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Characterization of the viability and the function of granulocytes used for transfusion

Mémoire

Andrea Murru

Maîtrise en sciences cliniques et biomédicales - avec mémoire Maître ès sciences (M. Sc.)

Québec, Canada

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Résumé

Les granulocytes neutrophiles sont les cellules immunitaires les plus abondantes dans l’organisme. Elles constituent notre première ligne de défense contre les infections microbiennes. Les personnes neutropéniques sont à haut risque de mortalité en cas d’infections microbiennes graves.

Lorsque les traitements antimicrobiens standards sont inefficaces, les patients peuvent recevoir une transfusion de concentré de granulocytes (CG) pour rétablir temporairement un nombre de neutrophiles en circulation permettant d’éradiquer l’infection.

Au Canada, les CG sont préparés par Héma-Québec à partir de donneurs stimulés avec une dose de prednisone augmentant le nombre de neutrophiles en circulation. Aux États-Unis le G-CSF sert d’agent de stimulation des donneurs. Les neutrophiles sont récoltés par leucaphérèse et certifiés pour la transfusion selon le nombre de neutrophiles récoltés. Or, ce critère ne reflète pas la viabilité et l’état fonctionnel des cellules transfusées.

Notre étude compare la viabilité, le phénotype et les fonctions antimicrobiennes des neutrophiles obtenus des CG dérivés de prednisone et de G-CSF. La prednisone mobilise des neutrophiles matures avec une phagocytose augmentée mais le nombre de neutrophiles est souvent proche de la limite transfusable. Le G-CSF augmente considérablement le nombre de neutrophiles récoltés, dont près de la moitié sont immatures, en plus d’abaisser la chimiotaxie et d’augmenter la production de cytokines.

D’après nos résultats, la durée d’entreposage des CG est le paramètre le plus critique pour le maintien de la viabilité et des fonctions antimicrobiennes des neutrophiles, particulièrement pour ceux dérivés du G-CSF alors que la leucaphérèse a un effet négligeable sur les deux types de CG.

L’amélioration des processus de fabrication, d’entreposage des CG et leur certification avec un critère fonctionnel permettra de maintenir la viabilité et les fonctions des neutrophiles et d’assurer leur efficacité in vivo.

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Abstract

Neutrophil granulocyte are the most abundant immune cells in the organism. They constitute our first line of defence against invasive pathogens. Neutropenic patients with life-threatening infections can be transfused with granulocyte concentrates (GCs) to temporarily restore a sufficient number of circulating neutrophils to eradicate infection.

In Canada, GC are prepared by Héma-Québec from donors pre-treated with a unique dose of prednisone enhancing the number of circulating neutrophils. In the U.S, G-CSF is used as mobilizing agent in the donors. Neutrophils are collected by leukapheresis and certified for transfusion according to the number of neutrophils collected. This criterion, however, does not reflect the viability and the functional capacity of the transfused cells.

Our study compare the viability, the phenotype and antimicrobial functions of neutrophils from prednisone and G-CSF-derived GCs. Prednisone mobilizes mature neutrophils with enhanced phagocytosis although the number of collected neutrophils is sometimes below the required transfusion dose of 1 x1010 granulocytes per unit. G-CSF significantly increases the number of collected neutrophils, though almost half are immature, along with decreasing chemotaxis and increasing cytokine production. According to our results, storage duration is the most critical parameter for the preservation of neutrophils viability and antimicrobial functions, particularly for G-CSF-derived GC neutrophils whereas leukapheresis had negligible effect for both type of GCs.

The improvement of GCs supply chain and storage as well as certification based on a functional criterion will allow to preserve the viability and effector functions of neutrophils and guaranty GCs in vivo efficacy.

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Table of contents

Résumé ... ii

Abstract ... iii

Table of contents ... iv

Tables ... vii

Figures ... viii

Abbreviations ... ix

Acknowledgments ... xiii

Foreword ... xv

Introduction

... 1

1. Types of transfusion ... 2

1.1. Labile blood products ... 2

1.1.1 Common transfused labile blood products ... 2

Red blood cells ... 2

Platelets ... 3

Plasma ... 3

1.2 Granulocyte concentrates ... 6

1.2.1 An overview of granulocyte transfusion therapy ... 6

1.2.2 Clinical efficacy of granulocyte concentrates ... 10

The “RING study” ... 10

Creation of an international register for GCs ... 11

1.2.3 Donor recruitment and pre-treatment ... 12

Glucocorticoids ... 14

G-CSF area ... 14

1.2.4 Collection methods for GCs ... 15

Continuous flow centrifugation ... 15

Buffy coat pooling ... 17

1.2.5 Certification of granulocyte concentrates... 19

1.2.6 Canadian context of granulocyte concentrate transfusions ... 20

Source of GCs in Canada ... 20

Processing of GCs in Canada ... 20

1.2.7 Management of the risk of adverse effect related to GC therapy ... 21

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Management of adverse effects of GC collection on donors ... 21

Management of adverse effects of GTX in the recipients ... 22

2. Antimicrobial immune response: Implications for GCs ... 23

2.1 Organization of immune antimicrobial response ... 24

Epithelial cells ... 24

Neutrophil life cycle and effector functions ... 25

Life cycle of neutrophils ... 25

Phagocytosis ... 32

Degranulation ... 34

Reactive oxygen species production ... 37

Formation of NETs ... 37

Neutrophils interaction with other immune cells ... 38

Neutrophil interaction with monocytes and macrophages... 38

Neutrophils as inducers of the adaptive immune response ... 42

2.2 Introduction to neutrophils diversity in health and disease ... 45

Chapter 1: Rationale, hypothesis and study design ... 48

1.1 Rationale ... 48

1.2 Hypothesis ... 48

1.3 Study design ... 49

Chapter 2: Comparison of neutrophil function in granulocyte concentrates from prednisone and G-CSF-treated donors: effect of stimulant, apheresis and storage. ... 51

2.1 Résumé ... 52

2.2 Abstract ... 54

2.3 Introduction ... 56

2.4 Materials and Methods ... 58

2.4.1 Donor recruitment and GTX collection ... 58

2.4.2 Gases and metabolites analysis ... 59

2.4.3 Neutrophils and peripheral blood mononuclear cells isolation ... 59

2.4.4 Flow cytometry analysis ... 59

2.4.5 Viability assay ... 60

2.4.6 Intracellular Calcium Mobilization ... 60

2.4.7 Chemotaxis... 60

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2.4.9 Superoxide Anion Measurement ... 61

