41). Furthermore, they clear antigens present in the alveoli through phagocytosis (33,34,42). Finally, AMs induce a tolerogenic response by inhibiting DC antigen-presenting functions (34).
1.2 Pulmonary Dendritic Cells
In addition to AECs and AMs, DCs participate in the first line of defense by sampling the airway environment, migrating to the lymph nodes (LN) after capturing antigens and providing signals for the differentiation of naïve T cells in either tolerogenic cells to induce tolerance or inflammatory T cells to initiate an immune response for the removal of pathogens (43,44). Airway DC subsets are divided based on their development lineage and marker expression into plasmacytoid DCs (pDCs), monocyte-derived DCs (moDCs), and conventional DCs (cDCs). Each subset possesses specific functions in airway homeostasis and immunity (both or either), depending on their environment. The following sections describe the markers and functions of DCs in mice, which are widely used as models to study airway immunology and which will be used to answer the main objectives of this thesis.
1.2.1 Development of pulmonary DCs
DC subsets all originate from precursors present in the bone marrow (BM) and the ligand for the receptor tyrosine kinase Flt3 (Flt3L) is involved in their development (45). Briefly, hematopoietic stem cells differentiate either in macrophage DC progenitors (MDPs) or common DC progenitors (CDPs) (34,46,47). MDPs differentiate into monocytes in the BM, which migrate to the periphery and then give rise to moDCs (34). CDPs give rise to fully differentiated pDCs in the BM (48,49) or to pre-DCs (34,45). The exact development of lung cDCs is not yet clear, as it was shown that they can originate from different precursors (pre-DCs, monocytes or unidentified precursors) (50,51). What is known is that airway cDCs are fully differentiated in the periphery (34), depending on the production of Flt3L by non-hematopoietic cells (52). cDCs can be further divided into distinct populations based on CD11b and CD103 expression, and CD11b+ cDCs development requires the transcription
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the transcription factors basic leucine zipper ATF-like transcription factor 3 (BATF3), IRF8 and inhibitor of DNA binding 2 (ID2) (54,55). Granulocyte-macrophage colony-stimulating factor (GM-CSF) is also suggested to be a key cytokine involved in airway cDCs development, although it remains debated if it is solely for CD103 expression (56,57).
1.2.2 pDCs
pDCs are mainly found in lymphoid organs, although a small population is present at steady-state in the airways (58,59). They express various markers, including CD45RA, SiglecH, Bst2, and lymphocyte antigen (Ly) 6G (34,59). Studies point that airway pDCs play a tolerogenic role in response to certain antigens by inducing the differentiation of naïve T cells into regulatory T cells (Tregs) via inducible costimulatory ligand (ICOS-L) (59–62). However, they are mainly known for their production of a large amount of type I interferon upon viral antigen stimulation, making them specialized cells to respond to viral infections (58,63,64).
1.2.3 moDCs
moDCs arise from C-C motif receptor (CCR) 2+ monocytes present in the bone marrow that are recruited to the airways by C-C motif ligand (CCL) 2 secreted by AECs after exposure to aerosols (65–67). The study of their functions is still difficult as they share expression of CD11b with CD11b+ cDCs. Furthermore, the marker Ly6C used to differentiate them from
CD11b+ cDCs does not allow complete separation (68). Other markers (CD64, MAR1 and
CD88) have been suggested for the specific identification of moDCs and were used to show that they produce cytokines in response to aerosols, are able to uptake aerosols, migrate to the LN for antigen presentation and induce CD4 T cell proliferation and cytokine production (68,69). They have been described to play a role in asthma and viral immunity (68,70).
1.2.4 CD11b+ cDCs
CD11b+ cDCs are a major subset of DCs in the airways and are thought to constitute the main pro-inflammatory DC population (71,72). Indeed, they are potent cytokine producers, and induce strong CD4 T cell proliferation (68,73–75). In a mouse model of asthma, they are the
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major population of DCs that capture aerosols in the lung and that migrate to the LN for antigen presentation (68). Yet, their specific contribution to airway immune responses remains to be studied because of a lack of good models to deplete them or to differentiate them from moDCs.
