Philippe LEMAITRE

Université Libre de Bruxelles

Faculté de Médecine

Early role of IL-17 and calcineurin inhibitor-mediated

Th2- and Th17-polarization of chronic trachea allograft

rejection pathways

Philippe LEMAITRE

Docteur en Médecine

Candidat-spécialiste en chirurgie générale

Thèse de doctorat présentée en vue de l’obtention du grade académique de

Docteur en Sciences Médicales

Promoteur : Pr. A. Le Moine Co-promoteur : Pr. M. Goldman

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Remerciements

Alain, ce que tu m’as apporté déborde largement le cadre de ce travail et je ne crois pas que quelques mots imprimés ici suffiront à t’exprimer ma gratitude. Tu m’as appris à maniper pour générer des résultats. Tu m’as appris à critiquer et interpréter ces résultats pour répondre, si possible, à la question posée. Tu m’as appris qu’il pouvait y avoir plusieurs réponses à une même question. Tu m’as appris qu’en prenant un problème par plusieurs bouts, on pouvait essayer de mieux le comprendre. En un mot, tu m’as appris à réfléchir. Et en plus, tu es devenu un ami. Que demander de plus ?

Professeur Goldman, lorsque je suis venu vous voir, il y a bien longtemps, pour vous demander s’il y existait un domaine commun à la chirurgie et à l’immunologie, vous m’avez répondu «bien entendu, c’est la transplantation », en souriant. Merci pour cette réponse qui a changé ma vie. Merci de m’avoir accueilli dans votre laboratoire et d’avoir supervisé ce travail.

Chloé et Benoit, durant ces quatre ans, vous m’avez aidé quand je me lançais dans une manip infaisable (même à trois), rassuré quand je pensais ne jamais y arriver, écouté quand je me posais mille questions (ce qui arrivait tous les jours), assuré mon équilibre alimentaire (un Quick par semaine, minimum), conduit sur des centaines de kilomètres… Mais surtout, on a beaucoup rigolé et on s’est bien amusés ! Quelle belle période ! Vous avez été fantastiques avec moi !

Louis, en arrivant au labo, je pensais que démontrer un effet suffirait amplement. Merci de m’avoir ouvert les yeux au sens « biologiste » des choses, ou comment chercher le « pourquoi » et le « comment » dudit effet.

Myriam, merci d’avoir accepté avec autant de gentillesse de revoir toutes les lames qui font l’objet de ce travail et d’avoir - en plus – bien voulu les revoir avec moi pour m’expliquer ce que tu en pensais.

Professeur Estenne, merci de m’avoir suggéré, lors d’une rencontre inopinée, l’idée d’un retour au laboratoire pour réaliser un travail de thèse. Merci aussi d’avoir supervisé ce travail.

Merci à tous les membres du labo, de l’animalerie et d’IPG car sans vous ce travail n’aurait jamais été possible et surtout l’ambiance au labo pas pareille.

David, Antoine, Elie et tous les autres, merci de m’avoir écouté avec autant d’attention parler de choses obscures (surtout pour les non-médecins) et d’avoir mis autant de bonne volonté à essayer de les comprendre.

Greg, Maman, Papa, merci de m’avoir soutenu avec autant de ferveur depuis le début de mes études (ce qui remonte au siècle passé déjà). Merci de si bien nous entourer, moi et ma petite famille.

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Contents

PART ONE

1. The innate and adaptive immune systems 14

1.1 Innate immunity 14

1.1.1 Innate sensing 14

Pattern recognition receptors 15

The complement system 16

1.1.2 Innate immune cells 17

Dendritic cells and macrophages 17

Neutrophils 18

Eosinophils 19

Mast cells 19

Gammadelta T cells 19

Natural Killer cells (NK) and NKT cells 20

1.2 Adaptive immunity 21 1.2.1 T cells 21 A. Cytotoxic CD8+ T cells 21 B. CD4+ helper T cells 23 Th1 cells 24 Th2 cells 24 Th17 cells 25

Reciprocal inhibitions of Th1/Th2/Th17 differentiation 26

The plasticity of effector T helper subsets 27

Regulatory T cells 27

1.2.2 B cells and antibodies 28

2. IL-17 biology 29

2.1 Discovery and family 29

2.2 The IL-17 receptor family and target cells 29

2.3 Cellular sources of IL-17 30

2.4 IL-17 is a proinflammatory cytokine 31

2.5 Th17 and IL-17-producing  T cells in health and disease 33

3. Immunity to transplants 35

3.1 Transplant antigens and presentation pathways 35

3.2 Implications of the innate immune system in rejection 37

PRRs in transplantation 37

The complement system in transplantation 37

Innate cells in transplantation 38

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3.4 Implication of B cells and antibodies in rejection 41

4. Ischemia-reperfusion injury 43

4.1 Pathological processes implicated in IRI 43

4.2 Innate and adaptive immune mechanisms in IRI 45

4.3 Post-transplant lung IRI = Primary Graft Dysfunction 47

5. The pathology of lung allografts 49

5.1 Hyperacute rejection 50

5.2 Acute lung rejection 50

5.3 Chronic lung allograft dysfunction 52

 Obliterative bronchiolitis and bronchiolitis obliterans syndrome

Lung pathology and functional tests 52

Risk factors 55

Multiple mechanisms contribute to OB development 55

Treatment 58

 Emergent forms of CLAD and the role of neutrophils

Neutrophilic reversible allograft dysfunction 59

Purely fibroproliferative BOS 60

Upper lobe fibrosis and restrictive allograft syndrome 60

Exudative /Follicular bronchiolitis 61

Large airway stenosis/malacia 61

6. Fibrosis 62

6.1 Innate immunity and fibrosis 64

6.2 Adaptive immunity and fibrosis 66

Th17 is proinflammatory and profibrotic 66

Th2 immunity is a potent driver of progressive fibrosis 66

Th1 regulates fibrosis 66

Tregs either suppress or promote fibrosis 67

7. Calcineurin inhibitors (CNIs) 68

Aim of the work 71

Experimental model 72

Results 73

Discussion and perspectives 77

1. The IL-17-mediated rejection 77

Innate and adaptive cells are implicated in IL-17-mediated rejection 77

Airway epithelial cells are allogeneic targets 78

Characteristics of the Th17-mediated rejection 78

Which mechanisms involve IL-17-mediated pathways in rejection? 79

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2. CsA-modulated alloreactive pathways 82

The ambiguous role of IFN- in allograft rejection 82

CsA differentially affects T helper subsets 83

Th2 and Th17 interrelationships in OAD development under CsA 85

Cyclosporine A as modulator of fibrosis 86

3. Clinical implications 87

1. Tacrolimus vs cyclosporine A 87

2. Targeting the IL-17-producing cells 87

Direct inhibition of IL-17 or its production 88

Strategies targeting Th17 differentiation 89

Strategies downstream IL-17 89

Targeting IL-17 in clinical lung transplantation 90

Adverse effects 91

3. Targeting the Th2 pathway in addition to IL-17 91

4. Considerations regarding BOS and emergent forms of CLAD 92

Conclusions 93 References 94

PART TWO

PART THREE

Reviews and collaborative articles

PART FOUR

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Abbreviations

Ab(s): antibody (-ies)

