New Non-microbial Immune-Modulatorsand Their Mode of Recognition by TLRs

Unravelling the Promiscuity of Toll-like Receptor 2 and 4:

New Non-Microbial Immune-Modulators and Their Mode

of Recognition by TLRs

Malvina Pizzuto

This thesis is presented for the degree of PhD in Science of

Malvina Pizzuto

The research thesis was done under the supervision of Dr. Michel Vandenbranden, Prof. Jean-Marie Ruysschaert and Dr. Caroline Lonez at the Department of Structure and

Function of Biological Membrane (SFMB) at the Free University of Brussels (ULB).

Part of this work was done at the University of Cambridge under the supervision of Dr. Monique Gangloff and Prof. Nicholas J. Gay (Department of Biochemistry) and

Prof. Clare Bryant (Department of Veterinary Medicine)

The study of lipopolyamines adjuvant properties was carried out in collaboration with Dr. Virginie Escriou at the CNRS, Unité de Technologies Chimiques et Biologiques pour la

Santé (UTCBS) at the Université Paris Descartes

Is that emergent web, full of feedback between levels, from the gene to the wider environment, that is life. It is a kind of music.

I Abstracts ... 1

I.I Abstract ... 3

I.II Resumé en français (French abstract) ... 4

1 Introduction ... 7

1.1 Toll-like Receptors ... 7

1.1.1 The innate immune system ... 7

1.1.2 Pathogen-associated molecular patterns and pattern recognition receptors ... 7

1.1.3 Toll-like receptors ... 9

1.1.3.1 Toll-like receptors classification and ligands ... 11

1.1.3.2 Toll-like Receptor signalling ... 12

1.1.3.3 Bacterial Lipopolysaccharide (LPS) and TLR4 ... 13

1.1.3.3.1 The Lipid A moiety and their derivatives ... 16

1.1.3.3.2 The LPS-sensing machinery: TLR4, MD2, LBP and CD14 proteins……….19

1.1.3.3.3 LPS signalling through TLR4/MD2 and CD14 ... 29

1.1.3.1 Lipopeptides and TLR2 ... 32

1.1.3.1.1 Lipopeptide binding to the TLR2/TLR6 and TLR2/TLR1 complexes ... 33

1.1.4 TLR4 and TLR2 agonists as vaccine adjuvants against infectious diseases and cancer ... 38

1.1.4.1 The adaptive immunity ... 38

1.1.4.2 Vaccine adjuvants ... 41

1.1.5 TLR4 and TLR2 antagonists as therapeutics anti-inflammatory drugs for inflammatory diseases and cancer ... 45

1.2 Other TLR2 and TLR4 modulators and the controversy between TLR specificity and promiscuity ... 46

1.2.1 Pattern-specificity and the actual molecular pattern ... 46

1.2.2 DAMPs ... 48

1.2.3 TLR modulations: more than PAMPs competitive binding ... 49

1.3 Cationic lipids ... 56

1.3.1 Structure ... 56

1.3.3 Signalling ... 58

1.3.4 Use as vaccine adjuvant ... 59

1.4 Other TLR/ lipids interactions ... 59

1.5 Cardiolipin ... 60

1.5.1 Biosynthesis and structure ... 60

1.5.2 Structural functions ... 61

1.5.3 Signalling ... 61

1.5.4 CL and immunity ... 63

1.5.5 CL-related diseases ... 64

2 Purposes of the work ... 67

3 Results and discussion ... 69

3.1 TLR2 promiscuity is responsible for the inflammatory properties of lipoplexes (Pizzuto et al. 2016) ... 71

3.2 Cationic Lipids As One-Component Vaccine Adjuvants: a Promising Alternative to Alum (in preparation paper) ... 101

3.3 Cardiolipin from TLR4-antagonist to agonist, an unsaturation tale (in preparation paper) ... 123

4 Overall conclusions ... 149

5 Perspectives ... 153

5.1 Further elucidation of the TLR-promiscuity ... 153

5.2 Biological significance of cardiolipins as DAMPs ... 153

5.3 Non-immunogenic lipopolyamines for gene therapy ... 153

5.4 Alternative mechanisms of TLR activation ... 154

5.5 From three- to one- component vaccines ... 155

6 References ... 159

7 Supplementary data ... 197

7.1 Supplemental of Toll-like receptor 2 promiscuity is responsible for the immunostimulatory activity of nucleic acid nanocarriers ... 197

7.2 Supplemental of “Cardiolipin from TLR4-Antagonist to Agonist, An Unsaturation Tale” ... 208

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I.I Abstract

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

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Figure 1: TLRs ligand recognition and signalling cascades (Gay et al., 2014) 1.1.3.1 Toll-like receptors classification and ligands

Toll-like receptor 4 (TLR4) was the first TLR to be identified; it recognizes bacterial

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TLR2 recognizes bacterial lipopeptides. Two possible heterodimers are formed by TLR2 with TLR1 or TLR6 to recognize triacylated or diacylated lipopeptides, respectively. The structures of both heterodimers have been solved (Jin et al., 2007; Kang et al., 2009). TLR3 senses double-stranded RNA (Alexopoulou et al., 2001). The structure has been elucidated by crystallographic study (Choe et al., 2005).

TLR5 recognizes bacterial flagellin (Hayashi et al., 2001; Uematsu et al., 2006).

Both TLR7 and the related TLR8 are the receptors for single-stranded virus RNA (Diebold et al., 2004; Heil et al., 2004; Lund et al., 2004).

TLR9 recognizes CpG-rich hypomethylated DNA motifs that are often found in bacteria but not very frequently in vertebrates (Hemmi et al., 2000). A couple of years later it was shown that this TLR also recognizes herpes virus (Krug et al., 2004a, 2004b; Lund et al., 2003).

TLR10 senses triacylated lipopeptides by forming a heterodimer with TLR2, but it does not seem to activate the typical TLR-dependent signalling cascades as other TLR2 subfamily members (Guan et al., 2010a). It was also reported to sense infections by the

Gram-positive Listeria monocytogenes in intestinal epithelial cells and in macrophages (Regan et al., 2013).

Murine TLR11 detect PAMPs from uropathogenic bacteria (Zhang et al., 2004a) and, in cooperation with murine TLR12, responds specifically to profilin from Toxoplasma gondii (Andrade et al., 2013; Koblansky et al., 2013; Yarovinsky et al., 2005).

Murine TLR13 senses bacterial ribosomal RNA (Hidmark et al., 2012; Li and Chen, 2012; Oldenburg et al., 2012).

