Role of NOX2 as regulator of the adaptive immune response

Thesis

Reference

Role of NOX2 as regulator of the adaptive immune response

CACHAT, Julien

Abstract

NOX2 is an enzymatic complex that produce oxygen radicals and is important for killing pathogens. A lack of function mutation in genes coding for NOX2 or its subunits cause a hereditary disease called chronic granulomatous disease (CGD). In addition to recurrent infection, CGD patients are also more prone to develop autoimmunity. This suggests an important role of NOX2 in controlling the adaptive immune response. The aim of this thesis was to study this role. We therefore used ovalbumin (OVA) as antigen and curdlan or alum as adjuvant to study the humoral and T cell response in NOX2-deficient and WT mice. We found that NOX2-deficient mice produced more OVA-specific IgG2c antibodies. This increased antibody production was associated with an enhanced Th1 response. We also demonstrated that NOX2-deficient dendritic cells are more efficient in activating T cells than WT DCs.

Finally, NOX2-deficient DCs produce more Th1-driving cytokine that WT DCs.

CACHAT, Julien. Role of NOX2 as regulator of the adaptive immune response. Thèse de doctorat : Univ. Genève, 2017, no. Sc. 5104

DOI : 10.13097/archive-ouverte/unige:96485 URN : urn:nbn:ch:unige-964858

Available at:

http://archive-ouverte.unige.ch/unige:96485

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITÉ DE GENÈVE

Département de pathologie et FACULTÉ DE MÉDECINE

immunologie Professeur K.-H. Krause

Section de chimie et biochimie FACULTÉ DES SCIENCE

Professeur T. Soldati

Role Of NOX2 As Regulator Of The Adaptive Immune Response

THÈSE

Présentée à la Faculté des sciences de l’Université de Genève Pour obtenir le grade de Docteur ès sciences, mention biochimie

Par

Julien CACHAT

de

Saint-Gingolph (VS)

Thèse N°5104

GENÈVE

Atelier impression Unimail 2017

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RÉSUMÉ

La maladie chronique granulomateuse (CGD) est une immunodéficience primaire causée par la mutation du gène codant pour NOX2 ou pour l’une des sous-unités de NOX2. La fonction de NOX2 est de transférer un électron depuis une molécule de NADPH cytosolique jusqu’à une molécule d’oxygène extra-cellulaire pour produire des anions superoxydes (O

).

Ces anions superoxides sont rapidement transformés en d’autres composés hautement réactifs, appelés espèces réactives de l’oxygène (ROS), tels que le peroxyde d’hydrogène (H

O

). La production de ROS par NOX2 est importante pour la destruction des pathogènes par les cellules immunitaires. Dans le cadre de la CGD, les patients sont incapables de produire des ROS via NOX2 et sont plus vulnérable aux infections. De nos jours, grâce aux avancées médicales et pharmaceutiques, les patients CGD vivent de plus en plus longtemps en étant sous traitement prophylactique d’antibiotique et d’antifongique. Avec cet accroissement de la survie des patients CGD, d’autres problèmes médicaux sont devenus de plus en plus important pour ces patients.

En effet, les patients CGD développent également souvent des syndromes hyperinflammatoire, se manifestant entre autre par la présence de granulomes, de maladies intestinales inflammatoires « Crohn-like » et de cystites. En plus de ces manifestations, les patients CGD sont également plus susceptibles de développer des maladies auto-immunes telles que le lupus ou l’arthrite. Finalement, une hypergammaglobulinémie est souvent mise en évidence dans le sérum de patients CGD.

Les modèles murins de la CGD récapitulent de manière fidèle la maladie humaine. Les souris CGD sont plus susceptibles aux infections. Il est également possible d’induire une hyperinflammation chez les souris CGD après injection intra-cutanée de curdlan au niveau de l’oreille externe. La prédisposition des patients CGD aux maladies auto-immunes se retrouve également chez la souris. En effet, les souris CGD développe plus facilement des arthrites auto- immunes et du lupus.

Ce tableau clinique de la CGD et les données animales évoquent un rôle de NOX2 dans la régulation globale de la réponse immunitaire, plutôt que uniquement un rôle dans le contrôle des infections. Pour appuyer ce constat, le transfert de cellule T de souris CGD souffrant d’arthrite est suffisant pour produire la maladie chez des souris de type sauvage saines.

Le sujet de mon projet de thèse était d’étudier la réponse immunitaire adaptative chez

les souris CGD. A cette fin, nous avons mené deux études : la première consistait à étudier la

réponse humorale et plus précisément les sous-types d’IgG produit chez les patients CGD et

chez la souris CGD. La deuxième étude consistait à étudier le rôle de NOX2 dans l’activation

des cellules T par les cellules dendritiques in vivo et in vitro.

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Notre première étude a montré une augmentation de la proportion des IgG2 et une diminution de celle des IgG1 chez les patients CGD. Nos expériences sur les souris ont étayé cet effet de NOX2 sur la production de sous-type d’IgG. Après immunisation des souris avec du curdlan et de l’ovalbumine, nous avons trouvé : i) une réponse hyperinflammatoire marquée, ii) une importante augmentation de la production d’IgG2c et iii) une production plus marquée d’IFNγ chez les souris déficientes en NOX2. Ces résultats montrent que NOX2 est important dans la régulation de la production des sous-types d’IgG.

Notre deuxième étude a montré une activation des cellules T plus importante quand

elles étaient activées par des cellules dendritiques déficientes en NOX2 que par des cellules

dendritiques de type sauvage, après leur activation par du curdlan. Des expériences avec des

cellules T déficientes en NOX2 ont montré que la présence de NOX2 dans les cellules T n’a pas

d’effet sur l’activation ou la prolifération de celle-ci. Egalement l’addition de H

O

exogène

durant l’activation des cellules T n’a pas d’effet sur leur taux d’activation. Finalement,

l’expression des molécules co-stimulatrices pour les cellules dendritiques matures, après

activation avec curdlan, n’est pas différente entre cellules déficientes en NOX2 ou de type

sauvage, mais le taux d’IL1β produit tend à être plus élevé avec les cellules dendritiques

déficiente en NOX2.

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TABLE OF CONTENTS

TABLE OF CONTENTS 4

LIST OF FIGURES AND TABLES 6

LISTE OF ABBREVIATIONS 7

9 9 10 11 11 12 13 13 13 14 14 14 14 15 15 15 15 15 16 16 17 17 17 18 18 18 18 19 20

A.

IV. Stimuli leading to NOX2 activation in immune cells

B.

i. Role of ROS in protein biosynthesis

a. DUOX and thyroid hormone biosynthesis b. NOX3 and otoconia biosynthesis

ii. Role of ROS in cell signalling a. Inhibition of phosphatase b. Activation of kinase c. Regulation of ion channel d. Gene expression

II. Other Sources of ROS

i. Mitochondria-derived ROS ii. Others ROS-producing enzymes

C.

I. Genetics of CGD

II. Clinical manifestation, diagnosis and treatment i. Infection

ii. Hyperinflammation a. Granulomas

b. Inflammatory bowel disease

c. Inflammation of the genitourinary system d. Chororetinitis

iii. Autoimmune disease in CGD patients III. Mice model of CGD

i. Phenotype of CGD mice 20

FIRST PART : INTRODUCTION 9-29

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D.

I. Dendritic cells (DC) 22

II. T lymphocytes 24

i. T helper (Th) lymphocytes 24

ii. Cytotoxic T cells 25

III. B lymphocytes 25

E.