1.2.5 CD103+ cDCs
CD103+ cDCs are a major population in the airways with CD11b+ cDCs (71). They are located at the basal surface of AECs via interaction of CD103 with its ligand, E-cadherin (71,76). They express the TJ proteins ZO-2, claudin-1 and claudin-7, allowing them to extend pseudopods into the airway lumen (71), a mechanism that is also observed in the gut (77). The main functions attributed to these cells in the airways are the induction of CD8 T cell mediated immunity and immune tolerance, although some studies reach contradictory conclusions, which will be described in the following paragraphs.
CD103+ cDCs are crucial for CD8 T cell immunity as their absence leads to an impaired
immune response to viruses or tumors (55,78,79). More precisely, they selectively express TLR3 (which recognizes double-stranded RNA (80)) and carry antigens to the LN for cross-presentation to CD8 T cells (75,81–85). Furthermore, a large population of airway CD103+ cDCs, but not CD11b+ cDCs, express X-C Motif Chemokine Receptor (XCR) 1 (86), the receptor for the chemokine X-C Motif Chemokine Ligand (XCL) 1, which is secreted by CD8 T cells upon antigen exposure and which is involved in their activation into cytotoxic T cells (87).
Many studies support a role for CD103+ cDCs in the maintenance of lung homeostasis and
the return to homeostasis. Their principal function of interest is that they induce Tregs, a mechanism depending on the production of retinoic acid (RA) and transforming growth factor (TGF)-β. This function was described in the lungs (88,89) and in the gut, where it was first assessed. The results suggested that CD103+ cDCs play a crucial role in intestinal tolerance to antigens (90–95). Other observations also support their role in lung homeostasis. Namely, that CD103+ DCs derived in vitro express less PRRs and proteins involved in
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phagocytosis than CD103- DCs (96); they secrete cytokines upon stimulation in lower
quantities than moDCs and CD11b+ cDCs (68,74); they are the main DC population
expressing ICOS-L (involved in the induction of Tregs) (68); they secrete CCL22 in the highest quantity (a chemokine for type 2 helper T (TH;TH2) cells and Tregs (97)), which
blocks CD4 T cell proliferation in vitro (74); they selectively secrete interleukin (IL)-2, which inhibits IL-17A production in activated CD4 T cells (98); they secrete IL-12 that dampens the allergic response (99); and they fail to sensitize mice to develop allergic response (68,100). Furthermore, they preferentially uptake apoptotic cells in the airways, suggesting that they play a role in efferocytosis (101), a process involved in lung homeostasis (39–41).
Contrarily, in vitro studies also show that CD103+ cDCs activate inflammatory cytokines
production in T cells. However, there is no consensus on which cytokines are preferentially induced by them (74,102,103). While possible, it was shown in vivo in a mouse model of asthma that CD11b+ and moDCs are the key T cell activators ̶ not CD103+ cDCs (68). Whether this is true in other airway diseases remains to be elucidated. Furthermore, it was shown that lack of CD103+ cDCs results in a decreased airway inflammatory response in asthma (102), contrarily to the data demonstrating that they fail to sensitize mice to develop an allergic response (68,100). Therefore, although literature mostly suggest a tolerogenic role for CD103+ cDCs, the induction of tolerance by these DCs remains controversial, and, as tolerance is an important mechanism involved in tissue homeostasis, the role for CD103 in lung homeostasis remains elusive.
