TLR signaling is initiated through the interaction of Toll/Interleukin-1 receptor (TIR) domains of the TLRs and recruited adaptor molecules. Depending on the adaptor molecule, TLR signaling may induce one of the two major downstream pathways, i.e., the MyD88-dependent and TRIF-dependent pathways. Most TLRs induce the MyD88-dependent pathway except for TLR3. In the MyD88-dependent pathway, tumournecrosis-factor-receptor-associated factor 6 (TRAF6) is recruited and activated by phosphorylation and ubiquitylation to induce sequential phosphorylations of MAP kinases and IB. These cascades lead to the activation of the transcriptional factors AP-1 and NF-B, respectively. Activated AP-1 and NF-B translocate to the nucleus and induce the expression of multiple pro-inflammatory genes including pro-IL-1. The MyD88-dependent pathway may also induce Type I interferons (IFNs) through transcriptional factor IRF7 activation, when it is induced by TLR7, TLR8 or TLR9. The TRIF-dependent pathway can be induced directly by TLR3 and indirectly by TLR4 via recruitment of an adapter molecule TRAM (TRIFrelated adaptor molecule), which results in the expression of the type I IFNs through activation of IRF3. NLRs induce signaling pathways through interaction of their Caspase recruitment domains (CARD)s with other CARD-containing molecules in the cytoplasm. NOD1 and NOD2 recruit a CARD-containing RICK resulting in NF-B activation. NALPs form a complex of CARD-containing molecules called inflammasome, which activates Caspase-1. Activated Caspase-1 processes pro-IL-1 into mature IL-1.

Fig. 1. Pathogen recognition receptors and their signaling pathways.

Innate Immunity: A Potent Target for Management of Inflammatory Bowel Disease 181

Vermeire *et al.*, 2011). For example, variants of the genes involving mucosal permeation and clearance of bacterial toxins such as mucin gene 19 (MUC19), organic cation transporter (OCTN) 1/2, multidrug resistance-1 (MDR1), have been reported to be associated with IBD (Barrett *et al.*, 2008; Ho *et al.*, 2005; Russell *et al.*, 2006; Waller *et al.*, 2006). Crohn's disease susceptibility genes NOD2, autophagy related protein 16-like 1 (ATG16), and immunityrelated GTPase family M (IRGM) are involved in production of anti-microbial peptides and intracellular bactericidal functions (Hampe *et al.*, 2007; Hisamatsu *et al.*, 2003; McCarroll *et al.*, 2008; Rioux *et al.*, 2007; Wehkamp *et al.*, 2005). Moreover, TLR4 gene variants have been associated with both Crohn's disease and ulcerative colitis (Browning *et al.*, 2007; Franchimont *et al.*, 2004; Ouburg *et al.*, 2005; Torok *et al.*, 2004b). Polymorphisms in TLR1, 2, and 6 (TLR2 forms heteromeric receptor complexes with TLR1 or TLR6 to induce signal transduction) have been associated with greater disease extension in both ulcerative colitis and Crohn's disease (Pierik *et al.*, 2006). Polymorphisms in regulatory gene elements of TLR5 (stop codon) and TLR9 (promoter region) have also been associated with Crohn's

Most of the IBD associated gene variants are loss-of-function type alterations. Therefore, in the pathogenesis of IBD, chronic inflammation due to aggressive T cell responses to commensal flora may be a result from the host's defective ability to maintain burdens of commensals and/or elimination of invading microbes and microbial toxins, rather than T

**3. Role of innate immunity in the pathogenesis of colitis – animal studies**

Mouse models of colitis are invaluable tools to determine the involvement of individual molecules in the pathogenesis of colitis. Gene recombination technology has provided ways to investigate the roles of individual PRRs in the pathogenesis of IBD by applying targeted gene knockout mice to animal models of IBD. Although none of the current murine models of IBD perfectly reproduce the human disease, each model has distinctive advantages to investigate a particular aspect of IBD pathogenesis. Most murine models of IBD require commensal flora to develop colitis, indicating that host response to commensal flora may play an important role in initiation of intestinal inflammation. However, the dextran sulfate sodium (DSS)-induced colitis, which is one of the well-established murine models of IBD, demonstrates more severe manifestations of colitis when depleted with the commensal flora (Kitajima *et al.*, 2001; Rakoff-Nahoum *et al.*, 2004). Since the underlying mechanism of DSSinduced colitis is associated with chemical damage of mucosal epithelium, this finding suggests a possible contribution of commensal flora to the protection of mucosa from damages induced by the noxious stimuli. This section details the roles played by the individual PRRs in induction and/or resolution of colitis based on knowledge obtained

Many knockout mice have been generated by targeting individual PRRs. Among them, only TLR5 and retinoid acid-inducible gene-I (RIG-I: a RNA helicase that recognizes intracellular RNA viruses) deficient mice develop spontaneous colitis, suggesting their regulatory roles in mucosal inflammation in the context of host-microbial interactions. Despite their importance in host defense against pathogens, most other PRR knockout mice do not

disease (Hawn *et al.*, 2003; Torok *et al.*, 2004a).

through studies in murine models of IBD.

**3.1 Spontaneous colitis observed in PRR-deficient mice**

cell function itself.

#### **2.1 Innate immune function in the intestine**

The rapid response of innate immunity to pathogens induces the production of antimicrobial peptides, phagocytic microbial killing and expression of cytokines, chemokines, and reactive oxygen species, leading to the recruitment of acute inflammatory cells to establish localized inflammation. Therefore, a defect in innate immunity may result in increased susceptibility of the host to pathogenic invasion even to commensals. In addition, proper induction of innate immunity is important to initiate an adaptive immune response that is antigen-specific and has an ability to temper ongoing inflammation through induction of regulatory immune properties. Abnormal innate immune signaling thus leads to inadequate induction of adaptive immunity that may result in disorganized immune responses to luminal pathogens.