I. In phagocytic cells 26

i. In dendritic cells 26

a. DC maturation 26

b. Antigen processing 27

II. In T lymphocyte 28

III. In B lymphocyte 29

A.

in CGD patients 34

B.

antigen-presenting cells 50

A.

B.

C.

SECOND PART : SCHEMATIC PROTOCOLS 30 - 33

FOURTH PART : GENERAL DISCUSSION 69 – 75

BIBLIOGRAPHIE 76 - 84

THIRD PART : RESULTS 34 -68

APPENDIX 85 - 141

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LIST OF FIGURES AND TABLES

FIGURES

Figure 1. The NADPH oxidase (NOX) family 9

Figure 2. NOX2 subunits and mechanism of NOX2 activation 10 Figure 3. Schematic representation of NOX2 expression 11

Figure 4. Reactive oxygen species 13

Figure 5. Schematic example of redox reaction with cysteine residue 14

Figure 6. ROS production by mitochondria 15

Figure 7. Granulomas 18

Figure 8. Hyperinflammation in CGD mice 21

Figure 9. Schematic representation of the immune response 22 Figure 10. Schematic representation of the immunological synapse 23 Figure 11. Effect of NOX2 on antigen processing 28 Figure 12. Hypothesised effect of NOX2 deficiency on global immune response 75

TABLES

Table 1. The genetics behind CGD 16

Table 2. Literature review on autoimmune disease in CGD patient 19 Table 3. Sub-groups of effector T helper (Th) cells and their main function 25 Table 4. Effect of cytokine on IgG subtype production in mince 26

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LISTE OF ABBREVIATIONS

ATP Adenosine triphosphate BCR B cell receptor

CGD Chronic granulomatous disease CTL Cytotoxic T lymphocyte

CVID Common variable immunodeficiency

Cys Cystein

DC Dendritic cell

DNA Deoxyribonucleic acid EBV Epstein-Barr-Virus FcγR Fc gamma receptor

fMLF Formyl-methionyl-leucyl phenylalanine H2O2 Hydrogen peroxide

His Histidine

IBD Inflammatory bowel disease IFNγ Interferon γ

IL4 Interleukin 4 IL5 Interleukin 5 IL6 Interleukin 6 IL10 Interleukin 10

IL12p40 Interleukin 12 p40 subunit

KO Knock-out

Lm Listeria monocytogenes LPS Lipopolysaccharide

MHC Major histocompatibility complex MPO Myeloperoxidase

mRNA messenger ribonucleic acid

NADPH Nicotinamide adenine dinucleotide phosphate

NFκB nuclear factor 'kappa-light-chain-enhancer' of activated B-cells NOX2 NADPH oxidase 2

O2.- Superoxide anions

PAMPS Pathogen-associated molecular pattern

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PID Primary immunodeficiency PKC Protein kinase C

PMA phorbol 12-myristate 13-acetate Pn Streptococcus pneumoniae PRDX Peroxiredoxin

PRR Pattern recognition receptor PTP Protein tyrosine phosphatase ROS Reactive oxygen species TCR T cell receptor

Tfh T follicular helper cell Th Helper T cell

TLR Toll like receptor TNF Tumor necrosis factor

WT Wild-type

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A.

The NADPH oxidase (NOX) is a family of proteins that is composed of 7 members in humans:

from NOX1 to NOX5, DUOX1 and DUOX2 (fig 1). NOX family members are well conserved in mammalians with one major difference, as there is no orthologue of NOX5 in mice and rats. All the members of the family share the same biochemical function: they transfer electrons from cytosolic NADPH molecules to extracellular oxygen molecules (O2). The result of this enzymatic reaction is the formation of superoxide anion (O2.-). As a consequence, the activation of NOX enzyme leads to consumption of large amount of oxygen, which is described as “respiratory burst”.

Figure 1: The NAPDH oxidase (NOX) family. (from Bedard et Krause, 2007 (Bedard and Krause 2007))

I. History of NOXes

The story of the NOX family begins in the first half of the 20th century with the description of this respiratory burst in, among others, phagocytes in 1933. An important line of research for the discovery of this protein family comes from clinical studies and the description of a new rare primary immunodeficiency disease in 1957 (Berendes, Bridges et al. 1957) : this disease is now known as chronic granulomatous disease (CGD). Ten years later, it was demonstrated that CGD is associated with loss of respiratory burst in phagocytes (Baehner and Nathan 1967). In 1978, the cytochrome b558 was found missing in leukocyte of CGD patients (Segal, Jones et al. 1978) and in 1986, the gene coding for NOX2 was cloned (Royer-Pokora, Kunkel et al. 1986). As production of ROS was detected in non- phagocytic cells, the search for NADPH systems in those cells lead to the discovery of the other member of the NOX family (Bedard and Krause 2007).

FIRST PART : INTRODUCTION

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II. NOX2 and its subunits

The NADPH oxidase 2 (also known as phagocyte NADPH oxidase) is formed of two transmembrane protein (gp91phox, usually and thereafter referred as NOX2, and p22phox) and 4 cytoplasmic subunits (p47 phox, p67 phox, p41phox and rac) (Fig. 2). The catalytic site of the enzyme is located on NOX2 and is formed by two hemes that are fixed on 6 well- conserved His residues in transmembrane domains. Those hemes support the transport of electrons from cytosolic NADPH through the membrane to extracellular oxygen. The NADPH binding domain is located at the C-terminus of the protein. The other transmembrane protein p22^phox acts as a chaperone and is necessary for NOX2 stability: as a consequence, NOX2 is rapidly degraded in absence of p22^phox (Huang, Hitt et al. 1995).

In phagocytic cells, NOX2 and p22^phox are localised at the phagosome and plasma membranes.

Figure 2: NOX2 subunits and mechanism of NOX2 activation. A. In resting cells, the cytosolic subunits p47^phox, p67^phox and p40^phox are spatially separated from the membrane subunits NOX2 (=gp91^phox) and p22^phox and RAC is associated with GDP.

B: Upon stimulation, p47^phox is phosphorylated and bring the cytosolic subunit to the membrane complex and RAC exchange is GDP with GTP. (From Bedard and Krause (Bedard and Krause 2007))

In resting conditions, p47phox, p67phox and p40phox form a cytosolic complex.

Therefore the catalytic unit is spatially separated from its activators under basal conditions, which avoids the unwilling production of ROS by NOX2. Upon stimulus, p47phox is readily phosphorylated and brings together p67phox and p41phox to the transmembrane heterodimer NOX2/p22phox (Han, Freeman et al. 1998, Groemping, Lapouge et al. 2003).

The last NOX2 subunit, RAC, is not restricted to NOX2 and is also involved in the function of other enzymes. However RAC is essential for NOX2 function.

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III. Expression of NOX2

The NOX2 gene is located on the X chromosome. It is mainly expressed in immune cells (eosinophils, neutrophils, macrophages, dendritic cells, B cells). The NOX2 mRNA has also been detected in total mRNA of many other tissues. Such tissue distribution data are difficult to interpret due to the probable contamination by immune cells. However it is now well accepted that NOX2 is expressed in non-immune cell such as endothelial cells (Jones, O'Donnell et al. 1996), neurons (Glass, Huang et al. 2006) and cardiomyocytes (Heymes, Bendall et al. 2003).

The amount of NOX2 expression vary greatly from cell type to cell type (fig. 3).