1.2.6 CD103: solely a cell subset marker? 1.2.6.1 CD103 expression
CD103, also known as αE integrin (ITGAE), is a membrane protein forming a dimer with the β7 subunit and its only known ligand is E-cadherin (104). In addition to being expressed by DCs in a variety of organs (lung, skin, spleen, gut) (105), CD103 is expressed by subsets of intraepithelial CD4, CD8 T cells and Tregs and plays a role in the adhesion of these cells to the epithelium, including in the airways (106–115). Many functions of these CD103+ T
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cells are described and seem tissue-specific, although the exact role of CD103 remains elusive. CD103+ CD8 T cells play a role in cancer, where their intratumoral presence is
associated with a better prognostic. More specifically, CD103 expression plays a role in CD8 T cell adhesion to tumors and in enhancing their cytotoxicity (116–118). Alternatively, CD103+ CD8 T cells can also be regulatory cells that inhibit CD4 mediated inflammatory response (119,120), a function also described in human CD8 T cells (110). Furthermore, CD103 is used as a marker for CD8 memory T cells (121,122). For Tregs, CD103 expression is involved in their retention in tissues, thus suppressing T cells mediated inflammation (107). It is suggested that CD103+ Tregs possess enhanced suppressive functions compared to their CD103- counterparts (123,124). The role for CD103 expression by Tregs was determined to be crucial for their suppressive function in the context of skin infection (107), but not in the context of colitis (92). The role for CD103+ CD4 T cells is still vague, but they are described as pathologic in ulcerative colitis (125). It is also suggested that CD103 expression on T cells plays a role in their proliferation (126,127), and that CD103 induces the formation of protrusions upon contact with E-cadherin, possibly contributing to the functions of CD103-expressing cells (DCs and T cells) that are described above (cytotoxicity, suppression, cross-presentation, etc.) (128).
1.2.6.2 CD103 and cell signaling
As mentioned, CD103 is the αE integrin forming a dimer with the β7 subunit (104). Twenty-four integrins have been identified in vertebrates, formed by a combination of one of the eighteen structurally diverse α subunit and one of the eight structurally conserved β subunit (129,130). They are transmembrane receptors, with an extracellular ligand-binding domain and a short cytoplasmic tail. The extracellular domain goes through conformational changes (low-affinity, intermediate-affinity and high-affinity) which is mediated by outside-in and
inside-out signaling (130,131). The intracellular signaling of integrins have been thoroughly
studied and is described as the adhesome, formed by 156 proteins, lipids and calcium ions linked by 690 interactions (129). The activation of the signaling pathways can lead to regulation of different functions, such as cell survival, growth and proliferation, and cell motility, spreading and migration (132). However, these signaling systems have been mostly studied using 1 and 3 associated integrins. Therefore, even though these signaling
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pathways have been thoroughly studied, the specifics of CD103 signaling remain largely unknown. The information accumulated so far indicate that the integrin-linked kinase (ILK) and the AKT/PKB pathway is involved in CD103 activation and is mediated by TGFβ (133). Otherwise, a study on the other integrin sharing the β7 subunit, α4β7, demonstrate that alterations in the conserved adjacent to metal ion-dependent adhesion site (ADMIDAS) in the β7 subunit leads to over activation of α4β7 (134). Sadly, the implication of this mutation for αEβ7 have not been studied, but is could be hypothesized that it leads to over activation of this integrin as well. Other than these two articles, nothing else on the signaling pathways involved in αEβ7 activation is reported.
1.2.6.3 CD103 deficient mice
A few breeds of transgenic mice are commonly used to study CD103+ cells, and more
precisely CD103+ DCs: Langerin-diphtheria toxin receptor (DTR) mice, BXH2 mice and
Batf3-/- mice. However, these mice lack entire DC population(s) (instead of removing solely
CD103 expression) as it either depletes the cells (DTR mice) or inhibits their development by removal of differentiation factors (IRF8 in BXH2 mice and BATF3 in Batf3-/- mice) (55,83,135–137). Furthermore, BXH2 mice have splenomegaly, myeloid leukemia, and do not produce normal amounts of IL12p40 (135,138), defects that can alter the studied immune response in a way that is not linked to the lack of CD103+ DCs.
