TLR signaling: a link between gut microflora, colorectal inflammation and tumorigenesis

Drug Discovery Today: Disease Mechanisms

DRUG DISCOVERY

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Vol. 8, No. 3–4 2011

Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – University of Rochester Medical Center, Rochester, NY.

DISEASE Mechanisms Underlying Gastrointestinal Cancers MECHANISMS

TLR signaling: a link between gut microflora, colorectal inflammation and tumorigenesis Rebeca Santaolalla1, Daniel A. Sussman1, Maria T. Abreu* Division of Gastroenterology, University of Miami Miller School of Medicine, Miami, FL, USA

A growing body of evidence supports the role of tolllike receptor (TLR) signaling in the intestinal mucosa and its role in inflammation and tumorigenesis.

Section editor: Xiaoxia Li – Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA.

Patients with chronic intestinal inflammation, as is the case with inflammatory bowel disease (IBD), and a subset of patients with inflammatory and sporadic colorectal neoplasia, have increased expression of TLRs, especially TLR4, on colonic epithelial cells. Mouse models of colitis and cancer are useful to understand the role of TLRs and bacteria in the development of colon cancer. Clear differences in bacterial colonization patterns are noted between normal and dysplastic colonic mucosa. TLRs offer a potential prognostic and therapeutic target, serving as the link between bacterial ligands and epithelial inflammation.

Introduction The gastrointestinal tract is in continuous contact with an enormous quantity and variety of microorganisms. This microflora has beneficial immune and metabolic functions, affecting the homeostasis of the epithelium and protecting the mucosa from pathogenic infections. However, many microbes can have deleterious effects, and the intestinal epithelium serves as an effective host protective barrier. The intestinal epithelial cells (IECs) serve in concert with the innate immune system to *Corresponding author: M.T. Abreu ([email protected]) 1 These authors contributed equally to this work. 1740-6765/$ ß 2012 Published by Elsevier Ltd.

DOI: 10.1016/j.ddmec.2012.02.002

shield the mucosa from potential infection. Toll-like receptors (TLRs) are expressed on the colonic and small intestinal epithelium, and function as part of this innate immune response. These receptors are known to recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) that initiate intracellular cell signaling that subsequently activates an inflammatory response and recruits inflammatory cells. Interestingly, in the healthy epithelium, most TLRs are expressed in low amounts creating a degree of immune tolerance to the high concentration of microorganisms that pass through the alimentary tract. Conversely, TLRs are upregulated in intestinal inflammatory diseases and some colonic neoplasias. We will review the current evidence for the role of TLR signaling in the intestinal mucosa and its role in inflammation and tumorigenesis highlighting the importance of this receptor in predicting prognosis or as a therapeutic target in a subset of tumors.

TLRs in the healthy and inflamed gut Commensal bacteria help to maintain IEC homoeostasis and cellular proliferation. For example, mice raised in an environment free of bacteria have reduced IEC proliferation compared with conventional colonized mice [1]. When these germ-free mice are colonized in a gnotobiotic environment with a single bacterial species, IECs undergo global intestinal proliferative transcriptional responses [2–4]. TLRs, members e57

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of a conserved interleukin (IL)-1 superfamily of transmembrane receptors that recognize PAMPs and DAMPs, may be a part of the mechanism by which IECs recognize and subsequently initiate an innate immune response to luminal bacteria. In the healthy gut, expression of most TLRs is observed in antigen-presenting cells, such as dendritic cells or macrophages while TLRs in IECs demonstrate much lower expression. Several members of the TLR family have been identified and their corresponding ligands and cellular locations elucidated (Table 1). In the healthy gut, IECs are exposed to a multitude of antigens from commensal organisms that do not elicit an inflammatory reaction. This observation may be explained in part by the phenomena of tolerance and cross-tolerance, whereby the cell becomes unresponsive to repeated stimulation with the same stimulus or other stimuli, respectively. These findings have been demonstrated with TLRs. For