1.1.3.2 Toll-like Receptor signalling

TLRs exist in the membrane as monomer or pre-formed dimers. In the latter, the

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adaptor molecules that initiate the signalling cascades recruiting and activating several kinases and ubiquitin ligases. Two main adaptor molecules and relative pathways have been described: the MYD88 adaptor-like protein (MAL) that induces myeloid differentiation primary response protein 88 (MYD88)-pathway and TRIF-related adaptor molecule

(TRAM) that induces TIR-domain-containing adapter-inducing IFNβ (TRIF)-pathway. The MyD88-dependent pathway is triggered by all TLRs, except TLR3, and results in the activation of transcription factors for pro-inflammatory cytokines: nuclear factor-κB

(NF-κB), cyclic AMP-responsive element-binding protein (CREB) and activator protein-1 (AP1). The TRIF-dependent pathway originates from TLR4 and TLR3 in the endosome and induces antiviral response and T-cells stimulation through the induction of interferon regulatory factor-3 (IRF3)(Youn et al., 2006).

The other endosomal TLRs induce a similar type-I IFN response through a MyD88-dependent induction of IRF7 and IRF5.

In this thesis, we will focus on TLR2 and TLR4 and their modulators. 1.1.3.3 Bacterial Lipopolysaccharide (LPS) and TLR4

Bacterial lipopolysaccharide (LPS), also called endotoxin, is the pathogen-associated molecular pattern (PAMP) recognized by TLR4. Even picomolar concentrations of LPS can be sufficient to be detected by the innate immune system (Gioannini et al., 2004; Jerala, 2007). LPS is an integral part of the Gram-negative bacteria outer membrane (Fig. 2). It contributes to bacterial membrane permeability, cell adhesion and cell stability and is released from bacteria by shedding or through bacterial lysis.

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Bacterial lipopolysaccharides are not homogenous molecules but rather a collection of molecules that have certain structural patterns in common. In general, the amphipathic molecule consists of three covalently linked components: a hydrophilic polysaccharide chain named the O-antigen, a core oligosaccharide and a hydrophobic lipidic part named Lipid A (Fig. 3)

Figure 3: The general structure of bacterial lipopolysaccharides (LPS). Modified from (Beutler and Rietschel, 2003) The exact composition and structure of O-antigen and core region can differ even in the same bacterial strain, and also the lipid A domain can vary in number and composition of its fatty acids, depending on their strain of origin. This variability is reflected in the range of biological activity found with the respective Lipid A components (Beutler and Rietschel, 2003; Czerwicka et al., 2013; Guo et al., 2013; Kim and Lee, 2013; Rietschel, 1975; Rietschel et al., 1994; Schromm et al., 1998) (see next paragraph).

The O-antigen can be detected by the adaptive immune system which has the capacities to deal with the huge number of possible variations of this antigen and which is able to induce the production of antibodies against the O-antigen (Erridge et al., 2002; Raetz and Whitfield, 2002; Rietschel et al., 1994).

The form of LPS with an O-antigen is known as "smooth" LPS (S-LPS). Specific growth conditions can also result in the synthesis of LPS without an O-antigen and shorter core oligosaccharides, which is named "rough" LPS (R-LPS). In addition to S-LPS and R-LPS there is semi rough LPS (SR-LPS) as well, a LPS with an O-antigen consisting of only one repeating unit (Fig. 4). R-LPS is not only a product of specific bacteria, each bacteria that produce S-LPS also always produce R-LPS resulting in a heterogeneous LPS mix

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part. The acidic KDO and phosphate residues in the inner core contribute to the negative charge of LPS (Czerwicka et al., 2013; Nikaido, 2003; Rietschel et al., 1994).

The length of the core oligosaccharide also serves as a means to distinguish different subforms of rough LPS from Ra-LPS (longest) to Re-LPS (shortest) (Fig. 4)(Huber et al., 2006).

Although in the crystallised complex between TLR4 and LPS the LPS structure is the Ra-LPS one (Park et al., 2009), Lipid A alone possesses all the immunostimulatory properties of LPS and is thus considered the minimal immunological active part of LPS (Niemetz and Morrison, 1977).

Evidence for an influence of the structure of the core oligosaccharide on immunological activity is, for the time being, sparse, but cannot be ruled out as at least one study claims that the core oligosaccharide is able to increase the binding of LPS to MD2 and thus enhance the immunological potential of Lipid A (Ittig et al., 2012). Furthermore, in the crystal, the phosphates of the core region participate in binding TLR4 (Park et al., 2009).

Figure 4: Schematic representation of the different LPS chemotypes: smooth LPS (S-LPS), semi-rough LPS (SR-LPS) and rough LPS (R-LPS) differ in the completeness of the LPS molecule. KDO,

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1.1.3.3.1 The Lipid A moiety and their derivatives

As pointed out before, Lipid A part is responsible for the biological activity of LPS by activating the innate immune system via TLR4. Its composition and structure determine how active it is and chemical modifications can even attribute an antagonistic effect to Lipid A (Schromm et al., 2000).

The number of acyl chains is variable and the actual degree of acylation can, as other structural variations, correlate with the ability of LPS from specific bacterial sources to trigger the immune system via the TLR4-receptor complex. Helicobacter pylori's,

Legionella pneumophila's or Yersinia pestis' LPS for example with their four to five C16-18 acyl chains are known to be less immunostimulatory than the hexa-acylated "standard" LPS with 12 to 14 carbons which seem to be one of the most active forms in terms of strength of the elicited immune response in humans. Other bacteria species have even been shown to be able to modify the composition of their Lipid A moiety to make it less potent during the process of infection and thus to evade detection by the immune system (Arpaia and Barton, 2012; Erridge et al., 2002; Guo et al., 1997, 1998; Moran et al., 1997; Rietschel et al., 1987; Robinson et al., 2008; Tran et al., 2005).

The polar head of Lipid A is the structurally most conserved and least variable part of the molecule: its diglucosamine backbone has two phosphate groups and is acylated with ester- and amide- linkers. Also the nature of these glycosyl residues is shared by most forms of endotoxin (Czerwicka et al., 2013; Jeannin, 2009).

The most-employed and most archetypical LPS (which is also the LPS used in this work) is bacterial lipopolysaccharide isolated from Escherichia coli. Its Lipid A carries six

acylated C12 and 14-chains linked to two phosphorylated glucosamine disaccharides (Fig. 5). The hydroxyl group at carbon 4 remains unoccupied and the one at position 6'

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Figure 5: Lipid A from Escherichia coli

Figure 6: Lipid A from Rhodobacter sphaeroides and capsulatus

The Lipid A of the most biologically potent LPS, i.e. of E. coli and most other agonist LPS, is acylated with saturated fatty acids. Unsaturated fatty acids are only rare in bacterial Lipid A, but they do exist, for example in the LPS synthesized by Rhodobacter

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LPS. (Erridge et al., 2002; Kim and Lee, 2013; Rietschel et al., 1994; Schromm et al., 2000).

These observations were used to design the synthetic LPS antagonist Eritoran (also known as E5564), which has four lipid chains and one double bond (Fig. 7).

This suggests that the saturation of the acyl chains is critical for TLR4 modulation. However, these molecules differ also in terms of number and length of acyl chains. Another difference in Eritoran is that the bonds between two of the acyl chains and the glucosamines in Eritoran are ether instead of ester or amide bonds.