2.4.10 Phagocytosis ... 61

2.4.11 Cytospin... 62

2.4.12 Cytokine production ... 62

2.4.13 Statistical analysis ... 62

2.5 Results ... 63

2.5.1 Cohort characteristics and GC collection... 63

2.5.2 Comparison of neutrophil counts in prednisone and G-CSF GCs ... 63

2.5.3 Viability and surface marker expression of prednisone and G-CSF mobilized neutrophils ... 64

2.5.4 Antimicrobial functions of prednisone and G-CSF-derived, GC neutrophils ... 65

2.5.5 Effect of leukapheresis on the function of prednisone and G-CSF-derived GC neutrophils ... 66

2.5.6 Effect of storage on neutrophil viability, cell-surface markers, pH and metabolite concentration ... 67

2.5.7 Effect of storage on neutrophil function ... 68

2.6 Discussion ... 69

Figures and legends ... 74

Tables ... 82

Supplementary data ... 84

2.7 References ... 93

Chapter 3: Discussion... 96

Conclusion ... 105

Bibliography ... 107

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Tables

Introduction

Table 1: The red blood cell antigens that define different blood types and cause transfusion reactions

... 5

Table 2: Adverse effects of GC collection on donors. ... 8

Table 3 : Adverse effects of GTX on the recipients ... 9

Table 4 : Serological tests for infectious agents routinely performed by Héma-Québec ... 13

Chapter 2: Comparison of neutrophil function in granulocyte concentrates from prednisone and G-CSF-treated donors: effect of stimulant, apheresis and storage

Table 1: Donors characteristics and collection regimens ... 82

Table 2 : Composition of donors peripheral blood and granulocyte concentrates ... 83

Supplementary table 1 ... 92

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Figures

Introduction

Figure 1 : Composition of whole blood ... 4

Figure 2 : Granulocyte collection on a Spectra Optia apheresis machine ... 16

Figure 3 : Granulocyte concentrate preparation from buffy coats ... 18

Figure 4 :Regulation of neutrophils life cycle ... 28

Figure 5 :Sequential granule maturation of neutrophils ... 29

Figure 6 : The temporal acquisition of effector functions during neutrophil differentiation ... 31

Figure 7 : Sequence of events during neutrophil recruitment from the circulation to the tissue ... 33

Figure 8 : Neutrophil fate after their interaction with pathogens ... 35

Figure 9 : Antimicrobial defences of neutrophils in affected tissue ... 36

Figure 10 : Neutrophil interactions with monocytes, M1 and M2 macrophages ... 40

Figure 11 :Neutrophil interaction with adaptive immunity ... 43

Figure 12 :Diversity of neutrophils ... 46

Chapter 2: Comparison of neutrophil function in granulocyte concentrates from prednisone and G-CSF-treated donors: effect of stimulant, apheresis and storage

Fig. 1 :Leukocyte composition in prednisone and G-CSF GCs ... 74

Fig. 2 : Viability, morphology, and cell-surface marker expression of prednisone- and G-CSF mobilized neutrophils ... 75

Fig. 3 :Viability and function of prednisone and G-CSF GC neutrophils ... 77

Fig. 4 : Effect of leukapheresis on neutrophil responses ... 79

Fig. 5 :Effect of storage on neutrophil isolation from GCs and GC metabolite composition ... 80

Fig. 6 : Variation in prednisone GC neutrophil viability and function after 24 hrs of storage ... 81

Supplementary figure 1 :Absence of significant effect of leukapheresis on neutrophil viability, chemotaxis, phagocytosis, and superoxide and interleukin-8 production ... 84

Supplementary figure 2 : Effect of 48 hours of storage on prednisone-mobilized neutrophil viability and anti-microbial functions ... 86

Supplementary figure 3 : Effect of storage on G-CSF GC neutrophils viability and anti-microbial functions ... 88

Supplementary figure 4 : Change in neutrophils cell-surface marker expression during storage. ... 90

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Abbreviations

7-AAD 7-aminoactinomycin D ACD Anticoagulant Citrate Dextrose

ANC Absolute Neutrophil Count

AnxA1 Annexin-A1

APC Antigen Presenting Cell

BSA Bovin Serum Albumin

CD Cluster of differentiation

CGD Chronic Granulomatous Disease

CMV Cytomegalovirus

CMP Common Myeloid Precursor

CXCL Chemokine (motif C-X-C) Ligand

CXCR Chemokine Receptor

DAMP Damage-Associated Molecular Pattern

DC Dendritic Cell

DMSO Diméthysulfoxyde

DNA Deoxyribonucleic Acid

EV Extracellular Vesicle

FBS Foetal Fovine Serum

fMLF N-Formylmethionyl-Leucyl-Phenylalanine

FMO Full Minus One

GC Granulocyte Concentrate

G-CSF Granulocyte-Colony-Stimulating-Factor

GM-CSF Granulocyte-Macrophages-Colony Stimulating Factor

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GMP Granulocyte Macrophage Precursor GTX Granulocyte Concentrate Transfusion GVHD Graft-Versus-Host Disease

HBSS Hanks' Balanced Salt Solution

HES Hydroxyethylstarch

HLA Human Leukocyte Antigen

IFN Interferon

Igs Immunoglubulins

IL Interleukin

LBP Labile Blood Product

LDN Low Density Neutrophils

LPS Lipopolysaccharide

MDP Monocyte Dendritic cell Progenitor MFI Mean Fluorescence Intensity

MMR Macrophage Mannose Receptors

MPO Myeloperoxidase

NADPH Nicotinamide Adenin Dinucleotide Phosphate

NE Elastase

NET Neutrophil Extracellular Trap

NK Natural Killer

O2 Oxygen

OD Optical Density

PAMP Pathogen-Associated Molecular Pattern PBMC Peripheral Blood Mononuclear Cells

PBS Phosphate-buffered saline

pH Hydrogen Potential

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PRR Pattern Recognition Receptor PMA Phorbol 12-myristate 13-acetate

RBC Red Blood Cell

RCT Randomize Clinical Trial

RNA Ribonucleic Acid

ROS Reactive Oxygen Species RPMI Roswell Park Memorial Institute

SLE Systemic Lupus Erythematosus

TGF Transforming Growth Factor

Th T Helper

TLR Toll-like Receptor

TNF Tumor Necrosis Factor

TRALI Transfusion Related Acute Lung Injury

US United States

WBC White Blood Cell

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L'expérimentateur est celui qui, en vertu d'une interprétation plus ou moins probable, mais anticipée des phénomènes observés, institue l'expérience de manière que, dans l'ordre logique de ses prévisions, elle fournisse un résultat qui serve de contrôle à l'hypothèse ou à l'idée préconçue. Pour cela l'expérimentateur réfléchit, essaye, tâtonne, compare et combine pour trouver les conditions expérimentales les plus propres à atteindre le but qu'il se propose. Il faut nécessairement expérimenter avec une idée préconçue. L'esprit de l'Expérimentateur doit être actif, c'est-à-dire qu'il doit interroger la nature et lui poser les questions dans tous les sens, suivant les diverses hypothèses qui lui sont suggérées. Claude Bernard, Introduction à l'étude de la médecine expérimentale.

