The present study demonstrates that TLRs are expressed in normal human intestinal mucosa. This same study demonstrates that epithelial cells rather than macrophages and other lamina propria populations are the predominant cells expressing TLRs. Further, this pilot study suggests that there is differential expression of different members of this receptor family. Thus, primary intestinal epithelial cells (IECs) of normal, nondiseased mucosa constitutively express TLR3 and TLR5, whereas TLR2 and TLR4 are present in much lower amounts as assessed by immunohistochemistry.
The intestinal epithelium serves as an essential barrier between microbes of the lumen and inflammatory cells of the lamina propria. It also plays a critical role in regulating the host immune defense reaction by recognizing and subsequently responding to invading pathogens by secretion of proinflammatory cytokines and chemokines (19
). We have recently demonstrated that various intestinal epithelial cell lines constitutively express several TLRs in vitro (5
). This expression is consistent with the emerging consensus that these receptors act as key mediators of host defense to bacterial challenges, linking innate and adaptive immune responses (9
). Thus these receptors appeared to be deployed to the true site of interface with lumenal bacteria and their products, the surface epithelium. The detection of TLRs at the mucosal surface in vivo is consistent with the detection of the TLRs on the apical surface of intestinal epithelial cell lines in vitro (unpublished observation).
Although the various TLRs seem to differ in their recognition of diverse bacterial products, TLRs' induced cell responses may be mediated by a common signaling pathway which shares many features with the IL1R pathway, including the involvement of MyD88, IRAK, TRAF6, and NF-κB. Stimulation of this pathway leads to production of inflammatory cytokines and costimulatory molecules (43
). It is very likely that LPS-induced TLR signals via a similar pathway in IECs, as intestinal epithelial activation of these proteins has recently been shown in response to IL-1β stimulation (2
In addition to the demonstration of TLR expression by normal intestinal epithelium, the present unicenter study suggests that expression of TLRs may be selectively altered in association with IBD and further that some of these alterations may be specific to the form of IBD, whether UC or CD. Thus, TLR3 and TLR4 are differentially modulated in the intestinal epithelium of patients with IBD. While TLR3 expression by IECs of UC patients is comparable to that of normal controls, TLR3 expression is significantly downregulated in CD. Of interest, reduced expression of TLR3 on IECs seems to be consistent in CD, irrespective of location or inflammatory activity. At a minimum, this implies that reduced TLR3 does not simply reflect the local effect of some inflammatory mediator. Inflammatory cells of the lamina propria remain positive for cell surface expression of TLR3, suggesting that the decrease of TLR3 may reflect a distinct intestinal epithelial cell-specific impairment in active CD but not UC. Thus, deficient TLR3 expression in the intestinal epithelium may be a distinctive feature of CD but not UC and could reflect a divergent dimension of pathophysiological mechanisms involved in these two disorders.
It is important to note that the functional role of TLR3 in mediating innate immune responses to specific microbes and their toxic constituents has not yet been definitively established. TLR3 mRNA may be significantly downregulated in response to LPS in mature dendritic cells (26
). In contrast, preliminary studies from this laboratory reveal that TLR3 protein is significantly upregulated in IECs in response to LPS in vitro (unpublished observation). Others have also recently demonstrated that expression of TLR3 mRNA may be upregulated by LPS and also tumor necrosis factor alpha in mature Langerhans cells (12
). Collectively, these divergent findings suggest that TLR3 can mediate cell-specific responses to LPS (26
). Further studies are needed to clarify the functional role of TLR3 as a specific pattern recognition receptor, its interaction with other receptor molecules, its regulation by cytokines in the intestinal epithelium as well as inflammatory cells, and finally, its causal relevance in the differential pathogenesis of inflammatory bowel diseases.
Interestingly, TLR3 is localized on chromosome 4 (q35) (33
) at the border of a large linkage region of a recently described IBD susceptibility gene, suggesting a potential pathogenic association of IBD with the TLR3 gene (14
). Thorough assessments of novel mutant polymorphisms in the TLR3 gene may provide insight into IEC-specific dysregulation of the receptor in CD.