Eosinophils and neutrophils express a large amount of NOX2 (Yagisawa, Yuo et al. 1996) while dendritic cells (Elsen, Doussiere et al. 2004) and B cells have a much lower expression level. For instance, NOX2 expression in B cells is thought to be approximately 5% of that in neutrophils (Chetty, Thrasher et al. 1995). Macrophages show an intermediate level of expression (Yagisawa, Yuo et al. 1996). The expression of NOX2 in T cells is still debated.

Although some studies have shown NOX2 expression and ROS production by T cells (Jackson, Devadas et al. 2004), these results were not confirmed in another publication (Gelderman, Hultqvist et al. 2006). Also, it is important to notice that this expression and ROS production could come from contaminating immune cells (van Reyk, King et al. 2001).

Figure 3: Schematic representation of NOX2 expression level. Neutrophils and eosinophils express a large amount of NOX2. B cells and dendritic cells express a much lower amount, and macrophage have an intermediate level of NOX2 expression. Presence of NOX2 in T cells is still debated.

IV. Stimuli leading to NOX2 activation in immune cells:

A large variety of stimuli can lead to NOX2 activation in immune cells. The best known and widely used agents to activate NOX2 are phorbol 12-myristate 13-acetate (PMA) and formyl-methionyl-leucyl phenylalanine (fMLF). PMA is a non-specific and potent activator of protein kinase C (PKC). Among other, PKC activation will lead to phosphorylation of p47phox and therefore to the activation of NOX2. fMLF is a chemoattractant that directs neutrophil migration toward the site of inflammation. fMLF bind to the formylpeptide receptor and leads to intracellular calcium rise and subsequent NOX2 activation (Foyouzi- Youssefi, Petersson et al. 1997). Arachidonic acid has also been used to activate NOX2.

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Note that high concentration of arachidonic acid are necessary for NOX2 activation (Ueyama, Tatsuno et al. 2007).

Whole bacteria and, in a more general way, pathogens are important activator of NOX2.

NOX2 activation by pathogens can be achieved via different receptors. Pathogen opsonisation by immunoglobulin is important for their phagocytosis. It has been shown that the binding of immunoglobulin to their Fcγ receptor (FcγR) on phagocytes leads to NOX2 activation (Ueyama, Tatsuno et al. 2007). Pathogen can also induce ROS production through recognition by pattern recognition receptors (PRR). Most Toll-like receptor (TLR) seem to activate NOX2: there is data of ROS production after TLR2 (Marcato, Ferlini et al. 2008), TLR3 (Yang, Kim et al. 2013), TLR4 (Marcato, Ferlini et al. 2008) and TLR8 (Makni- Maalej, Marzaioli et al. 2015) engagement. Dectin-1, a member of the C-type lectin family, is another PRR important for ROS generation. Indeed it has been shown that the dectin-1 agonist β-glucan is a potent activator of NOX2 (Schaeppi, Deffert et al. 2008).

Cytokines and complements factor are two other important elements of the immune response. The complement factors C3a (Elsner, Oppermann et al. 1994, Elsner, Oppermann et al. 1994) and C5a (Till and Ward 1985) are able to induce NOX2-derived ROS production. Many cytokines have also been involved in ROS production. Interferon γ (IFNγ) activates anti-microbial activity of macrophage, which implies, among others, NOX2 activation (Casbon, Long et al. 2012).

Finally, engagement of the B cell receptor (BCR) can stimulate ROS production through NOX2 activation. Some studies have shown ROS production upon T cell receptor engagement but ROS production by T cells is still debated.

Thus, many molecules involve in the immune response are able to activate NOX2, from pattern recognition receptors to cytokines.

B.

Activation of NOX2 leads to the production of superoxide anions (O2^.-), which have a short half-life and are rapidly metabolised into what is called reactive oxygen species (ROS). For example, O2^.- is metabolised either spontaneously or with the help of the superoxide dismutase (SOD), into hydrogen peroxide (H2O2), which is more stable and long-lived than O2^.-. It is also important to notice that, in contrast to O2^.-, H2O2 can cross the membrane and could theoretically have a paracrine action. Others ROS that are O2^.- metabolite includes hydroxyl radical (OH^.) and hypochlorous acid (HOCL) (fig. 4). These two last ROS are thought to be important for pathogen killing.

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Figure 4: Reactive oxygen species. Schematic view of the different reaction that can transform superoxide (O2^.-) into others ROS. (Adapted from Lambeth 2004 (Lambeth 2004))

I. Biological role of ROS:

Most ROS rapidly react with other biomolecules such as carbohydrates, lipids, proteins and DNA. Some of those reactions are irreversible and can be toxic to the cells. Therefore, ROS have been seen as deleterious for the cells for a long period of time. It was thought that ROS were only by-product of the respiratory chain that should be rapidly cleared by the anti-oxidant arsenal of the cell. At first glance, NOX2, the first NOX enzyme described, somehow confirmed this view of ROS as toxic molecules. Indeed, even though NOX2 produces ROS in a controlled way, the first described role of NOX2-derived ROS is to kill bacteria inside the phagosome. In line with this negative effect of ROS, it was thought that ROS play an important role in the pathogenesis of inflammatory diseases. Neutrophils and macrophages that are recruited to the inflammation site can produce large amounts of ROS, which will, in turn, cause the destruction of the local tissue. Those bystander effect of ROS are important for the recruitment of new inflammatory cells and help maintaining a high level of inflammation.

However, this view of ROS as uniquely undesirable deleterious agents has been challenged by the discovery of the other NOX members and by clinical data on CGD patients.

Similarly to NOX2, other NOXes are professional producer of ROS and a lot of effort have been made to discover new roles of ROS in the cells. Frome that research, the following roles of ROS have been suggested:

i. Role of ROS in protein biosynthesis

The role of NOX-derived ROS in biosynthesis is well recognised and best described with DUOX and NOX3.

a. DUOX and thyroid hormone biosynthesis: Thyroid hormones need an iodination process during their biosynthesis. DUOX enzymes are essential for this step, as they provide the hydrogen peroxide that is necessary for the function of the thyroid peroxidase and the iodination of thyroglobulin. This central role of DUOX- derived ROS in thyroid hormone biosynthesis is demonstrated by thyroid dysfunction and low production of thyroid hormone that is subsequent of a loss-of- function mutation in the DUOX2 gene (Moreno, Bikker et al. 2002).

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b. NOX3 and otoconia biosynthesis: Another evidence of the role of ROS in biosynthesis is given by the function of NOX3-derived ROS in the mice inner ear.

Indeed, NOX3 is essential for the biosynthesis of otoconia, a crystal that is important for the detection of movement and for equilibrium. NOX3-deficient mice have a phenotype called “head-tilt” which is characterised by a loss of equilibrium (Paffenholz, Bergstrom et al. 2004).

ii. Role of ROS in cell signaling

Beside their role in biosynthesis, ROS have also been involved in cell signalling as second messengers. Of importance, the range of ROS production by the other NOX members are much lower than those made by NOX2 in neutrophils. Such smaller amounts of ROS have a spatially limited range of action and might impact only molecules that are present in the close environment. Also, some redox reactions are reversible. This reversibility seems to be, at least in part, mediated by the peroxiredoxin (PRDX) family of proteins. Both confined and reversible actions are important characteristic for cell signalling.

One effect of ROS on cell signalling is obtained by acting on redox-sensitive elements in proteins. Cysteine residues are a privilege target for NOX-derived ROS, as they are sensitive to redox reactions (fig 5). The consequence of such redox reactions might lead to conformational changes, which could impact the function of the enzyme. The net effect on enzyme function can range from inhibition or activation of protein function to regulation of ion channel property.

