|Home | About | Journals | Submit | Contact Us | Français|
Recognition of microorganisms by pattern recognition receptors (PRRs) is the primary component of innate immunity that is responsible for the maintenance of host-microbial interactions in intestinal mucosa. Disregulation in host-commensal interactions has been implicated as the central pathogenesis of inflammatory bowel disease (IBD), which predisposes to developing colorectal cancer. Recent animal studies have begun to outline some unique physiology and pathology involving each PRR signaling in the intestine. The major roles played by PRRs in the gut appear to be regulation of the number and the composition of commensal bacteria, epithelial proliferation and mucosal permiability in response to epithelial injury. In addition, PRR signaling in lamina propria immune cells may be involved in induction of inflammation in response to invasion of pathogens. Because some PRR-deficient mice have shown variable susceptibility to colitis, the outcome of intestinal inflammation may be modified depending on PRR signaling in epithelial cells, immune cells, and the composition of commensal flora. Through recent findings in animal models of IBD, this review will discuss how abnormal PRR signaling may contribute to the pathogenesis of inflammation and inflammation-associated tumorigenesis in the intestine.
The innate immunity provides a primary host response to microbial invasion, which induces an inflammatory nidus to localize the infection and prevent systemic dissemination of pathogens. The key process in this is the recognition of microbial agents by PRRs. The PRRs include Toll-like receptors (TLRs), Nucleotide binding oligomerization domain (NOD)-like receptors (NLRs), RNA helicases (RIG-I, MDA5, and LGP2), C-type lectin Receptors, and cytosolic DNA sensors (DAI, AIM-2, LRRFIP1, RNA polymerase III, DExD/H box RNA helicases, and IFI16), which sense evolutionarily conserved pathogen-associated molecular patterns (PAMPs) of microorganisms (1). By detecting PAMPs, PRRs trigger sequential activation of intracellular signaling pathways leading to induction of a range of cytokines and chemokines that orchestrate the early host resistance to infection. Specifically, activation of NLRs results in the formation of a molecular scaffold complex (an inflammasome) that leads to the active release of IL-1β and IL-18 through caspase-1 activation (Figure 1). These PRRs signaling also initiate the differentiation of T cells and B cells to establish antigen-specific adaptive immunity.
Since the discovery of TLRs as a major family of PRRs, it has been of great interest whether or not they are functionally expressed in intestinal epithelial interface and what roles they play in the gastrointestinal tract. Because of the unique nature of the gut where diverse microorganisms coexist, microbial-sensing TLRs may have special roles in mucosal homeostasis. Among the thirteen TLRs discovered, TLR1 through TLR9 have been identified as being expressed in human IECs (2, 3). However, the functional consequences of these TLRs in healthy gut physiology have yet to be fully determined.
Although in vivo TLR responses are still uncertain at the epithelial surface of the gut, in vitro data has demonstrated hyporesponsiveness of IECs to TLR ligands (2, 3). The underlying mechanism of this observation comprises a decrease in TLR surface expression and the induction of an inhibitory molecule of TLR signaling after ligand stimulation. Antigen-presenting cells in the lamina propria also appear to be unresponsive to TLR ligands (4). Other TLRs are normally expressed in endosomes (TLR3, TLR7 to TLR9) or basolateral membrane (TLR5), where these TLRs are not exposed to pathogens unless pathogens get into the cells or invade mucosa. NOD and NLRs are also expressed in the cytoplasm and thus do not recognize extracellular pathogens unless pathogens inject the cells effector proteins (Table 1). These findings highlight a unique feature of PRRs in IECs that establishes tolerance to the commensal flora at the mucosal interface.