Furthermore, antagonist property does not necessarily require unsaturated fatty acids. Other bacterial saturated LPS have antagonist effects (Schromm et al., 2000). The Lipid A biosynthesis intermediate Lipid-IVa has also four saturated C14 acyl chains and is an antagonist in human but a weak agonist in mice (Walsh et al., 2008) (Fig. 8). Overall, the aforementioned heterogeneity of this molecule and the lack of synthetic comparable structures impede a proper structure-activity relationship analysis.

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Figure 8: Lipid-IVa and MPLA structures

1.1.3.3.2 The LPS-sensing machinery: TLR4, MD2, LBP and CD14 proteins

TLR4 is always associated with a soluble protein called Myeloid Differentiation protein-2 (MD2). MD2 is essential for the stability and the expression of TLR4 to the cell surface (Nagai et al., 2002).

The LPS recognition by the TLR4/MD2 complex is helped by two more proteins: the Lipid Binding Protein (LBP) found in serum, and the membrane anchored Cluster of

Differentiation-14 (CD14) (see Fig. 9). First LBP extracts LPS from the membrane of the invading Gram-negative bacteria second these molecules are transferred to a specific binding site on CD14. Finally, the LPS monomers are transferred to the TLR4-MD2

complex. All of the proteins participating in the LPS sensing complex are important in their own way for a normal and efficient LPS response and genetic deficiencies resulting in the mutation or absence of any of the involved molecules abolish LPS signalling (Kim and Lee, 2013).

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Figure 9: LPS-recognizing protein cascade: LPS-binding protein (LBP), cluster of differentiation 14 (CD14) in soluble (sCD14) and membrane-associated form (mCD14), Toll-like receptor 4 (TLR4) and myeloid

differentiation factor 2 (MD2) cooperate in the recognition of bacterial lipopolysaccharide (LPS) to trigger the innate immune response (Peri and Piazza, 2012).

1.1.3.3.2.1 Lipid Binding Protein (LBP)

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Figure 10: Lipopolysaccharide-binding protein (LBP) structure from two different angles (A, B): N-terminal (yellow), central (grey) and C-terminal (orange) domains, phospholipid-binding pockets with bound molecules (red)(Kim and Lee, 2013).

1.1.3.3.2.2 Cluster of Differentiation-14 (CD14)

CD14 is a glycoprotein that exists in two forms: a soluble form (sCD14) found in the serum and a form attached to the outer leaflet of the membrane via a GPI-anchor (mCD14)(da Silva Correia et al., 2001; Ulevitch and Tobias, 1995). CD14 is mainly expressed in monocyte- and macrophage-derived cells and on activated neutrophils (Kielian and Blecha, 1995).

Both CD14 forms have been shown to bind LPS, to transfer endotoxin monomers to TLR4-MD2 and to enhance the LPS response (Akashi et al., 2003; Moreno et al., 2004; Ohnishi et al., 2007). The constitutively expressed soluble form is found in the blood stream at a concentration of 2 to 6 µg/ mL in humans and in other body fluids such as urine, saliva and gingival crevicular fluid (Bazil et al., 1986; Bussolati et al., 2002; Duncan et al., 2004; Grunwald et al., 1992; Isaza-Guzman et al., 2008; Jin and Darveau, 2001; Nomura et al., 2003), but, as in the case of LBP, its concentration increases markedly after an infection as part of the inflammatory response (Ayaslioglu et al., 2005, 2012; Landmann et al., 1995).

The canonical role of the 55 kDa membrane-associated CD14 form in the LPS- sensing machinery is to concentrate LPS close to the cell membrane, acting as a necessary

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by their numerous leucine rich repeats and their overall horseshoe shape (Gay and Gangloff, 2007; Lee et al., 2012). CD14 with its eleven LRRs differs from most other members of the LRR family with regard to its ligand-binding site, which is located on the convex part of the molecule. The molecule has a hydrophobic pocket located between the LRRNT and the first LRR module (Fig. 11: "N"). A break in the inner, convex structure allows solvent access to the exposed hydrophobic residues there (Kim et al., 2005). It is this pocket that, based on mutagenesis and other experiments, is proposed to be the binding site for LPS molecules (Cunningham et al., 2000; Dziarski et al., 2000; Juan et al., 1995; Shapiro et al., 1997; Stelter et al., 1997; Viriyakosol and Kirkland, 1995). The interior of the pocket does not contain positively charged residues, suggesting an unspecific lipid-binding capability rather than specificity for negatively charged LPS. This is supported by reported binding of different lipid- containing molecules, some of them lacking negatively charged chemical groups (Kim and Lee, 2013). There is however a cluster of positively charged residues on the rim of the pocket, where the phosphorylated Lipid A part of LPS is bound (Kim et al., 2005).

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1.1.3.3.2.1 The TLR4/MD2 complex and the basis for TLR4 agonistic and antagonistic activity

Toll-like receptor-4 itself is a type I transmembrane protein with three domains: an extracellular domain for binding ligands, a single transmembrane domain and an

intracellular domain that serves as the signalling unit (Gay and Gangloff, 2007; Medzhitov et al., 1997). The extracellular TLR4 domain contains 22 LRR modules divided into three subdomains: N-terminal, central and C-terminal. The region separating the N-terminal from the central subdomain was shown to be important for the binding of MD2 to TLR4. This primary MD2-binding site lies on the concave site of the "horseshoe" (see Fig. 12, "primary interface"). Interactions on the TLR4-MD2-binding interface are mainly mediated by

charged residues, while hydrophobic residues are rare and do not seem to play a significant role in the formation of the heterodimer (Kim and Lee, 2013).

The binding of LPS to TLR4-MD2 induces the dimerization of the two heterodimers to an "m"-shaped heterotetramer in which the N-termini of the two TLR4s point outwards while the C-termini face each other on the inside of the complex (see Fig. 12). The

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.

Figure 12: Structure of human TLR4-MD2-LPS (Park et al., 2009) Red: lipid A, pink core region.

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The binding of LPS to MD2 induces conformation change on MD2 that gives the latter a second binding site to associate itself with a second TLR4. As mentioned before all six Lipid A chains are inserted into the hydrophobic cavity, but only five of them are really fully inside of it. The sixth one is not completely in the pocket and therefore partially exposed onto the surface of MD2. The interaction of this acyl chain with Phe126 of MD2 has been shown to play an important role in the conformational changes (Paramo et al., 2013; Park et al., 2009; Walsh et al., 2008). With the same acyl chain LPS also participates in binding the second TLR4 in a formed hydrophobic pocket between MD2 and secondary TLR4. This illustrates that the dimerization is a cooperative process between LPS and MD2 (Kim and Lee, 2013). The crystal structure of mouse TLR4/MD2 bound to Eritoran and human TLR4/MD2 bound to lipid-IVa show that the TLR4 antagonists bind MD2 but fail in

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Figure 13: comparison of agonistic vs. antagonistic conformations of ligands in the TLR4/MD2 crystal. Top: green: agonist lipid A (Park et al., 2009), orange: antagonist Eritoran (Kim et al., 2007). Bottom: blue: agonistic conformation of lipid-IVa in murine TLR4/MD2 (Ohto et al., 2012), pink: antagonistic conformation of lipid-IVa in human TLR4/MD2 (Ohto et al., 2007).