Another transgenic mouse breed that lacks ubiquitous CD103 expression can be used to study CD103: Cd103-/- (B6.129S2(C)-Itgaetm1Cmp/J) mice. These mice have the exon 10 of the Itgae gene replaced with a neomycin resistance gene (139), thus inhibiting the formation of the αEβ7 complex that binds E-cadherin without altering the formation of the other integrin requiring the β7 subunit, α4β7, an integrin that is crucial for T cell migration in the gut (140). Theoretically, this mutation does not deplete DC populations as CD103 is not known to be involved in their development. Supporting this, studies in these mice show normal frequencies and quantities of DCs in the gut (92,141) and the trachea (142). However, these mice lack intraepithelial T cells in the gut and skin, but not in the airways (139,143), and develop spontaneous skin lesions with age (up to 6 months of age) (144). Studies in these mice supporting a role for CD103 in homeostasis demonstrate an increased mortality rate and
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neutrophilic inflammation compared to WT mice in a model of lung fibrosis (145), and an exacerbated ear inflammatory response in a model of contact hypersensitivity (146).
Although a few functions for CD103+ cells have been described, the only clear functions described for CD103 are allowing the localization of CD103-expressing cells (DCs and T cell subsets) to the epithelium through binding to E-cadherin and the induction of cell protrusions upon contact with E-cadherin (106–115,128). It is therefore not known whether CD103 expression by airway cells is crucial for their functions or if it is solely a subset marker. In this thesis, the role of CD103 expression in lung homeostasis maintenance by DCs in response to bioaerosol-induced airway hypersensitivities will be evaluated.
The following sections will cover the role of bioaerosols in the induction of airway hypersensitivity responses, namely asthma and hypersensitivity pneumonitis (HP). Furthermore, the potential of bioaerosols containing archaea species, newly identified in high concentration in agricultural buildings, to induce an airway hypersensitivity response will be described.
1.3 Bioaerosols and Hypersensitivity Airway Diseases
1.3.1 BioaerosolsBioaerosols are complex air particles of biological origins (pathogenic or non-pathogenic microorganisms, animal or plant proteins), which can be found in a solid (dusts) or liquid (droplets) form (147–149). They are found in all environments, whether indoor or outdoor, and their composition can be of occupational or non-occupational character. For example, in a dairy barn, their composition can originate from cows (occupational) or from workers (non-occupational). Importantly, bioaerosols can be inhaled and enter the airways. The location of deposition in the airways depends on their physico-chemical properties (size, shape, density,
etc.) as well as the lung physiological properties of the host (breathing pattern, size of
respiratory tract, airway resistance, etc.) (148,150). After deposition of bioaerosols in the airways, mechanisms involved in the maintenance of lung homeostasis are responsible for their clearance. However, bioaerosols are not always cleared efficiently from the airways and
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can activate the immune system in an exaggerated manner, thus inducing airway diseases (147,151).
Bioaerosols are sources of infectious (tuberculosis, influenza, gastroenteritis, etc. (152–154)) and non-infectious airway diseases, such as airway hypersensitivity diseases (asthma and hypersensitivity pneumonitis (HP) (155)). Development of disease depends on the nature of the bioaerosol, its size, its concentration in the environment and the susceptibility of the host (151). The nature of many infectious and non-infectious airway diseases inducing bioaerosol (IB) components are well-defined (Mycobacterium tuberculosis (tuberculosis), influenza virus (influenza), pollen (asthma), Saccharopolyspora rectivirgula (SR; HP), etc.); however, not all IB components have been identified and there is a lack of study on potential IB components, such as those found in agricultural environments. The size of bioaerosols determines where it is deposited in the airways and where the pathology will develop. Usually, particles between 5 and 10µm will deposit in the upper airways (trachea), and smaller particles will enter deeper in the lung to deposit in the bigger bronchi or even the small bronchi (148,150). Some environments are more prone to induce airway diseases because of their high bioaerosol concentration, such as hospitals and agricultural buildings (147,151,156), and exposure to bioaerosols in the agricultural environment is associated with the development of airway diseases (157). However, recommendations on maximal exposure to bioaerosols remain to be determined, due to a lack of studies on exposure measurements (156).
Airway hypersensitivity diseases caused by IBs are of particular interests as they are common and have high social and economic impacts, as will be discussed in the following sections.
