TLRs are like the sentinels of our cells, they are located at the cell surface and alert the whole immune system of the presence of viruses or bacteria. They detect pathogens by recognizing their molecular patterns; this recognition is specific in order to avoid self-recognition, but they need some degree of promiscuity to remedy to pathogen heterogeneity or mutations. Promiscuity is generally defined as an indiscriminate association with molecules regardless their structure and is the contrary of specificity proper of the classic paradigm of key-lock receptor activation. My thesis results demonstrate that TLR4 and TLR2 are more promiscuous than what was believed and that this promiscuity leads to the

recognition of cationic lipids and cardiolipins.

Cationic lipids lipopolyamines are synthetic molecules nucleic acid nanocarriers proposed to be used for gene therapy, which consists in replacing a gene that is functioning improperly. This thesis

demonstrates that lipopolyamines activate TLR2 by forming conserved and/or alternative H-bonds with TLR residues, simulating the recognition of bacterial lipopeptides and inducing pro-inflammatory cytokines secretion; which is deleterious when we aim to use these nanocarriers in the context of gene therapy. We propose the use of unsaturated cationic lipids to avoid TLR2 recognition. TLR activation could be useful instead to prepare one-component vaccine adjuvants, for which both antigen carrier and TLR activation are needed to turn on the immune system and produce antibodies. The second chapter of this thesis investigates the pro-inflammatory properties of other cationic lipids and describes new lipopolyamines able to activate both TLR2 and TLR4. The study of their adjuvanticity properties showed that they are as efficient as the aluminium salts in stimulating antibodies production.

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I.II Résumé en français (French abstract)

Les récepteurs Toll-like (TLRs) sont des protéines transmembranaires qui constituent la première barrière de notre système immunitaire inné. Ils détectent la présence de bactéries et virus et alertent l’organisme via la sécrétion de molécules pro-inflammatoires appelés cytokines. Parmi les TLRs, TLR2 and TLR4 reconnaissent respectivement des lipides spécifiques aux bactéries, les lipopeptides et les lipopolysaccharides bactériens LPS. La reconnaissance de motifs moléculaires spécifiques aux pathogènes et absents dans notre organisme est essentiel afin d’éviter une réponse immunitaire venant du soi. Le but de notre thèse était de démontrer que les récepteurs Toll-like possèdent une certaine plasticité et peuvent reconnaître des ligands non identifiés jusqu’ici tels les lipides cationiques et la cardiolipine. Les lipides cationiques sont des molécules synthétiques utilisées comme agents de transfection. Notre travail démontre que les lipides cationiques dont la tête polaire est constituée par des polyamines peuvent mimer les propriétés des ligands naturels et induire la sécrétion de cytokines pro-inflammatoire via l’activation des TLRs. Cette interaction implique des interactions entre la chaine principale de la protéine et les lipides sans intervention des chaines latérales. Cette réaction inflammatoire est contre-indiquée en thérapie génique et nous proposons donc de remplacer les chaines acylées saturées par des chaines insaturées pour la synthèse des nouveaux agents de transfection non-immunogénique. D’autre part, l’activation des TLRs par des agents de transfections active le système immunitaire inné, ce qui permet l’activation du système adaptatif et la production d’anticorps. Nous avons étudié une large gamme des lipides cationiques et identifié des nouveaux activateurs á la fois de TLR2 et de TLR4. L’étude de leurs propriétés adjuvantes a démontré que les lipides cationiques sont des adjuvants comparables aux sels d’aluminium en terme de production d’anticorps. La cardiolipine est un lipide localisé dans la membrane des mitochondries et des bactéries. Le domaine hydrophobe est constitué de quatre chaines acylées qui chez les mammifères sont insaturées. Il a été démontré que la cardiolipine extracellulaire inhibe la sécrétion de cytokines induite par LPS. Notre travail de thèse démontre que cet effet inhibiteur est du à la capacité de la cardiolipine à bloquer le site de liaison du LPS. Le travail démontre aussi que lorsque les chaines acylées sont saturées, c’est le cas dans le Syndrome de Barth, la cardiolipine devient un activateur de TLR4 en interagissant avec TLR4 de façon similaire à LPS. Ce dernier résultat pourrait

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1 Introduction

1.1 Toll-like Receptors

1.1.1 The innate immune system

The innate immune system is the organism’s first line of protection against diseases. It enables the body to protect itself against potentially harmful external stimuli, to fight and to eliminate invading pathogens but also to repair and heal affected tissue (Medzhitov, 2008). Under normal circumstances the state of inflammation is not upheld for prolonged periods of time; however, it is possible that the protective response goes into overdrive in order to defeat the invading pathogens causing acute inflammation or tissue damage (Akira et al., 2006; Beutler et al., 2006). A state of acute inflammation can be easily recognized by its physical signs, which include heat, pain, redness, swelling, and loss of function of the affected tissue (Takeuchi and Akira, 2010). The persistence of an inflammatory state due to a dysregulated host response to infection cause life-threatening potentially fatal organ dysfunction called sepsis (10% mortality); the addition of particularly profound circulatory, cellular and metabolic abnormalities lead to septic shock and are associated with a higher risk of mortality than with sepsis alone (40%)(Singer et al., 2016). Another important medical aspect of inflammatory responses is their role in the development of autoimmune diseases such as lupus, type 1 diabetes, rheumatoid arthritis, multiple sclerosis and

psoriasis, which occur when the body is not able to discriminate between self and non-self recognition (Erridge, 2009; Takeuchi and Akira, 2010). The innate immune system

response is fast and non-specific, its role is to rapidly destroy or at least limit the spreading of pathogens while warning the body of their presence. This alarm triggers the adaptive immune system, which in contrast produces an adapted response to the type of invading pathogens (see 1.2 Vaccine).