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Figure 14: Structure of MD2 crystallised in absence of TLR4 modulators (Ohto et al., 2007). Magenta MD2, yellow: Myristic acid.

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1.1.3.3.3 LPS signalling through TLR4/MD2 and CD14

After LPS binding TLR4/MD2 is recruited into phosphatidylinositol 4,5- biphosphate (PIP2) and cholesterol rich regions of the plasma membrane. The adaptor MAL is anchored to the plasma membrane through a PIP2 binding motif, which determine its localisation in the aforementioned subdomains prior LPS stimulation. The LPS-dependent recruitment of TLR4 to these subdomains and the consequent dimerization of the intracellular TLR4 domains (TIR) induce the engagement of the adaptors MAL and trigger the MyD88 pathway. (Fig. 16) (Ruysschaert and Lonez, 2015).

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Figure 16: Signalling by membrane-bound TLR4 (Ruysschaert and Lonez, 2015)

Figure 17: Signalling by endocytosed TLR4 (simplified) [modified from Kagan, 2012].

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Figure 18: Activation of the MyD88- and the TRIF-dependent signalling cascades as a function of the presence or absence of CD14 and of the LPS chemotype ("smooth"/ sLPS or "rough"/ rLPS). Figure realized by Boris Schmidt ((Schmidt, 2014), thesis manuscript)

The reason for the TRIF pathway's requirement for CD14 is not completely clear. One hypothesis postulates that CD14 could have an effect on the supramolecular structure of TLR4-MD2 and induces a conformational change in TLR4-MD2 in the presence of LPS necessary for triggering the TRIF pathway (Jiang et al., 2005). As mentioned before, the TRIF-dependent pathway originates from the endosome. The LPS-induced endocytosis is then a prerequisite for the activation of the TRIF pathway and it was proposed that CD14 might control the TRIF-dependent signalling cascade by actually controlling the

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Figure 19: CD14-mediated transport events in TLR4 signalling (simplified). Modified from (Zanoni et al., 2011)

1.1.3.1 Lipopeptides and TLR2

A lipopeptide is a lipid whose polar head is made by an amino acid sequence (peptide). Bacterial lipopeptides have no shared sequence homology but are characterized by the N-terminal unusual amino acid cysteine whose S-atom link di-acylated lipidic moieties at the di-acylated glycerol backbone (Fig. 20). Lipopeptide moiety is the pathogen-associated molecular pattern (PAMP) recognized by TLR2.

The N-terminal end of the cysteine can be linked to a third acyl chain or not (Hantke and Braun, 1973). Such di- and tri-acylated lipopeptides are found in the cell wall of

mycoplasma, gram-positive and negative bacteria. The acyl chain lengths, as well as the composition of the amino acid sequence, are strongly variable. An example of tri-acylated lipopeptide is shown in Fig. 20.

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Lipopeptides are easy to synthesize, this has allowed extensive structure-activity relationship studies.

These studies revealed that TLR2 recognizes a large variety of lipopeptide structures, which differ in terms of chain length from C10 (or C12 depending on TLR2 species) to C18, peptide sequence and number of amino acids (Buwitt-Beckmann et al., 2005; Morr et al., 2002; Okusawa et al., 2004; Spohn et al., 2004). Synthetic Pam2CSK4 and Pam3CSK4

are the most utilised TLR2 ligands and are the ones used in this study.

TLR2 co-operates with TLR1 or TLR6 for binding lipopeptides. It is assumed that TLR2/TLR1 recognizes tri-acylated lipopeptides, whereas di-acylated lipopeptides use TLR2/TLR6 heterodimers for signalling (Barton and Medzhitov, 2003; Gay and Gangloff, 2007; O’Neill et al., 2013). This is supported by the inability of tri-acylated lipopeptides to activate TLR2/TLR6 (Takeuchi et al., 2002) and by the crystal structures of both TLR2 heterodimers bound to lipopeptides that have been resolved (Jin et al., 2007; Kang et al., 2009). However, di-acylated lipopeptides have been shown to activate TLR6 knock-out mice and cells expressing only TLR2 and TLR1 suggesting partially overlapping binding capacities of TLR2/TLR1 heterodimer (Buwitt-Beckmann et al., 2005; Omueti et al., 2005). Indeed TLR1 has a hydrophobic pocket that is essential for hosting the third acyl chain of tri-acylated lipopeptide but which does not obstruct di-acylated binding. Finally, one report describes di- and tri-acylated lipopeptides recognized by TLR2 in a both TLR1- and TLR6-independent manner (Buwitt-Beckmann et al., 2006), opening new questions about the role of TLR6 and TLR1. No other co-receptors are necessary for TLR2 signalisation, however CD14 and CD36 may enhance the immune response (Drage et al., 2009).

1.1.3.1.1 Lipopeptide binding to the TLR2/TLR6 and TLR2/TLR1 complexes

The crystal structures of TLR2/TLR6 bound to Pam2CSK4 (Kang et al., 2009) (Fig. 21) and TLR2/TLR1 bound to Pam3CSK4 (Jin et al., 2007) (Fig. 22) have been solved for mouse and human proteins, respectively. The binding to TLR2 is very similar in both structures: the two ester-bound lipids of Pam2CSK4 and Pam3CSK4 are inserted in an internal hydrophobic pocket of TLR2 located between the central and the C-terminal

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the TLR2 pocket is bigger of around 15% in murine and 10% in human than the volume theoretically required for binding the two palmitoyl groups of the ligand. The extra space in the pocket explains why modifications of the chemical structure and length of the ester-bound lipids of the ligands do not impair their ability to activate TLR2 (Buwitt-Beckmann et al., 2005; Morr et al., 2002).

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1.1.4 TLR4 and TLR2 agonists as vaccine adjuvants against infectious diseases and cancer

1.1.4.1 The adaptive immunity

Our immune system includes two subsystems, the innate and the adaptive ones. Innate immune cells are dendritic cells, macrophages, and neutrophils, among others; those cells recognize pathogens and activate inflammatory signals through cytokines secretion among others. This allows a fast initial containment of the infection during the development and differentiation of adaptive immune cells.

Innate immune cells, mainly dendritic cells (DC) and macrophages, are also able to present proteins or lipids from pathogens (antigens) at the surface of the cells through membrane protein complex (Major Histocompatibility Complex, MHC), and so are also called Antigen Presenting Cells (APCs). The concomitant activation of TLRs or other PPRs leads to cytokine secretion and co-stimulatory molecule expression at the cell surface (Fig. 24). Those signals enable cells from the adaptive system (T and B lymphocytes) to judge the antigen as belonging to an invading organism and so generate de novo specific

antibodies and TCR (T Cells Receptors) specific responses against the antigens. The type of the expressed co-stimulatory molecules or cytokines determines the precise form in which the adaptive immune system will respond to the threat [Xu et al., 2004].