In addition to being hyporesponsive, epithelial PRRs contribute to balancing the composition of luminal microorganisms by regulating the secretion of a range of antimicrobial peptides and mucosal IgA. Mice deficient in MyD88 have demonstrated a significant defect in production of multiple antimicrobial peptides in Paneth cells resulting in increased bacterial penetration to the mesenteric lymph nodes (5). TLR9−/− and NOD2−/− mice have impaired expression of Paneth cell cryptdin (mouse α-defensin) compared to WT mice (6, 7). Patients with Crohn’s disease (a chronic intestinal inflammatory condition) who carry NOD2 mutations have defective production of α-defensins (HD5, HD6) by Paneth cells along with an increased abundance of mucosa-associated bacteria (8, 9). In addition, signaling through TLR2, TLR3, TLR4, NOD1, NOD2 and NLR Protein 3 (NLRP3) have all been implicated with the expression of β-defensins in IECs (10, 11). Most TLR signaling in IECs induces B cell-activating factors that lead to immunoglobulin class switch recombination in lamina propria B cells without T cell activation, resulting in IgA secretion (12, 13). In addition, activation of TLR3 and TLR4 has been shown to induce the expression of polymeric immunoglobulin receptor (pIgR), an epithelial immunoglobulin transporter, by IECs that enhances luminal IgA secretion (14, 15). Therefore, TLR signaling in IECs is involved in multiple steps in intestinal IgA secretion. The antimicrobial peptides and the secretory IgA balance microbial composition limitting penetration of commensal bacteria in the intestine (16). Therefore, regulation of commensal bacterial burden and composition is likely to be a dominant function of PRRs to maintain intestinal homeostasis.
Targeted PRR gene knockout mice have provided important information regarding intestinal phenotypes of individual PRRs (Table 2). Despite their importance in regulation of commensal flora, only mice that are deficient either in TLR5, NLRP6, or retinoid acid-inducible gene-I (RIG-I) have shown to develop spontaneous intestinal inflammation (17–19). The hyporesponsive nature of PRRs at the intestinal host-microbe interface may allow absence of PRR signaling to keep normal intestinal integrity. Nevertheless, the lack of spontaneous phenotypes in most PRR-deficient mice suggests the possible involvement of compensatory mechanisms. Since many pathogens carry multiple PAMPs, a pathogen can be redundantly recognized by multiple PRRs within a single cell of the host. Because PRRs share several key innate immune signaling pathways, intestinal immune homeostasis may be preserved by providing a sufficient host response to commensal microorganisms in most PRR-deficient mice (Figure 2). However, many PRR-deficient mice develop more severe intestinal inflammation than WT mice in the setting of epithelial injury or abnormal cytokine environments such as the absence of IL-10, highlighting the requirement of proper PRR responses for danger signaling during pathologic conditions.
The role of each TLR signaling in the development of intestinal inflammation may vary depending on the cell types in mucosa and interactions between individual TLR signaling. The spontaneous intestinal inflammation in TLR5−/− mice implies a distinct role of TLR5 in maintenance of mucosal immune response to commensal bacteria. Induction of colitis in TLR5−/− mice is inhibited by cross breeding these mice with TLR4−/− mice indicating that the mucosal inflammation is TLR4-dependent without TLR5 regulation (17). It is still unclear which cell types in intestinal mucosa are responsible for the TLR4-mediated induction of colitis. However, epithelial TLR4 signaling is unlikely to be involved because constitutively active expression of epithelial TLR4 does not induce mucosal inflammation in the villin-TLR4 transgenic mice (13, 20). Though, selective deletion of MyD88 in IECs results in spontaneous inflammation of the small intestine (21), MyD88 signaling in myeloid cells is required for the development of intestinal inflammation during colonization of RAG2−/− mice with Helicobacter hepaticus (22). Consistently, TLR4 signaling in myeloid cells has been implicated with mucosal inflammation during enteric Citrobacter rodentium infection (23). Therefore, TLR signaling in the myeloid cell compartment plays more significant roles than epithelial TLR signaling in induction of mucosal inflammation in the intestine.