APC: antigen-presenting cell

AR: acute rejection

AZM: azithromycin

BOS: bronchiolitis obliterans syndrome

CF: cystic fibrosis

CLAD: chronic lung allograft dysfunction

COPD: chronic obstructive pulmonary disease

CNI: calcineurin inhibitor

CsA: cyclosporine A

CTL: cytotoxic T lymphocyte

DAMP: damage associated molecular pattern

DC: dendritic cell

DTH: delayed-type hypersensitivity

ECP: extra-corporeal photopheresis

FEF: forced expiratory flow

FEV1: forced expiratory volume in one second

HIF: hypoxia-inducible factor ()

HMGB-1: high-mobility group box 1

HSP: heat-shock protein

IBD: inflammatory bowel disease

IFN: interferon

IL-xx: interleukin xx

IL-xxR: interleukin xx receptor

IPF: idiopathic pulmonary fibrosis

IRI: ischemia-reperfusion injury

JAK: Janus-associated kinase

MHC: major histocompatibility complex

NRAD: neutrophilic reversible allograft dysfunction

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OB: obliterative bronchiolitis

PAMP: pathogen associated molecular pattern

PGD: primary graft dysfunction

PRR: pattern recognition receptor

RAS: restrictive allograft syndrome

RCT: randomized controlled trials

STAT: signal transducer and activator of transcription

TGF- transforming growth factor beta

Thxx: CD4+ T helper xx subset

TLI: total lymphoid irradiation

TLR: Toll-like receptor

TNF-: tumour necrosis factor alpha

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Summary

Lung transplantation is the only therapeutic approach for patients presenting end-stage pulmonary failure. Despite progress made in organ preservation and immunosuppression, primary graft dysfunction and obliterative bronchiolitis still hamper short-term and long-term outcomes, respectively. Interleukin-17 recently emerged as a major actor in several immuno-inflammatory disorders. Clinical and experimental evidence also suggest the implication of interleukin-17 or type 17 CD4+ T cells in lung rejection. We therefore investigated the contribution of this cytokine to graft pathology in a murine model of tracheal transplantation that recapitulates pathological features of lung rejection including the development of obliterative airway disease.

We first demonstrated that interleukin-17 contributes to inflammatory lesions in the early phase post-transplantation. Interleukin-17 was found to be produced by + T cells and CD4+ T cells infiltrating the graft and interleukin-17 neutralization significantly reduced the development of epithelial lesions together with inhibition of interleukin-6 and heat-shock-protein 70 gene transcription.

10 Finally, in vitro studies confirmed the resistance of type 2 CD4+ T cells and memory type 17 CD4+ T cells to cyclosporin-A mediated suppression.

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Résumé

La transplantation pulmonaire représente l’option thérapeutique de choix pour les patients présentant une insuffisance respiratoire terminale. Malgré les nombreux progrès réalisés en immunosuppression et en préservation d’organes, la dysfonction première du greffon et la bronchiolite oblitérante ont un impact très lourd sur la survie à court terme et à long terme des patients transplantés. L’interleukine 17 a récemment été décrite comme médiateur pro-inflammatoire. Des données expérimentales et cliniques incriminent également cette cytokine ainsi que les lymphocytes CD4+ de type 17 dans le rejet pulmonaire. Nous avons donc décidé d’étudier le rôle de cette cytokine dans les phénomènes de rejet survenant après greffe de trachée chez la souris. En effet, ce modèle génère des lésions similaires à celles survenant après greffe pulmonaire chez l’homme, aboutissant progressivement à une maladie fibro-oblitérante du greffon.

Dans la première partie de ce travail, nous avons démontré l’implication de l’interleukine 17 dans les lésions inflammatoires précoces survenant après la greffe. Dans ce cas, les lymphocytes T CD4+ ainsi que les lymphocytes T + infiltrant le greffon représentent les deux sources principales de l’interleukine 17. La neutralisation de cette cytokine réduit de manière significative les lésions épithéliales et est accompagnée d’une diminution de la transcription des gènes codant pour l’interleukine 6 et pour la protéine du choc

thermique 70.

12 diminuées dans des souris déficientes pour le gène de l’interleukine 17 ou de l’interleukine 4, ce qui prouve l’implication des lymphocytes CD4+ de type 17 et de type 2 dans la maladie fibro-oblitérante chronique du greffon survenant sous cyclosporine A. D’autre part, la déficience du receveur en interféron  aggrave les lésions, révélant un rôle protecteur de cette cytokine dans notre modèle. Enfin, des expériences de stimulation in vitro nous ont permis de mettre en évidence la résistance des lymphocytes CD4+ de type 2 et des lymphocytes mémoires de type 17 à la suppression par la cyclosporine A.

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Introduction

Lung transplantation is the only therapeutic approach for patients presenting end-stage lung failure. The major indications are chronic obstructive pulmonary disease (COPD, 35%), idiopathic pulmonary fibrosis (IPF, 23%) and cystic fibrosis (CF, 17%)1. Technically, bilateral transplantation accounted for more than 70% of the 3272 procedures worldwide performed in year 2009. Refinements in allocation, immunosuppression and patient care steadily improved recipients’ outcome over the last 30 years. However, the survival is still limited to a half-life of 5 years, contrasting with heart, liver or kidney transplants whose half-lives have been prolonged up to 10 years. The main reason for this limitation is chronic rejection, characterised by the obliterative bronchiolitis (OB). It affects around 60% of lung transplants after 5 years and remains the “Achilles’ heel” to the long-term success of lung transplantation. To date, intimate mechanisms underlying OB are poorly understood and no established treatments exist. Although very frequent, acute rejection (AR) is efficiently controlled by increasing the immunosuppression with corticosteroids and is considered with only little impact on graft outcome. Recent data demonstrate that primary graft dysfunction (PGD) represents a potential risk factor for OB in addition to its own life-threatening potential2. Indeed, PGD is a severe form of ischemia-reperfusion injury (IRI) that affects up to 25% of lung recipients and bears important morbidity and mortality rates. Since both OB and PGD currently represent major graft-oriented insults that impact survival after lung transplantation, a better understanding of the pathogenic mechanisms underlying these diseases may help to develop new therapeutic protocols.

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1. The innate and adaptive immune systems

In mammals, defence against pathogens is mediated by the early reactions of innate immunity and the later responses of the adaptive immunity. These systems are indissociable and the integration of their effector functions lead to a proper host defence but also to the various insults affecting a transplanted organ.

1.1 Innate immunity

Innate immunity is phylogenetically the oldest system and is highly conserved through evolution. This system represents the first line of defence against foreign aggression and is therefore specialised in pathogen recognition and/or destruction. The main components of the innate immune system are (1) physical and chemical barriers such as epithelia and antimicrobial substances produced at their surfaces, (2) rapid-response inflammatory cells comprising myeloid-derived cells (dendritic cells, monocytes/macrophages, neutrophils, mast cells and eosinophils) and lymphoid-derived cells (NK and NKT cells,  + T cells), (3) blood proteins including the complement system and other mediators of inflammation and (4) inflammatory cytokines. Some innate immune components are permanently active, such as epithelial barriers that prevent microbial entry, while others are normally inactive and poised to react quickly to microbes or cellular injury, such as phagocytes or the complement system.