Figure 24: The action of antigen presenting cells (APCs). Example of extracellular detection of PAMPs by TLRs. PAMPs activate TLRs and induce cytokine and co-stimulatory molecules expression while the antigen is transported to the cell surface. (Iwasaki and Medzhitov, 2010)

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The cytokine secreted together with the co-stimulatory molecules and the type of MHC determines the differentiation of naïve T cells into Helper (CD4, Th) or cytotoxic T cells (CD8, CTL). CTL cells eliminate pathogen-infected host cells by releasing cytotoxic molecules, while T Helper cells support the immune response by generation of cytokines that activate neighbouring cells to perform specific functions or chemokines that recruit new immune cell subsets to sites of pathogen encounter.

The type of cytokine secreted by macrophages and other innate cells determines the type of Th subset (Fig. 26) to adapt the immune response to the type of invading pathogen (against bacteria, viruses, parasites, allergy etc.). Many kinds of T helper cells have been characterized in the last years: Th1, Th2, Th9, Th17 and Th22. In addition, Treg

differentiation has mainly anti-inflammatory effect and a role in the resolution of

inflammation. (Zygmunt and Veldhoen, 2011). The study of new T-cell substes is still an emerging field, it is not clear how the same cytokine may induce a different subset and nor the function of each subset is completely understood.

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Helper T cells also migrate to the boundary between the T cell zones and the B cell follicle (Vinuesa and Cyster, 2011), where they activate B cells that similarly to APCs have

responded to pathogen antigens. Activated B cells secrete higher-affinity pathogen-specific antibodies able to directly neutralize extracellular pathogens by antigen binding (Zotos and Tarlinton, 2012).

Figure 27: B cells activation and antibodies production (adapted from (Rawlings et al., 2012)). Activated B cells present antigen to helper T cell. As from APCs T cells are activated by antigen and co-stimulatory molecules presentation. T cell activation induces B cells differentiation to plasma cells that produce antibodies.

Upon elimination of the invading pathogen, the majority of adaptive cells die and leave behind an “ever-growing” array of memory cell subsets. These memory cells are more sensitive to antigen stimulation through their TCR and are somewhat less dependent on co-stimulatory molecules to enable a rapid and protective immune response upon reinfection (Kaech et al., 2002).

This combination of antibodies specificity and memory are the mechanistic underpinnings for the clinical success of vaccination.

1.1.4.2 Vaccine adjuvants

The aim of a vaccine is to mimic the pathogen responsible for the disease and to develop an immunological protection against infection and other diseases such as cancer

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excessive immune response are the consequences of using components of living organism. Furthermore, these vaccines contain many unnecessary substances, whose adversary effects are difficult to predict. As a safer alternative, new generation vaccines are based on synthetic proteins as antigen mimetic (Berzofsky, 1993; Berzofsky et al., 2001) or transfected DNA sequences to allow expression of the antigen (Berzofsky et al., 2001; Kudrna and Ugen, 2015; Liu, 2010). However, those antigens are not or not enough immunogenic, so require the addition of immunostimulatory molecules to the vaccine formulation. Moreover, the antigen has to be protected from degradation and transported to the innate immune cells and so requires a third component that acts as a transporter. A vaccine has to mimic pathogen in simultaneously transporting the antigen and stimulating the innate immune system in the same cell. Co-delivery of the antigen and the

immunostimulatory molecule is therefore essential for vaccine formulation. A simple mixing without a stable association is not sufficient because components may be redistributed differently in the organism. The co-recognition is strongly facilitated by some transporters, such as liposomes, mineral oil and aluminium salts (Alum), which promote antigen

persistence and co-recognition. Alum adjuvanticity has been attributed to inflammasome activation as well. Both transporter and immunostimulatory molecules are defined as adjuvants in the vaccine formulation. Overall the ability of an adjuvant or of a mixture of adjuvants to target several immune receptors increases the efficiency and the versatility of a vaccine formulation (Awate et al., 2013; Christensen et al., 2011; Hennessy et al., 2010; Kaech et al., 2002; Petrovsky and Aguilar, 2004; Steinhagen et al., 2011)

Many preclinical and clinical studies have demonstrated the adjuvanticity of TLR agonists. In the following, we will focus on effective adjuvants used for human vaccines and based on synthetic derivatives from standard TLR2 and TLR4 ligands i.e. lipopeptides and LPS. For more details about other TLRs refer to the recent reviews (Basto et al., 2014; Dowling and Mansell, 2016; Steinhagen et al., 2011). Adjuvanticity of non-standard TLR2 and TLR4 modulators will be discussed in paragraph 1.2.

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However, almost no differences were observed in type I IFN cytokine secretion such as IP-10 (Mata-Haro et al., 2007). Type-I IFN response rather than MyD88 is needed for TLR4-induced adjuvant effects. Clinically relevant adjuvants other than MPLA, such as CpG oligonucleotides (Scheiermann and Klinman, 2014), also induce type I interferon but in a MyD88-dependent mechanism (Honda et al., 2005).

With the exception of Pollinex Quattro (Allergy Therapeutics, West Sussex, UK), that combines MPLA only with pollen antigen for the treatment of seasonal allergic rhinitis, MPLA is always combined with other adjuvants needed for antigen transport and uptake. Combinations include MPLA in conjunction with quillaja saponaria (QS21) and liposomes (AS01, GlaxoSmithKline GSK Vaccines, Middlesex, UK), QS21 and an oil-in-water

emulsion (AS02 (GSK)) or alum (AS04 (GSK)). (Dowling and Mansell, 2016)

AS04 has also been studied in combination with cancer vaccines. Melacine® consists of the lysates from two allogeneic melanoma cell lines combined with DETOX®, an adjuvant containing AS04 plus cell wall material from Mycobacterium Phlei. Melacine® was

approved for the treatment of metastatic melanoma by the Canadian FDA (Sondak and Sosman, 2003).

Other lipid A derivatives with potent adjuvanticity are deacylated lipooligosaccharide (dLOS) which consists of a core oligosaccharide lacking the terminal glucose residue, a glucosamine disaccharide with two phosphate groups, and two N-linked acyl groups (Han et al., 2014) and an aminoalkyl glucosaminide 4- phosphates, the CRX547 is in use as vaccine adjuvants and in cancer immunotherapy (Bowen et al., 2012). Very recently a hexa-acylated glucopyranosyl lipid A (GLA) in an oil-in-water emulsion (GLA-SE) has been tested in several preclinical and phase I clinical studies targeting herpes simplex virus-2 (HSV-2), Schistosomiasis, respiratory syncytial virus and the perioperative treatment of cancers to reduce metastasis. (Matzner et al., 2016; Odegard et al., 2016; Patton et al., 2015; Santini-Oliveira et al., 2016).