#### **2.2 Innate immune abnormalities in the pathogenesis of IBD**

Clinical and basic science studies have indicated that etiology of IBD is associated with a disregulated adaptive immune response characterized by an aggressive T cell response to commensal flora, which is triggered by environmental factors particularly in genetically susceptible individuals (Packey and Sartor, 2008). Both environmental factors and the genetic alterations seen in patients with IBD vary, further making identification of exact etiology of IBD difficult. What is interesting in the pathogenesis of IBD is that any combination of environmental triggers and genetic alterations still result in aggressive T cell response to commensal flora forming chronic inflammation in gastrointestinal mucosa. On the other hand, several reports have demonstrated abnormal innate immune functions as an important pathogenesis of IBD (Latella *et al.*, 2010). The expression of mucosal TLRs, especially in epithelial cells and lamina propria antigen-presenting cells, is normally downregulated, presumably to avoid inducing an excessive immune response to commensals (Melmed *et al.*, 2003; Otte *et al.*, 2004; Smythies *et al.*, 2005). However, several reports have shown increased expression of TLR4 and TLR2 in intestinal epithelial cells as well as lamina propria antigen-presenting cells in patients with both Crohn's disease and ulcerative colitis (Cario and Podolsky, 2000; Frolova *et al.*, 2008; Hausmann *et al.*, 2002; Szebeni *et al.*, 2008). Increased expression and *in vitro* cytokine responses of TLR2, TLR4, and TLR9 in peripheral blood mononuclear cells or dendritic cells have also been reported in IBD patients (Baumgart *et al.*, 2005; Baumgart *et al.*, 2009; Canto *et al.*, 2006; Jyonouchi *et al.*, 2010). Although increased TLR expression in peripheral blood cells has been found even in IBD patients in remission, up-regulation of these TLRs might not be a primary event in the pathogenesis of IBD, because expression of TLR4 and TLR2 is transcriptionally induced by inflammatory cytokines such as IFN- (Abreu *et al.*, 2002; Lin *et al.*, 2000; Rehli *et al.*, 2000). Similarly, NOD2 expression in intestinal epithelial cells is induced by inflammatory cytokines and increased in patients with IBD (Hisamatsu *et al.*, 2003; Rosenstiel *et al.*, 2003). Nevertheless, these findings strongly suggest a substantial involvement of innate immune abnormalities in the pathogenesis of IBD.

#### **2.3 Genetic links between innate immunity and IBD pathogenesis.**

Genome-wide association studies have demonstrated that most genetic alterations associated with IBD susceptibility relate to host mucosal barrier or anti-microbial functions, especially those involved in innate immune responses (Abraham and Medzhitov, 2011;

The rapid response of innate immunity to pathogens induces the production of antimicrobial peptides, phagocytic microbial killing and expression of cytokines, chemokines, and reactive oxygen species, leading to the recruitment of acute inflammatory cells to establish localized inflammation. Therefore, a defect in innate immunity may result in increased susceptibility of the host to pathogenic invasion even to commensals. In addition, proper induction of innate immunity is important to initiate an adaptive immune response that is antigen-specific and has an ability to temper ongoing inflammation through induction of regulatory immune properties. Abnormal innate immune signaling thus leads to inadequate induction of adaptive immunity that may result in disorganized immune

Clinical and basic science studies have indicated that etiology of IBD is associated with a disregulated adaptive immune response characterized by an aggressive T cell response to commensal flora, which is triggered by environmental factors particularly in genetically susceptible individuals (Packey and Sartor, 2008). Both environmental factors and the genetic alterations seen in patients with IBD vary, further making identification of exact etiology of IBD difficult. What is interesting in the pathogenesis of IBD is that any combination of environmental triggers and genetic alterations still result in aggressive T cell response to commensal flora forming chronic inflammation in gastrointestinal mucosa. On the other hand, several reports have demonstrated abnormal innate immune functions as an important pathogenesis of IBD (Latella *et al.*, 2010). The expression of mucosal TLRs, especially in epithelial cells and lamina propria antigen-presenting cells, is normally downregulated, presumably to avoid inducing an excessive immune response to commensals (Melmed *et al.*, 2003; Otte *et al.*, 2004; Smythies *et al.*, 2005). However, several reports have shown increased expression of TLR4 and TLR2 in intestinal epithelial cells as well as lamina propria antigen-presenting cells in patients with both Crohn's disease and ulcerative colitis (Cario and Podolsky, 2000; Frolova *et al.*, 2008; Hausmann *et al.*, 2002; Szebeni *et al.*, 2008). Increased expression and *in vitro* cytokine responses of TLR2, TLR4, and TLR9 in peripheral blood mononuclear cells or dendritic cells have also been reported in IBD patients (Baumgart *et al.*, 2005; Baumgart *et al.*, 2009; Canto *et al.*, 2006; Jyonouchi *et al.*, 2010). Although increased TLR expression in peripheral blood cells has been found even in IBD patients in remission, up-regulation of these TLRs might not be a primary event in the pathogenesis of IBD, because expression of TLR4 and TLR2 is transcriptionally induced by inflammatory cytokines such as IFN- (Abreu *et al.*, 2002; Lin *et al.*, 2000; Rehli *et al.*, 2000). Similarly, NOD2 expression in intestinal epithelial cells is induced by inflammatory cytokines and increased in patients with IBD (Hisamatsu *et al.*, 2003; Rosenstiel *et al.*, 2003). Nevertheless, these findings strongly suggest a substantial involvement of innate immune

**2.1 Innate immune function in the intestine**

**2.2 Innate immune abnormalities in the pathogenesis of IBD**

responses to luminal pathogens.

abnormalities in the pathogenesis of IBD.