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Acknowledgments

First of all, I would like to thank Dre Maria Fernandes, my research director. We met almost six years ago in a little French boulangerie in Québec City where I used to work. Several years later we met again at Laval University where Dre Fernandes was giving a lesson in my program. Later when I saw that she was looking for a student for an internship in her laboratory, I immediately wanted to work with her. We actually talked about this possibility at the same boulangerie we met. Three years later, I am still happy to be part of your wonderful team MJF. I sincerely thank you for giving me the chance to work on such exciting projects with such amazing team partners, Julien, Guillaume, Myriam and now Marie-Michèle. Thank you for being always supporting and proud of us and for always giving me the credit of my work. I have learnt a lot from you and your experience; I have met wonderful work collaborators and I, for sure, will learn and meet even more in the coming years in your laboratory.

I would also like to thank my co-director from Héma-Québec, Mélissa Girard. I am glad to have the opportunity to discover a different type of research at Héma-Québec. I sincerely thank you for all the work you have done along with Marie-Ève to make this project possible. Thank you to all the Héma- Québec team (Marie-Ève, Lucie, Patricia, Marie-Pierre, Marie-Joëlle) for the training you gave me and for your implication in my master project.

In the last four years, I did not only learn from the best (of course I am speaking of Julien Vitry, Myriam Vaillancourt and Guillaume Paré), but I also found friends I hope I will keep close in the future. Audrée, Stephan, Tania, Geneviève, Étienne, Sandrine, Julie, Marie and Yann, I thank you all for your support as friends and for all the good and bad moments we shared. These last years would not have been the same without all of you. I wish you all success and happiness during your study years and I hope to offer you the same friendship that you gave me during my master.

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Above anyone else, I would like to thank my, family. Mom, dad, you sacrifice almost everything for us.

To offer your children the best opportunities, you left your home, you brought us to this beautiful country that is Canada. You found a beautiful place in Québec City to rebuild our lives almost from zero. You gave us the best example of courage, tenacity and adaptability we could ever have. Baptiste and Callista you are the best partner in crime, I am very proud of you. Finally, thank you to my life partner, Jesus and his family. You always believe in me and support me. You are also an example of tenacity;

I have witnessed it many times and it really motivates me to reach my objectives every day.

To conclude, I thank the evaluation jury for their corrections and suggestions that improved my final thesis work.

Thank you, merci à tous pour votre aide et votre soutien, obrigado por me acompanhar e me apoiar, gracias a todos y nos vemos muy pronto para el doctorado !

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Foreword

Dre Maria Fernandes designed and governed the research project. She participated to the redaction and correction of the article included in this thesis.

Mélissa Girard co-directed the project. She organized, along with Marie-Ève Allard the research nurse, the logistic of donor recruitment, granulocyte concentrate collection and transport from Héma-Québec collection centre to our laboratory in Quebec City. They also participated to the correction of the article Héma-Québec research assistant participated to in vitro assays on neutrophils from pre-treated donors and granulocyte concentrates and to the correction of the article.

I conducted all the experimentation presented in the Chapter 2 except for the chemotaxis assay exclusively realised by Héma-Québec research assistant and by Guillaume Paré, research assistant in our laboratory. I realised all the data collection, analysis and interpretation of the results mentioned in the Chapter 2 including the statistical analysis. I, under the supervision of Dre Maria Fernandes, also wrote the article included in the same chapter entitled Comparison of neutrophil function in granulocyte concentrates from prednisone and G-CSF pre-treated donors: effect of stimulant, leukapheresis and storage.

In our laboratory, Guillaume Paré participated to in vitro experimentation and data collection. Julien Vitry, PhD, student, conducted in vitro experimentation on neutrophils from prednisone-derived granulocyte concentrates and Marie-Michèle Labrecque, MSc, student, participated to experimentation on neutrophils from non-stimulated, healthy donors. Myriam Vaillancourt, former research assistant in our laboratory, participated to the design of the neutrophils assays and trained me to realise them during my internship prior to my master. All of them participated to the correction of the article.

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During my master, I introduced and trained Marie-Michèle Labrecque during two internships on the granulocyte concentrate project. She is now a master student in our laboratory and her master project, in collaboration with Héma-Québec, will continue the work I realised during my master.

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Introduction

The history of transfusion medicine began with whole blood transfusions in the 17th century (1) and rapidly became a routine practice in the 20th century during the Second World War due to increased demand for blood donations to save people from traumatic injuries (2). Today, transfusions are also used to treat chronic diseases or even for prophylactic purposes in adults and children.

Transfusion medicine is practiced worldwide and is administered by blood bank centres and hospitals in each country. The use of blood products in transfusion medicine is standardized with guidelines followed in every part of the world. Collection centres organize donor banks, use high-performance collection machines, optimize storage protocols for transfusion products and their transport to assure transfusion product availability. Blood bank centres thus face daily challenges, such as donor recruitment or blood product storage, to provide safe, efficient quality products to save the lives of millions of people each year (3).

Transfusions are not always the therapy of choice due to potential risk such as transmission of infections, donor-recipient incompatibility or even rejection of the transfused product. In most cases, it is recommended to use available medication prior to transfusion.

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1. Types of transfusion

The objective of transfusions is to replace one or several of blood components in a recipient.

Transfused blood products include red blood cells (RBCs), platelets and plasma. In addition, stable products from plasma, human tissue and stem cells can also be transfused but will not be further discussed as the subject of my thesis is the transfusion of granulocytes.

1.1. Labile blood products

Whole blood is composed of 55 % plasma, 44 % red blood cells (RBCs) and 1 % white blood cells (WBCs) and platelets (Fig. 1). These perishable blood components are referred to as labile blood products (LBP) when used for transfusion. An overview of the most frequently used LBPs is provided below followed by a more detailed description of granulocyte concentrates (GCs), the subject of my thesis.

1.1.1 Common transfused labile blood products

Red blood cells

Red blood cells (RBCs) are the most common LBP transfused, with approximately 100 million RBCs transfused worldwide annually (4). RBCs transport oxygen (O2) throughout the body and are thus used to treat patients in need of an increase of oxygen in circulation following anaemia, caused by the reduction of RBCs number in circulation, or the decreased production or survival of RBCs. RBC transfusion is also often used during surgery or to restore RBCs after massive bleeding. Fortunately, RBCs have a long shelf life facilitating their distribution by blood centres. They can be stored up to 42 days at low temperature in additive solution, containing glucose and electrolytes, and anticoagulant without major loss of RBC viability and function. The most severe adverse effects following RBC

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transfusion are related to blood type mismatching. Testing the compatibility of blood types between the donor and recipient is thus indispensable prior to RBC transfusion (Table 1). Mismatching can lead to haemolytic transfusion reactions in the recipient involving the production of antibodies against transfused RBCs, a phenomenon called alloimmunization (5, 6). More common adverse effects of RBCs transfusion include fluid overload and infection following transfusion. Monitoring the recipient’s vitals and testing blood products for contamination prior to transfusion limit the occurrence of these adverse effects (7). RBCs are not the only cell type collected for transfusion medicine.