Dysbiosis of intestinal microbiota has been implicated as a prerequisite for development of IBD (24). Given the importance of mucosal PRR signaling in the maintenance of commensal flora in the normal intestine, PRR deficiency may alter the composition of commensal flora that may be involved in the pathogenesis of colitis. The colitic TLR5−/− mice show increased intestinal bacterial burden and variability of commensal flora compared to non-colitic TLR5−/− mice (17). An increase of the Enterobacteriaceae composition has been shown during chemically induced colitis in mice, which correlates with the severity of intestinal inflammation (25). The extent of this shift in commensal composition during colitis is less in TLR2/TLR4 double deficinet mice than WT mice (25). In addition, the TLR2/TLR4 double deficinet mice have significantly less burdens of Bacteroides, Prevotella, and Enterococcous species in the intestine compared to WT mice (25). IL-10−/− deficient mice is a well studied model of colitis as they develop spontaneous colitis due to uncontrolled pro-inflammatory cytokine production in response to commensal flora (26). In fact, different types of commensal bacteria cause intestinal inflammation with different onset, location, and degree in IL-10−/− mice (27). Interestingly, nullifying MyD88 in IL-10−/− mice results in the prevention of colitis induction (28). There are conflicting reports regarding inhibition or acceleration of colitis in TLR4/IL-10 double knockout mice, which may be explained by differences in flora across various facilities (29–31). Cross breeding IL-10−/− mice with TLR2−/− mice have shown exacerbation of colitis, but crossing IL-10−/− mice with TLR9−/− mice did not alter intestinal phenotype of IL-10−/− mice despite both TLR2 and TLR9 signal through MyD88 (29, 32). Nullifying MyD88 in another commensal dependent spontaneous colitis model, IL-2−/− mice show similar severity of colitis as their MyD88-sufficient IL-2−/− counterpart (28). Interestingly, treatment of TLR4/TLR5 double knockout mice with IL-10 receptor neutralising antibody (IL-10R mAb) results in development of colitis but IL-1R-deficient TLR5−/− mice does not show signs of colitis in response to IL-10R mAb treatment (33). Because IL-1R shares MyD88 signaling with other TLRs, these results indicate that IL-1 and MyD88 signaling may play a role for induction of colitis seen in TLR5−/− mice although in another study IL-1R−/− mice displayed worse colitis induced by dextran sulfate sodium (DSS) (34). These conflicting results suggest that the role of MyD88 in comensal dependent colitis may differ depending on its upstream inputs and cytokine profile.
Demand for PRR signaling becomes higher in the setting of epithelial damage than homeostatic conditions in the gut. Regardless of the cause, disruption of the epithelial barrier integrity leads to exposure of mucosal immune system to the mass of commensals. During epithelial damage, PRRs also recognize damage associated molecular patterns that are released from host tissues (35, 36). Mouse DSS-induced colitis is a well-established colitis model specifically relevant to investigate intestinal innate immune responses to epithelial injury as this model is induced by chemical damage of the epithelium and does not require adaptive immunity (37). In the DSS induced colitis, it has been shown that germ free mice demonstrate worse colitis compared to conventional mice, but similar sivirlity of colitis has been noted between conventional mice and the commensal-depleted mice that are orally supplemented with TLR2 and TLR4 ligands (38, 39). Consistently, TLR2−/− and TLR4−/− mice show increased severity of DSS-induced colitis (39, 40). MyD88 but not TIR-domain-containing adapter-inducing interferon-β (TRIF) deficient mice also demonstrated more severe disease than WT mice in this model suggesting the importance of MyD88-dependent TLR signaling in protection against mucosal damage (40, 41). However, the underlying mechanisms of protection differ between TLR2 and TLR4. TLR2 signaling in IECs induces tight junctional protein ZO-1 through activation of protein kinase C, which strengthen epithelial barrier integrity and resists cell apoptosis (42, 43). TLR2 signaling also induces cytoprotective trefoil factor (TFF3) production (44). By contrast, TLR4 signaling in IECs and subepithelial macrophages induces COX-2 that leads to PGE2 synthesis in IECs (45). PGE2 production supports epithelial survival and antagonizes apoptotic signaling. TLR4 signaling in IECs also induces expression and release of amphyregulin and epiregulin that activate epidermal growth factor receptor (EGFR) (46). Therefore, multiple cytoprotective functions in IECs are covered by several TLRs signaling, but defects in different aspects of the cytoprotection may result in similar manifestations in the intestine.