**2.3 Genetic links between innate immunity and IBD pathogenesis.**

Genome-wide association studies have demonstrated that most genetic alterations associated with IBD susceptibility relate to host mucosal barrier or anti-microbial functions, especially those involved in innate immune responses (Abraham and Medzhitov, 2011; Vermeire *et al.*, 2011). For example, variants of the genes involving mucosal permeation and clearance of bacterial toxins such as mucin gene 19 (MUC19), organic cation transporter (OCTN) 1/2, multidrug resistance-1 (MDR1), have been reported to be associated with IBD (Barrett *et al.*, 2008; Ho *et al.*, 2005; Russell *et al.*, 2006; Waller *et al.*, 2006). Crohn's disease susceptibility genes NOD2, autophagy related protein 16-like 1 (ATG16), and immunityrelated GTPase family M (IRGM) are involved in production of anti-microbial peptides and intracellular bactericidal functions (Hampe *et al.*, 2007; Hisamatsu *et al.*, 2003; McCarroll *et al.*, 2008; Rioux *et al.*, 2007; Wehkamp *et al.*, 2005). Moreover, TLR4 gene variants have been associated with both Crohn's disease and ulcerative colitis (Browning *et al.*, 2007; Franchimont *et al.*, 2004; Ouburg *et al.*, 2005; Torok *et al.*, 2004b). Polymorphisms in TLR1, 2, and 6 (TLR2 forms heteromeric receptor complexes with TLR1 or TLR6 to induce signal transduction) have been associated with greater disease extension in both ulcerative colitis and Crohn's disease (Pierik *et al.*, 2006). Polymorphisms in regulatory gene elements of TLR5 (stop codon) and TLR9 (promoter region) have also been associated with Crohn's disease (Hawn *et al.*, 2003; Torok *et al.*, 2004a).

Most of the IBD associated gene variants are loss-of-function type alterations. Therefore, in the pathogenesis of IBD, chronic inflammation due to aggressive T cell responses to commensal flora may be a result from the host's defective ability to maintain burdens of commensals and/or elimination of invading microbes and microbial toxins, rather than T cell function itself.

#### **3. Role of innate immunity in the pathogenesis of colitis – animal studies**

Mouse models of colitis are invaluable tools to determine the involvement of individual molecules in the pathogenesis of colitis. Gene recombination technology has provided ways to investigate the roles of individual PRRs in the pathogenesis of IBD by applying targeted gene knockout mice to animal models of IBD. Although none of the current murine models of IBD perfectly reproduce the human disease, each model has distinctive advantages to investigate a particular aspect of IBD pathogenesis. Most murine models of IBD require commensal flora to develop colitis, indicating that host response to commensal flora may play an important role in initiation of intestinal inflammation. However, the dextran sulfate sodium (DSS)-induced colitis, which is one of the well-established murine models of IBD, demonstrates more severe manifestations of colitis when depleted with the commensal flora (Kitajima *et al.*, 2001; Rakoff-Nahoum *et al.*, 2004). Since the underlying mechanism of DSSinduced colitis is associated with chemical damage of mucosal epithelium, this finding suggests a possible contribution of commensal flora to the protection of mucosa from damages induced by the noxious stimuli. This section details the roles played by the individual PRRs in induction and/or resolution of colitis based on knowledge obtained through studies in murine models of IBD.

#### **3.1 Spontaneous colitis observed in PRR-deficient mice**

Many knockout mice have been generated by targeting individual PRRs. Among them, only TLR5 and retinoid acid-inducible gene-I (RIG-I: a RNA helicase that recognizes intracellular RNA viruses) deficient mice develop spontaneous colitis, suggesting their regulatory roles in mucosal inflammation in the context of host-microbial interactions. Despite their importance in host defense against pathogens, most other PRR knockout mice do not

Innate Immunity: A Potent Target for Management of Inflammatory Bowel Disease 183

suggested to foster the recruitment and/or proliferation of regulatory T cells in the intestine

Since TLRs are expressed by most cell types in intestinal mucosa, the complexity of their function during chronic colitis may be due to the distinct roles played by TLR signaling in different cell types in the pathogenesis of chronic colitis. Compartmental differences of the role of TLR signaling in chronic colitis have been examined mainly in the MyD88 pathway (Asquith *et al.*, 2010; Fukata *et al.*, 2008; Gong *et al.*, 2010). Intestinal inflammation induced by *H. hepaticus* infection has been shown to be myeloid cell MyD88 dependent. In this infectious model, RAG2 x MyD88 double knockout mice as well as RAG2-/- chimeric mice that carry MyD88-deficient bone marrow demonstrate no intestinal inflammation but succumb to the infection, while MyD88 sufficient RAG2-/- counterparts show chronic colitis with splenomegaly but are protected from mortality (Asquith *et al.*, 2010). On the other hand, epithelial specific deletion of MyD88 results in spontaneous chronic inflammation in the small intestine (Gong *et al.*, 2010). These findings indicate that the MyD88 pathway in the myeloid compartment is necessary to induce intestinal inflammation against luminal

**3.4 Compartmental specificities of the role of PRRs in intestinal inflammation**

pathogens and sufficient to block invasion of *H. hepaticus* at the mucosal interface.

suppressive activities of regulatory T cells (Fukata *et al.*, 2008).

exploration.