Platelets

Platelet transfusions are also common with 2 million platelet products transfused in the US annually. Platelets are small, enucleated cells that are activated during bleeding to initiate coagulation and the formation of a clot (Fig. 1). Platelet transfusion can be administered for therapeutic or prophylactic purposes. Therapeutic platelet transfusions are administered when haemorrhage occurs to help coagulation. In contrast, prophylactic platelet transfusion is given to prevent haemorrhage in patients with induced or acquired thrombocytopenia (low platelet count) or platelet dysfunction. Platelet products are stored up to 5 days at room temperature and are thus at risk of bacterial contamination which could cause septic transfusion reactions in patients. Testing for bacterial contamination is therefore crucial as well as the development of new storage conditions to guarantee the sterility and safety of the product. Platelet products are also tested for compatibility with the recipient to avoid alloimmunization against the transfused platelets (8, 9).

Plasma

According to the Red Cross Blood Services, 10, 000 plasma units are needed daily in the U.S for transfusion. Plasma is the major component of the blood and contains several proteins such as albumin, coagulation factors and immunoglobulins (Igs) (Fig. 1). Similar to platelet transfusions, plasma transfusion can also resolve bleeding in patients with several types of coagulopathies and also be prophylactic to prevent bleeding prior to invasive surgery. Plasma products can be transfused fresh or thawed after freezing or cryoprecipitate-reduced plasma. Plasma can be stored frozen for one year, and thawed prior to transfusion. Adverse effects following plasma transfusion include allergic or

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Figure 1

Figure 1: Composition of whole blood. The blood is composed of a variety of cellular elements including RBCs, platelets and different types of leukocytes that are suspended in a liquid matrix called plasma that contains mostly water, proteins involved in coagulation and soluble elements such as gases, ions and nutrients.

Adapted from: https://www.medillsb.com/illustration_image_details.aspx?AID=9217&IID=174245

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Table 1

Table 1: The red blood cell antigens that define different blood types and cause transfusion reactions. The A and B antigens expressed on the surface of RBCs define the four blood types: A, B, AB and O blood type. The ‘O’ denotes RBCs that do not express either the A or B antigen. These antigens are also expressed on the surface of endothelial and epithelial cells. Since individuals with type A blood naturally produce antibodies against antigen B, they will suffer from a transfusion reaction if transfused with type B blood. Similarly, individuals of blood group B produce antibodies against antigen A. The AB blood type do not form antibodies against either antigen as they possess both A and B antigens. The O group (neither A or B antigen) develop antibodies against both A and B antigens. Adapted from: https://www.biolegend.com/en-us/blog/transplant-rejection-and-tnfrsf25.

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anaphylactic reactions, or transfusion related acute lung injury (TRALI) whose risk was recently lowered by transfusing plasma from primarily male and never pregnant female donors (10, 11). In addition to RBCs, platelets and plasma, granulocytes, can also be transfused, the main subject of this thesis. The clinical indications, collection and storage methods of granulocyte concentrates (GCs) for transfusion are different from those used for RBCs, platelets and plasma transfusions. There are currently no international guidelines for GC and local guidelines are used in each country.

1.2 Granulocyte concentrates

1.2.1 An overview of granulocyte transfusion therapy

Granulocyte transfusions (GTX) are used in a smaller patient population than other types of transfusions, namely, patients with life-threatening infections. GTX involves the transfusion of neutrophils, the most abundant leukocyte in circulation in humans that react quickly to infection by eliminating invasive pathogens. GTX is mostly indicated for the treatment of invasive bacterial or fungal infections in neutropenic patients with a low neutrophil count that are non-responsive to standard antimicrobial treatments (12, 13). Severe prolonged neutropenia is caused by the use of myelosuppressive treatments during cancer, auto-immune disease and stem cell transplantation. GTX can also be administered to patients with acquired neutrophil dysfunction such as chronic granulomatous disease (CGD) (14, 15). These patients are at high risk of infection due to their impaired production of reactive oxygen species (ROS). A general consensus is favouring GTX for treatment of severe bacterial or fungal infections in long-term neutropenic patients, determined by a neutrophil count lower than 0.5 x109 cells.L-1, with a good chance of neutrophil recovery within several weeks (16). A few centres also administer GTX as a prophylactic treatment for the patients cited above even though there is no clinical evidence that this treatment is beneficial (17).

In contrast to the previous LBPs, neutrophil concentrates used for GTX have a short half-life and have to be transfused within the 24 hrs after collection. Neutrophils are also easily activated. It is thus also recommended to store GCs at room temperature without agitation prior to transfusion (18-20).

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GCs are prepared from healthy donors administered with a pharmacological agent to increase the number of circulating neutrophils and to obtain a sufficient yield of at least 1 x1010 granulocytes for each transfusion (21, 22). Donors are pre-treated with a glucocorticoid and/or granulocyte-colony stimulating factor (G-CSF), usually the day before GC collection. After stimulation, GCs are collected by leukapheresis, the most common collection method in North America for GCs collection (23) This contrast with blood products presented previously collected by apheresis or derived from a whole blood donation. Alternatively, GCs are derived from whole blood donations by pooling the leukocyte layer of several blood donations as it is current practice mostly in Europe. Usually, blood from 10 healthy donors is required to prepare one GC with this method (24-26).

GC collection as well as GTX exposes donors and recipients, respectively, to adverse events that range from mild to severe. Donors are exposed to adverse events caused by the administration of the pre- treatment for neutrophil mobilization or during the leukapheresis procedure. Mild effects include bone pain, headache, fatigue, chills and nausea related to G-CSF. Glucocorticoids can cause mood changes and insomnia, especially when used together with G-CSF. The leukapheresis procedure along with the anticoagulant and sedimentation agent toxicity can cause hypocalcemia or hypotension. Severe side effects are rare and include posterior subscapular cataracts when high doses of glucocorticoids are administered repeatedly. In recipients, mild adverse effects are related to the transfusion of GCs sometimes causing fever, chills, hypervolemia or rigors. More severe adverse events include pulmonary complications and allosensitization related to ABO and human leukocyte antigen compatibility (HLA) or rejection of the transfused GC. The biological mechanism causing these mild to severe side effects in the recipients are not fully understood. Only fever is known to be associated with the release of pro-inflammatory mediators called cytokines by neutrophils in transfused GCs. Table 2 and 3 resume the most common adverse events occurring in donors and recipients respectively.