The DSS colitis model may rely on different mechanisms to induce acute vs chronic inflammation. For example, TLR9−/− mice have shown to be more susceptible to acute DSS colitis, but they demonstrate less severe manifestations of colitis during repeated cycles of DSS treatments compared to WT mice (7, 47). Suppression of TLR9 signaling by adenoviral-ODN, which is known to block the effect of bacterial CpG-ODN, have demonstrated a suppressive effect of intestinal inflammation in various mouse models of chronic colitis (47). Therefore, bacterial DNA from commensal flora is one of the factors inducing intestinal inflammation through activation of TLR9 during chronic colitis. These results indicate that TLR signaling during DSS colitis contribute to cytoprotection and mucosal restitution but at the same time it may be involved in sustained mucosal inflammation in response to commensal bacteria.
Intestinal inflammation in NLR-deficient mice have been extensively studied (Table 2). The most striking phenotype was observed in NLRP6−/− mice, which shows spontaneous development of colitis as well as greater susceptibility to DSS colitis than WT mice (19). Mice deficient in NLRP6 and its signaling adaptor apoptosis-asocated speck-like protein (ASC) show increased number of CD45+ cells in lamina propria, crypt hyperplasia in the colon and enlargement of Peyer’s patches with formation of germinal centers (19). These mice have increased mucosal permiability during DSS colitis and are unable to recover from colitis (48, 49). They have significantly elevated expression of CCL5 chemokine in the colon compared to WT mice, suggesting that NLRP6 plays an important role for the negative regulation of CCL5 to maintain intestinal host-commensal homeostasis (19). Interestingly, the susceptibility to intestinal inflammation and upregulation of CCL5 in NLRP6−/− mice is associated with alterations of commensal composition (especially high prevalence of genus Prevotellaceae and group TM7 bacteria) (19). Co-housing with NLRP6−/− mice and hence adopting similar flora, WT mice experienced up-regulation of CCL5 in the colon. Although this up-regulation of intestinal CCL5 did not cause spontaneous intestinal inflammation in WT mice, these mice had worse colitis in response to DSS than WT mice that are housed by themselves. Increased susceptibility to DSS induced colitis by co-housing with NLRP6−/− mice was not observed in CCL5−/− mice confirming CCL5 as an effector molecule in excerabation of colitis by the commensal alteration.
Since NOD2 gene mutations are associated with Crohn’s disease, mice with genetically modified Nod2 gene have been engineered and studied. Although NOD2-knockout mice (NOD2−/− mice) do not develop spontaneous colitis, these mice have increased burdens of Bacteroides, Firmicutes and Bacillus species in ileal mucosa compared to WT mice, and are more susceptible to 2,4,6-trinitrobenzenesulfonic acid (TNBS) induced colitis (50, 51). Bone marrow chimera model demonstrated that NOD2 deficiency in hematopoietic cells increases severity of TNBS induced colitis (50). Furthermore, transgenic mice that over-express NOD2 under MHC class II promoter (APCs) demonstrate increased resistance to TNBS induced colitis (52). Mice carrying the Nod2 mutation (Nod22939iC mice) noted in patients with Crohn’s disease (3020insC) have worse colitis secondary to DSS treatment compared to controls (53). In addition, NOD1−/− mice and Nod1/Nod2 double knockout mice have been reported to be more susceptible to DSS induced colitis compared to control mice partly due to increased mucosal permiability during colitis (54, 55). NOD2 has shown to regulate intestinal commnesal flora through the secretion of bacteria-killing factors (51). Therefore, impaired NOD2 signaling alters commensal composition and increases the susceptibility to intestinal inflammation that may be asociated with defective secretion of antimicrobial peptides in the intestine and abnormal immune cell response to the altered commensal flora. The increased mucosal permiability may facilitate mucosal immune cell exposure to luminal contents.