1.1.1 Innate sensing

15 patterns (DAMPs). These proteins are produced as a consequence of cellular stress or injury. DAMP sensing enables the clearance of injured cells and the restoration of tissue homeostasis. The concept of DAMP sensing also provides an explanation for the robust immune response occurring in the absence of obvious microbial PAMPs, principally generated against non-microbial non-self (cancers and transplantation)3,4.

Innate sensors comprise cellular PRRs (either membrane-bound or cytosolic) and soluble PRRs that may directly affect several cell types: innate immune cells (dendritic cells, monocytes/macrophages, neutrophils), T and B lymphocytes, endothelial and epithelial cells. Besides, the complement system represents the soluble effector arm of the innate immune system that can mediate pathogen recognition and/or direct destruction.

Pattern recognition receptors

The toll-like receptor (TLR) family represent the canonical trans-membrane PRR. Although very broad, TLR expression is cell-type specific, allowing allocation of recognition responsibilities and consequences to various cell types5. In addition to microbial patterns, TLRs may recognize endogenous DAMPs that are produced in large amounts by stressed or injured cells such as heat-shock proteins (HSPs) 60 or 70, the alarmin HMGB-1 (high-mobility

group box 1), heparan sulfate, fibronectin or hyaluronic acid. TLR signalling activates

pro-inflammatory mediators, and is therefore crucially implicated in the ischemia-reperfusion injury6. In addition, TLR activation stimulates key events that initiate and regulate the adaptive immune response and may therefore enhance allograft rejection and prevent tolerance. These include antigen uptake and presentation, DC maturation and migration, naive T cell activation through the upregulation of costimulation and cytokines (essentially towards Th1 via IL-12), control of T cell survival, control of effector T cells by regulatory T cells and shaping of B cell responses7.

Receptors of advanced glycation end-products (RAGE) are other membrane-bound signalling receptors expressed on hematopoietic and parenchymal cells. They recognize by-products of endogenous proteins, lipids and nucleic acids, pro-inflammatory mediators and the danger-associated protein HMGB-1. RAGE mediates human dendritic cell (DC) maturation and subsequent T helper polarization towards Th18,9.

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NOD-like receptors (NLRs) are cytoplasmic receptors comprising NODs (nucleotide-binding oligomerization domains) and NALPs (NACHT-LRR and pyrin domain-containing proteins).

NOD1 and 2 sense bacterial peptidoglycans. Their stimulation induces inflammation through the transcription of NF-B and AP-1. NALP-3 protein initiates inflammation in response to various forms of cellular stresses such as breach in membrane integrity or potassium efflux caused by a high extracellular ATP concentration. NALP-3 is crucial for the formation of an inflammasome that controls the activation of the caspase-1-dependent inflammatory cytokines IL-1 and IL-18. Of note, the adjuvant alum that is widely used in human vaccines also activates the NALP-3 inflammasome.

Finally, surfactant proteins A and D are collectins, a family of soluble PRRs which best characterized members are the mannose binding lectins (MBLs). These proteins are implicated in bacterial or fungal opsonization, inflammatory cytokine secretion and free radicals production by phagocytes.

The complement system

17 several units of C9). Finally, C3b is degraded into iC3b and C3dg, which both mediate leukocyte adhesion.

Conversely, soluble or cell-bound complement regulators such as the C1 esterase inhibitor or the decay accelerating factor (DAF or CD55) protect the host against self-attack.

Altogether, the consequences of complement activation are (1) opsonization of pathogens and their phagocytosis by neutrophils or macrophages, (2) initiation of an inflammatory response through anaphylatoxin release and (3) lysis of target organisms by the MAC.

1.1.2 Innate immune cells

Innate immune cells may be divided in two groups: myeloid-derived cells (dendritic cells, monocytes/macrophages, neutrophils, mast cells and eosinophils) and lymphoid-derived cells (NK and NKT cells,  T cells). Their role in transplantation has been widely addressed but no study incriminates these cells as being absolutely necessary for solid organ rejection. They may however have a direct noxious impact on the graft, e.g. in IRI, or represent the effector arm of the adaptive immune system. Innate cells recognize a limited number of endogenous (DAMPs) or exogenous (PAMPs) motifs but monocytes and NK/NKT cells have also been shown to discriminate between self and non-self. Important features of these cells are to be rapidly activated upon stimulation and quickly recruited to injured tissues.

Dendritic cells and macrophages

18 present antigens from another cell and activate T cells specific for this antigen (Joffre NRI 2012). These events all concur to the initiation of anti-donor adaptive responses.

Circulating monocytes are incompletely differentiated cells. These maturate upon tissue entry into macrophages that are then characterized according to their specific location (e.g. lung resident macrophages are called alveolar macrophages). Macrophages are classically activated by IFN- and the engagement of PRRs, or activated through the binding of immunoglobulins. Phagocytosis of necrotic particles is the major function of these classically activated macrophages (M1 type), which also secrete proinflammatory cytokines (IL-1, 12, 18, TNF- and IFN-) and reactive oxygen and nitrogen species. IFN--activated macrophages may also participate in delayed-type hypersensitivity (DTH). This phenomenon typically occurs in recall Th1 adaptive responses. DTH is characterized by tissue swelling due to capillary leak, T cell and macrophage infiltration, and fibrin deposition, resulting in tissue induration. Chronic DTH and fibrosis may ensue if the immune response fails to eradicate the pathogen. In contrast, macrophages may become alternatively activated (M2 type) if they develop in the context of IL-4 or IL-1312. Pro-inflammatory M1 macrophages have been implicated the pathogenesis of kidney, liver or lung IRI (see below). These cells also efficiently present antigens to effector T cells and may thus enhance alloreactivity. As addressed page 64, M1 and M2 macrophages have opposed effects in fibrosis. Finally, macrophages are implicated in tissue repair. Indeed, phagocytosis of apoptotic cells deactivates macrophages, which acquire anti-inflammatory properties, such as the production of IL-1013,14.

Neutrophils

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Eosinophils

Eosinophils are bone marrow derived granulocytes that are abundant in the inflammatory infiltrates of late-phase reactions and contribute to many of the pathologic processes in allergic diseases. Th2 cytokines (especially IL-5) are potent eosinophil-activating cytokines. Eosinophils can cause tissue damage by the release of the cytotoxic cationic granule proteins (major basic protein (MBP) and eosinophil cationic protein), reactive oxygen species, arachidonic acid metabolites. These functions were considered beneficial in the defense against parasitic infections but detrimental in the context of allergic responses. It is now established that eosinophils possess characteristics of APCs and also produce cytokines that further promote inflammation and Th2 polarization (IL-1, 3, 4, 5, 8 and TNF-). In parallel, eosinophils have also regulatory properties, notably through IL-4 that may control Th1 reactivity and through TGF-, IL-10, IDO and galectin-10 that regulate effector T cell responses. Finally, TGF- and other mediators contribute to airway remodelling in asthma. These regulatory and repair properties have led the “LIAR hypothesis”, which proposes a central role for eosinophils in “Local Immunity And/or Remodeling/repair” in order to maintain tissue homeostasis15. Whether eosinophils are directly involved in the damage to foreign tissue and thus actively contribute to rejection, or are recruited in allografts in response to tissue damage and engaged in LIAR activities, is currently unknown.