Figure 5: Schematic example of redox reaction with cysteine residue. Cysteine oxidation in presence of H2O2 can lead to conformational change in proteins, which could impact protein function. In our example, the creation of a disulphide bound lead to hiding the catalytic site (in red).

a. Inhibition of phosphatases: The catalytic region of protein tyrosine phosphatase (PTP) is composed of cysteine residues. It has been shown that ROS are able to oxidise these cysteine residues, which leads to the inactivation of PTP's (Meng, Fukada et al. 2002, Barford 2004).

b. Activation of kinases: the precise mechanism by which ROS can activate kinases is not known. However there is convincing evidence that NOX- derived ROS can activate the MAP kinase system (Paulsen, Truong et al. 2011).

Such activation of kinase activity might also be explained by indirect effect of ROS through inactivation of phosphatases.

c. Regulation of ion channels: Ion channels sensitive to NOX-derived ROS are K⁺ channels (Lee, Yu et al. 2003) and intracellular Ca²⁺ channels (Thiels,

C H₂O₂

+

C SH C _SH C C ^S C

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Urban et al. 2000). ROS can have a direct effect on ion channels or regulate their activity indirectly through changes in cell signalling.

d. Gene expression: Production of ROS has been shown to increase transcription of certain genes. This rise in gene transcription can be achieved either by an increased activity of kinase signalling or by direct regulation of transcription factors, through redox-sensitive cysteine. NFκB is one of the transcription factors that is targeted by NOX2-derived redox alteration.

II. Other Sources of ROS:

We discussed above the role of NOX enzymes as professional producers of ROS.

However, NOXes are not the only system that is able to produce ROS in a cell.

Figure 6: ROS production by mitochondria. During electron transport through the respiratory chain, some leakage of electron (e^-) lead to formation of superoxide anion (O2.-). (Adapted fromLi, Fang et al. 2013))

iii. Mitochondria-derived ROS

ROS are by-product during the process of ATP generation by electron transfer to an oxygen molecule to form a molecule of water (figure 6). The amount of ROS generation by mitochondria and their importance in physiology remains mainly unknown.

Historically, mitochondrial ROS have been seen as an unwilling product of the respiratory chain or of cellular metabolism. ROS were therefore seen as damaging agents for the cells, which should be cleared by the extensive anti-oxidant arsenal.

Mithochondria-derived ROS have been involved in ageing, even though this view is now debated (Baranov and Baranova 2017).

iv. Other ROS-producing enzymes:

NOX enzymes and mitochondria are the main producer of ROS but other enzymes such as xanthine oxidase, cyclooxygenase, lipoxygenase and cytochrome P450 can also produce ROS. Peroxisomes are another organelle known to produce ROS. In that context, ROS have also been categorised as by-products. Nevertheless, this view is also questioned. Indeed cytochrome P450 produces hydrogen peroxide that seems to be important in diurnal cycle of corticosteroid production (Kil, Lee et al. 2012).

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C.

Chronis granulomatous disease (CGD) is a rare (̴ 1: 250 000 birth) human congenital disease due to a lack-of-function mutation in any of the genes coding for NOX2 subunits. Therefore, patients suffering from CGD are unable to produce the oxidative burst that follows pathogen phagocytosis.

The main consequence is a lack of clearance of certain pathogens and CGD patients are more susceptible to develop some bacterial and fungal infections. Thus, the disease is classified as a primary immunodeficiency disease (PID) of the innate immunity with phagocyte defects. CGD was described at the same time as common variable immunodeficiency (CVID) (Janeway, Apt et al.

1953), another PID. At the time CGD was described, two clinical features helped to distinguish it from CIVD. Firstly, granulomas formation is typical for CGD patients and even gave its name to the disease. Secondly, CGD patients have a tendency to develop hypergammaglobulinemia (Janeway, Craig et al. 1954). This higher level of serum immunoglobulin was a surprise, because CIVD was characterised with agammaglobulinemia.

Table 1: The genetics behind CGD.

I. Genetic of CGD

The genetics behind CGD is diverse due to the number of genes encoding for NOX2 subunits (Table 1). The gene that encodes gp91phox is called CYBB and is located on the X chromosome. The gene that encodes the other subunits are autosomal: CYBA for p22^phox on chromosome 16, NCF1 for p47^phox on chromosome 7, NCF2 for p67^phox on chromosome 1 and NCF4 for p40^phox on chromosome 22. Most of the patients have a mutation in CYBB and have therefore an X-linked hereditary disease, which explains why the CGD population is composed in the vast majority of male individuals ( ̴80%). This genetic diversity of the disease has also an impact on the clinical manifestations. For example, mutations in NCF1 are often associated with a milder phenotype, while mutations in CYBB seem to be associated with a more pronounced phenotype (Koker, Camcioglu et al. 2013). This categorisation of CGD patients based on the genetic defect is not perfect to predict the severity of the disease. Indeed, it seems that the presence of residual ROS production by the mutated enzyme is more predictive of the severity than the genetic defect itself. However, as X-linked genotypes are often associated with less Protein Gene Chromosome

location

Mode of inheritance Frequency

NOX2 (gp91^phox)

CYBB Xq21.1 X-linked 70%

P22^phox CYBA 16q24 Autosomal recessive 5%

P47^phox NCF1 7q11.23 Autosomal recessive 20%

P67^phox NCF2 1q25 Autosomal recessive 5%

p40^phox NCF4 22q13.1 Autosomal recessive 1 individual

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residual ROS than the other genotypes, the genotype often offers a good approximation to the disease severity.

II. Clinical manifestations, diagnosis and treatment

The main manifestations of CGD patient are recurrent infections, cutaneous abscess (granulomas), lymphadenitis and inflammatory bowel disease (IBD). Recurrent and life- threatening infections during the first year of life are often the first manifestation of the disease.

However, as I mentioned above, milder CGD phenotypes exist: in those cases the disease is sometimes diagnosed later in life and the presenting manifestation is IBD rather than infection.

In any cases, the diagnosis consists in the absence of ROS detection in patient’s whole blood after stimulation with PMA or fMLP. This phenotypic diagnosis is then confirmed by genotyping the mutation. Rapid diagnosis and onset of a rapid prophylactic treatment with antibiotics and antifungals are essential to improve the survival. The only curative treatment is bone marrow transplantation.

Nowadays, thanks to efficient prophylactic antibiotherapy, CGD patients are living longer and new clinical manifestations become important. Many reports have described hyperinflammation and higher susceptibility to autoimmune diseases in CGD patients.

i. Infection

CGD patients are not susceptible to all pathogens and only a group of pathogens are often found in patients. Basically, catalase-positive bacteria are commonly found during infection in CGD patient. The most frequent bacteria are Staphylococcus aureus, Serratia marcescens, Burkholderia cepacia, Nocardia and Salmonella (Segal, Leto et al. 2000, Martire, Rondelli et al. 2008). Infection with Mycobacterium tuberculosis and BCG are also important in countries where tuberculosis is endemic (such as Iran, China) (Deffert, Cachat et al. 2014). Finally, Aspergillus is an important pathogen in CGD condition as infection with this fungi is still an important cause of mortality in CGD patient (Winkelstein, Marino et al. 2000).