Table 1. TLRs and their ligands TLR

Ligand

References

1

 Triacylated lipoproteins  Mycobacterial products

[55]

2

 Gram-positive cell-wall components (peptidoglycan, lipoteichoic acid)  Mycobacterial cell-wall components (lipoarabinomannan, mycolyl-arabinogalactan)  Yeast cell-wall (zymosan)  Herpes simplex viruses

[10,56–58]

3

 dsRNA (virus)  Poly I:C

[10,59,60]

4

        

[10,56,61–66]

Pairs with 1 or 6 to recognize PAMPs

Gram-negative lipopolysaccharide (LPS) Heat shock proteins Hyaluronic acid High-mobility group protein 1 (HMGB1) Fatty acids Fibronectin Low density lipoprotein Surfactant protein A b-Defensin

 Flagellin

[10,67]

6

Pairs with 2 to recognize PAMPs

[55]

7

 GU nucleosides  Antiviral guanine analogs  ssRNA viruses

[68–70]

8

 GU nucleosides  ssRNA

[68]

9

 Microbial nucleic acids specifically (unmethylated CpG dinucleotides)  Mitochondrial damage-associated molecular patterns (DAMPs)  Herpes simplex viruses

[58,71,72]

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example, apical activation of TLR9 inhibits the inflammatory response of IECs to basolateral PAMPs, consistent with crosstolerance [5]. Molecules that inhibit TLR signaling can also contribute to tolerance through evasion of TLR activation interacting protein (TOLLIP), an intracellular protein that inhibits TLR2 and TLR4 signaling. Single immunoglobulin IL-1R-related molecule (SIGIRR/TIR8), a negative regulator of TLR4 and TLR9 signaling, induces tolerance to the TLR4 ligand LPS [6–8]. Finally, peroxisome proliferator activated receptor-g (PPARg) is induced by TLR4 signaling and may diminish TLR-mediated inflammation by negatively regulating NF-kB activation [9]. Alternatively, patients with chronic intestinal inflammation, as is the case with inflammatory bowel disease (IBD), have increased expression of TLRs, especially TLR4 and TLR2, on IECs [10,11]. In colon cancers observed in patients with ulcerative colitis (colitis associated cancers or CACs), we have shown increased protein expression of TLR4 [12]. Mouse models of colitis are useful to understand the role of bacteria in the development of CAC. Several rodent models require the presence of bacteria in the intestine to develop dysplasia or cancer, while germ-free rats given carcinogens are protected. Long-term or repeated cycles of dextran-sodium sulfate (DSS) treatment can induce chronic colitis leading to dysplasia and cancer in rodents. As in human IBD, the incidence of neoplastic lesions in this model is associated with the severity of mucosal injury [13,14]. Another experimental method to induce CAC is by administration of DSS and the carcinogen azoxymethane (AOM). TLR4-deficient mice are significantly protected from colorectal neoplasia, and treating mice with an anti-TLR4 antibody protects against inflammation-induced neoplasia [12]. Likewise, mice deficient in a TLR adaptor protein (MyD88) crossed to APCmin/+ mice, an animal model of familial adenomatous polyposis, develop smaller intestinal polyps when compared to APCmin/+ mice [15]. Thus, TLR-signaling in the intestinal mucosa is a double-edged sword – important for epithelial proliferation and repair but promoting neoplasia.

Colitis-associated cancer – role of the intestinal microbiota and TLR signaling

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The clearest link between inflammation and colorectal cancer (CRC) is found in patients with IBD. CACs comprise 5% of all CRC and are the cause of one-sixth and one-twelfth of all deaths in UC and CD patients, respectively. Clinically, the severity of colonic inflammation in IBD patients correlates with the risk of CRC [13,14]. Several groups have already reported an association between TLRs and colorectal neoplasia. In contrast to healthy mucosa, TLR4 is up-regulated in the colonic epithelia of patients with both Crohn’s disease and ulcerative colitis [10]. Additionally, the quantity of TLR4 expression correlates with the degree of colonic dysplasia in ulcerative colitis. Furthermore, mouse models support these findings, as TLR4-deficient mice are significantly protected