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properties only in a vaccine against dust mite allergy (Jacquet et al., 2005) but fail in inducing antimicrobial antibodies production (Sasaki et al., 1997; Wilmar et al., 2012). Finally, OM-174 is a lipid A derivatives with agonistic activity on TLR2 and antagonistic on TLR4. In a Phase I trial completed in 2013 it was efficient against refractory solid tumours in adults (Isambert et al., 2013).

Molecules able to activate exclusively TLR2, when used only for their immunostimulatory properties were not effective adjuvants (Basto et al., 2014). Although Pam3CSK4 induced good antibody responses against influenza antigens (Caproni et al., 2012), another work showed Pam3CSK4 as not able to enhance antibodies production against OVA (Bal et al., 2011). Furthermore, no TLR2-based adjuvants have been approved nor involved in clinical trials. However, very recently Pam2CSK4 has been shown as inducing an antibody

response against parasite Leishmania major and Brugia malayi (Halliday et al., 2016). A major advantage of TLR2 ligand (limited to protein based vaccine) is that by linking the peptide antigen to an acylated s-glycerylcysteine moiety, it may generate a TLR2 ligand with the antigen in its own structure (Moyle, 2017). LYMErix vaccine contains Pam3Cys linked to the C terminus epitope of outer surface protein A (OspA) of Borrelia burgdorferi, the causative agent of Lime disease. It was licenced in 1998; but following autoimmune and inflammatory side effects the vaccine was withdrawn from the market (Abbott, 2006). On the other hand, linking a TLR2 ligand to a synthetic antigen is relatively easy, allowing simplified production of completely synthetic and safer vaccines. In a vaccine against HIV-1, Tat protein in conjunction with MALP-2 lipopeptides showed long-lived antigen-specific CTL and humoral responses in preclinical tests (Borsutzky et al., 2006).

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Vaccigrade™ from InvivoGen significantly enhanced both systemic and mucosal immunity against a vaccine antigen. This activity seems to be mediated by an optimal DC maturation process, which induces strong B and T cell stimulation and autophagy (Pavot et al., 2014).

1.1.5 TLR4 and TLR2 antagonists as therapeutics anti-inflammatory drugs for inflammatory diseases and cancer

TLR4 and TLR2 antagonists have been used in clinical trials to treat TLR-related diseases such as autoimmune diseases, arteriosclerosis, sepsis, chronic inflammation, asthma, chronic obstructive pulmonary disease exacerbation, acne and atopic dermatitis (Erridge, 2009; Hennessy et al., 2010; Zuany-Amorim et al., 2002). In addition, TLR are involved in cardiovascular disorder, multiple sclerosis, diabetes, obesity, metabolic syndrome,

neuropathic pain, Alzheimer Disease (AD), psychiatric diseases, skin inflammations (dermatitis), psoriasis and some tumours (Mai et al., 2013; Miranda-Hernandez and Baxter, 2013; Peri and Calabrese, 2014; Takeuchi and Akira, 2010). The interest of these approaches has been largely discussed but only a limited number of TLR2 and TLR4 inhibitors have been investigated (Dowling and Mansell, 2016; Hennessy et al., 2010; Peri and Calabrese, 2014; Zuany-Amorim et al., 2002). Among these, only Eritoran is based on standard TLR-ligand structure and inhibit LPS binding to TLR4/MD2 by occupying MD2 pocket, so acting as a competitive antagonist. Eritoran passed successfully Phase II clinical trials for the treatment of sepsis, but failed in decreasing patient’s mortality during Phase III (https://www.drugs.com/clinical_trials/phase-iii-study-eritoran-does-not-meet-primary-endpoint-11082.html). Sepsis is a notoriously difficult indication; perhaps new TLR4-antagonists or trials utilizing TLR4 antagonists to treat other inflammatory disorders may be more successful.

TLR pro-inflammatory activity can also be suppressed by using small molecule able to block the cytoplasmic signalling cascades. For instance, AV411 (or Ibudilast) blocks LPS signalling by inhibiting phosphodiesterases that participate in the TLR4 downstream pathway. It is in Phase II clinical trials for the treatment of neuropathic pain (Rolan et al., 2008) and already used in Asia for the treatment of asthma and post-stroke disorders (Rolan et al., 2009). Another way to block a receptor activity is by using blocking

antibodies: antibodies bind the receptor and avoid dimerization by steric encumbrance. TLR4 targeted monoclonal antibody NI-0101 (Novimmunne) passed Phase I clinical trials for safety and tolerability. It has several potential indications including asthma and

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delayed renal graft function and for prevention of low- and intermediate-risk myelodysplastic syndrome, a disease that affects normal blood cell production in the bone marrow

(http://adisinsight.springer.com/drugs/800029873). TLR2 antagonists were not appreciated until 2014 (see paragraph 1.2), hence their mechanism and potential application need more studies and yet there is no TLR2 antagonist licensed for human use.

1.2 Other TLR2 and TLR4 modulators and the controversy between TLR specificity and promiscuity

Promiscuity is generally defined as an indiscriminate association with molecules

regardless their structure and is the contrary of specificity. Specific receptors are proteins that recognize only one specific ligand because it is able to perform the specific

interactions necessary for receptor activation. This is the case of enzymes and is known as the key-lock paradigm. On the contrary, promiscuous receptors recognize a large variety of structures because, according to the structures, alternative (promiscuous) interactions can be made to induce receptor activation. Given that PRRs of the innate immune system need sensitivity and specificity to discriminate between “self” and “non-self” and avoid autoimmune responses, a promiscuous receptor appears to be a

contradiction. On the other hand, TLRs have to deal with exogenous living organisms able to undergo chemical modifications to escape TLRs recognition and promiscuity might be the strategy to maintain their recognition. The following paragraphs try to resume what is known about TLR2 and TLR4 modulators in order to address three main arguments pro and con TLR specificity/promiscuity: the specificity to molecular patterns; the recognition of damage associated molecular patterns and the modulation by synthetic molecules without similarity with bona fide ligands.

1.2.1 Pattern-specificity and the actual molecular pattern

As discussed in section 1.1.3, the LPS and lipopeptide microbial structures recognized by TLR4 and TLR2 seem to be strongly variable. However, when testing molecules extracted from pathogens the exact composition of the lipidic extract is difficult to evaluate and presence of small quantity of the bona fide TLR ligand can be misleading in evaluating structure-activity relationships.

Although these contamination concerns are the main argument against TLR promiscuity, the activity of synthetic lipopeptides and lipid A derivatives (see section 1.1.3.1 and

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the recognition remains specific to a class of molecules that share a molecular pattern as a mark of a specific pathogen. Usually, this pattern is a feature indispensable for pathogen life. In contrast to this pattern-specificity, TLR2 have been suggested to recognize an extremely broad spectrum of structurally unrelated molecules from bacteria, fungi, yeast and viruses, in addition to lipopeptides: glycolipids (LPS and lipoteichoic acids (LTA) among others), GPI-anchored structures, peptidoglycans, polysaccharides (zymosan), proteins (hemagglutinin, porins among others), glycoprotein and lipoproteins; all listed in (Zähringer et al., 2008).