The main site of infection are lungs, lymph nodes, skin, liver and colon. Aggressive antibiotic treatment is often needed to cure these infections. Surgical approaches to drain abscesses or to excise infected lymph nodes are sometimes necessary. Once infections are cleared, it is important to maintain a prophylactic antibiotic and antifungal therapy to prevent new infections. Addition of interferon-γ (IFNγ) to antibiotic and antifungal therapy has been tested but the benefit of such combined prophylactic therapy is still controversial. One study showed a better prevention of infection with IFNγ (1991) while a retrospective study showed no benefit of the combined therapy with IFNγ compared to antibiotic and antifungal alone (Martire, Rondelli et al. 2008).

ii. Hyperinflammation

Discrimination between infection and hyperinflammation is not an easy task and many inflammatory phenotypes in CGD patients were first thought to be due to recurrent infections (Harris and Boles 1973). However it is now well-recognised that hyperinflammation can develop without causing agent.

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a. Granulomas: The name of the disease comes from the skin granulomas often found in CGD patients (figure 7). Granulomas in CGD patients are often large- sized and can develop in virtually all organs. They become clinically important depending on their localisation: granulomas can cause bowel or urethra occlusion.

Importantly, no pathogens could be revealed from those lesions, which respond to immunosuppressing drug but not to antibiotics: this characteristic greatly points toward an inflammatory mechanism rather than a consequence of recurrent infections (Schappi, Klein et al. 2003, Levine, Smith et al. 2005).

Figure 7: Granulomas. Skin granulomas found in CGD patients are of large size (macroscopic) (Available from: http://www.medindia.net/patients/patientinfo/chronic- granulomatous-disease.htm)

b. Inflammatory bowel disease (IBD): CGD patients often suffer from diarrhoea, abdominal pain and rectal bleeding, which is often associated with weight loss (Schappi, Smith et al. 2001). IBD presents in CGD patient is similar to Crohn disease in its symptomatic presentation and also in its histopathology features.

However, it is important to distinguish Crohn disease and IBD in the context of CGD as treatment should be different. Indeed, anti-TNF are often used to manage Crohn disease but such a treatment leads to increased and serious infections in CGD patients (Deffert, Olleros et al. 2011). Treatment with steroids is recommended in the CGD context.

c. Inflammation of the urogenital system: The presentation of cystitis in CGD patients can be referred to an uncommon paediatric disease called eosinophilic cystitis (Kontras, Bodenben.Jg et al. 1971, Bauer and Kogan 1991, Barese, Podesta et al. 2004). Although they are similar, an important difference between CGD cystitis and eosinophilic cystitis should be emphasised: in CGD patitents, cystitis are recurrent and need a long term management in contrast to eosinophilic cystitis which is in general a transient disorder. Cystitis in CGD patients respond well to corticosteroids, which confirms its inflammatory nature (Claps, della Corte et al.

2014).

d. Chororetinitis: Chorioretinitis is an important inflammatory disease to consider in CGD patients as this pathology is easily undiagnosed by general examination and can lead to blindness (Kim, Kim et al. 2003). A study reports up to 44% of CGD patients that suffer from this chorioretinitis (Al-Muhsen, Al-Hemidan et al. 2009).

19

Table 2: Litterature review on autoimmune disease in CGD patient. (From Cachat and Krause, 2014 (Cachat, Deffert et al. 2015))

iii. Autoimmune diseases in CGD patients:

The most often described auto-immune diseases in CGD patient are cutaneous and systemic lupus erythematosus, idiopathic thrombocytopenic purpura and arthritis (table 2). In addition to the number of reports of autoimmune disease in CGD patients, two other data in human are putting weight on the association between NOX2 deficiency and autoimmunity. i) Mothers and sisters of X-linked CGD patients are heterozygous carriers of the mutation. Interestingly, lupus has also been extensively reported in X- linked CGD carriers (Cale, Morton et al. 2007). ii) A recent genetic study in human showed a correlation between a polymorphism of NCF1 –resulting in impaired NOX2- derived ROS generation- and diverse autoimmune diseases (Zhao, Ma et al. 2017).

autoimmune and/or inflammatory disease

# published cases

references

discoid lupus erythematosus (cutaneous presentation)

33

Winkelstein JA (Winkelstein, Marino et al. 2000); Van der Berg (Van den Berg, Van Koppen et al. 2008);

Martire B (Martire, Rondelli et al. 2008); Foster (Foster, Lehrnbecher et al. 1998); De ravin SS (De Ravin, Naumann et al. 2008)

idiopathic thrombolytic

purpura 10

Winkelstein JA (Winkelstein, Marino et al. 2000);

Shamsian BS (Shamsian, Mansouri et al. 2008);

Foster (Foster, Lehrnbecher et al. 1998); Van der Berg (Van den Berg, Van Koppen et al. 2008); Fattahi F (Fattahi, Badalzadeh et al. 2011); Trelinski

(Trelinski, Chojnowski et al. 2005) juvenile rheumatoid

arthritis 9

De Ravin SS [106]; Van der Berg (Van den Berg, Van Koppen et al. 2008); Fattahi F (Fattahi, Badalzadeh et al. 2011); Lee BW [110]

systemic lupus

erythematosus 7

Winkelstein JA (Winkelstein, Marino et al. 2000);

Foster (Foster, Lehrnbecher et al. 1998); Martire B (Martire, Rondelli et al. 2008); Van der Berg (Van den Berg, Van Koppen et al. 2008); Manzi S (Manzi, Urbach et al. 1991); Badolato R (Badolato,

Notarangelo et al. 2003)

Kawasaki disease 3

Yamazaki-Nakashimada MA (Yamazaki- Nakashimada, Ramirez-Vargas et al. 2008);

Muneuchi J (Muneuchi, Ishimura et al. 2010); Tsuge M (Tsuge, Shigemitsu et al. 2012)

IgA nephropathy 2 De Ravin SS (De Ravin, Naumann et al. 2008) recurrent pericardial

effusion 1 De Ravin SS (De Ravin, Naumann et al. 2008) antiphospholipid syndrome 1 De Ravin SS (De Ravin, Naumann et al. 2008)

autoimmune pulmonary

disease 1 De Ravin SS (De Ravin, Naumann et al. 2008) myasthenia gravis 1 Winkelstein JA (Winkelstein, Marino et al. 2000)

20

These data suggest that the link between auto-immune diseases and NOX2 is not restricted to CGD patients but in a broader way to diminished NOX2 function.

Autoantibodies are one of the diagnosis criteria of lupus erythematosus and arthritis and they have also been involved in the physiopathogenesis of lupus (Jacob and Stohl 2010). Interestingly, screening for a range of auto-antibodies in CGD patients and X- linked carriers established a higher presence of auto-antibodies in the CGD condition, even when no auto-immune disease was diagnosed (Ammann, Wara et al. 1979, Martin- Villa, Corell et al. 1999).

III. Mice model of CGD

Different knock-out mouse models exists to mimic the human CGD. The first models created are knock-out for the CYBB gene (referred as NOX2^-/- or NOX2KO) (Jackson, Gallin et al. 1995) and for the NCF1 gene (referred as p47^-/- or p47KO) (Pollock, Williams et al. 1995).

A third model is based on a naturally occurring loss-of-function mutation in the NCF1 gene (referred as p47*/*) (Huang, Zhan et al. 2000).

i. Phenotype of CGD mice

The human disease is well recapitulated by those animal models. NOX2^-/- mice are more susceptible to infections (Chang, Segal et al. 1998, Bingel 2002): the autopsy of 72 mice show that 36 of them had bacterial pneumonias and 13 had suppurative adenitis.