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from colorectal neoplasia and treating mice with an anti-TLR4 antibody protects against inflammation-induced neoplasia [16]. A transgenic mouse model expressing a constitutively active TLR4 gene under the control of the villin promoter (villin-TLR4 mice) is highly susceptible to colorectal polyps in response to the AOM and DSS [16]. Moreover, TLR4 promotes the development of CAC by mechanisms including enhanced Cox-2 expression and increased epidermal growth factor receptor (EGFR) signaling; Cox-2 and EGFR are known contributors to CRC [12] (Fig. 1). Furthermore the TLR4-NFkB pathway has been exploited as a target to prevent inflammation and carcinogenesis in a model of CAC [17]. To further support the involvement of TLRs in CRC, Uronis et al. have shown how intestinal bacteria promote tumor progression in IL-10 deficient mice treated with AOM [18].

The authors have taken the conventional model of colitisassociated neoplasia in IL-10 / mice and administered AOM to induce more colonic tumors. Conversely, germ-free IL-10 / mice treated with AOM presented with normal mucosa and an absence of tumors. However, mono-association with Bacteroides vulgatus decreased colitis and tumorigenesis in these mice. This study described that MyD88, a common adaptor molecule in TLR signaling, is essential to induce colorectal tumorigenesis, as conventionalized double knock-out IL10 /  MyD88 / mice showed no tumors after AOM. We have recently shown that TLR4 is over-expressed in human UCassociated dysplasia and cancer, which suggests that these observations in mouse models have relevance to human disease [19]. Conversely, some authors have suggested that there may be an inverse relationship between TLRs and CRC. Salcedo et al.

LPS CD14

TLR4

PGE2 Basolateral Cell membrane

MD2

EP2 or EP4

AR MyD88

EGFR AR Proliferation

NF-κB

MAP Ks

ATF2

mRNA

Cytoplasm

Nucleus

COX2

EGFR Ligands

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Figure 1. TLR4 activates EGFR and COX2 signaling. TLR4 and its cofactors CD14 and MD2 recognize LPS, triggering intracellular signaling through MyD88. Downstream activation of nuclear factor-kB (NF-kB) and mitogen-activated protein kinases (MAPKs) in the cytosol of the IECs lead to the release of EGFR ligands. One of these ligands, amphiregulin (AR), activates EGFR signaling and leads to cell proliferation. TLR4 signaling also induces COX2 expression and PGE2 secretion, which, through the EP2 or EP4 receptors, can further stimulate the expression and release of AR by IECs. Also shown, but not directly involved in activation of EGFR by TLR4, are the sub-cellular locations of other TLRs. COX2: cyclooxigenase 2; EGFR: epidermal growth factor receptor; PGE2: prostaglandin E2; ATF2: activating transcription factor 2. Adapted from: Abreu MT. Nat. Rev. Immunol. 2010;10:131–44.

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suggested that MyD88 signaling prevents development of colon adenocarcinoma in the AOM-DSS model [20]. This particular study did not distinguish whether the effect of MyD88 is mediated by epithelial cells or hematopoietic cells. This observation is in contrast to the finding that the absence of MyD88 prevents CAC when crossed to IL-10 / mice and decreases the size of polyps when crossed to Apcmin/+ mice [15]. Often this study is misinterpreted that the mice were protected against polyposis when in fact the study found that the animals continued to have microadenomas, suggesting that TLR signaling may regulate initiation versus progression of dysplasia. Other TLRs are also likely to play a role in colonic inflammation, tumorigenesis, and/or tumor promotion. TLR2 also seems to play a protective role in tumorigenesis, as TLR2deficient mice have an increase in the number and size of tumors [21]. In addition, TLR5 signaling has been shown to have anti-tumorigenic properties in a tumor xenograft model that used the human colon cancer cell line DLD-1 injected in CD-1 nude mice [22]. Moreover, Vijay-Kumar et al. showed that TLR5 / mice develop spontaneous colitis [23], suggesting a protective role of TLR5. TLRs have been shown to play an important role in inflammation-induced cancer, although there is still much to be elucidated. Bacterial recognition through TLRs is also crucial to maintain intestinal homeostasis, helping in renewal of the epithelium and repair after injury. Depending on the degree of TLR signaling and its context, signaling may protect or induce intestinal tumorigenesis.

