Toll-like receptors

Toll-like receptors (TLRs) play a pivotal role in host defence, as they recognize microbial ligands and are members of the group of PRRs. As already mentioned, 11 mammalian TLRs have been identified. Characteristically they show three common structural features: a divergent ligand-binding extracellular domain with leucin-rich repeats, a short trans-membrane region, and a highly homologous cytoplasmatic toll / interleukin-1 receptor domain, which is essential for the initiation of signalling cascades (AKIRA 2003).

TLRs have been detected on various cell types. In our study we want to put the focus on the expression of TLRs on the epithelium of the esophagus. In the literature, several authors discussed the occurrence of TLRs in a number of organs. Most of the studies conducted concentrate on TLR expression by human intestinal cells, as many authors are of the opinion that TLRs play an important role in the pathogenesis of inflammatory bowel disease (RAKOFF-NAHOUM et al. 2004; VORA et al. 2004;

CARIO and PODOLSKY 2006). Furthermore, the existence of TLRs on human keratinocytes (KOELLISCH et al. 2005; NAGY et al. 2005; SUMIKAWA et al. 2006;

BUECHAU et al. 2008), the cornea (KUMAR et al. 2006), lung epithelial cells (BIRCHLER et al. 2001; DROEMANN et al. 2003) and vaginal epithelial cells has been described (PIVARCSI et al. 2005).

The function of the TLRs as PRRs results in an activation of defence mechanisms of the cell. As already mentioned previously, one of the first line defence mechanisms is the production of antimicrobial peptides. Several authors assumed a correlation for the expression of TLRs and the production of antimicrobial peptides (APs) by the cell (BIRCHLER et al. 2001; VORA et al. 2004; SUMIKAWA et al. 2006; BUECHAU et al.

2008). BIRCHLER et al. (2001) were the first to mention that TLRs mediate induction of the synthesis of APs. They found out that human lung epithelial cells constitutively express TLR2 and produce ß-defensin 2 in response to bacterial lipoprotein.

Subsequently many studies on different epithelial cell types followed.

In the intestine, triggering of TLRs is mainly caused by commensal bacteria, and the crucial function of the TLRs in the intestine is to provide epithelial homeostasis and integrity (HOOPER et al. 2001; RAKOFF-NAHOUM et al. 2004).

Not all of the 12 different TLRs seem to be activated under normal steady-state conditions; moreover TLR2 and TLR4 are in the focus of attention and seem to contribute to the production of APs (VORA et al. 2004). TLR2 is required for the

recognition of bacterial lipopeptide, and, in combination with TLR6, for the recognition of peptidoglycan and lipoteichoic acid, which are components of Gram-positive bacteria (TAKEDA et al. 2003). Additionally, TLR2 is able to detect the yeast cell wall component zymosan (AKIRA 2003). TLR4 mediates the recognition of lipopolysaccharides (LPS), found on the outer membrane of Gram-negative bacteria (AKIRA 2003).

In order to connect the activation of TLRs and the resulting production of APs, signalling pathways are necessary. Signals from TLRs can be mediated into the cell via different pathways. The nuclear factor kappa-B (NF-κB) pathway plays the key role in this manner (VORA et al. 2004). NF-κB is a transcription factor and possesses an important role in immunity. It is localised in the cell cytoplasm and is hold in its inactive form through its association with the unphosphorylated IκB proteins, thus NF-κB cannot move to the nucleus or activate genes. One way to activate the transcription factor is via triggering TLRs with PAMPs. This results in an alteration of the shape of the TLR. Subsequently the TLR binds several adaptor molecules, of which the myeloid differentiation primary response gene 88 (MyD88) is the most important one; following various kinases are activated. Through their activation in the last step, the IκB protein is phosphorylated leading to its destruction and the release of the active NF-κB (O'NEILL 2006).

TLR activation can be a potent stimulus of AP production, but also for the synthesis of other factors, such as interleukin 6 or tumor necrosis factor. These substances are involved in cytoprotection and tissue repair in the intestine (RAKOFF-NAHOUM et al.

2004) and other organs, for example in the lung (WARD et al. 2000).