Hyperinflammation can be induced in these models by injection of heat-inactivated Aspergillus fumigatus (Morgenstern, Gifford et al. 1997, Petersen, Hiran et al. 2002).

Of importance, only certain stimuli are able to induce hyperinflammation in CGD mice.

Among this stimuli, curdlan is a potent inducer of hyperinflammation in CGD mice (figure 8) (Schaeppi, Deffert et al. 2008). Curdlan is a component of fungi wall. This compound belongs to the family of β-curdlan and can induce an immune response by interacting with the pattern recognition receptor (PRR) dectin-1.

21

Figure 8: Hyperinflammation in CGD mice. Injection of curdlan intradermally in ears of mice produce a massive inflammation in NOX2KO mice but not in WT mice as shown by histology.

Finally, there is also evidence that CGD mouse are more susceptible to auto-immune disease. The first line of evidence comes from the group of Prof. Holmdahl: as they search for gene that are associated with higher susceptibility to arthritis in rats (Olofsson, Holmberg et al. 2003, George-Chandy, Nordstrom et al. 2008) and mice (Hultqvist, Olofsson et al. 2004), they found a correlation between p47^phox mutations and arthritis in both species, which was unexpected at that time. The correlation was then corroborated by animal experiments (Hultqvist, Backlund et al. 2007, George- Chandy, Nordstrom et al. 2008, Lee, Won et al. 2011). Importantly, it was also shown that T cell adoptive transfer from NOX2KO mice that suffered from arthritis was sufficient to initiate the disease in wild-type mice, demonstrating the importance of auto-reactive T cells (Olofsson, Holmberg et al. 2003). The link between CGD and auto- immune disease has also been extended to animal models of lupus (Campbell, Kashgarian et al. 2012). Concerning the development of experimental autoimmune encephalitis (EAE) in a CGD animal model, the situation seems to be more complex.

EAE is induced in mice by immunisation with the myelin-oligodendrocyte glycoprotein (MOG) full protein or the immunogenic MOG peptides. A study has shown an increase susceptibility of CGD mice to autoimmune encephalitis in mice (Hultqvist, Olofsson et al. 2004), but other publications did not find such correlation between NOX2-deficiency and increased EAE development (van der Veen, Dietlin et al. 2000, Allan, Tailor et al.

WT

mice NOX2KO

mice

22

2014). These differences might be due to the different mouse background used in these study. Indeed, Hulqvist et al used mice in a B10Q background while Van der Veen et al and Allan et al used mice in a C56BL6 background.

D.

In the introduction on CGD above, we have seen that NOX2 is not only important for innate immune functions but also for the control of the adaptive immune response. Indeed, the increased susceptibility to autoimmune diseases of CGD patients and mice strongly suggest a deregulation in the control of the adaptive immune system. In the next paragraphs, we will introduce the adaptive immune system and present the data available on the role of NOX2 in adaptive immune cells. The immune response is a complex and well-orchestrated biological system (Figure 9), with the main function of protecting the body again pathogens. This function makes of the immune system a very powerful system to recognise and destroy invading pathogens. As consequence, if this system is not kept under check, it can lead to tissue damage, as seen in hyperinflammation and autoimmune diseases. The adaptive immune system is composed of B and T lymphocyte. Antigen presenting cells, such as dendritic cells, are central for the activation of T lymphocyte. Although macrophages and B lymphocytes are also able to present antigen to T lymphocytes, I will focus on dendritic cells to discuss the antigen presentation and the activation of T lymphocytes.

Figure 9: Schematic representation of the immune response. Dendritic cell (DC) actives CD8+ and CD4+ T cell into cytotoxic (CTL) or effector CD4+ cell. Effector CD4+ T cell helps then B cell activation into plasmatic cells or help macrophage (Mϕ) activation. (Adapted from Pacheco and Prado, 2012 (Pacheco, Contreras et al. 2012))

I. Dendritic cells (DCs)

Dendritic cells have a special role to play during the immune response as they connect the innate immunity to the adaptive immunity: they are the most potent cells to activate naïve T

23

cells. But they are not the only immune cells able to present antigens: macrophages and B cells are also able to activate T cells to some extent. These cells capable to present antigen to T cells are called antigen-presenting cells (APCs).

DCs are phagocytes and they sense the environment for pathogens. Three events occur when DCs encounter an extracellular pathogen: the pathogen is recognised by some endocytic PRR (Janeway and Medzhitov 2002), digested and presented on major histocompatibility complex (MHC) molecules.

Basically, antigens issued from intracellular pathogens are presented by MHCI molecules and antigens from phagocytosed pathogens are presented on MHCII molecules (Germain 1994). There is also a difference in antigen processing between MHCI and MHCII antigens. Antigens presented by MHCII molecule are 13-17 amino acid long and are mainly produced in the phagosome through the action of phagosomal proteases. Antigens presented by MHCI molecules are 8-10 amino acids long and are mainly processed by the proteasome. A fraction of phagocytised peptides are presented by MHCI molecules instead of MHCII by a process called cross-presentation. One of the mechanism leading to cross presentation involve that the antigenic protein leaves the phagosome and be directed to the proteasome (Cresswell, Ackerman et al. 2005) (fig. 11). Similarly, intracellular peptides can be presented on MHCII molecule instead of MHCI by a process called autophagy (Munz 2016).

Figure 10: Schematic representation of the immunological synapse. The centre of the immunological synapse (c-SMAC) is composed of MHCII molecule interacting with TCR and of co-stimulatory molecule bound to their ligands. The periphery (p- SMAC) is composed of integrin and cell-adhesion molecule. (Adapted from Murphy, K.P., et al. 2008)

Immunological synapse

Antigen-MHC/TCR Co-stimulatory molecule/ligands

LFA-1

ICAM-1

24

In the meanwhile, DCs initiate their migration toward lymph nodes. During this journey, they up-regulate the expression of co-stimulatory molecules as well as the level of antigen- loaded MHC molecules on their cell surface (Worbs, Hammerschmidt et al. 2017). Once in the lymph node, DCs are fully matured. The role of co-stimulatory molecules is to ensure full T cell activation. At steady state, DCs mainly present self-antigens and exposure of co-stimulatory molecules on their surface is low: in that context, antigen presentation leads rather to regulatory T cell (Treg) induction, anergy or clonal deletion.

During T cell activation, APCs are closely interacting with T cells and an immunological synapse is established between those two cells (Dustin 2014). This immunological synapse is centred on the interaction of both antigen-loaded MHCII molecule with TCR and co-stimulatory molecules with their ligands in the central core, and by interaction of the integrin LFA-1 with the cell-adhesion molecule ICAM-1 in the periphery to stabilise the synapse (Figure 10).

In addition to this direct interaction with T cells, DCs can also produce cytokines.

Cytokines mostly influence T cell differentiation during the antigen presentation process. The main cytokines produced by mature DCs are IL-12, TNFα, IL-6 and IL-1β (Blanco, Palucka et al. 2008).

II. T lymphocytes

T lymphocytes are classified in two subgroups depending on their functions: T helper lymphocytes and cytotoxic T lymphocytes:

i. T helper (Th) lymphocytes

Th lymphocytes are characterised by the expression of the co-receptor molecule CD4 on their cell surface and respond to antigens presented by MHCII molecules. Therefore, they mainly respond to extracellular pathogens. Their function is to regulate and support the immune response: they help B lymphocytes to mature, lead to full activation of infected neutrophils and macrophages and they also help restraining the inflammation.