Mast cells

Mast cells reside at host-environment interfaces. As ultimate differentiation occurs in situ, only immature progenitors arisen from bone marrow precursors may be found in the circulation. Upon danger sensing, mast cells elicit the fastest immune response through the release of cytokines (TNF-α, IL-4, 5, 13), enzymes (tryptase), lipid mediators (prostaglandin D2, leukotrienes, PAF) and biogenic amines (histamine). Depending on the tissue, the inflammatory response may result in vascular leak, broncho-constriction or intestinal hypermotility. In addition to their own antigen-presenting capacities (in MHC class I and II), mast cells promote the interaction between APCs and T cells.

Gammadelta T cells

+ _{T cells represent the prototypic “unconventional” or “innate-like” T cells. In adults, }+ _T

20 polarization of T cells. + T cells are activated by stress-induced stimuli. These may be MHC-related ligands, microbial or endogenous phosphoantigens, natural killer receptors ligands (NKRs, e.g. NKG2D) or PRR ligands (TLRs or dectin-1). After activation, + T cells quickly upregulate memory markers, allowing the rapid induction of a “lymphoid-stress surveillance response” that is not delayed by clonal expansion or de novo differentiation16. The functional specialisation of + T cells often correlates with the expression of particular TCR variable genes, which underlie specific developmental programmes and peripheral plasticity. As a consequence, individual subsets are tissue-specific and have restricted effector properties and activation status. Altogether, + T cells can kill infected, activated or transformed cells, have bacteriostatic properties or induce antibacterial functions in other immune cells or epithelia. Furthermore, they produce cytokines that promote protection against viruses or intracellular pathogens (TNF- and IFN-), extracellular bacteria and fungi (IL-17) and parasites (IL-4, 5 and 13). Finally, + T cells can downmodulate innate and adaptive effector cells (through the production of TGF- and IL-10), and promote tissue healing and epithelial regeneration through the release of epithelial growth and survival factors. Owing to these various capacities, + T cells are implicated in anti-tumour immunity, allergy and asthma. As they represent a dominant innate source of IL-17, the biological roles of IL-17-producing + T cells will be specifically addressed below.

Natural Killer cells (NK) and NKT cells

21 regulatory impact on the adaptive B and T cell response.

The slight difference between NK and NKT cells is that the latter express  TCRs with very low diversity, which predominantly recognize lipid antigens (such as gal-cer) presented in the context of class I-like MHC molecules called CD1 molecules. Upon stimulation, these cells are capable of rapidly producing cytokines such as IFN-, IL-4 or IL-17.

1.2 Adaptive immunity

The adaptive immune system comprises antigen-presenting cells that display antigens to and activate the effector T and B lymphocytes. Dendritic cells, macrophages and B cells are the principal APCs and are specialized to capture antigens and present them to lymphocytes. T and B lymphocytes are endowed with the characteristics of the adaptive immunity: they express diverse and highly specific antigen receptors brought about by somatic gene rearrangement (allowing the recognition of as much as 109 different motifs), they expand clonally and they generate immunological memory.

1.2.1 T cells

T lymphocytes arise from bone marrow precursors and subsequently migrate and mature in the thymus (hence their name). Naive mature CD4+ and CD8+ lymphocytes then home to the T cell zone of the secondary lymphoid organs (lymph nodes and spleen) where they may encounter antigens presented by mature DCs and become activated. T lymphocytes are central in the various cell-mediated immune reactions that serve as the defence mechanism against pathogens.

A. Cytotoxic CD8+ T cells

22 Subsequent activation of CD8+ T cells then requires a second signal. In the setting of a strong innate immune response (to a microbe or to a graft in the context of IRI), the APCs are able to deliver this signal. However, in the weak context of a latent viral infection, cancer or long-term organ transplants, CD8+ activation may require an additional help from CD4+ T cells. These second signals from CD4+ T cells may be either cytokines directly acting on the CD8+ T cells (IL-2) or the expression of CD40 ligand (CD40L), which interaction with CD40 on the APC enhances its CD8+-activating capacities (upregulation of costimulatory molecules, IFN- production)19.

Differentiated CTLs classically express the transcription factors T-bet and eomesodermin, and deliver a “lethal hit” that results in apoptotic target cell death. This is mediated by the release of granules containing granzymes and perforin, which cause cell death through activation of caspases, or by creation of aqueous pores in cellular membrane, respectively. CTLs also express FasL that binds to the ubiquitous death receptor Fas (a membranous analogue of TNF receptors) and promotes caspase-8-mediated apoptosis. Importantly, these effects are antigen-specific, contact-dependent and occur in an immunological synapse created between the CTL and the target cell, which all concur to the specific death of target cells without affected adjacent normal cells.

In addition, CTLs secrete IFN-, lymphotoxin and TNF- that can further activate macrophages and promote inflammation. Finally, their early IFN- production acts as a critical Th1-orienting factor20,21.

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B. CD4+ helper T cells

After TCR engagement (also referred as signal 1), class II-restricted CD4+ T cells acquire helper function (Th). Costimulation delivered by the APC (signal 2), as well as the cytokine microenvironment (signal 3), are the additional signals that will influence their differentiation into cells bearing diverse functional capacities. Indeed, four main Th subsets are well established, each with a unique transcription factor and cytokine signature, referred as Th1, Th2, Th17 and Treg (Figure 1). Recently, Th9 and follicular helper T cells (Tfh) have also been described.

Figure 1: CD4+ T helper subsets

Schematic view of the four main T helper subsets. Differentiating and inhibitor factors, signature cytokines and principal effector mechanisms are presented.

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Th1 cells

Th1 lymphocytes are central in cell-mediated immune responses. Their defining features are the expression of IFN- and the transcription factor T-bet. Their differentiation is mediated via the signal transducers and activators of transcription (STAT) 1 and STAT4 proteins. Indeed, in both human and mouse, Th1 differentiation upon TCR engagement is initiated by the binding of IFN- to its receptor, which induces the expression of T-bet through STAT1 phosphorylation. In turn, T-bet, which is considered as the master regulator of Th1 differentiation, mediates the expression of a functional IL-12 receptor, potentiates IFN- expression and inhibits IL-4 and IL-17 production. In concert with TCR engagement, IL-12 signalling through STAT4 generates an increase in IFN- transcription and T-bet, which both reinforce Th1 commitment. IFN- and IL-12 may be produced upstream Th1 cells by NK/NKT cells and APCs, respectively, in the context of intracellular bacterial (Listeria or Mycobacteria), parasite (Leishmania) or viral infection. IL-12 production by APCs may also be mediated by CD40-CD40L interaction or PRR recognition of DAMP release during IRI.

In addition to IFN-, Th1 cells also classically produce IL-2 and TNF-. In the context of rejection, this will result in CD8+-mediated cytotoxicity, macrophage-dependent delayed-type hypersensitivity (DTH), neutrophil activation and the isodelayed-type switch towards the complement-fixing IgG2a antibody production by B cells. Furthermore, Th1 cells may become directly cytotoxic through the expression of Fas-ligand (FasL).

Th2 cells

Naive T helper cells differentiate in Th2 cells if TCR engagement occurs in the presence of IL-4. Indeed, IL-4 activates STAT6, which, together with TCR signalling, induces the expression of GATA-3. GATA-3 is the master regulator of Th2 commitment and enhances the expression of IL-4, IL-5 and IL-13 (the signature Th2 cytokines). GATA-3 further stabilizes the Th2 phenotype by enhancing its own expression (positive feedback loop) and by preventing the production of the IL-12R required for Th1 differentiation. As IL-4 may be produced by eosinophils, mast cells, basophils and NKT cells, Th2 differentiation occurs in response to helminths and allergens. Of note, IL-4 is also secreted in small amounts by antigen-activated T helper cells before differentiation and may thus be amplified if persistent antigenic stimulation occurs with little inflammation or macrophage activation (i.e. IFN- or IL-12). Indeed, IFN- potently represses Th2 differentiation.