1.3.2 Hypersensitivity Responses: Definition
The classification of the immune mechanisms in four types of hypersensitivity responses (Type I - IV) is the work of Gell and Coombes in 1963, in an attempt to categorize inflammatory responses induced after exposure to medications (158). Although this classification has its limitations (it is mainly a very general classification that does not take
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into account specific mechanisms of the possible inflammatory cascades elucidated after their work), it has been used widely to categorize inflammatory diseases. Today, hypersensitivity diseases are defined as exaggerated inflammatory responses developed after sensitization to non-infectious molecules, whether endo- or exogenous, that usually induce immune tolerance. A wide variety of these hypersensitivity-inducing molecules acting in different organs have now been identified such as pharmacological molecules, venom, and IB components in the airways (which is the focus of this thesis) (159) although some remain to be elucidated.
Type I hypersensitivity, also called the immediate or allergic response, is caused by the development of an immune response where mast cells and immunoglobulin (Ig) E play a crucial role. IgE are produced after the first sensitization to the inducing molecule and are ligated to the membrane of mast cells. Upon following exposures to the inducing molecule , there is mast cell degranulation, which leads to the release of immune mediators that can cause vasodilation and contraction of smooth muscles (159–161).
Type II hypersensitivity is based on the ligation of the inducing molecule to cell membrane, and then the ligation of antibodies (IgG and IgM) to the inducing molecule. This opsonization process induces the killing of the marked host cells by phagocytes (macrophages, neutrophils and eosinophils) and by anaphylotoxins (159,160).
Type III hypersensitivity is caused by the formation of immune complexes between IgG or IgM and the inducing molecule that precipitates in the blood vessels or tissues where the inflammatory response occurs. Such complexes are called precipitins and they amplify the inflammatory response where they precipitate (159–161).
Type IV, or delayed, hypersensitivity is cell-mediated, in contrast to the other three types of hypersensitivity, which are antibody-mediated. This hypersensitivity response involves a strong activation of cytotoxic CD8+ T cells or CD4+ T
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and TH cells produce the inflammatory mediators interferon-gamma (IFN-γ) and IL-17A,
damaging the tissues (160).
Important airway diseases fall into one or more categories of hypersensitivity responses, such as asthma (type I hypersensitivity disease) and hypersensitivity pneumonitis (HP; mixed type III/IV hypersensitivity disease). However, there is a lack of study of the immune mechanisms induced by potential airway hypersensitivity disease IB components, such as methanogenic archaea species found in agricultural environments. Furthermore, induction of airway hypersensitivity responses in a chronic manner damages the lung tissues in a way that current therapies cannot reverse, reinforcing the need to elucidate mechanisms involved in lung homeostasis, such as the one surrounding CD103 expression, in order to prevent the development of these diseases.
1.3.3 Asthma
Asthma is a complex chronic respiratory disease that develops in susceptible individuals. Numerous genetic (genes mostly involved in the epithelial barrier and immune response) and environmental (pollution, tobacco smoke, etc.) susceptibility factors have been identified (162–168), of which many differed between the multiple cohorts studied, rendering this disease highly heterogeneous. It is a fairly common disease, affecting more than 235 million people worldwide, and the hospitalizations and treatments are an economic burden for the healthcare system (169–171).
1.3.3.1 Pathology
Asthma is classified based on clinical phenotypes and subsets, which include allergic asthma, non-allergic asthma, occupational asthma, etc. (172,173). Allergic asthma is the best defined subset and involves the activation of a type I hypersensitivity response after exposure to IB components. However, not all asthma subsets are characterized by a type I hypersensitivity, as noted by the presence or absence of granulocytes in the bronchoalveolar lavages (BAL) of patients and by the cytokines present in the airways (173). Nevertheless, the general
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immunopathology involves an inflammatory response, a remodeling of the airways and airway hyperresponsiveness (AHR).