Sporadic colorectal cancer, TLRs, and the microbiome Sporadic CRC lacks a known heritable or familial component and occurs in patients without IBD. However, many similarities may be noted between sporadic CRC and CAC. Both malignant endpoints share common molecular pathways, including b-catenin, p53, K-ras, and B-raf [24]. In animal models, the development of both malignancies is influenced by characteristics of the intestinal flora [24]. Inflammation may also be seen on histologic examination of either tumor type, with microsatellite unstable sporadic tumors being infiltrated with lymphocytes [25]. Yet, these distinct tumors are clearly divergent on gross examination, with CACs progressing from aberrant crypt foci (ACF) through varying degrees of dysplasia to produce flat, pre-malignant lesions, while sporadic CRCs typically follow a predictable adenoma to carcinoma sequence with three-dimensional polyps acting as the dysplastic intermediaries between ACF and cancer [24]. Given the prevalence of inflammation noted in certain sporadic CRCs and the high bacterial load presented to colonic epithelial cells, attention has naturally turned to the role of commensal organisms and the microbiome in influencing cancer risk. Indeed, markers of inflammation such as C-reactive protein are elevated in a subset of patients with sporadic CRC and highlight the role of inflammation [26]. In response e60

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to the role of bacteria in human disease, the National Institutes of Health created the Human Microbiome Project. The goal of this venture is to improve health and understand the underlying etiologic processes of disease by monitoring and manipulating this microbiome [27]. Using these molecular approaches to identify organisms has importance as most of the species populating the GI tract cannot be cultivated with current microbiological techniques. Intestinal microbiota may inflict epithelial damage as an intermediary between the host’s dietary choices and the colonic epithelium. For example, colonic bacteria produce short chain fatty acids (SCFAs) from dietary fiber that are used as fuel by IECs [28]. These SCFAs create anti-inflammatory prostaglandins and chemokines within IECs [29], supporting the role for SCFAs or probiotic agents such as Bifidobacteria and Lactobacilli in altering diet-mediated CRC risk [30]. Some early studies have demonstrated intriguing results with respect to elucidating the role of bacterial diversity in colorectal neoplasia. Adenomas, the intermediary dysplastic histology between ACF and sporadic CRC, were found to have higher bacterial diversity and richness than normal control tissues in humans; differences in the quantities of specific flora were also noted, with adenomas having a higher quantity of Proteobacteria and a lower number of Bacterioidetes species than controls [31]. Polyp formation has also been associated with an increased colonization by microbes [32]. This disparity in microbial presence is true not only for adenomatous polyps, but also for sporadic colon cancers, where distinct products of metabolism are observed compared to controls [33]. Clear differences in colonization patterns are noted not only between individuals with neoplasia and controls, but also observed within individuals between the microbiomes colonizing colon tumor tissue and the adjacent non-malignant mucosa [34]. These findings support the study of the tumor microenvironment on CRC disease risk. This distinction in microbial composition among CRC patients is likely to have an impact on mucosal immune responses [35]. Variability in the bacterial flora and in the host’s immune response to these commensal organisms raises the question of how the colonic epithelium interacts with these microbes. Because TLRs recognize bacterial products, there is the possibility that changes in bacterial composition will trigger activation of distinct TLRs. This relationship may also function in the reverse direction, because the presence or absence of TLR molecules in experimental models changes the colonic flora [36,37]. In the case of TLR4, its ligand, lipopolysaccharide (LPS), is a major component of Gram-negative bacteria. Gram-negative bacteria are the most abundant bacteria in the colon. Evidence supporting TLR4 as a mediator in CRC exists not only for CAC, but also for sporadic CRCs. As discussed above, TLRs participate in the immune response to invading pathogens, leading to activation of both the innate and adaptive immune responses. In particular, TLR4 has been associated