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unmethylated nucleic acids (Beutler, 2009; Blasius and B., 2010; Kumar et al., 2009; Medzhitov, 2007; Takeuchi and Akira, 2010). These motifs are called Pathogen Associated Molecular Patterns (PAMPs)(Janeway, 1989).

The PAMPs are well-suited targets for the innate immune system. Across one specific class of microorganisms the patterns are highly conserved (Kimbrell and B., 2001; Medzhitov and A., 1997), due to their essential roles in the physiology of the

microorganisms their potential to counter the immune detection system by adaptive evolution is severely limited (Beutler, 2004; Janeway, 1989; Medzhitov, 2007). The TLR-specificity to microorganisms allows the defence system to distinguish between host ("self") and invasive ("non-self") molecules (Janeway, 1992). However, more recent studies suggest that PRRs also target endogenous molecules released from damaged or dying cells via specific damage-associated molecular patterns (DAMPs), which of course relativizes the argument of uniqueness to microbes to some extent (Bianchi, 2007;

Takeuchi and Akira, 2010).

The innate immune system's pattern recognition receptors are mainly expressed on dendritic cells, macrophages and neutrophils (Beutler, 2009; Blasius and B., 2010; Kumar et al., 2009; Medzhitov, 2007; Takeuchi and Akira, 2010). These PRRs can be categorized into three types on the basis of their function and subcellular localization (Hargreaves and Medzhitov, 2005; Iwasaki and Medzhitov, 2010; Takeuchi and Akira, 2005): secreted, cytoplasmic and transmembrane PRRs. Families of PRRs that fall into these three groups include the Toll-like receptors (TLRs), the C-type lectin receptors (CLRs), the Retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) and the NOD-like receptors (NLRs) (see Table 1)(Takeuchi and Akira, 2010).

The secreted receptor category comprises plasmatic proteins such as collectins,

pentraxins and ficolins that bind to pathogen cell surfaces and trigger the classical and the lectin complement pathway. The activation of the complement system leads to the direct elimination of invading pathogens, opsonization which in turn facilitates phagocytosis by the macrophages and neutrophils, and the recruitment of inflammatory cells (Iwasaki and Medzhitov, 2010; Janeway and Medzhitov, 2002; Takeuchi and Akira, 2005).

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and stress signals such as peptidoglycan degradation products, microbial products, UV-irradiation, but also non-pathogenic crystal particles such aluminium salts (Martinon et al., 2009). The other big family are the RLRs, which are expressed by most cell types and which detect viral pathogens such as viral RNA that is detected by RIG-I and MDA5 (Pichlmair and Sousa, 2007; Saito et al., 2007; Takeuchi and Akira, 2009; Yoneyama and Fujita, 2008).

The last category includes the transmembrane receptors, which are scavenger receptors, C-type lectins and the Toll-like receptors (TLRs). The antigen-capturing scavenger

receptors act as phagocytosis receptors. The C-type lectins Dectin-1 and Dectin-2 detect molecular patterns found on fungal cell walls (Brown, 2006; Iwasaki and Medzhitov, 2010; Robinson et al., 2009). The TLRs will be discussed in depth in the next chapter.

In general, after the recognition of the PAMP-containing ligands by their respective receptors the pathogen is phagocytized and subsequently degradated in the

phagolysosome. Some of the resulting molecular fragments are then presented by the major histocompatibility complexes (MHCs) on the surface of antigen-presenting cells (APCs) (see 1.2 Vaccines).

Another important effect of the sensing of PAMPs or DAMPs by PRRs is the production of proinflammatory cytokines (whose role is to amplify the inflammatory signals by interacting with specific receptor located in other cells), chemokines (whose role is to guide the

migration of cells to the site of the inflammation by acting as chemoattractants), type I interferons (IFNs) (similar to cytokines but important for antiviral response), modulators of PRR signalling and other antimicrobial proteins. The specific pattern and the types of produced proteins depend on the activated PRRs (Takeuchi and Akira, 2010). But in general, these molecules trigger a state of inflammation in the affected tissue and thus summon other cells to the infected site.

1.1.3 Toll-like receptors

Toll-like receptors (TLRs) are expressed on a variety of cells (dendritic cells,

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leucine rich repeat (LRR) motifs, a transmembrane stretch only recently characterized (Mineev et al., 2017) and a cytoplasmic region Toll/Interleukin 1 receptor (TIR). Broadly speaking the LRR regions are responsible for the recognition of the ligands while the TIR domain is crucial in triggering the signalling cascades inside of the cell (Gay et al., 2014). From the late 1990s/ early 2000s onwards more than twelve mammalian TLR family members have been discovered and numerous of their ligands have been identified (see Fig.1). TLR1 to 10 are conserved in both human and mouse while TLR11 to 13 on the other hand are not present in the human genome. TLR5, TLR2/TLR1 and TLR2/TLR6 activate cascades from the cell surface, TLR3, TLR7, TLR8 and TLR9 from the

Table of contents

I.I Abstract

I.II Résumé en français (French abstract)

1 Introduction