Organizing a supply chain for GCs and assuring the quality and safety of the product thus requires compatible donors, the administration of a potent neutrophil mobilizing agent, and the standardization of collection methods and storage conditions that preserve the quality of the LBP from its collection to its transfusion into the recipient. While GTX is still used as a last resort, life-saving therapy for

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Table 2

Table 2: Adverse effects of GC collection on donors. List of adverse potential events caused by, G-CSF or glucocorticoid administration and t leukapheresis. Adapted from (27)

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Table 3

Table 3: Adverse effects of GTX on the recipients. List of potential adverse events in recipients of GTX related to the infusion of GCs, pulmonary complications and to allosensitisation of the recipients. Adapted from (27)

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neutropenic patients, its clinical benefit remains scientifically unproven. Several studies have attempted to address this question but the insufficient number of participants, the variability in GCs preparation and the diversity of the diseases treated render it challenging to draw a conclusion on the efficacy of GTX. The most complete and informative studies on the clinical efficacy of GTX are presented below.

1.2.2 Clinical efficacy of granulocyte concentrates

The “RING study”

The RING study (Resolving Infection in people with Neutropenia with Granulocytes) published by Price (13) provided information on GTX efficacy from multiple centres from 2008 to 2013. The aim of this study was to gather evidence of clinical efficacy by randomizing a group of daily GTX recipients and a control group that did not receive GTX. Both groups were administered standard antimicrobial therapy. Unfortunately, when the study ended in 2013, not enough participants were enrolled and randomized to draw precise and meaningful conclusions. Nevertheless, this study is currently a reference in the GTX area since no other study has been performed on a larger patient cohort.

Initially, the study was designed to recruit subjects that would either receive standard antimicrobial therapy or granulocyte concentrates prepared by leukapheresis from healthy donors pre-treated with 480 µg of intravenous G-CSF and 8 mg of dexamethasone taken orally 8 to 16 hours prior to collection.

Donors were recruited from the general population by blood collection centres or among friends and family of the patients. Donors were matched for ABO with recipients and HLA or granulocyte compatibility, and verified for cytomegalovirus (CMV) serology. The dose of granulocytes transfused was approximately 0.6 x109 cells.kg-1 for a mean recipient weight of 70 kg. The dose was proportionally adjusted for patients with a weight inferior to 30 kg. The patients were stratified depending on the high infection risk in patients with stem cell transplant or relapsed leukaemia or low infection risk in patients with other types of neutropenia and depending on the type of infection. The GCs were administered daily (up to 42 days) and discontinued if an improvement in the clinical profile was observed such as

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neutrophil recovery or resolution of the infection. Information was gathered on serious adverse effects possibly caused by the transfusion.

The main conclusion was drawn from the first analysis evaluating the survival of the GC recipients after 42 days of randomization. The clinical response of the infection treated showed no significant difference between the GC recipients and the control group regardless of the level of infectious risk of the patients and the type of infection treated. Patients’ survival after 90 days was also not affected by GTX. Since the number of participants was lower than expected, the power of the statistical analysis could not reach 80 %, particularly for the analysis of infection subgroups. Moreover, the minimal dose of 0.6 x109 cells.kg-1 was not reached for one quarter of patients that thus received a lower dose. A comparison of patients receiving a high dose to those that received a lower dose of neutrophils revealed that the probability of survival of the group receiving a higher dose was significantly increased compared to patients receiving lower doses of neutrophils. This result was nuanced by the fact that the control group showed intermediate survival between high and low dose group. Nevertheless, the study underscored the importance of verifying the absolute neutrophil count (ANC) prior to transfusion considering the variability in the collected and transfused doses.

The RING study and other smaller clinical trials do not currently support or refute the clinical use of GCs. The same is true for studies on the use of GC for prophylaxis. The limits often cited include the limited number of participants needed to reach a statistical power of 80 % with a significance level of 5 %. A cohort of 2,748 participants is required to detect a significant decrease in mortality due to GTX. It is thus unlikely that any study conducted will reach such a large number of participants (28).

Creation of an international register for GCs

The limitations of randomized clinical trials (RCTs) has not discouraged the use of GTX. On the contrary, it led to the establishment of a common register known as the PROspective Granulocyte usage and outcomes Survey (ProGrES), to gather information on the patients treated, their follow-up and their participation in randomized studies as well as on the GC production. The main objectives of this register are to gather information on the different methods of GCs preparation across the world as well as the clinical outcome of GTX for the different diseases treated. Eventually, the register will

(28)

register was extended to an international level by the Biomedical Excellence for Safer Transfusion (BEST) Collaborative. The participating countries are France, Canada, Brazil and the US and thus include GCs prepared from buffy coat pools and by apheresis. The information collected includes the number of granulocytes transfused, demographic and serological data of the donors, the type of granulocyte mobilization agent used and the type of infection treated, the duration of neutropenia, the resolution of the infection, the risk of mortality or the alloimmunization of the patients. The major advantage of the international register system is that it increases the number of participants and the quantity of information available at a much lower cost than RCTs carried out by a single country or centre. It is also an indispensable source of information for rarer conditions that could not be studied on a large scale in a single-centre study. In addition, the information available through this register will also be classified and standardized to be accessible at the administrative and scientific level for research (28).

1.2.3 Donor recruitment and pre-treatment

One of the main challenges of GTX is donor recruitment that is administered by blood centres.

Depending on the blood centre, GC donors are recruited from a bank of regular donors or from friends and relatives of the patients. Since patients usually need transfusion daily for at least a week, several donors are required per patient (29, 30). Prior to transfusion, donors are tested for several infectious diseases to avoid transmission to the recipient as well as for ABO and HLA compatibility (Table 4).

To obtain the minimal dose of 1 x1010 granulocyte per GC unit, donors are pre-treated with a mobilizing agent to increase the ANC in circulation by favouring the release of neutrophils from the marginated pool (from the peripheral tissues) or the release of new neutrophils from the bone marrow into circulation. Glucocorticoids and/or G-CSF are administered to donors 12 to 24 hrs before GC collection.

(29)

Table 4

Table 4: Serological tests for infectious agents routinely performed by Héma-Québec. List of the different tests used to detect the listed infectious agents. Adapted from the Circular of information – For the use of labile blood products, December 2015 Edition, Héma-Québec

(30)

Glucocorticoids

Glucocorticoids are known for their anti-inflammatory effect on leukocytes (31). They preferentially mobilize neutrophils from the marginated pool from the tissues and also from the storage pool in the bone marrow (32). Glucocorticoids such as dexamethasone, prednisone and prednisolone have been used alone to increase neutrophil count. They are usually administered orally 12 to 18 hrs before granulocyte collection in a unique dose, usually 8 mg for dexamethasone and 50 to 100 mg for prednisone and prednisolone (33-35). On average, glucocorticoid pre-treatment increases the circulating neutrophil count by 2-fold (36). This increase was not considered sufficient by several studies as the minimal dose of granulocytes was not always reached. Interest in GTX thus diminished until G-CSF was introduced as a mobilizing agent.