25 promotes B-cell proliferation, immunoglobulin class-switch to IgE (helminths opsonisation), and the survival and differentiation of T cells. IL-4 has also direct effects on parenchymal cells, such as bronchial epithelial cells, in which it can induce inflammation, fibrosis or EMT24. In addition, 5 promotes the recruitment and activation of eosinophils. Finally, 4 and IL-13 lead to an alternative activation of macrophages that promotes collagen synthesis and fibrosis, which underscores some redundancy between IL-4 and IL-13 properties.

Th17 cells

In contrast to Th1 and Th2 differentiations, which depend on their respective effector cytokines (IFN- and IL-4, respectively), IL-17 is not required for Th17 commitment. Instead, full Th17 differentiation is a complex three-step process requiring the presence of the immunoregulatory cytokine TGF-β and the pleiotropic pro-inflammatory cytokine IL-625. 1. The combined actions of TGF-β and IL-6 together with TCR engagement on a naive T cell

activate STAT3, which induces the transcription factor RORt (retinoic orphan receptor

t), considered as the master Th17 regulator. Isolate TGF-β signalling would have led to the expression of Foxp3 (the master regulator of Tregs) and IL-6 acts via HIF to repress Foxp3 and promote RORt. By inducing HIF, hypoxia thus promotes Th17 commitment and inhibits Tregs (as discussed below). STAT3 also induces ROR, the expression of IL-23R and the production of IL-17A, IL-17F, IL-22 and IL-21.

2. Next, IL-21 amplifies the Th17 response in an autocrine fashion by reinforcing the activation of STAT3. Of note, IL-21 is a member of the IL-2 family produced by Th17 cells but also by NK/NKT cells.

3. Finally, binding of IL-23 to its functional receptor expressed by differentiated Th17 stabilizes the Th17 phenotype. IL-23 is an IL-12 family member produced by APCs in inflammatory conditions. It also promotes effector functions of Th17 cells via GM-CSF. The importance of this cytokine is illustrated by the fact that IL-23 deficient mice are resistant to Th17 mediated autoimmune diseases and susceptible to Th17 inducing infections (Candida, Klebsiella, Porphyromonas).

IL-26 1 in humans. Mature Th17 cells express the chemokine receptor CCR6 that senses CCL20 and attracts Th17 to inflamed tissues.

From a mechanistic viewpoint, the proinflammatory effector functions of Th17 cells are cytokine-mediated. Indeed, in addition to IL-17 (A, F and heterodimers A/F, which have been discussed separately), Th17 cells also secrete IL-6, IL-9, IL-21 and IL-22, TNF- and GM-CSF. Of note, IL-17 and IL-22 cooperate in various inflammatory conditions28. As already discussed, neutrophils are the principal innate effector Th17-mediated immune responses.

Reciprocal inhibitions of Th1, Th2 and Th17 differentiation pathways

Differentiation of CD4+ T cells into functionally distinct helper T subsets is crucial for proper host defence and normal immunoregulation29.

Many reports demonstrate that the development of Th1 and Th2 pathways are mutually exclusive. Indeed, by blocking the expression of the IL-12 receptor β2 chain, IL-4 inhibits Th1 differentiation because IL-12 is crucial for their development30. In addition, IL-4 reduces IL-12 production by DCs31. Reciprocally, IL-12 and IFN- induce T-bet, which acts in concerts with Runx3 to inhibit the transcription of IL-432,33.

27

The plasticity of effector T helper subsets

Although CD4+ cell lineages have elements of stability and have been referred as distinct lineages, increasing evidences point to their substantial phenotypic flexibility. Underlying this flexibility is chromatin remodelling. Mechanistically, histone modifications may be associated with transcriptional activation or repression (H3K4me3 is an activator signal, whereas H3K27me3 represents a repressor signal)43. T helper master regulators may be associated with activator and repressor signals resulting in three states: activated, repressed or bivalent44. Subsequent analysis revealed that T-bet is bivalent in Th2 and Th17 cells. Both subsets may thus express T-bet and produce IFN-, as shown by Hegazy and colleagues for Th2 cells in response to viral infection, and by Hirota and colleagues for Th17 cells in EAE45,46. GATA3 is also bivalent in Th17 cells, allowing for the reprogramming of Th17 into Th2 subsets26. This plastic state, however, is not absolutely reciprocal because RORt and IL-17A genes in Th1 and Th2 cells are potently repressed which blocks their reconversion to Th17 cells47.

Regulatory T cells

28 Treg properties are mediated by the Foxp3-induced production of regulatory cytokines such as IL-10, TGF-β and IL-35. Antigen-specific or cross-reactive Tregs also express cytotoxic T lymphocyte antigen-4 (CTLA-4), which may inhibit APC activity. This may occur either by competition with positive costimulation signals such as CD28 or by inducing the production of indoleamine 2,3-dioxygenase, which results in the local deprivation of tryptophan and the production of inhibitory molecules (kynurenines). In addition, Tregs may also limit IL-2 availability and control energy metabolism via CD39 and CD73. Indeed, CD39 and CD73 are nucleotidases both capable to degrade AMP, ADP or ATP into adenosine, which further suppresses effector T cell functions and DC maturation by binding to its A2A receptor49.

2.2.2 B cells and antibodies

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2. IL-17 biology

2.1 Discovery and family

IL-17 was originally described and cloned by Rouvier and colleagues in a mouse T cell hybridoma and hence named “murine cytotoxic T lymphocyte associated antigen-8” (mCTLA8)51. A few years later, the human homologue was described in CD4+ T cells and named IL-1752. Based on their very high structural homology, 6 other IL-17 family members have subsequently been described. The initial cytokine is now known as IL-17A and the others IL-17B, C, D, E and F (plus the viral homologue ORF13 or vIL-17). IL-17A and F are the main and the closest members of the family as they present 55% of structural homology. Though all of them have pro-inflammatory properties, their biologic actions as well as induced responses differ: whereas IL-17A and F are typically related to Th17 responses, IL-17E (also known as IL-25) is a Th2-promoting cytokine. Similarly, their cellular sources are very different: IL-17A and F are produced by immune cells while IL-17B, C and D may be found in non-immune cells. Altogether, by virtue of both its cellular sources and signalling properties, I7-17A acts as a bridge between adaptive and innate immunity.

2.2 The IL-17 receptor family and target cells

The IL-17 receptor family comprises five receptor subunits (IL-17RA – IL-17RE). They are type I transmembrane proteins, which efficient intracellular signalling requires homo- or heterodimeric association of two subunits53. Therefore, in addition to IL-17RA, IL-17RC is required for cell signalling in response to both IL-17A and IL-17F54 (Figure 2). IL-17RA is by far the largest member of the family and is ubiquitously expressed on various tissues including spleen, kidney, lung, liver, brain, heart, skeletal muscle and testes. Its expression spans both immune cells (T, B, NK cells and monocytes) and non-immune cells (fibroblasts, epithelial cells, osteoblasic cells and stromal cells). In contrast, IL-17RC is less expressed on myeloid cells, which may explain why fibroblasts, epithelial cells and endothelial cells are major targets of IL-17A and IL-17F55.