As already mentioned previously, correlation between the occurrence of TLRs on an epithelium and the resulting production of APs has not only been described for intestinal epithelium, but also for other organs. The expression of TLR2 and a resulting production of APs has additionally been observed in human keratinocytes of the skin (KOELLISCH et al. 2005; NAGY et al. 2005; SUMIKAWA et al. 2006;

BUECHAU et al. 2008), in lung epithelial cells (BIRCHLER et al. 2001), in vaginal epithelial cells (PIVARCSI et al. 2005) and also in corneal epithelial cells (KUMAR et al. 2006). Not all APs seem to be triggered by the activation of TLRs, as from most studies conducted can be concluded that mainly ß-defensin production is upregulated by the activation of TLR2 or TLR4 (BIRCHLER et al. 2001; VORA et al.

2004; NAGY et al. 2005; KUMAR et al. 2006; SUMIKAWA et al. 2006). Concerning intestinal epithelial cells, MUKHERJEE et al. (2008) mentioned that hBD-2 is normally low in cultured epithelial cells and can be induced in response to microbial influence in a TLR2 dependent manner.

Only for the skin a relationship between TLR2 expression and enhanced cathelicidin production was shown (BUECHAU et al. 2008).

Most of the observations mentioned beforehand have been obtained from human epithelial cells, whereas publications about TLRs expression and their functional relationship to APs for animals are sparse. For the dog, TLR expression has been reported in different uninfected tissues. Thus, TLR4 was detected in the epithelium of the lung, small intestine, cornea and renal tubules by performing immunohistochemistry (WASSEF et al. 2004). mRNA of TLR4 was found in peripheral blood leukocytes (PBL), in the spleen, stomach and small intestine, and was moderately expressed in the liver. However, it was not detected in the kidney, large intestine and skin (ASAHINA et al. 2003). Applying polymerase chain reaction (PCR), mRNA of TLR2 was found in blood monocytes, lymph nodes, lung, liver, spleen, bladder, pancreas, small intestine and skin of the dog (ISHII et al. 2006), and also in canine heart tissue (LINDE et al. 2007). Intriguingly in one study it was mentioned that a dysregulation of TLR2 and TLR4 may contribute to the pathogenesis of inflammatory bowel disease (IBD) in dogs, which is characterised by a chronic inflammation of the small intestine (SWERDLOW et al. 2006).

TLR9 occurrence in canine tissue has been maintained by real-time PCR for lymph nodes and spleen (HASHIMOTO et al. 2005). The wide spread of TLRs in the dog might also indicate the occurrence of APs in the mentioned tissues (LINDE et al.

2007).

Descriptions about feline TLRs are made in context with the feline immunodeficiency virus (FIV), and the authors primarily mention TLR expression on lymphocytes (IGNACIO et al. 2005). Additionally, mRNA of feline TLR4 was highly expressed in lung, bladder and peripheral blood monocytes, moderately in kidney, liver, spleen and large intestine, and with low levels in pancreas and small intestine (ASAHINA et al. 2003).

Selected bovine and ovine tissues have been tested for the occurrence of various TLRs. As a result, quantitative real-time PCR confirmed expression of 10 TLRs within the ovine jejunum, Peyers patches and lymph nodes. Whereas TLRs 3, 5 and 6 were abundant in the jejunum, all TLRs had been detected in bovine skin; here TLRs 2 and 7 were most abundant (MENZIES and INGHAM 2006).

In cattle, a relationship between ß-defensin 5 production and TLR2 and TLR4 expression in mastitis infected mammary glands has been observed (GOLDAMMER et al. 2004).

Occurrence of TLR2 in pigs has been verified for epithelial cells lining the tracheobronchial and intestinal tracts, bile ducts in the liver and renal tubules.

Additionally it was found in the basal cell layer of the epidermis (ALVAREZ et al.

2008). Furthermore, TLR4 mRNA has been detected in tissue samples of the bone marrow, thymus, lymph node, spleen, brain, liver, kidney and ovary (ALVAREZ et al.

2006).

Information about the expression of TLRs in equine tissue is scarce, although TLR9 was detected in the spleen and lymph nodes (ZHANG et al. 2008). Furthermore, tissue expression profiles showed that TLR3 was highly expressed in the kidney, duodenum, spleen and liver, and moderately expressed in bone marrow, lung, and skin (SANG et al. 2008).

In summary, TLR signalling pathways might be involved in commensal-induced antimicrobial peptide production, therefore helping to prevent pathogenic bacteria from break through the epithelial barrier. TLR distribution is consistent with a surveillance function at entry sites, allowing early detection of microbial invasion.