Although there is a conflicting report, we and others have shown a defective colitogenic function of MyD88-/- T cells in an adoptive T cell transfer model of colitis, in which MyD88- /- naïve T cells have less ability to induce chronic colitis compared to MyD88 sufficient T cells after transfer to RAG-/- mice (Fukata *et al.*, 2008; Tomita *et al.*, 2008). In addition, we have shown that MyD88-/- regulatory T cells are unable to sufficiently suppress T cell mediated colitis, indicating that TLR signaling in T cells is important to elicit the full

Ligation with TLR2 in combination with T cell receptor stimulation has also been reported to expand regulatory T cells in a MyD88-dependent manner, while it induces transient loss of their suppressive function (Sutmuller *et al.*, 2006). Therefore, TLR signaling in T cells seems to act as a co-stimulatory factor. Although antigen-presenting cells are thought to be a major cell type expressing TLRs, their TLR signaling is normally down regulated in the intestine (Rescigno and Matteoli, 2008; Smith *et al.*, 2005). Nevertheless, There unique roles for TLRs in individual cell types in intestinal inflammation may be revealed with future

**3.5 Animal studies of phenotypic testing of disease susceptibility genes in human IBD**  Testing the phenotype of individual susceptibility genes in IBD using mouse models is one of the priorities in the field of IBD research. Most IBD susceptible genes carry loss-offunction mutation, but by now we have not seen spontaneous intestinal inflammation in any mouse models that carry a functional deletion of the disease candidate gene. NOD2 deficient mice and those with an insertion of the human disease-associated mutation 3020insC do not demonstrate spontaneous intestinal inflammation but are more susceptible to DSS-induced colitis (Kobayashi *et al.*, 2005; Maeda *et al.*, 2005). ATG16L1 hypomorphic mice have abnormal Paneth cell granules and defective cryptdin production similar to Crohn's disease patients with ATG16L1 mutation, but these mice do not develop spontaneous colitis

during colitis (Heimesaat *et al.*, 2007).

develop spontaneous colitis in the presence of commensal flora. Therefore, some compensatory mechanisms may exist involving both host and microbial factors. For instances, many pathogens carry multiple PAMPs. Host antigen-presenting cells can recognize surface PAMPs of a bacterial pathogen by TLR2 (and TLR1 or TLR6), TLR4, or TLR5, and the same pathogen can be recognized by TLR9 or Nod-like receptors after being digested in the cytoplasm. Importantly, these PRRs share several key innate immune signaling pathways including NF-B and MAP-kinases. This redundancy of the pathogen recognition system may be reasonable to reduce the risks of outbreaks of pathogens that acquire evolutional changes to evade host immune responses. Cross regulations between PRRs exist as TLR4 deficiency protects TLR5-/- mice from developing spontaneous colitis (Vijay-Kumar *et al.*, 2007a). What we need to take into account is that the roles of PRRs during mucosal damage or inflammation may differ from their roles in homeostatic maintenance of mucosal integrity. Therefore, the lack of intestinal spontaneous phenotypes in the PRR knockout animals does not simply negate their contribution to the pathogenesis of IBD.

#### **3.2 Role of PRRs in acute murine colitis**

Consistent with the finding in germ-free mice, most TLR knockout mice demonstrate increased susceptibility to DSS-induced acute colitis. For example, deficiency in TLR2, TLR4, TLR5, and TLR9 has been individually associated with the increased susceptibility to DSSinduced colitis (Fukata *et al.*, 2005; Ivison *et al.*, 2010; Lee *et al.*, 2006; Rakoff-Nahoum *et al.*, 2004). Mice deficient in MyD88, a major downstream molecule of most TLRs except for TLR3, also demonstrated more severe disease than WT mice in this colitis model suggesting the importance of MyD88-dependent TLR signaling in protection against chemically induced mucosal damage (Fukata *et al.*, 2005; Rakoff-Nahoum *et al.*, 2004).

#### **3.3 Role of PRRs in chronic murine colitis**

TLR signaling may act differently during chronic colitis where sustained inflammation exists. Unlike their increased susceptibility to the acute DSS model, most TLR-deficient mice are protected in the chronic models of colitis. For example, TLR9-deficient mice demonstrate less severe manifestations of chronic colitis induced by four cycles of DSS treatments compared to WT mice (Obermeier *et al.*, 2005). As I mentioned earlier, TLR9-deficient mice should be more susceptible to each cycle of DSS treatment than WT mice, while mechanism inducing chronic inflammation may differ from acute mucosal damage in this model. MyD88 deficiency protects mice from development of chronic colitis in several IBD models including the IL-10-/- model (Rakoff-Nahoum *et al.*, 2006). IL-10-/- mice are known to develop spontaneous colitis due to uncontrolled pro-inflammatory cytokine production in the presence of commnesal flora (Berg *et al.*, 1996; Kuhn *et al.*, 1993).