G-CSF area

G-CSF directly stimulates granulopoiesis in the bone marrow (37). It can also induce the release of immature neutrophils from the bone marrow into the circulation when its level increases. G- CSF is the most potent mobilizing agent as it increases neutrophil count by 7 to 10-fold depending on the dose administered and the number of successive G-CSF injections. Moreover, the concomitant administration of G-CSF and a corticosteroid, most commonly 8 mg of dexamethasone, further increases the neutrophil yield (36). This combination of mobilizing agents is mostly used in the US where G-CSF is administered intravenous by a nurse at the blood collection centre and dexamethasone is taken per os, both between 12 and 24 hours before GC collection. Although G-CSF has proven its efficacy to increase neutrophil count in the circulation, there is considerable variability in how and when this drug is administered in different blood centres. In some, G-CSF is administered as a unique dose the day before collection while in others, the dose is calculated according to the weight of the donor for an unique dose or repeatedly for several days before GC collection (38).

In countries where the use of G-CSF is not approved for GC production, glucocorticoids are used alone, as it is current practice in Canada. Alternatively, when mobilizing agents are not authorized neutrophils are harvested from pooled buffy coats.

(31)

1.2.4 Collection methods for GCs

Continuous flow centrifugation

GC collection requires a method that separates neutrophils from other blood components.

Leukocyte populations have distinctive size, shape and density. Neutrophils measure about 12-15 µm and are usually larger than lymphocytes 6-18 µm, but smaller than monocytes 12-20 µm. The presence of numerous granules in the neutrophil cytoplasm increases the density of these leukocytes compared to mononuclear cells (39, 40). These characteristics are exploited to separate leukocytes from whole blood by centrifugation according to their density. The most common method used for GC collection is called leukapheresis and is based on continuous flow centrifugation. Leukapheresis machines harvest donor blood and separate it into various components by high speed centrifugation. Each targeted component is then directed into a collection bag while the remaining components are returned to the donor (Fig. 2). During this process, the blood is mixed with an anticoagulant and a sedimentation agent to increase red blood cells sedimentation and reduce their number in the final product. In most centres, citrate is used as an anticoagulant and hydroxyethylstarch (HES) as a sedimentation agent (41, 42).

Since leukapheresis returns blood components to the donor, donors can donate more often ensuring a reliable source of neutrophils for GCs.

The implementation of the leukapheresis procedure increased the quality of the GCs collected compared to previous collection methods (43). Prior to leukapheresis, GCs were collected by continuous flow filtration leukapheresis on nylon filters. Although this method allowed to harvest the required number of neutrophils, the time (3 hrs) of adherence of neutrophils to nylon filters caused activation and decreased neutrophil viability and function. Although the administration of glucocorticoid prior to filtration and the reduction of filtration time seemed to lower this negative effect, continuous flow centrifugation is still the preferred method to collect GCs as it causes less cellular damages (44).

Although leukapheresis is optimized to enrich neutrophils for GCs, the final yield may vary due to interdonor variability or the type of leukapheresis machine used (45-47). While leukapheresis is the

(32)

Figure 2

Figure 2: Granulocyte collection on a Spectra Optia apheresis machine. Leukapheresis involves centrifuging whole blood to separate its components into three different layers: the RBCs, platelet-rich plasma and buffy coat layers. Leukocytes are harvested from the buffy coat layer through the collection port that is connected to the collection bag, while RBCs and platelet-rich plasma are returned to the donor through different port. Adapted from PMN collection brochure, https://www.terumobct.com/spectra-optia/protocols.

(33)

preferred method in North America, buffy coat is the main source of GC neutrophils in Europe, the United-Kingdom and France (23, 24, 48).

Buffy coat pooling

The production of GCs by pooling buffy coats from several healthy donors is an alternative to leukapheresis. Briefly, granulocytes are collected from the buffy coat layers located at the interface of plasma and RBCs in centrifuged whole blood (Fig. 1). Buffy coats from 10 to 20 donors are then pooled to obtain the required ANC for transfusion. Another centrifugation is often run after pooling the buffy coat to further reduce the number of RBCs and to enrich the product in granulocytes. The enriched granulocyte pool is stored in autologous plasma and/or platelet additive solution until transfusion (Fig.

3) (24, 25, 48).

Buffy coat-derived GCs are prepared in European countries that do not have a licence to administer an anticoagulant or sedimentation agent to donors considering that both have related adverse effects.

Other constraints include the need of trained personnel to perform leukapheresis and a sufficient number of GC donors. Moreover, leukapheresis is a time-consuming procedure compared to regular whole blood donations used for buffy coat collection (28). Even if the two collection methods differ, they are used for similar clinical indications and their certification is based on the same criteria.

(34)

Figure 3

Figure 3: Granulocyte concentrate preparation from buffy coats. Buffy coats obtained from 10 ABO-matched healthy donors are used to create two pool of 5 buffy coats. These pools are diluted with additive solutions and centrifuged to separate leukocytes, residual RBCs and supernatant. The residual RBCs are then reduced from the leukocyte layer by an Optipress II system before being pooled with 70ml of plasma from one of the donations. Adapted from (24).

(35)

1.2.5 Certification of granulocyte concentrates

For blood bank centres to provide safe and efficient blood products, certification criteria need to be established prior to transfusion to the recipient (6, 23, 49, 50). Assuring the safety of the product for the recipients is also indispensable to avoid adverse effects caused by the transfusion. Since the granulocyte concentrate production procedure is optimized to collect the maximum number of neutrophils, the ANC in the final product is an important criterion. ANC, with sterility (if bloods products is tested positive for contamination, they are discarded), are the only criteria used to certify GC for GTX regardless of the GC preparation or storage protocol and of the disease treated. Several studies determined that a minimal dose of 1 x1010 granulocytes per GC unit is required for GTX (21, 22). This criterion, however, is not used to standardize the dose administered to the patients as a higher dose can also be transfused.

Certification of the quality and effectiveness of a blood product requires an understanding of each step in the production chain that can affect its efficacy. GCs, unlike other blood products, cannot be stored for an extended time due to the short half-life of neutrophils. GCs obtained from leukapheresis procedure are often transfused within 24 hours after collection although this delay can be extended to 48 hours for granulocyte concentrates obtained by pooling healthy donors’ buffy coats. Although this limited storage is usual practice by blood centres, there is no established method to verify and monitor GC antimicrobial properties, based on neutrophils viability and function, during storage (between collection and transfusion).

GC preparation varies among blood centres (45, 48, 49, 51). Since my thesis focuses on GCs prepared in Canada by Héma-Québec, the specific collection conditions used in Canada are presented below.

(36)

1.2.6 Canadian context of granulocyte concentrate transfusions

The informations in the following paragraphs were obtained from Héma-Québec public reports such as Circular of information – For the use of labile blood products, December 2015 Edition, Héma-Québec and Annual Report 2018-2019, Héma-Québec as well as from the National Institute of Scientific Research report entitled La géographie du don du sang au Québec: une analyse exploratoire, Apparicio P., Charbonneau J., Dussault G, Janvier 2009.