IBs of allergic asthma cover a broad class of molecules, such as the common allergens from pollens, molds, animal proteins and house dust mites (HDM) (174,175). The classical type I hypersensitivity response in allergic asthma develops after sensitization and re-exposure to these IBs that activate the AECs, leading to secretion of IL-4, IL-5 and IL-13 by innate (type 2 innate lymphoid cells; ILC2s) and adaptive TH2 immune cells. These cytokines are
mediators involved in the recruitment and activation of eosinophils, production of IgE and activation of mast cells. Remodeling of the airways and exaggerated airway smooth muscle (ASM) contractility are also characteristics of the asthmatic response. These three components (inflammation, remodeling and increased ASM mass), which have been thoroughly studied in both humans and animal models, are interlinked and lead to the development of symptoms and signs, including wheezing, shortness of breath, cough and airway limitation. These symptoms and signs can usually be well-controlled through proper intake of medication and are reversible; however, subsets of patients are difficult to treat, such as patients with uncontrolled allergic asthma, also called severe asthma.
1.3.3.2 Immune response
Upon exposure to IB components in allergic asthma, there is recruitment of TH2 cells and
eosinophils in the airways and mast cells are activated. The activation of these mechanisms depends on a complex immune cascade involving the release of cytokines typical of a hypersensitivity type I response by AECs, DCs, ILC2s, basophils and B lymphocytes.
1.3.3.2.1 TH2 cells
Naïve T cells proliferate into TH2 cells in the LN after activation by mature DCs that migrated
from the airways, IL-4 and IL-2. When the IB components are present near the epithelium, immature DCs are able to capture these, which initiates their maturation and migration to efferent LN. Furthermore, AECs exposed to IB components increase their secretion of DAMPs (adenosine triphosphate (ATP), uric acid) and cytokines (GM-CSF, thymic stromal
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lymphopoietin (TSLP), IL-33 and IL-25) (176–183) that activate immature DCs and signal them to increase their expression of CD252, a protein involved in TH2 cells differentiation
(184–186). In the LN, the presence of IL-4, possibly produced by basophils and CD4 T cells themselves (187,188), is required in addition of DCs for the proper differentiation and proliferation of TH2 cells (189). IL-4 activates signal transducer and activator of transcription
(STAT) 6 and IL-2 activates STAT5, which activates the transcription factor GATA binding protein 3 (GATA3) in naïve T cells (190–192). GATA3 and STAT5 further activate IL-4 secretion for the retroactive activation of TH2 cells (193,194), and GATA3 binds promoters
of the Il5 and Il13 genes that are involved in TH2 inflammatory functions (195). After
proliferation in the LN, TH2 cells migrate to the airways in response to the chemokines
CCL17 and CCL22 that bind CCR4 (196,197). This mechanism depends on the increased expression of CCL22 and CCL17 in response to IL-13 (196,197). ILC2s, innate T cells present at steady-state in the airways, secrete IL-13 in response to IL-33 and IL-25 produced by AECs (198), making them possible inducers of CCL17 and CCL22 for TH2 recruitment.
In the airways, the secretion of IL-4, IL-5 and IL-13 by TH2 cells plays a major role in the
development of the eosinophilic response and mast cell activation.
1.3.3.2.2 Eosinophils
Eosinophils develop in the bone marrow before migrating to the airways and their development and survival depend on the cytokines IL-3, GM-CSF and IL-5 (199,200). IL-5 is produced by ILC2s (in response to IL-25 and IL-33) and by TH2 cells after exposure to IB
components, and its presence in the serum allows for the activation of eosinophil differentiation in the bone marrow (200,201). Migration of eosinophils to the airways is then mediated by eotaxins (eotaxin-1: CCL11; eotaxin-2: CCL24; and eotaxin-3: CCL26) (200,202,203), chemokines mainly produced by AECs in response to IL-13 (204–206). Eosinophils are activated during their migration to the airways and in the airways by a variety of inflammatory mediators, including IL-5 and GM-CSF, leading to cytokine secretion and degranulation of the cells (207). The inflammatory mediators produced by eosinophils, such as IL-4 and IL-13, further enhances TH2 and eosinophilic response (199,208,209). The
granules contain cationic proteins and enzymes that mainly contribute to airway remodeling and the activation of mast cells (210,211).