There are conflicting data in IL-10 x TLR4 double knockout mice demonstrating either increased or reduced colitis compared to IL-10-/- mice depending on the facilities (Biswas *et al.*, 2011; Gonzalez-Navajas *et al.*, 2010). In the presence of *Helicobacter hepaticus*, IL-10 x TLR4 double knockout mice generate atypical regulatory T cells which possess pro-inflammatory properties (Matharu *et al.*, 2009). Therefore, TLR4 deficiency may be colitogenic or protective in the IL-10-/- model depending on the presence or absence of *H. hepaticus*. Regulatory T cells are indispensable for termination of ongoing inflammation, and TLR4 has been

develop spontaneous colitis in the presence of commensal flora. Therefore, some compensatory mechanisms may exist involving both host and microbial factors. For instances, many pathogens carry multiple PAMPs. Host antigen-presenting cells can recognize surface PAMPs of a bacterial pathogen by TLR2 (and TLR1 or TLR6), TLR4, or TLR5, and the same pathogen can be recognized by TLR9 or Nod-like receptors after being digested in the cytoplasm. Importantly, these PRRs share several key innate immune signaling pathways including NF-B and MAP-kinases. This redundancy of the pathogen recognition system may be reasonable to reduce the risks of outbreaks of pathogens that acquire evolutional changes to evade host immune responses. Cross regulations between PRRs exist as TLR4 deficiency protects TLR5-/- mice from developing spontaneous colitis (Vijay-Kumar *et al.*, 2007a). What we need to take into account is that the roles of PRRs during mucosal damage or inflammation may differ from their roles in homeostatic maintenance of mucosal integrity. Therefore, the lack of intestinal spontaneous phenotypes in the PRR knockout animals does not simply negate their contribution to the pathogenesis

Consistent with the finding in germ-free mice, most TLR knockout mice demonstrate increased susceptibility to DSS-induced acute colitis. For example, deficiency in TLR2, TLR4, TLR5, and TLR9 has been individually associated with the increased susceptibility to DSSinduced colitis (Fukata *et al.*, 2005; Ivison *et al.*, 2010; Lee *et al.*, 2006; Rakoff-Nahoum *et al.*, 2004). Mice deficient in MyD88, a major downstream molecule of most TLRs except for TLR3, also demonstrated more severe disease than WT mice in this colitis model suggesting the importance of MyD88-dependent TLR signaling in protection against chemically

TLR signaling may act differently during chronic colitis where sustained inflammation exists. Unlike their increased susceptibility to the acute DSS model, most TLR-deficient mice are protected in the chronic models of colitis. For example, TLR9-deficient mice demonstrate less severe manifestations of chronic colitis induced by four cycles of DSS treatments compared to WT mice (Obermeier *et al.*, 2005). As I mentioned earlier, TLR9-deficient mice should be more susceptible to each cycle of DSS treatment than WT mice, while mechanism inducing chronic inflammation may differ from acute mucosal damage in this model. MyD88 deficiency protects mice from development of chronic colitis in several IBD models including the IL-10-/- model (Rakoff-Nahoum *et al.*, 2006). IL-10-/- mice are known to develop spontaneous colitis due to uncontrolled pro-inflammatory cytokine production in

There are conflicting data in IL-10 x TLR4 double knockout mice demonstrating either increased or reduced colitis compared to IL-10-/- mice depending on the facilities (Biswas *et al.*, 2011; Gonzalez-Navajas *et al.*, 2010). In the presence of *Helicobacter hepaticus*, IL-10 x TLR4 double knockout mice generate atypical regulatory T cells which possess pro-inflammatory properties (Matharu *et al.*, 2009). Therefore, TLR4 deficiency may be colitogenic or protective in the IL-10-/- model depending on the presence or absence of *H. hepaticus*. Regulatory T cells are indispensable for termination of ongoing inflammation, and TLR4 has been

induced mucosal damage (Fukata *et al.*, 2005; Rakoff-Nahoum *et al.*, 2004).

the presence of commnesal flora (Berg *et al.*, 1996; Kuhn *et al.*, 1993).

of IBD.

**3.2 Role of PRRs in acute murine colitis**

**3.3 Role of PRRs in chronic murine colitis**

suggested to foster the recruitment and/or proliferation of regulatory T cells in the intestine during colitis (Heimesaat *et al.*, 2007).

#### **3.4 Compartmental specificities of the role of PRRs in intestinal inflammation**

Since TLRs are expressed by most cell types in intestinal mucosa, the complexity of their function during chronic colitis may be due to the distinct roles played by TLR signaling in different cell types in the pathogenesis of chronic colitis. Compartmental differences of the role of TLR signaling in chronic colitis have been examined mainly in the MyD88 pathway (Asquith *et al.*, 2010; Fukata *et al.*, 2008; Gong *et al.*, 2010). Intestinal inflammation induced by *H. hepaticus* infection has been shown to be myeloid cell MyD88 dependent. In this infectious model, RAG2 x MyD88 double knockout mice as well as RAG2-/- chimeric mice that carry MyD88-deficient bone marrow demonstrate no intestinal inflammation but succumb to the infection, while MyD88 sufficient RAG2-/- counterparts show chronic colitis with splenomegaly but are protected from mortality (Asquith *et al.*, 2010). On the other hand, epithelial specific deletion of MyD88 results in spontaneous chronic inflammation in the small intestine (Gong *et al.*, 2010). These findings indicate that the MyD88 pathway in the myeloid compartment is necessary to induce intestinal inflammation against luminal pathogens and sufficient to block invasion of *H. hepaticus* at the mucosal interface.

Although there is a conflicting report, we and others have shown a defective colitogenic function of MyD88-/- T cells in an adoptive T cell transfer model of colitis, in which MyD88- /- naïve T cells have less ability to induce chronic colitis compared to MyD88 sufficient T cells after transfer to RAG-/- mice (Fukata *et al.*, 2008; Tomita *et al.*, 2008). In addition, we have shown that MyD88-/- regulatory T cells are unable to sufficiently suppress T cell mediated colitis, indicating that TLR signaling in T cells is important to elicit the full suppressive activities of regulatory T cells (Fukata *et al.*, 2008).