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with the process of tumor progression via the NF-kB pathway [38]. Indirect evidence from animal models supports this relationship. The TLR adapter molecule MyD88 is involved in tumor growth in a mouse model of APC-dependent CRC [15]. Blockade of the TLR4 receptor in mice with CRC xenografts decreases the growth of colon tumors [39]; blockade of this receptor complex has also prolonged survival in mice with intestinal tumors [15]. Additionally, silencing TLR4 in models of sporadic CRC decreases metastatic tumor burden [40]. Thus, TLR4 may play a role in promoting the development of a tumor and metastases. It may also play a role in immune surveillance. Findings from animal models of CRC are corroborated by human studies. Specific polymorphisms of toll receptors are also associated with an increased CRC risk and influence prognosis [41,42]. Polymorphisms of TLR3 are independent prognostic markers for stage II CRC [43]. The TLR4/MyD88 co-receptor complex is over-expressed in CRCs compared to the normal and adenomatous colonic epithelium, confirming that this signaling pathway is important in human sporadic CRC [44]. In both murine models and human samples, TLR4 and IL-6 expression in the tumor microenvironment are associated with the presence of adenocarcinoma, and higher levels of TLR4 expression in the tumor stroma are noted with disease progression. TLR4 expression in the stroma of patients with stage 3 CRCs predicts early relapse, suggesting the importance of this marker in predicting prognosis or as a therapeutic target among a subset of tumors [45]. Downstream molecular markers and chemokines associated with TLR4 activation are commonly implicated in sporadic CRC, further suggesting the role for TLRs in colorectal neoplasia. High serum IL-6 levels, a downstream product of NF-kB, are associated with a poor prognosis in patients with CRC [30,33]. NF-kB and IL-6 are both induced by TLRs, and TLR4 in particular [34,35]. Furthermore, it has been suggested that commensal bacteria-induced IEC inflammation and TLR4 activation could result in damage to genomic DNA, chromosomal translocation, and chromosomal instability [46,47].

therapy with LPS has already demonstrated anti-tumor efficacy in murine models. The luminal presence of TLR4 in inflamed intestinal epithelium may also provide an opportune route for cellular drug delivery across the mucosal barrier.

Conclusions A growing body of evidence supports the role of colonic inflammation in carcinogenesis. The complicated interaction between the gut microbiota and colonic epithelial cells is mediated by the innate immune system. TLRs offer a potential prognostic and therapeutic target, serving as the link between bacterial ligands and epithelial inflammation. Current efforts to classify bacterial flora by the Human Microbiome Project and further clarification of the microbiological and inflammatory tumor microenvironments will help us to understand this complex process. Elucidation of these mechanisms should prove useful in identifying novel drug targets and further study of existing modifiers of TLRs.

References 1

2 3 4 5 6 7 8 9

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Therapeutic targets for CRCs The pharmacologic prevention of colon cancer is a burgeoning field. Common anti-inflammatory agents prevent the formation of adenomas and CRCs via the Cox-2 pathway [20–23]. Dietary supplementation with SCFAs has also shown anti-neoplastic promise [46,48]. Several agents have been developed to target the anti-tumor potential of TLRs [22,49,50]. In addition, TLR4-directed therapies show significant promise as targets in the adjuvant treatment of tumors [51]. Rapamycin decreases both TLR4 expression and NF-kB levels which could decrease tumor invasion [52]. Furthermore, TLR4 inhibition by a molecule initially developed as an anti-sepsis agent selectively targets TLR4 signaling and may be exploited for its potential anti-neoplastic and anti-inflammatory properties in the future [53,54]. TLR4-targeted

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