The role of NLRP3 in colitis has been studied in several mouse colitis models with various results. Multiple reports have demonstrated that mice deficient in NLRP3 or its downstream signaling molecules ASC and caspase-1 have increased severity of DSS induced colitis compared to WT mice (56–59). NLRP3−/− mice are also more susceptible to TNBS induced colitis than WT mice (56, 57). Bone marrow chimera experiments highlight the importance of NLRP3 in intestinal stromal cells rather than hematopoietic cells in resistance to DSS induced colitis (57). Both NLRP3−/− mice and caspase-1−/− mice show increased mucosal permeability accompanied by impaired epithelial proliferation during DSS colitis (57). The number of proliferating epithelial cells and mucosal permeability in these mice are similar to WT mice at baseline indicating that these mice have defective repair responses to mucosal injury. Since intraperitoneal injection of recombinant IL-18 during DSS treatment reduced colitis severity in caspase-1−/− mice, NLRP3-mediated protection from DSS-induced mucosal injury might be mediated by IL-18 production through caspase-1 activation.
Earlier studies, however, have shown that blocking IL-18 hampers mucosal induction of pro-inflammatory cytokines and chemokines and attenuates severity of DSS induced colitis in mice (60, 61). IL-18 transgenic mice exhibit an increased susceptibility to DSS induced colitis (62). The other report has described that absence of caspase-1 protects mice from DSS induced colitis especially in chronic phase (60). Finally, two reports have demonstrated that NLRP3−/− mice develop a less severe intestinal inflammation during DSS induced colitis and that pharmacological inhibition of caspase-1 attenuated DSS induced colitis (19, 63).
There appears to be two phenotypes of NLRP3 in colitis. Worse DDS induced colitis in NLRP3−/− and caspase-1−/− mice are likely due to impaired epithelial proliferation after epithelial injury (57, 59). The precise mechanism by which NLRP3 regulates epithelial proliferation is still obscure, though this possibly involves IFN-γ and IL-18 in regulation of cell proliferation (64–66). By contrast, NLRP3−/− mice have reduced mucosal expression of IFN-γ and impaired neutrophil activity during DSS induced colitis, which may attenuate intestinal inflammation in this model (56, 67). IL-18 is known to induce IFN-γ production directly by NK cells and synergistically with IL-12 by T cells (66, 68). Therefore, NLRP3 may play both promotive and suppressive role in DSS induced colitis.
Which of these opposite NLRP3 phenotype dominates may be dictated by the composition of the intestinal flora. As demonstrated by experiments of WT mice co-housed with NLPR6−/− mice, severity of DSS induced colitis can be substantially influenced by the composition of commensal flora (19). NLRP3−/− mice have shown a unique expression profile of intestinal antimicrobial peptides and distinct intestinal microbiota from WT mice (56). Since development of intestinal commensal flora depends on maternal gut flora and immunity that differ between breeding facilities, it is possible that mice raised in different facilities have distinct flora profiles that lack some of the key populations responsible for each NLRP3-associated intestinal phenotype (25, 69, 70).
NLR inflammasome is activated in cells of intestinal mucosa during colitis. DSS has been shown to activate NLRP3 inflammasome and releases IL-1β from LPS stimulated macrophages through potassium efflux, lysosomal maturation, and production of reactive oxygen species (ROS) (63). Effective cytokine release by inflammasome requires TLR signaling that induces precursors of IL-1β and IL-18 (Figure 1). Commensal bacteria through TLR signaling may individually activate NLR inflammasomes by inducing adenosine triphosphate (ATP), lysosomal leakage and ROS production (71–73). Recently we have described that oxidized mitochondrial DNA induced by the above stimuli binds to NLRP3 and induces inflammasome activation during cell apoptosis, but this is inhibited by autophagy activation (74). Therefore, host microbial interactions in intestinal mucosal interface are orchestrated by TLRs and NLRs through regulation of the balance between autophagy and inflammasome activation.