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1.3.3.2.3 Mast cells
The activation of mast cells plays a role in type I hypersensitivity through the release of mediators stored in their granules or through the production of inflammatory molecules. The activation of mast cells depends on a variety of mechanisms, IgE production being involved in allergic asthma. IL-4 and IL-13 produced by IB component-induced TH2 cells are
responsible for isotype switching of IB component-specific IgG1 and IgE in B cells
(199,212). IB component-specific IgEs bind to the high-affinity receptor of the Fc region of IgE (FCεRI) expressed by mast cells (199). When IB components bind to these IgEs, there is cross-linking of FCεRI, inducing degranulation of the cells (213). Granules stored in mast cells contain a variety of inflammatory mediators, such as histamine, heparin, and tryptase (213). Activation of mast cells also induces production and secretion of lipid mediators (prostaglandins, thromboxanes, leukotrienes, etc.) and cytokines (IL-4, IL-13, tumor necrosis factor-alpha (TNF-α), etc.) (213–216). These mediators act on inflammatory cells (recruitment and activation), ASM cells (contraction or relaxation), fibroblasts (activation) and endothelial cells (vasodilation and angiogenesis), thus contributing to the exacerbation of the inflammatory response, airway remodeling and AHR.
1.3.3.3 Remodeling and ASM contractility
Remodeling of the airways in asthma involves desquamation of epithelial cells, subepithelial fibrosis, smooth muscle cell hyperplasia and hypertrophy, goblet cells hyperplasia and angiogenesis. All these mechanisms, which will be covered in more details in the following sections, contribute to the narrowing of the airways through an increase of the thickness of the epithelium, the loss of respiratory functions in asthmatic patients, and further enhance the immune response via the secretion of inflammatory mediators.
1.3.3.3.1 Epithelial desquamation
Epithelial desquamation involves the loss of mucous or ciliated cells on top of basal cells. It is described in the airways of asthmatic patients (217,218), although whether it is a consequence of mechanical damage caused by the biopsy remains debated (219).
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Nevertheless, epithelial damage is induced in the airways of asthmatic patients, inducing apoptosis of AECs. Granule proteins from eosinophils have also been shown to kill mammalian cells and are thought to play a role in damaging AECs (199,220,221). Many IBs (HDM, pollen, Aspergillus, cockroaches) possess proteinase activity (activating PARs) and exposure of the epithelial cells of asthmatic patients to these IBs leads to loss of TJ and AJ, disrupting the integrity of the epithelial barrier (217,222–225).
1.3.3.3.2 Subepithelial fibrosis
Subepithelial fibrosis involves the deposition of extracellular matrices (ECMs) and components, such as fibronectin and collagen (types III and V), under epithelial cells (226,227). It stiffens the airways, reducing its capacity to return to steady-state after inspiration (i.e. increases the elastance of the airways) (228). TGF-β plays a role in the deposition of ECMs in asthma and it is induced by IL-5 and IL-13 (229–234). IL-13 induces latent TGF-β and TGF-β in AECs, monocytes, and macrophages (232,235,236). IL-5 induces fibrosis through recruitment of eosinophils in the airways, which produce TGF-β (237–239) and activate AECs to produce pro-fibrotic molecules (210).
1.3.3.3.3 Goblet cell hyperplasia
Goblet cell hyperplasia is the increase of goblet cells in the airways. In AECs, IL-13 induces the expression of the transcription factor Sam pointed domain-containing ETS that, in turn, inhibits Foxa2, leading to differentiation of AECs into mucous cells and hypersecretion of mucus (232,240,241). It is MUC5AC, not MUC5B, that is increased in asthmatic patients, and hypersecretion of this mucin plays a crucial role in the development of AHR by occluding the airways (242–244). In fatal asthma, hypersecretion of mucus causes occlusion of the airways, which is thought to contribute to the death of patients (245).