Ligation with TLR2 in combination with T cell receptor stimulation has also been reported to expand regulatory T cells in a MyD88-dependent manner, while it induces transient loss of their suppressive function (Sutmuller *et al.*, 2006). Therefore, TLR signaling in T cells seems to act as a co-stimulatory factor. Although antigen-presenting cells are thought to be a major cell type expressing TLRs, their TLR signaling is normally down regulated in the intestine (Rescigno and Matteoli, 2008; Smith *et al.*, 2005). Nevertheless, There unique roles for TLRs in individual cell types in intestinal inflammation may be revealed with future exploration.

#### **3.5 Animal studies of phenotypic testing of disease susceptibility genes in human IBD**

Testing the phenotype of individual susceptibility genes in IBD using mouse models is one of the priorities in the field of IBD research. Most IBD susceptible genes carry loss-offunction mutation, but by now we have not seen spontaneous intestinal inflammation in any mouse models that carry a functional deletion of the disease candidate gene. NOD2 deficient mice and those with an insertion of the human disease-associated mutation 3020insC do not demonstrate spontaneous intestinal inflammation but are more susceptible to DSS-induced colitis (Kobayashi *et al.*, 2005; Maeda *et al.*, 2005). ATG16L1 hypomorphic mice have abnormal Paneth cell granules and defective cryptdin production similar to Crohn's disease patients with ATG16L1 mutation, but these mice do not develop spontaneous colitis

**4.1 TLR2**

(Hennessy *et al.*, 2010).

colitis (Bunting *et al.*, 2011; Cheng *et al.*, 2011).

**4.2 TLR3**

**4.3 TLR4**

Innate Immunity: A Potent Target for Management of Inflammatory Bowel Disease 185

Oral administration of a TLR2 agonist Pam3CSK4 has been suggested to have therapeutic potential in DSS-induced colitis (Cario *et al.*, 2007). The protective effect of the TLR2 agonist is associated with preservation of epithelial tight junctions and reduced apoptosis, and largely depends on the expression of the gap-junctional protein Connexin 43 in epithelial cells (Cario *et al.*, 2007; Ey *et al.*, 2009; Podolsky *et al.*, 2009). TLR2 stimulation also increases the colonic production of trefoil factor (TFF) 3 that facilitates wound healing and blocks apoptotic signaling (Podolsky *et al.*, 2009). Moreover, oral TLR2 agonist has been shown to delay induction of spontaneous colitis in MDR1-/- mice (Ey *et al.*, 2009). The central pathogenesis of MDR1-/- colitis is an impaired epithelial barrier function (Collett *et al.*, 2008). Therefore, the TLR2 agonist can be a cytoprotective rather than immunomodulatory strategy for acute as well as chronic phases of colitis. TLR2 agonists have not yet entered clinical trials for human diseases. Conversely, a TLR2 antagonist (OPN-305) has been developed, but its effects have not been tested on murine colitis

Interesting data has been reported regarding the preventive effect of a TLR3 agonist on murine models of IBD (Vijay-Kumar *et al.*, 2007b). Pre-treatment of mice with a synthetic TLR3 ligand, polyinosinic:polycytidylic acid (poly I:C) protected mice from development of DSS-induced acute colitis (Vijay-Kumar *et al.*, 2007b). Similar effects of poly I:C treatment were observed in IL-10-/- mice. In this report, poly I:C was subcutaneously injected and the oral application did not show any protective effect on the colitis probably due to degradation of poly I:C by abundant RNAse in the gastrointestinal lumen. Although the exact mechanism underlying poly I:C-mediated preventive effect on colitis has been obscure, type I interferons which are predominantly induced by TLR3 signaling may have an immuno-regulatory capacity. Since intraperitoneal administration of high dose poly I:C (about ten times more than the dose in the former report) has been reported to cause mucosal destruction in the small intestine, we have to adjust the dose and route of poly I:C administration before clinical application (Zhou *et al.*, 2007a; Zhou *et al.*, 2007b). Because TLR3 expression has been shown to be significantly down regulated in the inflamed and non-inflamed mucosa of Crohn's disease as well as the mucosa of ileal pouches in patients with ulcerative colitis, stimulating TLR3 may restore an immunological defect caused by its down regulation (Cario and Podolsky, 2000; Heuschen *et al.*, 2007; Toiyama *et al.*, 2006). The existence of clinically tested poly I:C analogue (poly I:C12U: Ampligen) is advantageous for the development of a TLR3 stimulation strategy (Nicodemus and Berek, 2010). Ampligen has been proven to have less toxicity than poly I:C and completed a phase III clinical trial for chronic fatigue syndrome (Nicodemus and Berek, 2010). Several antagonists for TLR3 signaling has been reported, but have not been developed as a therapeutic strategy for

TLR4 is one of the most studied PRRs as a therapeutic target of IBD. A TLR4 antagonist (CRX-526; a synthetic lipid A mimetic molecule) has been shown to prevent the

(Cadwell *et al.*, 2008). The phenotypic discrepancies in these IBD susceptibility genes between human and mouse may be due to differences of species, but these results remind us the fact that the pathogenesis of IBD is complex of abnormalities of genetic, immunological, and environmental factors.