In human IBD, accumilation of IL-17 expressing helper T cells (Th17 cells) in intestinal mucosa has been observed (75). It is known that the gastrointestinal tract is the major organ generating Th17 cells which depends on commensal bacteria (71, 76). Recently, intestinal specific role of IL-1β in Th17 cell generation has been shown (77). Flow cytometric analysis demonstrates that CD11b+F4/80+CD11c−/low macrophages is the main source of IL-1β in the intestine in the presence of commensal bacteria (77). Macrophages deficient in Crohn’s disease-associated autophagy-related 16-like 1 (ATG16L1) have shown averrant IL-1β production (78). The importance of commensal bacteria mediated IL-1β has been further demonstrated in the T cell-induced mouse colitis model, in which IL-1R−/− naïve T cells failed to induce colitis after transfer to RAG1−/− mice due to the abscence of Th17 cell accumulation and reduced pro-inflammatory cytokine production in colonic mucosa (79). Although the effector role of IL-17A expressing T cells in induction of intestinal inflammation has been controvertial, these results explain a significant part of the pathogenesis of IBD in the context of host-commensal interactions, especially of Crohn’s disease that is associated with ATG16L1 gene mutations (71, 80–83).
Chronic intestinal inflammation has long been suggested to trigger tissue neoplastic transformation as higher incidence of intestinal cancer has been observed in patients with IBD. Extensive studies have identified the molecular pathogenesis of sporadic colon cancer based on genetic alterations. Although the process of developing colitis-associated cancer (CAC) involves some of those genetic abnormalities, unique aspects of colon carcinogenesis in the setting of chronic colitis have been illuminated (84, 85). Since inflamed intestinal cells in patients with IBD have these genetic alterations before developing histological features of dysplasia, genetic alterations in CAC may be a secondary step rather than primary cause of tumorigenesis (86). It is likely that abnormal signaling in some PRRs leads to uncontrolled expression of the genes and enzymes regulating cell proliferation, apoptosis, and DNA repair prior to the gene alterations. While precise mechanisms underlying initiation and/or promotion of the inflammation-associated intestinal cancer have yet to be fully determined, frequent cycles of injury and repair of the epithelium in the presence of tumorigenic cytokines, chemokines, and prostaglandins may predispose to genetic mutations, which increases neoplastic risk (87, 88). Since conventionalization of germ-free animals accelerates epithelial proliferation in the intestine, PRR signaling may play an important role in regulation of epithelial proliferation (89). It has been reported that TLR-mediated MyD88 signaling in subepithelial macrophages regulates crypt stem cell differentiation and epithelial proliferation through expression of Cyclooxygenase 2 (COX-2) and Prostaglandin E2 (PGE2) (90, 91). TLR4 stimulation has also been shown to induce proliferation of human intestinal epithelial cells via induction of EGFR ligands (92, 93). The inflammatory milieu may enhance surface expression of TLR2 and TLR4 leading to IECs responsiveness to their ligands (94, 95). These results suggest that abnormal TLR signaling, especially TLR2 and TLR4 in both IECs and subepithelial macrophages, may induce aberrant epithelial proliferation and thus may contribute to cancer development in the setting of chronic inflammation.
Recent reports regarding PRR phenotypes in mouse models of colitis-associated (AOM-DSS) and spontaneous multiple intestinal (Apc/Min) neoplasia appear to show some important pathways in the regulation of intestinal tumorigenesis (Table 3). The AOM-DSS (a single injection of azoxymethane followed by repeated DSS treatment and recovery) model mimics human CAC as it represents repeated mucosal injury and repair leading to epithelial proliferation and dysplastic transformation in the colon (96). In this model, TLR4−/− mice are protected from tumor development due to decreased expression of mucosal COX-2, PGE2, and amphiregulin (46). Supplementation of PGE2 during the recovery of colitis bypasses the protective phenotype of TLR4−/− mice against intestinal tumors along with sustained up-regulation of COX-2 in subepithelial macrophages and epithelial amphiregulin production (45). Therefore, the up-regulation of PGE2 during the recovery phase of colitis is a key for colitis-associated tumorigenesis involved in TLR4 signaling. TLR4 expression only in stroma but not in myeloid cells restored tumor incidence in TLR4−/− mice (97). In addition, transgenic expression of constitutively active TLR4 results in increased mucosal PGE2 production and tumor development in this model (20). These results indicate that epithelial TLR4 signaling contribute to tumor development through mucosal production of PGE2 in the setting of chronic colitis.