30 through ACT1 and TRAF6, culminating in the activation of pro-inflammatory mediators such as nuclear factor κB (NF-κB)56. While NF-κB activation by IL-17 remains quite low compared to the activation generated by TNF- or IL-1β, IL-17 has been shown to stabilize short-lived inflammatory mRNAs such as those induced by NF-κB and TNF-. This post-transcriptional mechanism underlies the pro-inflammatory synergy between IL-17A and TNF-57,58.

Figure 2: The IL-17 receptor and signaling

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2.3 Cellular sources of IL-17

Cells from the adaptive immune system as well as from the innate immune system may produce IL-17 (figure 3). IL-17 producing CD4+ helper T cells, namely Th17 cells, represent the predominant adaptive source of the cytokine (their commitment was addressed page 25). However, CD8+ T lymphocytes have also been shown to secrete IL-17. These are called Tc17.

The predominant innate producers of IL-17 cells are + T lymphocytes. Indeed, they represent the principal (60%) IL-17-producing population in mesenteric lymph nodes59. In contrast to Th17 cells, the IL-17-producing capacities of + T cells could arise from early thymic selection. In addition, invariant natural killer T cells (iNKT) and NK cells, lymphoid-tissue inducer (LTi)-like cells, neutrophils and mastocytes have all been shown to produce IL-1760.

Most IL-17 producing cells express the transcription factor ROR-t and the surface marker CCR6. Its ligand, CCL20, may be released by injured epithelial cells, allowing for the recruitment of IL-17-producing cells to mucosal tissues. Furthermore, IL-17-producing cells express pattern recognition receptors (PRRs) (such as dectin-1 or 2) that promote IL-17A production on pathogen encounter16,61. These cells are typically found at intestinal, cutaneous or lung mucosal interfaces where they participate to tissue surveillance and first-line defence mechanisms.

2.4 IL-17 is a proinflammatory cytokine

32 molecules (ICAM-1) in keratinocytes as well as iNOS and cyclooxygenase-2 in chondrocytes. On immune cells such as dendritic cell (DC) progenitors, IL-17 has been shown to increase MHC and costimulatory molecule expression. This promotes the functional maturation of DCs, optimizing alloreactive lymphocyte sensitization62,63. In addition, IL-17 has been shown to directly promote IL-23, IL-1, IL-6 and TGF production by antigen-presenting cells (APCs), thereby influencing the differentiation of helper T cells64. Finally, IL-17 has been shown to help B cells and promote antibody production by contributing to germinal center formation and immunoglobulin class switch recombination65.

Figure 3: IL-17 is a proinflammatory cytokine

IL-17 may be produced by various cell types from the innate and adaptive immune systems. The broad expression of its receptor is responsible for its multiple

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2.5 Th17 and IL-17-producing  T cells in health and disease

While Th17 cells have received much of the attention since the discovery of IL-17, + T cells are now being recognized as important players in IL-17-mediated immune reactions. The great overlap between both subsets makes it difficult to decipher their respective roles (Figure 4). Indeed, they may both react to the same initiating signals (especially IL-23) and can be quickly recruited to inflamed tissues. Because they produce the same effector cytokines, there is a great redundancy in their biologic actions.

The primary function of Th17 cells is the clearance of extracellular pathogens. They are indeed critical in the defence against Klebsiella pneumoniae, Staphylococcus aureus,

Citrobacter rodentium, Salmonella and Shigella sp. and Bordetella pertussis. Th17 cells may

also augment Th1 responses against intracellular pathogens such as Mycobacterium

tuberculosis and Francisella tularensis66,67. Th17 cells are also involved in protection against

Candida albicans68. Their importance in this context is best illustrated in patients suffering Figure 4: Similarities between Th17 and IL-17-producing + T cells

Similarities between Th17 cells and + T cells generate a great overlap in their biologic actions.

34 hyper-IgE syndrome (Job’s syndrome), who have a dominant-negative mutation in STAT3 responsible for a targeted deficit in Th17 cells. These patients show an increased susceptibility to lung or cutaneous infections by Candida albicans (mucocutaneous candidiasis or CMCD), Klebsiella pneumoniae or Staphylococcus aureus69,70. Furthermore, memory Th17 clones specific for these pathogens can be found in healthy patients71.

The IL-23/Th17 pathway has also been associated with the pathogenesis of various experimental and human autoimmune diseases such as inflammatory bowel diseases (IBDs), multiple sclerosis, psoriasis, rheumatoid arthritis or uveitis68. Accordingly, IL-17 or anti-IL-17R antibodies are currently successfully tested in patients suffering some of these diseases72. Genetic polymorphisms in the IL-23R signalling pathway have been linked with the susceptibility to IBDs and psoriasis73,74. Depletion of IL-23 has also been efficiently tested in Crohn patients75. Various experimental data and the above-mentioned aspects in humans suggest IL-23 rather than IL-17 may be central in Th17-mediated autoimmunity. Furthermore, pathogenic Th17 cells usually evolve and display a Th1-like phenotype in this context68,76.

Whereas recent data also implicate Th17 cells in allergic diseases such as asthma or contact dermatitis77-79, their role in tumour progression or protection remains controversial68. As detailed page 81, sustained IL-23/Th17-mediated inflammation has also been shown to promote fibrosis.

Similarly to Th17 cells, IL-17-producing + T cells are implicated in pathogen clearance. Indeed, they promote the early response and subsequent Th1-mediated protection against

Mycobacterium tuberculosis in the lung and are critical to control S. aureus in the skin80-82. In autoimmune diseases, IL-17-producing + T cells have been implicated in both the induction and the effector phase of a collagen induced arthritis83. In addition, these cells could cooperate with Th17 cells to enhance the pathogenesis of experimental autoimmune uveitis or encephalitis64,84. In this latter context, IL-17-producing + T cells represented a feedforward loop that enhanced autoimmune Th17-mediated diseases. Finally, IL-17+ +T cells have been implicated in brain or kidney IRI and in lung inflammation85-87.

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3.

Immunity to transplants

3.1 Transplant antigens and presentation pathways

36 rejection89. With time, the frequency of direct alloreactive T cells declines while the continuous influx of the processed donor antigens by the recipient APCs through the indirect pathway increases the number of indirect alloreactive T cells that may contribute to chronic rejection. Because the antigenic source cannot be eliminated, the natural history of allograft rejection resembles chronic DTH.

Figure 5: Direct and indirect pathways of alloantigen recognition in transplantation

a) The direct pathway is unique to transplantation. In this setting, the TCR directly recognises an intact allo-MHC molecule expressed on donor cells. The allo-MHC class I molecules are recognized by the TCR of CD8+ T cells whereas the allo-MHC class II molecules are recognized by the TCR of CD4+ T cells.

b) The indirect pathway corresponds to the natural processing of extracellular proteins by APCs, in which donor MHC antigens (either class I or II) are engulfed and processed by recipient APCs before being presented as MHC-derived peptides to CD4+ T cells in the context of recipient MHC class II molecules.