Source of GCs in Canada

Héma-Québec is the only blood bank centre to produce GCs for Canada. Québec is located in Eastern Canada and GCs transported to other provinces could require 4000 km of travel prior to transfusion. Optimal transport and storage of GCs is, therefore, crucial to ensure the preservation of GC viability and antimicrobial capacity. In Canada, GCs reach recipients within 24 hours of their collection through express transport services.

A key strategy adopted by Héma-Québec to avoid donor availability issues was to establish a GC donor bank. Moreover, the distance between the donor’s place of residence and the collection centre has a major impact on donor recruitment. For the 1.6 million of blood donations performed in Québec in the past five years, this distance was lower than 20 km on average and lower than 6.4 km for 50 % of donors. The donors’ proximity from the blood collection centre is therefore a main asset of donor recruitment for regular whole blood and GC donations.

Processing of GCs in Canada

GCs in Canada are prepared by leukapheresis at the Héma-Québec blood centre from healthy donors pre-treated with a glucocorticoid known as prednisone. In the last 5 years, Héma-Québec produced more than 460 GCs for Canada. GC donors are screened and recruited among a bank of regular apheresis donors established by Héma-Québec that ensures the availability of compatible donors at all times. Héma-Québec limits apheresis donation frequency to 6 to 8 donations per year for

(37)

each donor. Leukapheresis donation requires the administration of a single dose of 50 mg of prednisone per os 12 to 18 hours to the donors prior to the donation. Each GC unit must contain the minimal dose of 1010 granulocytes with a residual and variable number of red blood cells, lymphocytes and platelets. The volume of the final product is standardized at 350 ml and leukapheresis lasts approximately 2 hours. During, leukapheresis, sodium citrate is used as an anticoagulant and HES to sediment of red blood cells as is current practice worldwide for leukapheresis procedure for granulocytes. After collection, GCs are stored at ambient temperature (between 20 and 24oC) without agitation prior to transfusion. The guidelines for GTX in Canada indicate that patients should be transfused daily until the fever subsides and the infection is controlled or the neutrophil count reaches at least 0.5 x109 neutrophils.L-1 of blood.

1.2.7 Management of the risk of adverse effect related to GC therapy

As mentioned earlier, GTX is associated with mild to severe adverse events in both donors and GC recipients (Table 3 & 4). In order to minimize the occurrence of these effects, blood collection centres use medication to provide care to donors and apply several preventive measures to avoid GC toxicity in recipients.

Management of adverse effects of GC collection on donors

Reported adverse effects related to GC donations can be caused by the leukapheresis procedure or the mobilization agent (Table 3). To prevent these effects, physiological parameters such as temperature and blood pressure must always be monitored during collection and reported if abnormal. Some mild effects such as bone pain are associated with the release of neutrophils from the bone marrow (52). All these effects seem to be proportional to the dose of G-CSF administered and can be partially modulated with analgesics (52, 53). More severe effects include the risk of posterior subscapular cataracts after repeated use of high doses of glucocorticoids (54).

(38)

Management of adverse effects of GTX in the recipients

Adverse events caused by GTX in recipients range from mild to severe. Most reactions to GTX can be prevented or managed by monitoring the patient during infusion to provide immediate care when necessary. Side effects reported in Table 4 can be prevented by decreasing the speed of infusion and by administering medication prior to GC collection. For example, amphotericin B used for the management of fungal infection in neutropenic patients is now administered at least 2 hrs before GC infusion to avoid pulmonary reactions due to neutrophil aggregation (52).

More serious transfusion related adverse effects can be prevented by recruiting ABO and HLA compatible donors. After collection of blood products, processing usually includes leukocyte reduction, irradiation or washing to prevent certain adverse transfusion effects. Since RBCs and granulocytes have a similar granularity, GCs are contaminated with RBCs during centrifugation which exposes the recipient to haemolytic transfusion reactions. The recruitment of ABO-matched donors prevents haemolytic reaction without adding major constraints on donor recruitment since ABO testing is routinely practiced in blood bank institutions. When ABO-matched donors are not available for donation, blood products containing less than 2 ml of RBC can be transfused with a low risk of developing alloantibodies against donor RBCs in the recipient. In some blood centers. donors are tested for HLA compatibility with the recipient to avoid the formation of anti-HLA and granulocyte antibodies that could impair the post-transfusion increment response (54). After GC collection, further processing includes irradiation to prevent adverse transfusion effects.

Irradiation is preconized for blood products that contain residual presence of lymphocytes, RBCs and platelets that expose recipients to potential immunologic reactions against those cells. Directly after the collection procedure, GC are thus irradiated 25 to 30 Gy to prevent T-lymphocyte proliferation and replication. Granulocytes are not affected by irradiation as they don’t undergo proliferation once they exit the bone marrow. Bashir et al., studied the effect of irradiation on GCs obtained from whole blood donation and concluded that the viability and the function of neutrophils were not affected by irradiation

(39)

(24). Overall, irradiation lowers the risk of graft-versus-host-disease (GVHD) in transfused immunosuppressed patients (55).

Neutropenic patients are myelosuppressed and are thus at a higher risk of a worse clinical outcome following infection. CMV infection following transfusion has been reported in 4% of GC-transfused patients, although all of them were previously exposed to CMV (56). Considering that leukoreduction, used to reduce the risk of leukocyte-transmitted infections such as CMV in other blood products (57), is not an option for GTX, serological testing is needed prior to transfusion. In Héma-Québec, serological testing of GCs involves detecting the presence CMV although serology is not yet routinely practiced worldwide in all blood institutions, especially when the local population has a high probability of previous CMV exposure. Future RCTs on CMV infection in CMV seropositive recipients are thus needed to help establish optimal guidelines for prevention of CMV transmission and bad clinical outcome in GC recipients.

2. Antimicrobial immune response: Implications for GCs

The success of GTX therapy relies on the antimicrobial defences of the most abundant leukocyte in circulation, the neutrophil. The neutrophil together with eosinophils and basophils as well as monocytes, macrophages, mast cells and dendritic cells provide what is known as innate immunity.

It’s called innate immunity since we are born with these defenses (58). This type of immunity is activated rapidly by a wide variety of pathogens. In contrast, adaptive immunity responds more slowly and is targeted towards specific pathogens. Adaptive immunity is acquired after birth explaining its slower response due to the need for cells to become activated, to differentiate and proliferate prior to providing protection from stimuli considered as non-self. The key cells in acquired immunity include B and T lymphocytes (59).

To provide an effective antimicrobial response, neutrophils possess several effector functions such as phagocytosis, production of reactive oxygen species, degranulation and secretion of microbicidal

(40)

proteins, neutrophil extracellular traps (NETs) formation and production of pro-inflammatory mediators such as cytokines and lipids (60-64). Neutrophils, however, don’t operate alone to eradicate infection.

