Toll-like receptor 2

Toll-like receptors (TLRs) comprise a class of transmembrane pattern recognition receptors that play a key role in microbial recognition, induction of antimicrobial genes and control of adaptive immune responses (JANEWAY and MEDZHITOV 2002). Eleven different TLRs have been identified in mice (LOTZ et al. 2007) and ten in humans (AKIRA 2003; JANEWAY et al. 2005). One aim of this study was to provide the first proof of TLR expression in epithelial cells of the esophagus of the species studied. After an analysis of the literature available, we decided to put our

focus on TLR2, as several authors emphasised the occurrence of this TLR on intestinal epithelial cells of the human and murine gut (CARIO and PODOLSKY 2006; CARIO et al. 2007; TIZARD 2008), and on epidermal keratinocytes (KOELLISCH et al. 2005; LEBRE et al. 2007). For the intestine it was demonstrated that TLRs are involved in tissue regeneration and inflammation, as well as in controlling the integrity of the intestinal epithelial barrier (RAKOFF-NAHOUM et al. 2004; CARIO and PODOLSKY 2006; CARIO et al. 2007).

Regarding the skin, LEBRE et al. (2007) showed that TLRs actively contribute to the induction of an immune response.

In this dissertation thesis the existence of TLR2 on epithelial cells of the esophagus was verified by IHC. Thus, we provide the first comprehensive evidence for the expression of the latter TLR in the nine different species studied.

So far no information concerning the expression of TLRs in the esophagus epithelium was available from other mammals including humans. In our study we were able to demonstrate the existence of TLR2 in all species, which indicates a constitutive expression of TLR2. These observations are in line with the findings of CARIO et al. (2007) who emphasised the functional relevance of TLR2 to control tight junction associated intestinal barrier integrity and to balance mucosal homeostasis. Furthermore, a constitutive expression of TLR2 was observed on human keratinocytes in the skin (LEBRE et al. 2007). A constitutive expression of TLRs in cells creating an epithelial barrier against invading microorganisms is comprehensible as these PRRs enable the organism to continuously screen the microbial composition, readily recognize potential imbalances and rapidly initiate innate immune responses.

In regard to the specific locations of TLR expression in the esophagus epithelium, it could be observed in all species that the staining intensity of TLR2 was very high in the stratum basale and decreased towards the stratum spinosum. The cells of the stratum granulosum and the stratum corneum showed no positive reaction.

Only in the porcine esophagus a positive staining was observed in the stratum granulosum. It appears logical that the stratum corneum cells do not express TLR2, as it can be assumed that such cells do not possess the full ability to respond, i.e. by the production of antimicrobial products. The fairly isolated

expression of TLR2 in the basal part of the epithelium may have a functional background. It is conjecturable that TLR expression at this site of the esophagus epithelium is correlated with high production of APs in the stratum basale (compare 5.3.1). Several authors described a functional relation between the expression and activation of TLRs and the inducible production of APs via a TLR dependent pathway (BIRCHLER et al. 2001; VORA et al. 2004; SUMIKAWA et al.

2006; BUECHAU et al. 2008; LINDE et al. 2008). The findings of the latter authors correspond with our results. It was only possible within the scope of this study to demonstrate a co-expression of TLR2 and APs, leaving the evaluation of the direct functional relation of both to further studies. However, we could not accurately differentiate which AP is induced due to a TLR2 response. BIRCHLER et al.

(2001) argued that only hBD-2 was induced via a TLR2 dependent pathway, whilst hBD-3 was not inducible via TLR2. Later, the findings of BIRCHLER et al. (2001) were confirmed for intestinal and epidermal cells (VORA et al. 2004; KUMAR et al.

2006; MUKHERJEE et al. 2008). To our knowledge, only SUMIKAWA et al. (2006) demonstrated a TLR2 dependent hBD-3 production in human keratinocytes.

Nevertheless, a TLR2 dependent expression of CAT was only demonstrated for the skin (BUECHAU et al. 2008). It is an interesting finding that TLR2, hBD2 and -3 and CAT expression was shown for the stratum granulosum of the porcine esophagus. This feature might indicate a TLR2 triggered production of the latter APs in the stratum granulosum. Further studies should concentrate on the expression of TLR4 in the esophagus epithelium, as this TLR is considered to participate in the surveillance of the intestinal tract, and co-expression with APs has already been demonstrated (CARIO et al. 2000; CARIO et al. 2002; VORA et al. 2004).

Functions of TLR2 in the esophagus epithelium

Besides being responsible for triggering an innate immune response, i.e. induction of AP production, TLRs are thought to fulfil further tasks to help maintain epithelial homeostasis.

RAKOFF-NAHOUM et al. (2004) demonstrated the functions of TLR2 by simulating intestinal injury and inflammation in TLR2 and MyD88 (TLR signalling protein) knock-out mice. First, they showed a dysregulation of proliferation and

differentiation in the intestinal epithelium in the absence of TLR signals. TLR absence resulted in an increased number of proliferating cells. Secondly, the authors demonstrated a decreased production of cytoprotective and reparative factors (IL-6 and TNF) in such knock-out mice. IL-6 and TNF play a pivotal role in protecting the epithelium from injury and in the initiation of repair responses.

Moreover, it was shown that the production of such cytokines is dependent on the presence of commensals, which stimulate TLRs. It can be assumed that the loss of commensal-mediated cytoprotection of the intestinal barrier, due to TLR2 / MyD88 dysfunction, results in the destruction of epithelial homeostasis. After the breakdown of epithelial integrity normally innocuous bacteria, such as Escherichia coli, can cross the Tunica mucosa, invade the blood stream, and cause fatal systemic infection (JANEWAY et al. 2005). Besides functioning as PRRs, TLR2 might also act as regulatory feature of the epithelial integrity in the esophagus epithelium, i.e. in case of injury. This could be one explanation for our finding that herbivorous mammals revealed stronger reaction intensities to TLR2, in comparison to the omnivorous and carnivorous species studied. The epithelium of the herbivorous species is exposed to higher mechanical stress than that of the other two nutrition groups. It remains to be elucidated in future experiments, if IL-6 and TNF are also present in the esophagus epithelium of the animal species studied, in order to support or refute the latter theory.

Since TLRs play a pivotal role in innate immunity and are the front liners in pathogen recognition, we hypothesised that TLRs are also expressed in the esophagus epithelium. Furthermore, we aimed to reveal whether a co-existence with APs occurs, as previously shown for other epithelial surfaces. After demonstrating a colonisation of the esophagus surface with microorganisms by cryo SEM, we had strong evidence that PRRs are in fact necessary in the esophagus epithelium. In this context we were clearly able to demonstrate TLR2 expression in the nine species studied. Moreover, we can support our hypothesis of a simultaneous expression of TLR2 and the APs hBD-2, -3, and CAT. It has to be taken into account that TLR2 is stimulated by the commensal bacteria and induces the production of APs by the epithelial cells. Furthermore, the regulation of antimicrobial responses by TLR2 activation could prevent over production of antimicrobial proteins that could interfere with intestinal homeostasis. More

specific information about the functional relationship of TLR2 and the APs has to be gained from future experiments, also meaning that the signal pathway of TLR2 (e.g. NF-κB) in the epithelial cells of the esophagus has to be studied in more detail.