On the other hand, TLR2−/− mice have demonstrated increased tumor development in the AOM-DSS model (98). Although IECs in TLR2−/− mice are less proliferative and more apoptotic in normal mucosa, they become more proliferative and less apoptotic during chronic colitis compared to WT mice (98). Underlying mechanism of increased tumor burden in TLR2−/− mice is associated with strong activation of epithelial STAT3 (signal transducer and activator of transcription 3) and higher expression of tumorigenic cytokines (IL-6, TNF-α, and IL-17A) in intestinal mucosa. Mucosal TNF-α signaling and IL-6-mediated STAT3 activation are known to be indispensable during tumor development in this model (99). In addition, STAT3-mediated Th17 cell generation has been shown to facilitate tumor development in Apc/Min mice (100). It has been reported that increased expression of these cytokines in TLR2−/− mice is due to compensatory activation of TLR4 (23, 101). Therefore, enhanced tumorigenesis in TLR2−/− mice may involve TLR4 signaling.
MyD88−/− mice have demonstrated multiple phenotypes in development of intestinal tumors. MyD88−/− mice develop more tumors in the AOM-DSS model than WT controls but fewer tumors in AOM-treated IL-10−/− model compared to their WT counterparts (102, 103). This discrepancy is likely due to differences in inflammation between the models, since MyD88 deficiency results in increased severity of colitis in the AOM-DSS model but reduced colitis in AOM treated IL-10−/− model (102, 103). It is important to note that there is no increase in IEC proliferation after AOM-DSS treatment in MyD88−/− mice (102). The underlying mechanism of the increased susceptibility of MyD88−/− mice to the AOM-DSS induced intestinal tumor is upregulation of the genes associated with Wnt signaling, DNA repair, and angiogenesis (102). In addition, MyD88−/− mice showed more frequent clonal mutations in the β-catenin gene in IECs during AOM-DSS treatment (102). Without chronic inflammation, MyD88 deficiency results in resistance to intestinal tumor development in the Apc/Min and AOM (without DSS) models (102, 104). Therefore, MyD88 signaling may act both in a tumorigenic and anti-tumorigenic capacity depending on the presence and the types of chronic inflammation in the intestine. This is possible because MyD88 transduces multiple receptor signaling pathways including most TLRs, IL-1R, and IL-18R, which individually lead to the induction of a distinct set of genes (105). Therefore, tumorigenesis induced by AOM-DSS treatment in MyD88−/− mice may differ from that in TLR2 or TLR4 deficient mice.
Besides TLRs, IL-18R, another upstream of MyD88 signaling, appears to be an important regulator of inflammation-associated tumorigenesis in the AOM-DSS model (102). Mice deficient in IL-18 and IL-18R but not IL-1R exhibit similar susceptibility to tumor development as MyD88−/− mice in the AOM-DSS model (102). Similar to the MyD88−/− mice, these mice that are deficient in IL-18 and IL-18R exhibit severe inflammation but do not have increased IEC proliferation in response to AOM-DSS, suggesting that IL-18-induced IL-18R activation is responsible for the protective role of MyD88 signaling during colitis-associated tumorigenesis in this model.
Higher incidence of intestinal tumors in the AOM-DSS model has been observed in mice deficient in NLRs (NLRP3, NLRP6, and NLRC4), who are unable to produce a mature form of IL-18 (48, 58, 67, 106). Similar tumorigenic phenotype has been reported in caspase-1−/− mice (58). Since caspase-1 activation is the final step of NLR signaling, NLR-dependent protection against colitis-associated tumorigenesis is mediated by their endproducts. It is likely that IL-18 signaling integrates the role of NLRs in the resistance to intestinal tumor development as IL-1R−/− mice have similar susceptibility to intestinal tumor in the AOM-DSS model compared to WT mice (102). The major defect in these mice lies in the regulation of epithelial cell proliferation. NLRP6−/− mice have shown greater expression of the genes associated with Wnt-signaling pathway in tumor tissue than tumors in WT mice in the AOM-DSS model (49). Although the precise mechanism has not been explored in IL-18-mediated protection against colitis-associated tumorigenesis, NLR signaling in myeloid cells may be responsible for this protection as it has been demonstrated in bone marrow chimera experiments (48, 58). Since IL-18 is known to facilitate anti-tumor immunity through induction of Th1 and cytotoxic T cell responses, IL-18-mediated suppression of intestinal tumorigenesis may also be involved in the regulation of anti-tumor immunity (107).