### **4. Challenges manipulating innate immunity (stimulate or suppress)**

Manipulation of a particular immune pathway especially within a targeted organ is challenging. In this regard, manipulating an innate immune response as a therapeutic target of IBD has some advantages and disadvantages. The major advantage includes the nonspecific nature of the innate immune responses, which provides broader effects than targeting adaptive immune responses which are antigen-specific. The rapid responses in both induction and resolution of innate immune signaling imply that exogenous control may be relatively easy. On the other hand, the multiple effects that can be induced by activation of a PRR may be a difficult point to enhance or suppress a specific function of the innate immune responses. In this section, possible targets in the innate immunity that may be utilized to treat or prevent intestinal inflammation and their potential pitfalls will be discussed through reviewing previously reported challenges in murine models of IBD (Table 2).

#### **4.1 TLR2**

184 Colitis

(Cadwell *et al.*, 2008). The phenotypic discrepancies in these IBD susceptibility genes between human and mouse may be due to differences of species, but these results remind us the fact that the pathogenesis of IBD is complex of abnormalities of genetic, immunological,

Manipulation of a particular immune pathway especially within a targeted organ is challenging. In this regard, manipulating an innate immune response as a therapeutic target of IBD has some advantages and disadvantages. The major advantage includes the nonspecific nature of the innate immune responses, which provides broader effects than targeting adaptive immune responses which are antigen-specific. The rapid responses in both induction and resolution of innate immune signaling imply that exogenous control may be relatively easy. On the other hand, the multiple effects that can be induced by activation of a PRR may be a difficult point to enhance or suppress a specific function of the innate immune responses. In this section, possible targets in the innate immunity that may be utilized to treat or prevent intestinal inflammation and their potential pitfalls will be discussed through reviewing previously reported challenges in murine models of IBD

**4. Challenges manipulating innate immunity (stimulate or suppress)**

**PRRs Agonist / Antagonist Model of IBD used Major effect**

TLR3 Agonist (poly I:C) Acute DSS colitis Prevention.

Chronic MDR1-/-

Acute DSS colitis

Chronic MDR1-/-T cell transfer colitis

TLR5 Agonist (flagellin) Acute DSS colitis Prevention if it is administered

Acute DSS colitis TNBS colitis

Chronic DSS colitis IL-10-/-, T cell transfer

colitis

TNBS colitis

Table 2. Therapeutic challenges of PRR manipulation for murine models of IBD.

Prevention and treatment. Strengthen epithelial barrier.

Involvement of Type I IFN?

Blocking acute inflammatory

Blocking cytokine responses.

Prevention. Anti-apoptotic effect. Immuno-modulatory effect. Induction of tolerance.

Blocking host response to luminal bacterial CpG.

Down-regulation of multiple

Increase TFF3.

Prevention.

intraperitoneally.

TLR responses.

infiltrate.

TLR2 Agonist (Pam3CSK4) Acute DSS colitis

and environmental factors.

(Table 2).

TLR4 Antagonist (1A6)

TLR9 Agonist (CpG-ODN)

Antagonist (CRX-526)

Antagonist (AV-ODN)

NOD2 Agonist (MDP) Acute DSS colitis

Oral administration of a TLR2 agonist Pam3CSK4 has been suggested to have therapeutic potential in DSS-induced colitis (Cario *et al.*, 2007). The protective effect of the TLR2 agonist is associated with preservation of epithelial tight junctions and reduced apoptosis, and largely depends on the expression of the gap-junctional protein Connexin 43 in epithelial cells (Cario *et al.*, 2007; Ey *et al.*, 2009; Podolsky *et al.*, 2009). TLR2 stimulation also increases the colonic production of trefoil factor (TFF) 3 that facilitates wound healing and blocks apoptotic signaling (Podolsky *et al.*, 2009). Moreover, oral TLR2 agonist has been shown to delay induction of spontaneous colitis in MDR1-/- mice (Ey *et al.*, 2009). The central pathogenesis of MDR1-/- colitis is an impaired epithelial barrier function (Collett *et al.*, 2008). Therefore, the TLR2 agonist can be a cytoprotective rather than immunomodulatory strategy for acute as well as chronic phases of colitis. TLR2 agonists have not yet entered clinical trials for human diseases. Conversely, a TLR2 antagonist (OPN-305) has been developed, but its effects have not been tested on murine colitis (Hennessy *et al.*, 2010).

#### **4.2 TLR3**

Interesting data has been reported regarding the preventive effect of a TLR3 agonist on murine models of IBD (Vijay-Kumar *et al.*, 2007b). Pre-treatment of mice with a synthetic TLR3 ligand, polyinosinic:polycytidylic acid (poly I:C) protected mice from development of DSS-induced acute colitis (Vijay-Kumar *et al.*, 2007b). Similar effects of poly I:C treatment were observed in IL-10-/- mice. In this report, poly I:C was subcutaneously injected and the oral application did not show any protective effect on the colitis probably due to degradation of poly I:C by abundant RNAse in the gastrointestinal lumen. Although the exact mechanism underlying poly I:C-mediated preventive effect on colitis has been obscure, type I interferons which are predominantly induced by TLR3 signaling may have an immuno-regulatory capacity. Since intraperitoneal administration of high dose poly I:C (about ten times more than the dose in the former report) has been reported to cause mucosal destruction in the small intestine, we have to adjust the dose and route of poly I:C administration before clinical application (Zhou *et al.*, 2007a; Zhou *et al.*, 2007b). Because TLR3 expression has been shown to be significantly down regulated in the inflamed and non-inflamed mucosa of Crohn's disease as well as the mucosa of ileal pouches in patients with ulcerative colitis, stimulating TLR3 may restore an immunological defect caused by its down regulation (Cario and Podolsky, 2000; Heuschen *et al.*, 2007; Toiyama *et al.*, 2006). The existence of clinically tested poly I:C analogue (poly I:C12U: Ampligen) is advantageous for the development of a TLR3 stimulation strategy (Nicodemus and Berek, 2010). Ampligen has been proven to have less toxicity than poly I:C and completed a phase III clinical trial for chronic fatigue syndrome (Nicodemus and Berek, 2010). Several antagonists for TLR3 signaling has been reported, but have not been developed as a therapeutic strategy for colitis (Bunting *et al.*, 2011; Cheng *et al.*, 2011).