This group of lymphocytes can be further subdivided in five main sub-groups (Table 3) (Abbas, Murphy et al. 1996). The first group is formed of Th1 T lymphocytes and is characterised by IFNγ productio. The second group is characterised by the production of IL-5 and is referred as Th2 T lymphocytes. The third group of T lymphocytes mainly produce IL17 and is known as Th17 T lymphocytes (Tabarkiewicz, Pogoda et al. 2015).

Another group is formed of T regulatory (Treg) cells and is characterised by the expression of the transcription factor FOXP3 (Roncarolo, Bacchetta et al. 2001). The last group is composed of T follicular helper (Tfh) cells (Cannons, Lu et al. 2013)

25

Table 3: Sub-group of effector T helper (Th) cell and their main function.(Adapted from Murphy, K.P., et al.2008)

ii. Cytotoxic T lymphocytes (CTL)

CTL are characterised by the expression of the co-receptor molecule CD8 on their cell surface and respond to antigen presented on MHCI molecule. Therefore, they mainly respond to intracellular pathogens and their function is to kill infected cells through an antigen-specific mechanism (Barry and Bleackley 2002).

III. B lymphocytes

B lymphocytes produce immunoglobulins, which are important immune effectors to clear infection and also to give long term protection to re-infection by the same pathogen. They can produce 5 different classes of immunoglobulins based on the situation (Sogn and Kindt 1988): IgD, IgM, IgG, IgA and IgE. IgD and IgM are a membrane-bound immunoglobulins and acts as B cell receptors (BCR). Antigen recognition by IgD or IgM leads to internalisation of the immunoglobulins, digestion of the complex formed by the antigen and the immunoglobulin, and antigen processing and loading on MHCII molecules. B cells need to interact with effector Th cells through their antigen-loaded MHCII molecules to be activated. Of note, IgM can also be secreted and are the first immunoglobulin produced after B lymphocyte activation. Therefore, IgM offers a first protection before the production of more potent and more antigen-specific immunoglobulins. IgA is a secreted immunoglobulin which is produced in mucosal (gut, respiratory and urogenital tract) area and avoids colonisation of this area by pathogens (Kerr 1990). IgE are also secreted immunoglobulin and they are specialised in the response against parasite and in reaction to allergens(Kawakami, Kitaura et al. 2005). IgG is the main seric immunoglobulin involved in protection again invading pathogens. IgG can be further divided in 4 subclasses: IgG1 to IgG4 in human and IgG1, IgG2c (or IgG2a), IgG2b and IgG3 in mice (Galzie 1991). The signal that leads to IgG subclass production in human is not clear. In mice, the situation is better understood: IFNγ drives the production of IgG2c while IL5 drives an IgG1 response (Banerjee, Klasse et al. 2010) (table 4).

26

Table 4: effect of cytokine on IgG subtype production in mice.

E.

I. In phagocytic cells:

As mentioned before, phagocytosis is followed by a respiratory burst due to NOX2- derived ROS production. As emphasised by the recurrent infections seen in CGD patients, this oxidative burst is important for pathogen killing. Our understanding of the mechanism of action of the killing effects of ROS has evolved during the last 20 years. Nowadays, beside a direct killing effect of ROS, it is thought that ROS also kill pathogen by modulating the phagosomal pH or by disturbing key pathogen signalling pathways.

Hydrogen peroxide is a weak base and it impacts on the phagosomal pH differently depending on the cell type. ROS production is not the only factor that influences phagosomal pH and the activity of the vacuolar ATPase (V-ATPase) is also very important. In neutrophils, ROS are produced in large amounts for a short period of time, which leads to a transient alkalinisation of the phagosome. CGD neutrophils have more acidic phagosomes (Segal 2005).

In macrophages, ROS production is lower and also the activity of the V-ATPase is higher, which leads to rapid acidification of their phagosomes. In dendritic cells, both V-ATPase and NOX2 activity are low and the overall result is an alkalinisation of the phagosome. Absence of NOX2 in dendritic cells also leads to more acidic phagosomes. (Savina, Jancic et al. 2006).

i. In dendritic cell:

It is well-established that dendritic cells produce low levels of NOX2-derived ROS (Elsen, Doussiere et al. 2004). Different condition have been reported to induce ROS production by DCs, such as pathogen phagocytosis, DC stimulation with LPS, contact allergens (Byamba, Kim et al. 2010) or cationic liposome adjuvants (Yan, Chen et al. 2008).

ROS production might have a role in two main functions of DCs:

a. DC maturation: Data in the literature on an effect of NOX2-derived ROS production on DC maturation are not consistent. The effects of ROS change as a function of the study design. Studies that used small-molecules NOX2-inhibitors rather suggest a dampened DC maturation phenotype after NOX2 inhibition. Indeed, treatment of the DC line XS52 with ebselen – a NOX2 inhibitor that has also off- target effects- leads to a decreased secretion of IL-1β, IL-6, IL12p40 and TNFα and

27

a decreased expression of CD86 after LPS treatment (Matsue, Edelbaum et al. 2003).

Similarly, treatment with superoxide dismutase (SOD) decreased the expression level of the co-stimulatory molecules MHCII, CD80 and CD86 during skin inflammation (Kwon, Han et al. 2012). Finally, secretion of inflammatory cytokines by human monocyte-derived DCs was diminished by free radicals scavengers (Yamada, Arai et al. 2006). On the other hand, studies that used DC from CGD mouse models reported different results: p47-deficient and NOX2-deficient DC were reported to produce more IL12p70 after activation with IFNγ plus LPS (Jendrysik, Vasilevsky et al. 2011) or with UV-killed Streptococcus pneumoniae (Pn) and Listeria monocytogenes (Lm) (Vasilevsky, Liu et al. 2011). Furthermore, activation with UV-killed Pn or Lm showed an increased upregulation of co-stimulatory molecules. A last study did not report any differences in DC maturation when comparing p47^*/* and WT DC after immunisation with collagen type II and complete Freund’s adjuvant (Gelderman, Hultqvist et al. 2007). This discrepancy in the literature might be due to the different dendritic cell activator that are used or to the use of NOX2 inhibitor or mouse model of CGD.

b. Antigen processing: DCs present small peptide antigens issued from digested pathogen proteins, on their MHC molecules. Antigen processing is dependent on the activity of phagosomal proteases and exacerbated protease activity can lead to antigen degradation. ROS have been reported to be able to regulate proteolytic activity inside the phagosome by inhibiting some proteases (Lockwood 2000). In practice, this change in proteolytic activity has be shown to cause a higher antigen degradation in NOX2-deficient DCs (Rybicka, Balce et al. 2012). As a consequence, the fraction of phagocytosed antigen displayed on MHCI molecules through cross-presentation is reduced, leading to a decreased activation of CD8+ T cells from phagocytosed antigen in mice (Savina, Jancic et al. 2006) and human (Rybicka, Balce et al. 2012). Although studies agreed on the role of NOX2 in modulating cross-presentation efficiency, the precise mechanism that causes this difference is still debated: changes in phagosomal pH and direct redox mechanisms have been reported to be important in cross presentation. Indeed, it is known that phagosomal proteases are pH-sensitive but those proteases are also redox sensitive (fig. 11). Results obtained with dendritic cells on cross-presentation have also been verified in macrophages (Rybicka, Balce et al. 2010, Balce, Li et al. 2011).