1.3.3.3.4 Angiogenesis
Angiogenesis consists in an increase in the pre-existing blood vessels network and is a normal wound healing process that supplies nutrients. In asthma, many immune mediators, such as TGF-β and vascular endothelial growth factor (VEGF), and ECM proteins contribute to
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angiogenesis (246,247). In addition to increasing the thickness of the airway epithelium, angiogenesis increases plasma exudation in the lungs in response to immune mediators causing vasodilation (for example, histamine), thus increasing elastance of the airways (213,246). Furthermore, it contributes to immune cells trafficking in the airways from the periphery, enhancing the chronic inflammatory response (246).
1.3.3.3.5 ASM cells hyperplasia, hypertrophy and ASM contractility
Smooth muscle mass in asthmatics patients is increased via ASM cell hyperplasia and possibly hypertrophy, and is a major contributor to airway narrowing and AHR in asthma (231,248,249). Many cytokines secreted in the inflammatory response (histamine, tryptase, TGF-β, etc.) and mediators involved in extracellular matrix deposition can cause this phenomenon by inducing proliferation of ASM cells (250). Furthermore, hyperplasia of ASM cells is thought to induce remodeling of the airways by mechanically stressing the epithelium (leading to secretion of fibrotic molecules by AECs) and by secreting pro-fibrotic cytokines themselves (250,251). ASM cells are also able to release inflammatory mediators that recruit eosinophils and activate mast cells, contributing to the development/maintenance of the inflammatory response (252). There are two school of thoughts to explain the exaggerated ASM contractility, one stating that there are defects in ASM cells and the other postulating that it is a consequence of the ASM environment. Considering these two school of thoughts, an intrinsic defect in ASM cells, increased sensitivity and increased contractility in response to immune mediators contribute to AHR development in asthma (253,254). IL-13 is a spasmogen acting directly on ASM cells by enhancing their calcium influx and sensitivity (232,255), and indirectly by inducing secretion of immune mediators by other cells, such as histamine from mast cells (199,213).
1.3.4 Hypersensitivity Pneumonitis
HP is an interstitial lung disease (ILD), a disease where the tissue around the alveoli is damaged (256,257). It is developed after exposure to an IB component in susceptible individuals. The precise susceptibility factors remain unknown to this day although genetics and the environment (presence of viruses in the airways or exposure to pesticides (258,259))
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are strongly suggested to play a role in the induction of HP (260–262). A few polymorphisms have been associated with HP, in genes mainly involved in antigen processing and presentation (263–265). Polymorphisms in the gene for TNF-α have also been studied, with contradictory results (262,266). Interestingly, a polymorphism in metalloproteinase inhibitor 3 has been shown to have a protective role in HP, although the mechanism remains unknown (267,268). Unfortunately, the prevalence of this disease is not well-defined, mainly due to difficulty of diagnosis and the lack of epidemiological studies (256,269,270). Nevertheless, it can be estimated that 4-15% of ILDs reported in Europe is HP, and that 0.5-3% of farmers will develop HP (271,272).
So far, up to two hundred HP-IB components have been identified, which can be of biological or chemical origin (269,273); however, there are a lot of cases where the IB component is not identified (274–276). Two common forms of HP for which the immunopathology is well-described are farmer’s and bird fancier’s lung diseases. Farmer’s lung is caused by inhalation of the bacteria SR, an actinomycete found in moldy hay, and bird fancier’s lung disease is caused by inhalation of proteins from bird droppings and feathers (270,273).
1.3.4.1 Pathology
Classically, HP is divided in three-forms (acute, subacute and chronic) based on the clinical features (277). However, others argue that a two-form model (acute and chronic) would fit best (256,278). The form which the disease takes is suggested to depend on the nature of the IB component, and the exposure concentration and rate (279). To support this, farmer’s lung disease patients typically show clinical signs of acute HP, while bird fancier’s lung disease patients show chronic symptoms (270). Whether the different forms are developmental stages of the disease remains to be debated (269,270), although it has been previously reported that the chronic form developed in patients suffering first from acute HP (280).
In the acute form, the symptoms (influenza-like) appear shortly after an intense exposure to the IB component (270,279). This form is usually non-progressive and, upon cessation of exposure to the IB component, symptoms can be reversed (269). The subacute and chronic