(Seong and Matzinger, 2004). In view of the fact that PRRs of the innate immune system need sensitivity and specificity, a promiscuous receptor appears to be a contradiction. The aim of this work is to describe an unambiguous structure–function relationship for TLR2 and its PAMPs. In this review, we concentrate exclusively on TLR2 and its agonist isolated from Gram-positive S. aureus (Fig. 3).

TLR2 and LPS

The first agonist identified for TLR2 was LPS (Kirschning et al., 1998; Yang et al., 1998), which subsequently was found to be due to contaminating lipoproteins of Gram-negative bacteria in commercially available LPS preparations (Hellman et al., 2003; Lee et al., 2002). However, experiments using TLR4-knock out mice demonstrated that TLR4 (together with MD-2), not TLR2, is the LPS receptor (Poltorak et al., 1998). The best example demonstrating how contamination in natural PAMP preparations is responsible for the assignment of TLR2 activity is that of Porphyromonas gingivalis (formerly Bacteroides gingivalis) LPS, which was identified as a TLR2 agonist (Hirschfeld et al., 2001). However, upon further purification contaminat-ing TLR2 activity was eliminated (Hirschfeld et al., 2000). Hashimoto and coworkers subsequently

identi-fied the TLR2-active substance, which surprisingly co-purified with lipid A in several chromatographic, thin-layer systems. Using preparative thin-layer chromatography they identified a ninhydrin staining lipopeptide, which was separated from the lipid A, and which they identified as the molecule expressing TLR2 activity (Hashimoto et al., 2004). Despite the demonstration of TLR2-activating contaminants in highly purified lipid A preparations of P. gingivalis, discussions assuming that LPS or lipid A is an agonist for TLR2 continue. Despite the clear demonstration that lipoprotein/lipopeptide contamination is responsible for TLR2 activity, LPS isolated from various Gram-negative bacteria have subsequently been published as TLR2 agonists. These include LPS from Leptospira interrogans (Werts et al., 2001), P. gingivalis (Hirschfeld et al., 2001), Legionella pneumophila (Girard et al., 2003), Flavobacterium menigosepticum (Tanamoto et al., 2001), Prevotella intermedia(Kirikae et al., 1999) and others (Table 1).

As described earlier, the most likely candidates expressing high TLR2 activity are lipoproteins or lipo-peptides. Since these molecules share physicochemical behavior similar to LPS (but also to LTA) co-extraction and co-purification of such compounds with LPS or lipid A is not unexpected, especially when only two extraction steps are employed, as is usually the case for LTA and LPS. In the LPS of all Gram-negative bacteria, lipid A represents a unique structural entity

ARTICLE IN PRESS

Fig. 1. Schematic structures of reported non-proteinous agonists of TLR2.

U. Za¨hringer et al. / Immunobiology 213 (2008) 205–224 208

Figure 28: schematic structure of putative TLR2 ligands, from (Zähringer et al., 2008) Zhiringer et al. performed an extensive work that demonstrated that only

lipoproteins/lipopeptides are sensed at picomolar levels by TLR2 and claimed that the activity of all other bacterial compounds so far reported as TLR2 agonists was most likely due to contamination by highly active natural lipoproteins and/or lipopeptides (Zähringer et al., 2008). Interestingly, the crystal structure of monomeric TLR2 bound to LTA, as well as to known inactive lipids phosphatidylcholine (PC) and

1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid

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Furthermore, Jiménez-Dalmaroni et al. demonstrated that human TLR2 ectodomain in solution binds synthetic diacylglycerol lipopeptides and glycolipids phosphatidylinositol mannosides (Pim2 and Pim4) but does not bind gp120 from HIV-1 nor monoacylated lipopeptides (Jiménez-Dalmaroni et al., 2015). This means that TLR2 is promiscuous in binding di-acylated lipids, but that binding does not necessarily imply activity.

Co-purification with natural lipids without receptor related biological activity is reminiscent of the myristic acids observed in the MD2 pocket (see above). It is likely that hydrophobic pockets are often (or always) filled by natural lipids that stabilize the pocket waiting for the real modulator. The hydrophobic interaction is strong, but it is relatively nonspecific, it is necessary but not sufficient for activity, whereas a specific interaction pattern of the head group is essential for a robust immune response by TLR2 heterodimers.

1.2.2 DAMPs

In contrast to the pattern-specificity argument, TLR2 and TLR4 have been suggested to recognize natural DAMPs (Damage Associated Molecular Patterns) with structures unrelated to LPS or lipopeptides. DAMPs are endogenous molecules expressed or released as a consequence of injury that drives sterile inflammation to contribute to the repair of damaged tissues (Bianchi, 2007). This is illustrated with the IL-1 cytokines, whose receptor is IL-1R (Kim et al., 2013a; Rider et al., 2011). Numerous endogenous molecules such as α-synuclein, β-defensin, lysozyme, High-Mobility Group Protein-1 (HMGB1), heat shock proteins (HSP), and saturated fatty acid among others, have been suggested to be TLR2 (Gustot et al., 2013, 2013; Tsan and Gao, 2007) and TLR4 (Lucas and Maes, 2013) ligands. So far there is no evidence that DAMPs are TLRs ligands and again such

promiscuity with self-molecules is in contrast with the role of TLRs. Despite this apparent contradiction, these molecules are mostly segregated in cellular compartment from which they can not interact with TLRs. Localisation of TLRs is indeed an additional mechanism that allows self/non-self discrimination (Barton and Kagan, 2009; Barton et al., 2006) and the release or not of DAMPs more than specificity, would regulate endogenous TLR modulation. Alternatively, α-synuclein, and lysozyme have been shown to activate TLR2 and NLRP3 inflammasome only when folded as cross β-amyloid fibril, structures

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that TLRs had not had to deal with such molecules when developing their specificity and that cellular disorders lead to the interaction of such endogenous molecules with TLRs, with potential links to the pathogenesis of chronic inflammatory diseases, and

autoimmunity (Rifkin et al., 2005). However, similar to putative TLR2 ligand, several works have shown that the TLR-immunostimulatory proprieties of these DAMPs are lost when they are high purified (Bausinger et al., 2002; Erridge and Samani, 2009; Erridge et al., 2007; Gao and Tsan, 2003; Tsan and Gao, 2007), hence attributing all the previous results to contaminations. Nevertheless, a role of TLR on high purified HMGB1, cross β-amyloid, and saturated fatty acids mediated inflammation seems to be established (Bertheloot and Latz, 2017; Erridge, 2010; Gustot et al., 2013) and several hypotheses, some alternative to the one of TLR ligands, are still under investigation. For instance, an unpublished work performed by Prof. M.A. Febbraio (Garvan Institute of Medical Research, Sidney, Australia) and G.I. Lancaster (CMML, Baker Heart and Diabete Institute Melbourne, Australia) (Keystone symposium communication) demonstrates that saturated fatty acids do not activate TLR4 directly but induce an inflammatory response that is modulated by TLR4-induced genes in a sort of cross talk between bacterial infection and lipid

metabolism. In a similar way, unsaturated fatty acids have anti-inflammatory activity, not because they bind MD2/TLR4, but because they block transcription factors of

pro-inflammatory cytokines (Calder, 2005; Pereira et al., 2014; Zhao et al., 2005). 1.2.3 TLR modulations: more than PAMPs competitive binding