Furthermore, the importance of NOX2 in antigen processing seems not to be restricted to cross-presentation, as a study also showed an effect of NOX2-derived ROS on generation of MHCII-dependent antigens. NOX2-deficient DCs produce a different epitopic repertoire for MHCII molecules (Allan, Tailor et al. 2014). Such

28

differences in the repertoire of MHCII antigens has also been reported in B lymphocytes (Crotzer, Matute et al. 2012).

Figure 11. Effect of NOX2 on antigen processing. A) Extracellular foreign proteins are internalised and fused with the phagosome (1). Inside the phagosome, NOX2-derived ROS control protein degradation either by direct inhibition of proteases or indirectly through phagosome alkalinisation. Digested protein is then loaded on MHCII molecule (2). But a fraction of protein are not digested inside the phagosome and are directed to the proteasome where they are digested to produce antigen loaded to MHCI molecule. This process is called cross-presentation (2’). B) In absence of NOX2, there is a loss of control of protease activity, which lead to higher protein degradation and decreased cross-presentation.

II. In T lymphocytes:

As we discussed above, CGD patients and mice are more susceptible to autoimmune diseases and autoimmune phenotype scan be induced by adoptive T cell transfer from CGD mice (Olofsson, Holmberg et al. 2003). Concerning T lymphocyte biology, the effect of ROS on T cell signalling has been reported in many studies and reviewed in (Pani, Colavitti et al.

2000, Reth 2002). Treatment of T cells with antioxidants lead to enhanced ERK activation after anti-CD3/anti-CD28 T cell activation (Kwon, Devadas et al. 2002). Similar experiments with NOX2KO T cells revealed the same enhanced ERK activation after ant-CD3/anti-CD28 stimulation and correlated this change in T cell signalling with a Th1 biased phenotype (Jackson, Devadas et al. 2004). However, these observations were not confirmed in another study (Belikov, Schraven et al. 2014). Therefore, it is well recognised that ROS can influence cell signalling, however the biological consequence of this change in T cell signalling is still debated.

Based on my experience in my host laboratory, I rather favour the hypothesis that NOX2 is not expressed by T cells. However, due to the difficulty to obtain a pure population of isolated T

A B

29

cells, this question is difficult to be answered. Although, I thing that it is also possible that NOX2 might be expressed in some T cell subtypes after differentiation, while absent in naïve T cells.

I. In B lymphocytes:

There is also important human and mouse data that suggest a role of NOX2-derived ROS in B cell biology. Indeed, hypergammaglobulinemia is often found in CGD patients. Also CGD-carrier mothers and CGD patients have a tendency to develop autoantibodies even in the absence of diagnosed autoimmune disease (Cale, Morton et al. 2007). Finally, immunisation studies in mice have shown an increased level of antibody production in CGD mice after immunisation with UV-killed Streptococcus pneumoniae (Pn) and Listeria monocytogenes (Lm) (Vasilevsky, Liu et al. 2011) or with collagen type II (Gelderman, Hultqvist et al. 2007).

In contrast to T cells, the presence of NOX2 in B cells is well established: NOX2- derived ROS production has been measured in EBV-transformed B cell lines, and repeated in tonsillar B lymphocytes (Maly, Cross et al. 1988, Maly, Nakamura et al. 1989). NOX2 expression is quite high in B cells, but a post-transcriptional degradation of the gp91 mRNA explains the lower ROS production in B cells compared to neutrophils (Chetty, Thrasher et al.

1995). NOX2 activators in B cell are various and range from anti-CD40 antibody (Lee and Koretzky 1998, Ha and Lee 2004), BCR crosslinking (Vene, Delfino et al. 2010) to LPS (Vene, Delfino et al. 2010).

The consequence of NOX2-derived ROS production on B cell biology is not clear.

Indeed, the literature is divergent concerning the impact of NOX2 on B cell proliferation. A study showed enhanced B cell proliferation in NOX2KO mice (Richards and Clark 2009), but another showed no difference in proliferation of p47-deficient B cells compared to WT (Wheeler and DeFranco 2012). Similar divergences are found with studies comparing the effect of antioxidants on B cell proliferation: a study showed no impact of antioxidants on B cell proliferation (Morikawa and Morikawa 1996), while another showed a diminished B cell proliferation under similar conditions (Wheeler and DeFranco 2012). However, the reservoir of memory B cells might be impacted by NOX2-deficiency, as diminished memory B cell pool has been reported in CGD patients. However, this decreased in memory B cells seemed to have no effect on the humoral response, as the response of memory B cell toward influenza remained identical in CGD patients (Moir, De Ravin et al. 2012)

Similarly to T lymphocytes, ROS can impact cell signalling in B lymphocytes, especially after B cell activation with anti-CD40 antibody: antioxidants decrease NF- κB, JNK and MAPK signalling (Lee and Koretzky 1998, Ha and Lee 2004).

30

The materials, buffers, media and other chemicals used for the experiments are described in the material and method section in the manuscript of the third part of this thesis. However, the protocols are sometimes complex and it might be difficult to understand them easily from a text. Therefore, before I present my result in the next section, I will draw a schematic summary of the central protocols I followed during my thesis.

I. Mice immunisation:

NOX2KO and WT mice were immunised with ovalbumin and curdlan or alum as adjuvant.

For this purpose, 50 l of ovalbumin/curldan or of ovalbumin/alum in sterile PBS buffer were injected sub-cutaneoulsly in the outer ear of mice at day 0. Blood was then collected in an eppendorf from the caudal vein of each mice before the injection and 10 and 14 days after injection. After blood coagulation and centrifugation for 5 minutes at 2500 rpm, the serum was transferred into a new Eppendorf and stored at -20°C until the ELISAs were performed.

The immunisation produced a local inflammation of the ear which was characterised by an ear swelling and redness. The ear swelling was measured before and 10 and 14 days after the injection to monitor the local inflammatory response.

II. Generation of bone marrow-derived dendritic cells (BMDCs)

After mice euthanasia, bones from hind legs were dissected and cleaned. The two extremities of the bones were cut and the bone marrow was flushed out with a syringe filled with culture medium into a 50 ml falcon. After centrifugation, the cells were resuspended in 2

SECOND PART : SCHEMATIC PROTOCOLS

31

ml of red blood cell lysis buffer for 1 minute. Fresh culture media was then added to stop the action of the lysis buffer. After another centrifugation, the cells were resuspended in 30 ml of medium that was complemented with GM-CSF and plated in a petri-dish. GM-CSF was renewed at day 3 and day 8 and the cells were split at day 5. BMDCs were ready to be used for the co-culture or BMDC activation assay at day 10.

III. Co-culture experiment:

The co-culture experiment aims at recapitulating the antigen presentation by dendritic cells to T cells. For that purpose, 10⁴ BMDCs were activated with 5 g/ml of curdlan and loaded with the ovalbumin peptide (OVA(323-339)). After 16 hours, BMDCs were harvested to remove the curdlan and the unloaded OVA(323-339). Then 10⁵ of isolated T cells, that were labelled with CFSE or not, were added to the activated and OVA-loaded BMDCs. T cell activation was assessed by the up- regulation of CD69 which is a marker of early T cell activation. T cell proliferation was measured by CFSE dilution.

IV. In vivo T cell activation/proliferation:

Injection of 50 l of a solution composed of curdlan and OVA(323-339) in the outer ear of NOX2KO mice or WT mice led to activation of dendritic cells, uptake of the ovalbumin and their migration toward the draining lymph nodes. One day after this injection, T cells that were isolated from OTII mice, were labelled with a fluorescent dye and were injected intra-veinously