Neutrophils constantly interact with other innate and adaptive immune cells to regulate the immune response (65). While neutrophils are the most abundant leukocytes in GCs, monocytes, eosinophils, basophils and some lymphocytes have been reported in this blood product (47). Lymphocytes are the second most abundant cells in GCs. Since they are a major player in adaptive immunity, GCs are irradiated prior to transfusion to inhibit their differentiation and proliferation. Thus, it is possible that other innate and adaptive immune cells found in GCs also contribute to the overall antimicrobial effect of GCs in vivo.

2.1 Organization of immune antimicrobial response

As previously mentioned, our immune system provides both innate and adaptive immunity.

These seemingly separate entities function together to mount a robust and efficient immune response.

Epithelial cells

The epithelium is the first barrier against pathogens and is also part of innate immunity. To prevent the entry of bacteria, fungi and viruses, epithelial cells can produce mucus to trap and eliminate pathogens for example in the respiratory track (66, 67).

Epithelial cells recognise pathogens through pathogen recognition receptors (PRR) expressed on their surface. These receptors recognize pathogen associated molecular patterns (PAMPs) through different types of PRR such as toll-like receptors (TLRs) (68). Each TLR is specialised in the recognition of different PAMPs from exogenous pathogens as well as damage associated molecular patterns (DAMPs) that can be released by endogenous damaged cells. Bacterial components such as lipopolysaccharide (LPS), a component of the surface membrane of Gram-negative bacteria, is recognized by TLR4. Components of fungal pathogens such as mannans and beta-glycans can be

(41)

recognized by TLR4, TLR1/TLR2, and TLR6/TLR2 and C-type lectin receptors such as Dectin-1 and SIGNR1 (69). Viruses are also recognized through their RNA or DNA by intracellular TLRs (TLR9, TLR7, and TLR3) at the surface of endosomes (70). Recognition of pathogens through PRRs and co- receptors causes the activation of intracellular signalling cascades involving adaptor molecules, translocation of transcription factors to the nucleus and expression of cytokines such as interleukin (IL)- 8, a potent chemoattractant for neutrophils and monocytes.

Neutrophil life cycle and effector functions

Neutrophils comprise 50-70 % of all circulating leukocytes, and are one of the first leukocytes to be recruited to the site of infection (61). Neutrophils have a short life span in circulation of 8-12 hrs on average that can be extended to 5.4 days when activated (71-74). Since they were described by Elli Metchnikoff who was awarded the Nobel Prize in 1908 (75), neutrophil biology has evolved considerably. At first, neutrophils were only considered short-lived phagocytic cells often causing aggregation and inflammatory tissue damage. Neutrophils not only phagocyte pathogens and kill them by producing reactive oxygen species (ROS) and antimicrobial peptides but they also trap them extracellularly in NETs during acute infection. Furthermore, neutrophils are now considered essential in the regulation of innate and adaptive immunity through cross-talk with monocytes and macrophages to eradicate pathogens (76).They also link the innate and adaptive immune response by interacting with dendritic cells and by inducing adaptive T-cell immune response against pathogens and tumour antigens as well as the B-cell response (62, 64, 65). New features of neutrophil biology also include the emergence of neutrophil sub-populations in health and disease characterized by a pro- or anti- inflammatory phenotype (60).

Life cycle of neutrophils

Neutrophils, eosinophils, basophils and monocytes, are cells of the myeloid lineage whereas lymphocytes such as T and B cells are form the lymphoid lineage. Neutrophils originate from a self- renewing hematopoietic stem cell that in the bone marrow differentiates into a common myeloid progenitor (CMP). The CMP further differentiates into granulocyte and macrophage progenitors (GMP)

(42)

that gives rise to neutrophils and macrophage and dendritic cell (DC) progenitors (MDP) (77). GMPs differentiate into promyelocytes, myelocytes, metamyelocytes, band cells prior to becoming segmented mature neutrophils. Each step of differentiation is carefully regulated by specific growth factors and cytokines such as G-CSF (64, 71).

Neutrophils express the chemokine receptor CXCR4 at their surface to maintain their homing in the bone marrow by binding with chemokine ligand CXCL12 expressed on the surface of stromal cells. G- CSF directly stimulates the proliferation of neutrophil precursors and neutrophil release from the bone marrow by interfering with the CXCR4/CXCL12 interaction. During infection, neutrophil production is partly regulated by Th17 lymphocytes that produce IL-17. This cytokine increases the secretion of G- CSF and as a consequence, granulopoiesis and induces the release of neutrophils from the bone marrow (Fig. 4) (78-80).

Once neutrophils exit the bone marrow, their cytoplasmic granules and secretory vesicles are fully formed. The primary granules (azurophilic) are formed during the promyelocyte stage and contain myeloperoxidase (MPO), elastase (NE) and defensins. Secondary granules (specific) are formed at the myelocyte stage and contain metalloproteinase, lactofferin, cathelicidin, lipocalin 2 and olfactomedin 4. The tertiary granules (gelatinase) are the last granules to form when neutrophils differentiate into band cells and express arginase 1, gelatinase and lysozyme. Secretary vesicles are only found in mature segmented neutrophils. They contain several membrane receptors, components of the nicotinamide adenin dinucleotide phosphate (NADPH) oxidase complex as well as albumin and soluble mediators (Fig. 5) (63, 81). The release of the content of granules and secretory vesicles increases when neutrophils destroy pathogens through a process known as degranulation (81, 82).

After neutrophils complete their antimicrobial functions, they are removed from the site of infection to avoid collateral damage and to control the immune response. The interaction of neutrophils with

(43)

pathogens can result in different outcomes. Classically, neutrophils undergo spontaneous apoptosis after the internalization of pathogens by phagocytosis and are removed from the site of infection by

(44)

Figure 4

Figure 4: Regulation of neutrophils life cycle. Neutrophils mature in the bone marrow from GMP to mature segmented neutrophils and then exit the bone marrow under the action of G-CSF that inhibits the interaction between CXCL12 and the CXCR4 receptor on neutrophils. During inflammation, neutrophils produce cytokines such as CCL2 and CCL20 that promote the secretion of IL-17 by Th17 cells. The release of G-CSF by Th17 cells induces neutrophil differentiation and release from the bone marrow creating a positive regulation loop. When apoptotic neutrophils are cleared by macrophages, the production of IL-23 that stimulates Th17 cells is inhibited diminishing the production of G-CSF and consequently the release of neutrophils from the bone marrow.

Adapted from (64)

(45)

Figure 5

Figure 5: Sequential granule maturation of neutrophils. Neutrophils acquire the various granules in a sequential manner during differentiation from the promyelocyte state to mature neutrophil.

Granule formation coincides with the temporal expression by the neutrophil of the proteins stored in each type of granule. Azurophilic primary granules are the first to mature followed by specific secondary granules and gelatine tertiary granules. The secretory vesicles mature at the last stage of neutrophil differentiation. Adapted from (83)

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