Guardians of the Gut: Newly Appreciated Role of Epithelial Toll-Like Receptors in Protecting the Intestine

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It has long been recognized that, in addition to “learning” to defend the host against pathogens to which it has been exposed, the immune system possesses an innate means by which it can recognize select microbial molecules so as to mount a protective responses to potentially dangerous organisms. A key component of this system is the Toll-like receptors (TLR), which were named based on their homology to the toll protein in Drosophila. TLR recognize a variety of conserved microbial patterns including lipopolysaccharide, peptidoglycan, and flagellin (recognized by TLR-4, -2, and -5 respectively) produced by bacteria and nucleic acids produced by bacteria or viruses (TLR-3, -7, -8, and -9). Much of the research on TLR has been performed on immune cells, especially macrophages and dendritic cells. These cells broadly express a number of TLR, thus permitting them to recognize, and thus protect the host against, a variety of microbes.

Specifically, ligation of TLR on these cells induces expression of a panel of genes that maximize that cell's ability to combat microbes and, furthermore, result in recruitment/activation of other immune cells to the site of infection, especially neutrophils. However, it is now well-appreciated that some TLR are also expressed on intestinal epithelial cells (IEC). Because IEC are normally in close proximity to commensal bacteria, whose basic components are structurally very similar to pathogens, TLR must function somewhat differently to avoid the host being in a constant state of acute inflammation. In the case of TLR-5, localization of the receptor to the basolateral surface of the IEC reduces the potential for activation to instances where bacteria have breached the epithelium.¹, ² In the case of TLR-2, ligation of this receptor on IEC does not cause robust induction of proinflammatory gene expression, but rather causes a rapid increase in IEC barrier function.³ In contrast, some other TLR, including the lipopolysaccharide receptor TLR-4, seem to not be functional on IEC in that they are expressed at low levels and/or lack coreceptors necessary for their function. However, under select proinflammatory conditions, particularly states in which IEC are exposed to interferon (IFN)-γ, TLR-4, and perhaps other TLR, can become functional on these cells.⁴ Because IFN-γ has a variety of effects on multiple cell types, the consequences of IEC TLR signaling that occurs in such scenarios has been difficult to decipher, but seems likely to play a role in states of chronic gut inflammation. However, newly generated mice have made it possible to investigate this question and, consequently, reveal a previously unappreciated role of TLR in IEC.

To study the consequences of IEC TLR-4 activation, which may occur in chronic inflammatory conditions in vivo, Limin Shang et al⁵ generated mice that expressed a constitutively active version of TLR-4 in their IEC. Specifically, they expressed a chimeric protein consisting of CD4 fused to the cytoplasmic region of TLR-4 under the control of the villin promoter. CD4-TLR chimeras have long been observed to function as constitutively active TLRs presumably because of the ability of CD4 to dimerize. Because the intracellular region of TLRs is highly homologous, the phenotype of the transgenic mice sheds light on events that may occur when there is chronic expression and activation of any IEC TLR. That the transgenic mice indeed had evidence of such TLR activation was confirmed by observing increased activation of the proinflammatory transcription factor nuclear factor (NF)-κB, which is activated by most TLRs. Interestingly, such chronic TLR activation in IEC by itself did not result in histopathologic evidence of acute inflammation (ie, neutrophil influx), but rather resulted in a striking increase in both intestinal B lymphocytes and, consequently, fecal immunoglobulin (Ig)A. This phenotype correlated with epithelial production of 2 chemokines, CCL20 and CCL28, that promote B-cell recruitment and the cytokine APRIL that promotes class-switching of B cells to IgA-producing cells. Blocking expression of these chemokines prevented the increase in both B cells and fecal IgA suggesting a pivotal role for these cytokines in the phenotype of these mice (Figure 1). Although not specifically examined in their study, such increased IgA production is likely to provide broadly increased protection against a variety of potentially pathogenic microbes.

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• Figure 1. 

  Chronic epithelial TLR activation promotes expression of fecal IgA. Constitutive activation of epithelial TLR signaling results in NF-κB–mediated production of B-cell chemoattractants, CCL20 and CCL28, that recruit B lymphocytes. TLR signaling may also result in production of APRIL, which promotes B-cell differentiation into IgA-producing plasma cells (PC).

Interestingly, in accordance with this notion, some mouse strains that have been suggested to develop spontaneous colitis owing to excessive exposure to microbial products, secondary to alterations in gut microflora and/or loss of epithelial barrier function have, in fact, been reported to have both substantial elevations in IgA and be resistant to enteric pathogens.⁶, ⁷ Moreover, in humans, increased IgA responses to a variety of antigens correlate well with inflammatory bowel disease. Thus, it seems reasonable to speculate that there may be a potential role for up-regulated epithelial TLR signaling in maintaining the chronic phase of this disorder. Understanding the precise role of IEC TLR signaling in promoting adaptive immunity in the gut remains an important research challenge.

