Toll-like receptors and their agonists

Cells of the innate immune system act through the recognition of pathogen associated molecular patterns (PAMPs), which are regions of highly conserved motifs displayed on the surface of most pathogens (Elward and Gasque, 2003; Werling and Jungi, 2003). Upon invasion of a host, these motifs are recognized by pattern recognition receptors (PRRs).

Among these are a class of molecules known as toll-like receptors or TLRs (Kawai and Akira, 2005). These receptors are expressed on a variety of immune cells, such as macrophages, dendritic cells (DCs), B cells, specific T cells and even on some non-immune cells such as fibroblasts and epithelial cells (Akira et al., 2006). A phylogenetic analysis conducted by Purcell et al. (2006) supports the evidence that fish are able to respond to many known mammalian TLR agonists, and also to produce a variety of cytokines in response to stimulation. So far, a total of 11 TLRs have been identified in mammals (Kawai and Akira, 2005; Meylan and Tschopp, 2006), and several more have been found in fish (Meijer et al., 2003; Jault et al., 2004). The TLRs are highly specific, as each responds to different TLR agonists (table 1.2). These may be either live pathogens, or simply isolated PAMPs acting as immunostimulants and activating cells of the immune system. Bricknell and Dalmo (2005) define an immunostimulant as “…a naturally occurring compound that modulates the immune system by increasing the host’s resistance…”. However, synthetic compounds such as imiquimod are known to possess immunostimulatory properties (Hemmi et al., 2002;

Gorden et al., 2005). It is important to note the use of the term “modulate”, as a substance with potential immunostimulatory properties may lead to a down regulation of the immune response if administered in excess amounts (Sakai, 1999).

Table 1.2 – A selection of toll-like receptors and their agonists. Agonists written in italic have been explored in this thesis and will be further described later in the text. Toll-like receptors 3, 7, 8 and 9 all recognize PAMPs in endosomal/lyzosomal compartments, while the rest are expressed on the cell surface.

Properties of the receptor Agonists

TLR 2

The TLR2 receptor is dependent on the formation of dimers with TLR1 or TLR6 (Wetzler, 2003; Bricknell and Dalmo, 2005). Due to this heterodimerization, the TLR2 is able to recognize a great variety of ligands.

Microbial lipopeptide

Binding of agonists to the receptor will induce synthesis of type I interferons (IFNα and IFNβ), which then exert antiviral and immunostimulatory activities (Kaisho,

TLR4 is unique in the TLR family in that it requires the molecule MD-2 in addition to LPS in order to initiate signaling. This molecule is indispensable for TLR4 but will not affect the response to other bacterial

components such as peptidoglycans or CpG DNA (Viriyakosol et al., 2001; Nagai et al., 2002).

LPS

TLR 5

The TLR5 receptor recognizes bacterial flagellin (Hayashi et al., 2001). The receptor is expressed on the basolateral surface of intestinal epithelia, and will therefore activate pro-inflammatory gene expression only if flagellin crosses the epithelium (Gewirtz et al., 2001; Kaisho and Akira, 2002).

Flagellin

TLR 7/8

Along with TLR3 and 9, TLR7 and 8 recognize PAMPs in endosomal/lysozomal compartments, though the natural ligand for the receptors is still not known (Hemmi et al., 2002; Lee et al., 2003; Kaisho, 2005).

Single stranded RNA

Imidazoquinoline compounds - Imiquimod

- Resiquimod

TLR 9

Despite a variety of effects concerning this receptor, they are all dependent on the TLR9-MyD88-mediated pathway (Hemmi et al., 2003).

Bacterial and viral CpG (pDNA)

1.2.1. Signaling pathways of toll-like receptors

The ability of TLRs to recognize different PAMPs comes down to two domains that are characteristic for the TLR family. One is an extracellular domain of leucine-rich repeats, the other is a cytoplasmic Toll-IL-1 receptor (TIR) domain which is similar to that found in the interleukin 1 family (Akira, 2003; Akira and Hemmi, 2003). In order to initiate a signaling pathway, the TLRs depend upon the recruitment of TIR containing adapter proteins such as MyD88, TIRAP, TRIF and TRAM, in addition to the recognized PAMP (Barton and Medzhitov, 2003; O'Neill et al., 2003). These adapters also contain TIR domains and are essential for activation of MAP (mitogen activated protein) kinases and NF-κB (nuclear factor κB) translocation (Medzhitov et al., 1997; Li et al., 2004). This will in turn stimulate gene expression, especially of those that promote immune and inflammatory responses (Baeuerle and Baltimore, 1996; Baldwin Jr, 1996). Figure 1.1 shows how TLR signaling may be divided into MyD88-dependent and MyD88-independent pathways (Yamamoto and Akira, 2005).

Signaling through TLR4 is both MyD88-dependent and independent. For TLR2 and 4, TIRAP activation elicits MyD88 activation downstream of the receptors. Only TLR3 signaling is independent of the MyD88 adapter, and depends instead on the binding of TRIF (Yamamoto et al., 2002; Oshiumi et al., 2003). Recruitment of TRIF activates the MyD88-independent pathway, and leads to activation of NF-κB, and the transcription factor IRF3. IRF3 activation is necessary for induction of type 1 interferons (IFNs), especially IFN-β (Kawai and Akira, 2007). Type 1 IFNs in turn activate genes for production of defense-mediating molecules, such as cytokines (Stark et al., 1998). For TLR4, TRAM participates in activation of the pathway downstream of the receptor. Bonizzi and Karin (2004) also demonstrated a distinction between two different NF-κB pathways, where one is thought to be mostly involved in innate immunity and the other in adaptive immunity. This will not be adressed any further here.

Figure 1.1 – The MyD88-dependent and MyD88-independent pathways and the participation of TIR-domain containing adaptors in eliciting TLR signaling (from Yamamoto and Akira, 2005).

Different endogenous signals can be induced through TLR activation, depending on the type of PAMP that is bound. These signals can be grouped into three categories (Medzhitov and Janeway, 1997):

1. Signals that mediate an inflammatory response. These include type 1 interleukins (ILs), tumor necrosis factor α (TNF-α), IL-6, type 1 interferons and also various chemokines. 2.

Signals functioning as co-stimulators for T-cell activation, though few have been recognized so far. 3. Signals that control the induction of effector functions. These include IL-4, IL-10, IL-5, IL-12 and IFN-γ, and help regulate the differentiation of naïve helper T-cells into either T-helper 1 (T_H1) or T-helper 2 (T_H2). The different signals will be described further in the following sections.

1.3. Cytokines in activation of immune responses