Several TLR agonists and antagonists have been applied for the prevention and/or treatment of murine models of colitis (Table 4). In general, PRR signaling contributes to induction of regional inflammation but may promote cytoprotective and repair responses during mucosal injury in the gut. In addition, a portion of TLR4 signaling and most nucleotide-sensing TLRs may contribute to immunomodulation through induction of type I IFNs. Therefore, selection of signaling targets and the timing of intervention are important to establish successful therapeutics for colitis.
Oral administration of a TLR2 agonist Pam3CSK4 has shown its therapeutic potential in DSS induced colitis (42). The protective effect of Pam3CSK4 is mediated through preservation of tight junctional epithelial barrier (42, 44, 108). TLR2 stimulation also increases the colonic production of TFF3 that facilitates wound healing and blocks apoptotic signaling (44). Increased susceptibility of TLR2−/− mice to colitis-associated tumor further indicates a possible beneficial effect of Pam3CSK4 for cancer prevention during chronic colitis. Although TLR2 agonists have not yet entered clinical trials for human diseases, these results suggest that TLR2 signaling may be an ideal target for management strategy of IBD.
TLR4 antagonists (CRX-526; a synthetic lipid A mimetic molecule, 1A6; a specific monoclonal antibody) have been applied to prevent murine colitis (109, 110). Although TLR4 antagonist (1A6) suppressed induction of acute inflammatory infiltrate by blocking the expression of chemokines in the intestine when administered prior to colitis, it delayed mucosal healing when administered to established colitis (110). The TLR4 blocking strategy did not ameliorate the chronic model of T cell transfer colitis, but administration of 1A6 during recovery phase of colitis significantly prevented tumor development in the AOM-DSS model (20, 110). Therefore, combination therapies with cytoprotective agents may be required for TLR4 blocking strategy to avoid the delay of mucosal healing. TLR4 antagonists are currently evaluated in clinical trials for sepsis cases (111).
In vivo manipulation of NLRs have also been examined. NOD2 stimulation by systemic administration of MDP appears to be beneficial in mouse models of colitis (112). Glyburide (glibenclamide), a sulfonylurea drug for the treatment of type 2 diabetes, is known to inhibit ATP-sensitive K+ channels and thus can prevent NLRP3 inflammasome activation (113). Glyburide has been shown to prevent ventilator-induced lung injury in mice and delay endotoxin-induced lethality in mice, but it has not been applied for animal models of colitis (113, 114).
PRR signaling plays significant roles in intestinal homeostasis which is mainly composed of the maintenance of commensals. Extensive studies in animal models of colitis have provided several key points of the contribution of individual PRRs and their effector pathways in the pathogenesis of colitis. Epithelial PRR signaling mainly promotes mucosal protection through induction of pathways that leads to cell proliferation and survival or cytoprotection in response to mucosal injury. By contrast, it is likely that PRR signaling in hematopoietic cells is responsible for induction of local inflammation in response to invading pathogens. These PRR signals may be enhanced during chronic inflammation in the intestine. Continuous activation of abnormal PRR signaling leads to the development of neoplasms. In this regard, PRR signaling is involved in multiple aspects of intestinal tumorigenesis as well as host anti-tumor immunity. Manipulation of PRR signaling as therapeutic strategies of human diseases has just begun. Not only targeting particular PRR signaling, but targeting upstream signaling or downstream effector molecules such as caspase-1, IL-1β or IL-18 may be beneficial to develop novel strategies for managing IBD and prevention of colorectal cancer in those patients.
This work was supported by NIH grants 1R01HL66436 and 1R01AI 067995 for M.A, and Senior Research Award from Crohn’s Colitis Foundation of America (3391) and a NIH grant 1R01AI095255-01A1 for M.F. The authors have no conflicting financial interests.