In contrast to TLR3, TLR4 is significantly increased in IECs throughout the lower gastrointestinal tract regardless of whether assessed in active or inactive disease of both CD and UC patients. However, the subcellular distribution of TLR4 differed between CD and UC epithelia (apical versus basolateral). Recent findings from animal models and genetic complementation studies have suggested that TLR4 can serve as a major transducing subunit of the LPS receptor complex (31
). Lumenal LPS is usually well tolerated in large quantities within the healthy intestine. This tolerance could result from TLR4 downregulation minimizing LPS recognition, given that primary IECs in normal tissue appear to express very little, if any, TLR4 (28
). However, in IBD, host tolerance towards lumenal bacterial toxins may be broken (8
), which could reflect increased LPS recognition as a result of TLR4 upregulation. Acute injury of the intestinal mucosa may also lead to recruitment of TLR4-positive macrophages into the mucosa. These inflammatory cells highly express the TLR4 coreceptor CD14, which could play an important linking role in enhancing hyperresponsiveness of the intestinal mucosa to LPS in IBD (1
Spontaneous mutations of TLR4 could prime individuals experiencing acute infections to develop especially severe disease (3
). It has previously been shown that C3H/HeJ mice which have a single point mutation of TLR4 (16
) are highly susceptible to developing a more severe form of dextran sodium sulfate-induced colitis (9
). Interestingly, the TLR4 gene is localized on chromosome 9 (q32-33) (33
), another genomic region in which a CD susceptibility gene has been implicated (6
). In active IBD, variant alleles in the TLR4 gene could induce functional dysregulation of the receptor to LPS. In this study, we also found that during long-standing, quiescent disease, IECs constitutively overexpress TLR4 compared to normal controls. This observation could result from a “gain-of-function” mutation in this receptor which could functionally exhibit proinflammatory effects in response to physiological concentrations of LPS. However, it remains to be shown whether upregulated TLR4 confers functional hyperresponsiveness of the intestinal epithelium to LPS or rather reflects a loss of response. Moreover, TLR4 upregulation could also result from the effects of ligands other than LPS (20
). The factors and mechanisms regulating TLR4 expression in IECs in IBD remain to be further elucidated.
Our study suggests that TLR2 and TLR5 expression in IECs remain unchanged in active IBD. Neither control nor IBD tissues exhibit significant TLR2 expression in IECs. Upregulation of TLR2 is restricted to scattered inflammatory cells of the lamina propria in active IBD. While TLR4 appears to be important for LPS signaling, recent in vitro studies suggest that TLR2 mainly transduces signals by gram-positive ligands such as lipoteichoic acid, peptidoglycan, and lipopeptides (22
). The lack of any significant alteration in intestinal epithelial expression of TLR2 in IBD suggests that such bacterial cell wall components of gram-positive microbes may not play a major role in modulation of innate immune responses in these disorders.
Similar to TLR2, TLR5 also appears not to be significantly regulated in acute intestinal inflammation in IBD. TLR5 is constitutively expressed on all surface IECs, regardless of whether derived from normal or IBD mucosae. However, the functional role of TLR5 in the gastrointestinal immune system needs to be further defined.
This is the first report suggesting that TLR expression may be altered in disease. However, the conclusiveness of this preliminary report is limited by the fact that it has been performed in a unicenter setting. It is evident that larger multicenter studies are needed to further specify differences in TLR expression between these entities of IBD and, more importantly, other inflammatory diseases of the gastrointestinal tract. So far only 10 members of the TLR superfamily have been identified. It is expected that more than 30 different TLRs are expressed in mammals; hence, at this point we cannot exclude the possibility that our newly generated antibodies might cross-react with any other, so far unknown TLR which might show homologies in the extracellular domains with TLR2, TLR3, or TLR4.
Based on the results of this initial study, we note that epithelial cells may be the predominant site of TLR expression in intestinal mucosa and postulate that IBD may be associated with distinctive changes in selective TLR expression in the intestinal epithelium. However, it remains unclear whether immune imbalance in IBD may either lead to or result from TLR dysregulation in IEC. Further studies are needed to focus on the direct pathogenetic relevance and immune consequences of TLR dysregulation in active IBD.