Contamination issues are overcome when investigating synthetic molecules. However, there are several ways to block the inflammatory activity of a receptor that do not involve receptor promiscuity: suppression of dimerization by extracellular antibodies or compound that bind the TIR intracellular domain; inhibition of enzyme involved in intracellular

signalling; disruption of protein conformation; covalent binding that suppresses agonist binding.Hence, it should be always pointed out that signalling is a complex network of events and do not involve exclusively receptor/ligand binding; especially for inhibitors, competition test should be used to determine the antagonism of a molecule.

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Green tea polyphenols do not prevent LPS- induced dimerization of TLR4 but inhibit the TLR4 signal downstream by blocking IKKβ and TBK1 kinases (Youn et al., 2006). Sulforaphane, present in cabbages, caffeic acid phenethyl ester, 6- shoagol, the most bioactive component of ginger and chalcone isoliquiritigenin, a component of liquorice, bind covalently the Cys133 in the hydrophobic pocket of MD-2, blocking the interaction with LPS and lipid-IVa (Ahn et al., 2009; Kim et al., 2013b; Koo et al., 2013; Park and Youn, 2010).

Short synthetic peptides corresponding to the minimal TLR4-binding region of MD-2, can replace MD2 in binding TLR4 and inhibit LPS induced TLR4-dependent expression of proinflammatory cytokines both in human and in murine cells (Slivka et al., 2009). In support of competitive TLR2 and TLR4 modulation by non-microbial compound with structures unrelated to PAMPs, several work investigated the TLR-immunomodulatory activity of small hydrophobic molecules (Cheng et al., 2012, 2015; Durai et al., 2017; Guan et al., 2010b; Mistry et al., 2015; Murgueitio et al., 2014; Peri and Calabrese, 2014; Wang et al., 2013). Glycyrrhizin, a triterpene saponin (liquorice) suppresses

TLR4/MD-2-mediated NF-κB and MAPK activation, resulting in decreased production of

proinflammatory cytokines (Kim et al., 2008); it inhibited the formation of the LPS/MD-2/TLR4 complex in an experiment of co-precipitation with biotinylated LPS (Honda et al., 2012). A series of pyrimido[5,4-b]indoles stimulate TLR4 signal in HEK293 reporter cell lines. Interestingly, the activities of these compounds seem to be CD14- independent, as assessed by the capacity to stimulate cytokine production also in CD14- defective

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Some recently studied TLR4-active compounds from natural sources.

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Some recently studied TLR4-active compounds from natural sources.

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simpler, much stronger and approximately equipotent agonists, respectively designated Neoseptin-3 and Neoseptin-4 (Fig. 1B). Neoseptin-3, which induced TNFα production by macrophages in a concentration-dependent manner (Fig. 1C), was selected for more detailed studies.

Further SAR analysis indicated that few chemical substitutions were compatible with retention of biological activity (Fig. S1). Even subtle modifications such as the substitution of fluorine for hydrogen at the para position of the phenyl ring, or transfer of

the amino group of the aniline ring to an adjacent position led to a dramatic loss of activity. However, some of the modified com-pounds could antagonize Neoseptin-3.

In vitro dose–response experiments demonstrated an EC50of

18.5 μM for Neoseptin-3. Despite lower potency, Neoseptin-3 efficacy approximated that of LPS in promoting macrophage TNFα production (Fig. 1C). Neoseptin-3 also activated IL-6 and IFN-β production in a dose-dependent manner (Fig. 1 D and E). Responses to Neoseptin-3 were similar for mouse bone

A B C E D F G

Fig. 1. Neoseptin-3 induces TNFα, IL-6, and IFN-β secretion in different mouse cells. (A) Screen of peptidomimetic molecules (400 wells, 20 compounds per well; ref. 40) for stimulation of TNFα production by mouse peritoneal macrophages, a subset of the full set of compounds examined. (B) Chemical structures of Neoseptin-1, -3, and -4. (C–E) TNFα (C), IL-6 (D), or IFN-β (E) in the supernatants of mouse peritoneal macrophages after treatment with Neoseptin-3 or LPS for 4 h. (F and G) TNFα in the supernatants of mouse BMDM (F) or BMDC (G) after treatment with Neoseptin-3 for 4 h. In C–G, the means of triplicate samples are plotted; P values were determined by Student’s t test and represent the significance of differences between responses of unstimulated cells and stimulated cells. Results in C–G are representative of two independent experiments (error bars represent SEM). *P≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Wang et al. PNAS | Published online February 1, 2016 | E885

IMMUNOLO GY AND INFLAMMA TION PNAS PLUS Figure 4.

Some recently studied TLR4-active compounds from natural sources.

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Some recently studied TLR4-active compounds from natural sources.

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C18:2 cardiolipin

Figure 29: TLR4 modulators suggested as interacting with MD2

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cytokines comparable to LPS in terms of efficacy but in a CD14-independent manner. The crystal shows that two Neoseptin-3 molecules bind MD-2 pocket similar to lipid A despite their completely different chemical structures and induce local conformational changes and a nearly identical dimerization interface between the two mTLR4/MD-2 heterodimers. In particular, the MD-2 Phe126 loop region undergoes a conformational change similar to that observed in the lipid A complexed structure. Surprisingly, the two Neoseptin-3 molecules (Neo-3A and Neo-3b) occupy less than half the total volume of the MD-2 pocket. Nevertheless, the interactions are sufficiently strong and specific to anchor the ligand near the entrance of the hydrophobic pocket of MD-2 for dimerization with the secondary mTLR4. Like lipid A, Neoseptin-3 participates in creating the dimerization interface. The t-butyl group of Neo-3B overlaps with the position of R2′′ chain of lipid A, whereas the two terminal benzene rings from the two Neoseptin-3 molecules overlap with the R3 chains; the Neo-3B phenol ring shows no overlap with lipid A creating a new key contact area between MD-2 and TLR4* through hydrophobic and hydrophilic interaction that are not present in the TLR4/MD-2/lipid A complex (Fig. 30).

Neoseptin-3 binds also human MD-2 but fails both activation and inhibition of human TLR4/MD2. The requirement for binding of two Neoseptin-3 molecules along with an overall smaller molecular size compared with lipid A may account for the failure of