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3.2 Implications of the innate immune system in rejection (figure 6) PRRs in transplantation

Both direct and indirect pathways of allorecognition may be greatly influenced by innate immune sensing. In this context, PAMPs and DAMPs may be compared to vaccine adjuvants. Indeed, skin or cardiac allograft survival is improved in mice deficient in signalling proteins downstream TLRs and tolerance can be abrogated by TLR ligands90-92. In humans, loss-of-function polymorphisms in TLR-4 were associated with a reduction in acute lung rejection severity and frequency, without affecting the infectious complications93.

While in mice, RAGE inhibition has been shown to delay cardiac allograft rejection, evidences of such an impact still lack in humans, as selected polymorphisms of the RAGE gene does not seem to affect kidney rejection94,95. Similarly, the decreased Th17 response in patients bearing the dectin-1 Y238X loss-of-function polymorphism does not directly affect graft versus host disease (GVHD) or outcome after stem cell transplantation96.

In clinical lung transplantation, however, RAGE represents an epithelial injury marker whose measurement has been linked to PGD risk97,98. Moreover, in a very recent report, Sharma and colleagues confirmed that a crosstalk between alveolar macrophage-produced HMGB1 and RAGE-mediated iNKT activation represents a critical event for IL-17 production, neutrophil activation and subsequent lung IRI99.

Finally, MBLs can bind immune complexes and apoptotic debris, as released during transplantation. Patients bearing genes coding for low MBL production or activity are at risk for infectious diseases in childhood. In the context of lung transplantation, donor-derived low-level genes have been associated with greater outcomes100.

The complement system in transplantation

38 immune response (because anaphylatoxins may promote APC functions, T cell proliferation, cytokine release and survival)11. Reflecting the neuroimmune dysregulation occurring in a brain death donor, Damman and colleagues have demonstrated that C3 can be upregulated already before procurement101. Pratt and colleagues have subsequently shown that C3-deficient kidneys are not rejected by wild-type recipients. This demonstrated that donor parenchymal or immune cells represent a critical source of C3 and confirmed the detrimental potential of complement activation before procurement102. In addition, Pavlov and colleagues have shown that donor deficiency in complement regulatory proteins, such as the decay-accelerating factor (DAF, CD55) enhanced cardiac allograft rejection103. In models of lung rejection, complement has been shown to promote early microvascular loss and ischemia, as well as late airway fibro-obliteration104,105. Recently, Suzuki and colleagues demonstrated that IL-17 and C3a were implicated in a feed-forward loop leading to experimental OB106.

Innate cells in transplantation

39 neutrophilic reversible allograft dysfunction (NRAD), which may represent a new type of chronic lung allograft rejection111.

Eosinophils have been identified as effectors of Th2-mediated rejection, which occurs when rejection is diverted from Th1, as in the absence of CD8+ effector functions. In this context, eosinophil inhibition may prolong allograft survival112. In clinical lung transplantation, tissue eosinophilia serves as severity marker for acute rejection113. In addition, increased (>2%) BAL eosinophilia has recently been associated with worse late outcome114.

In the context of acute alloreactive responses, mast cells have recently been shown to have a protective role, through the promotion of Treg cell function and the direct attenuation of CD4+ and CD8+ T cell responses115,116. However, through the release of profibrotic mediators (TGF-β, bFGF, histamine…), mast cells have also been incriminated in chronic rejection and fibrotic processes.

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3.3 Implication of T cells in rejection

T cells are critical in mediating allograft rejection. Indeed, animals that lack T cells are unable to reject fully mismatched transplants, whereas adoptive transfer of purified T cells from WT animals restores allograft rejection122. Accordingly, therapies that deplete peripheral leukocytes (including T cells), such as ATG or alemtuzumab, are effective in preventing and reversing episodes of acute rejection and promote long-term graft survival and patient outcomes123. Such drugs are therefore essentials in many induction protocols.

To date, the various known subsets of T cells have been attributed diverse implications in rejection. Globally, CD4+ helper T cells have a greater implication in rejection than CD8+ T cells, as shown by the inability of CD4-/-, but not CD8-/- recipients to reject solid organ allografts124. Yet, CD8+ T cells are implicated in acute rejection mechanisms, and memory CD8+ T cells represent a major barrier to tolerance induction125,126.

The initial paradigm of helper T cell implication in rejection was based on the recognition of only two populations: the Th1 subset and the Th2 subset.

Early reports demonstrated that donor-specific T cell responses elicited after transplantation were typically dominated by IFN-. Similarly, elevated T-bet mRNA levels have been found in kidney transplant biopsies during acute rejection126-129. In addition, Th1-attracting Figure 6: Implications of the innate immune system in transplantation

41 chemokines (CXCL9 and CXCL10), Fas-FasL interactions as well as DTH have all been implicated in rejection20. The historical belief was thus that Th1 cells mediated rejection. IFN--/- and T-bet-/- recipients have however been shown to reject cardiac or kidney allografts36,130,131, suggesting that both are dispensable for rejection. Of note, as neither

IFN- nor T-bet are entirely Th1-specific, these reports did not individually address the role of Th1 cells in rejection.

At the time Th1 cells were seen as detrimental players in transplantation, Th2 were considered protective. Arguments therefore were that IL-4 blunts the Th1 pathway and has been shown to promote growth and function of natural and induced Tregs132,133. Th2 cells were also known to produce IL-10, which is a regulatory cytokine (it is now well established that many other cell types (including Th1 cells) secrete IL-10). In addition, both IL-4 and IL-13 can prevent macrophage-mediated damages, increase cytoprotective molecules (HO-1) and downregulate DC responses to innate stimulation (i.e. by LPS)134,135. However, the Th2 pathway is now recognized to mediate an alternative rejection. Indeed, Th2 cells, IL-4, IL-5 and eosinophils have been implicated in allograft rejection in rodent models, especially when CD8+-related IFN- production is missing21,136. Furthermore, IL-4 has been shown to break experimental liver allograft tolerance137. Finally, IL-4 and IL-13 mediate fibrosis and an alternative activation of macrophages towards the profibrotic M2 type. In clinical transplantation, elevated IL-4 and IL-13 levels are found during kidney and lung allograft rejection138,139.

Regarding the more recently described subsets, Tregs have a critical role in the induction and maintenance of tolerance140. However, under specific conditions, these cells have also been implicated in Th17 differentiation and may therefore promote rejection110. Indeed, as discussed later in this work, Th17 cells are being increasingly incriminated in rejection.

3.4 Implication of B cells and antibodies in rejection

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4. Ischemia-reperfusion injury (IRI)

Ischemia and reperfusion is a pathological condition characterized by an initial restriction of blood supply to an organ, followed by the restoration of perfusion and reoxygenation. Perhaps surprisingly, reperfusion and reoxygenation are frequently associated with an exacerbation of tissue injury and inflammation. This phenomenon may occur in various conditions, such as altitude, arterial occlusion, cardiac arrest, sleep apnoea or sickle cell disease and is, by definition, unavoidable in solid organ transplantation. Cardiopulmonary bypass also technically causes an isolated lung IRI. However, transplantation IRI differs in two aspects. First, while advances are made in preservation protocols, the collected organs are submitted to a cold ischemic period, which may bear some drawbacks148. Second, despite careful sterility rules, the surgical act may not be 100% sterile and lead to some degree of bacterial contamination and/or translocation. Ischemia and reperfusion of a transplanted organ lead to an inflammatory injury, which consequences may be early graft failure or an increase in subsequent rejection149. Moreover, an impaired tissue oxygenation sets a vicious circle because (1) hypoxia-induced inflammation increases the metabolic demands of cells that are in a low metabolic substrates environment and (2) this occurs in tissues where inflammation and oedema further decrease the local tissue oxygen concentration. Furthermore, if ischemia is prolonged, microvascular changes may lead to persistent blood flow obstruction resulting in a “no reflow phenomenon” despite reperfusion.