It is now recognized that innate immunity, particularly inflammation, also plays an important role in cancer. Specifically, a number of epidemiologic studies indicate that, depending on the type of cancer and specific underlying causative factors, it is likely that innate immune responses are able to both drive and/or protect against tumor development. To mechanistically understand how innate immunity, and in particular TLR, affect tumor development/progression, a number of recent studies have turned to murine models of cancer.

For example, Medzhitov et al reported that tumor development/progression in APC^Min/+ mice was substantially reduced by genetic ablation of the TLR signaling adaptor protein MyD88,⁸ which is required for signaling by most TLR, supporting the notion that TLR-mediated NF-κB activation promotes cancer, perhaps in large part by preventing apoptosis. However, because causes of cancer are so diverse, it is quite reasonable to predict that different results might be obtained in different models. Indeed, Rhee et al² observed that, in a model system that might provide insights into colon cancer, TLR-5 signaling was protective against tumor development. Specifically, when colon carcinoma-derived DLD-1 cells were subcutaneously implanted in nude mice, blockade of TLR signaling via use of stably expressed MyD88 shRNA in the transplanted cells substantially increased tumor development, particularly the size of the tumors that were formed. Consistent with the fact that TLR-5 is the dominant TLR in most epithelial cell lines, including DLD-1, this effect could be mimicked by shRNA targeting TLR-5. These results suggest that growth of epithelial-derived tumors may normally be retarded by TLR-5 signaling in nascent tumors. Such ablation of MyD88/TLR-5 signaling correlated with reduced production of chemokines, concomitantly reduced neutrophil infiltration to the vicinity of the tumors, and reduced tumor necrosis. Together, these findings indicate that, at least in this instance, any anti-apoptotic effects of TLR-5–mediated NF-κB activation on the tumor-forming cells are more than counteracted by the resulting NF-κB–mediated neutrophil and the subsequent ability of these cells to promote tumor necrosis. It should be noted that inherent difficulties in designing appropriate controls for shRNA-based experiments make it important that similar results are obtained via alternate approaches (eg, neutralizing antibodies). Additionally, the specific nature of the experimental system utilized leaves some questions to be answered; for example, how does TLR-5 become activated in the subcutaneous environment, which one would presume is sterile and thus lacking flagellin? Nonetheless, the findings are very exciting and, indeed, fit well with much of what is known about the function of epithelial TLR-5. Determining how broadly the results apply to other models of epithelial based tumors will be an exciting area of future research.

A potentially very important implication of this work is that one might be able to prevent tumor growth/promote tumor necrosis via administration of exogenous flagellin. Indeed, Pothoulakis et al went on to demonstrate that localized delivery of flagellin in the vicinity of the tumor could retard tumor development that resulted from implantation of DLD-1 cells. Given its broad expression in epithelial cell lines, TLR-5 is likely to be functionally expressed on most carcinomas, suggesting a novel therapeutic paradigm whereby colon cancer can be slowed, in early stages, with flagellin treatment. Such flagellin might also find utility as adjunct therapy to more traditional cancer therapies (chemotherapy and radiation), in part because it likely functions via distinct mechanism and because flagellin has recently been reported to prevent much of the damage to normal tissues that occurs in response to radiation and chemical treatment.⁹, ¹⁰ Thus, perhaps flagellin may both, simultaneously, boost the efficacy of radiation/chemotherapy and prevent adverse events that make these therapies difficult to tolerate. However, it should be noted that, in contrast with radiation/chemotherapy, which retard the progression of a wide variety of tumors, flagellin may actually promote growth of some tumors; Sfondrini et al¹¹ observed that flagellin administered to transplanted mammary tumor cells promoted tumor development in mice. Given the potent effects of flagellin on cell survival, which may promote oncogenesis, and immune cell recruitment, which may promote tumor necrosis, it is perhaps not surprising that flagellin has opposite effects in different cancer models. Thus, any potential development of flagellin, or synthetic TLR-5 ligands for cancer treatment, will need to carefully define the range of tumor types/stages it is effective against as well as define conditions under which it might promote tumor progression (Figure 2). Although defining such conditions will be challenging, given the mortality resulting from epithelial-derived tumors, the potential benefit may be enormous.

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• Figure 2. 

  TLR signaling can promote both survival and tumor necrosis. Activation of TLR-5 in carcinomas may simultaneously (1) promote cancer cell survival via activating NF-κB–mediated anti-apoptotic gene expression and (2) recruit neutrophils that promote tumor necrosis. Whether necrosis or survival prevails will likely depend on the specific characteristics of a particular tumor.

Thus, as shown in this issue of Gastroenterology, gut epithelial TLRs do more than drive acute inflammation in response to breech of the epithelium. Rather, they seem to play a key role in a variety of gut processes including development of the IgA-mediated responses, which have been associated with chronic inflammation and cancer. Consequently, understanding the mechanisms by which such TLRs drive these responses may offer a wealth of strategies to treat a variety of intestinal disorders including infection, IBD, and the toxicity/adverse effects associated with chemical/radiation-based cancer therapy.

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