4.1 Pathological processes implicated in IRI

44 significant alterations in the transcriptional control of gene expression, a phenomenon called “reprogramming”. These cellular adaptations rely on the hypoxia-mediated nuclear translocation of the hypoxia-inducible transcription factor (HIF). Under hypoxic conditions, HIF and NF-κB cooperate. On the one hand, NF-κB induces inflammatory proteins (acute phase proteins, adhesion molecules and proinflammatory cytokines) and HIF itself. In an amplification loop, HIF mediates the transcription of NF-κB, plus TLR-, metabolic- and angiogenic-related genes. Members of the HIF family thus interact with the members of the NF-κB family in ways that link hypoxia with inflammation. In addition, LPS and ROS can activate HIF even under normoxic conditions, initiating an inflammatory response before tissue hypoxia. Metabolically, HIF mediates the switch from fatty acid oxidation to more oxidation-efficient glycolysis152. Furthermore, it enhances innate immune cell functions while regulating the adaptive response and promoting epithelial protection. Indeed, by allowing myeloid cells to generate ATP in oxygen-deprived inflamed tissues, HIF regulates several functions of these cells, such as aggregation, motility, invasiveness, bactericidal activity and antigen presentation. HIF also prolongs the lifespan of neutrophils in hypoxic conditions, by inhibiting apoptosis. In T cells, HIF has been recently shown to enhance Th17 development through direct transcriptional activation of ROR-t153. Concurrently, HIF attenuates Treg development by targeting Foxp3 for proteasomal degradation154. While promoting inflammation in immune cells, HIF has been shown to rather promote epithelial cell barrier function and protection. As this protection occurs through adenosine upregulation and signalling, which attenuates immune responses, vascular fluid leakage and neutrophil accumulation, HIF also shows regulatory properties that may help the termination of an ischemic insult149.

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4.2 Innate and adaptive immune mechanisms in IRI

As described above, innate and adaptive immunities may be intrinsically influenced by hypoxia. In turn, both are involved in extrinsic damages to ischemic cells or tissues (figure 8). DAMPs such as HMGB1, HSPs or ATP, and PAMPs such as LPS (released during the surgical procedure) may trigger TLR signalling, which has been implicated in kidney and liver IRI6,159,160. Experimental evidence also suggests that neo-epitopes in ischemic membranes can trigger both the classic (see below) and the lectin pathways of complement cascade. Recognition of damaged cells can further amplify the injury, through anaphylatoxin release and immune cell recruitment161. Among these, innate immune cells dominate the infiltrates during the early phase of reperfusion. Indeed, neutrophils are among the first cells that infiltrate ischemic kidneys and their depletion or migration blockade decrease kidney IRI in mice162. Of note, IL-17 has been implicated in neutrophil recruitment in liver and kidney IRI163,164. In addition, platelets may enhance IRI through various mechanisms155. However, by clearing tissue debris, macrophages and neutrophils may also contribute the resolution of injury.

Adaptive immunity may be implicated in IRI as the consequence of inflammatory bystander T cell activation, or due to more complex autoimmune interactions caused by unmasking of cryptic epitopes. These may be recognized by antigen-specific cells or bound by specific

Figure 7: Biological processes implicated in IRI

Ischemia and reperfusion lead to various cell-intrinsic and extrinsic damages that all contribute to the pathology of IRI.

46 antibodies that subsequently activate the complement cascade. Supporting the former aspect, Bobadilla and colleagues incriminated Th17 anti-ColV mechanisms in lung PGD165. Consistently, depletion of CD4+ T cells has been shown to prevent experimental lung IRI166. Regarding the latter, self-antibodies are implicated in cardiac ischemia (IgM against myosin) and lung PGD (IgG against colV and K-1 tubulin)143,167,168.

T cells are also important regulators of IRI, as evidenced by a delayed recovery from hind limb IRI in CD4-/- and CD8-/- recipient mice. Indeed, regulatory subsets may be impacted in these transgenic animals169.

Figure 8: Immune actors implicated in IRI

Innate and adaptive immunities are involved in IRI. Neutrophils are central in this process. IL-17 represents a key immune mediator in this phenomenon.

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4.3 Post-transplant lung IRI = Primary Graft Dysfunction

Despite refinements in lung preservation and improvements in surgical techniques and perioperative care, IRI may lead to an early allograft dysfunction of variable intensity. This syndrome, known as primary graft dysfunction (PGD), remains a significant cause of early morbidity and mortality after lung transplantation. PGD typically occurs within the first 72 hours after transplantation and affects 10 to 25% of the recipients. It is characterized by nonspecific alveolar damage, lung oedema and hypoxemia. The clinical spectrum can range from mild hypoxemia associated with few infiltrates on chest X-rays to a picture similar to full-blown acute respiratory distress syndrome (ARDS). Therefore, the International Society for Heart and Lung Transplantation (ISHLT) proposed a standardized definition of PGD based on PaO2/FiO2 (P/F) ratio and chest infiltrates occurring in the absence of cardiogenic

pulmonary oedema, hyperacute rejection, venous anastomotic occlusion or infection (Table 1)170,171.

Assessed at various time-points after reperfusion, PGD grade has been shown to correlate with plasmatic lung injury biomarkers (ICAM-1, PAI-1 and sRAGE) and survival, with grade 3 PGD being related to the worst outcome172,173. Indeed, severe PGD bears a 30-day mortality

close to 50% and the patients require longer mechanical ventilation and global hospital stay. Furthermore, PGD has recently been shown to represent a risk factor for the development of OB2.

Risk factors include inherent (age, African-american race, female sex, history of smoking) and acquired (mechanical ventilation, aspiration, trauma, hemodynamic instability after brain death) donor variables, recipient variables (pulmonary hypertension, diffuse parenchymal lung disease) and operative variables (cardiopulmonary bypass, blood transfusions)171. These are often related to inflammation. Recently, pretransplant antibodies to ColI and V and K-1 tubulin were also correlated to PGD risk165,167,168.

Adapted from Lee et al. Proc Am Thor Soc 2009.

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Preventive strategies have focused on reducing ischemic times and improving lung preservation techniques (volume, temperature, pressure and composition). A few randomized controlled trials also examined agents that could reduce PGD, such as inhaled nitric oxyde (iNO), the soluble complement receptor-1 inhibitor (sCR1) and the platelet-activating factor inhibitor antagonist BN52021, but these had only a modest early impact on clinical parameters171.

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5. The pathology of lung allografts

Rejection of an allograft results from various injuries, which eventually lead to loss of function. Four entities are usually recognized following lung transplantation, according to the time-course of their appearance (Figure 9):

1. Primary Graft Failure (which has been discussed above)

2. Complement-mediated hyperacute rejection may develop within hours after implantation

3. Acute rejection usually occurs within one year after transplantation 4. Chronic rejection develops beyond the first year

Figure 9: Adverse events following lung transplantation