#### **4.3 TLR4**

TLR4 is one of the most studied PRRs as a therapeutic target of IBD. A TLR4 antagonist (CRX-526; a synthetic lipid A mimetic molecule) has been shown to prevent the

Innate Immunity: A Potent Target for Management of Inflammatory Bowel Disease 187

immunostimulatory effect of CpG-ODNs, respectively. Since abundant bacterial ODNs naturally exist in the colorectal lumen that play a part of inflammatory stimuli in the setting of mucosal damage, pretreatment with CpG-ODNs has been suggested to induce host immune tolerance against endogenous bacterial ODNs (Obermeier *et al.*, 2003). Preventive effect of CpG-ODNs on induction of colitis has also been shown in other models such as 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis and can be achieved by oral administration, making this strategy further relevant (Rachmilewitz *et al.*, 2002). Although CpG-ODNs are generally thought as immunostimulative, CpG-ODN stimulation suppresses TNF- and IL-1 expression in *ex vivo* colonic mucosa taken from active UC patients (Rachmilewitz *et al.*, 2006). A subset of patients with ulcerative colitis responds to Type I INF therapy through a yet unknown mechanism (Jacobs *et al.*, 2000; Madsen *et al.*, 2001; Nikolaus *et al.*, 2003). Since TLR9 signaling may induce Type I interferons which may act as immunomodulatory property during intestinal inflammation, emphasizing this aspect of TLR9 signaling may be

Suppression of TLR9 signaling has also been beneficial for treating chronic murine colitis. Intraperitoneal as well as oral administration of adenoviral-ODN (AV-ODN) known to block the effect of bacterial CpG-ODN effectively suppressed intestinal inflammation during chronic DSS colitis (Obermeier *et al.*, 2005). The protective effect of AV-ODN was also observed in other chronic colitis models such as IL-10-/- and T cell transfer colitis models (Obermeier *et al.*, 2005). The protective effect of blocking TLR9 signaling during chronic colitis is consistent with the fact that TLR9-deficient mice have reduced severity of chronic colitis (Obermeier *et al.*, 2005). These results indicate that bacterial CpG-ODN abundantly existing in colorectal lumen is one of the indispensable factors inducing intestinal

NOD2 signaling is a plausible therapeutic target as NOD2 mutations are associated with susceptibility to Crohn's disease. The mutations are known to be loss-of-function type mutations and thus the backup of this signaling is a reasonable idea. The use of NOD2 knockout models is consistent with the human disease setting. NOD2 transgenic mice are resistant to TNBS colitis (Yang *et al.*, 2007). In addition, intraperitoneal injection of a NOD2 ligand MDP protected mice from induction of acute colitis in DSS and TNBS models (Watanabe *et al.*, 2008). The mechanism underlying MDP-mediated protection is associated with down regulation of multiple TLR responses by NOD2 stimulation, as pretreatment of dendritic cells with MDP has been shown to inhibit cytokine responses induced by TLR stimulation. One concern is that the NOD2-deficient mice introduced NOD2 construct with a CARD15 frame-shift mutation were not protected from colitis by MDP (Watanabe *et al.*, 2008). Nevertheless, development of NOD2-stimulation strategy is advantageous because MDP has already been applied to several clinical trials of cancer treatments (Meyers *et al.*,

We discussed the unique aspects of innate immunity in the context of intestinal homeostasis and the pathogenesis of colitis. It is important to understand intestinal-specific innate

another therapeutic target in the management of IBD (Katakura *et al.*, 2005).

inflammation during chronic colitis.

**5. Conclusion and future direction**

**4.6 NOD2**

2008).

development of acute (DSS-induced) and chronic (MDR1-/-) colitis (Fort *et al.*, 2005). We have further detailed the effect of a specific TLR4 antagonist monoclonal antibody on induction and resolution of acute DSS-induced colitis (Ungaro *et al.*, 2009). Consistent with the former report, our TLR4 antagonist antibody suppressed induction of acute inflammatory infiltrate by blocking the expression of several chemokines in the large intestine when administered prior to DSS treatment (Ungaro *et al.*, 2009). However, blocking TLR4 signaling after colitis was established delayed mucosal healing from the DSS-induced injury (Ungaro *et al.*, 2009). These results indicate that there are multiple roles of TLR4 signaling in the pathogenesis of DSS-induced colitis; it is responsible for acute inflammatory infiltrate through induction of chemokines during induction of colitis, but also contributes to mucosal repair during resolution of colitis. The mechanism underlying the contribution of TLR4 to mucosal repair is associated with TLR4-dependent induction of cyclooxygenase 2 and following production of prostaglandin E2 in response to mucosal damage (Fukata *et al.*, 2006). In addition, the therapeutic effect of the TLR4 antagonist antibody was not found in chronic model of T cell transfer colitis (Ungaro *et al.*, 2009). Therefore, blocking TLR4 may be beneficial in interfering with a particular aspect of colitis pathogenesis i.e., acute inflammatory infiltration, and thus combination therapies with cytoprotective agents may be required to proceed to the clinical stage using this strategy to treat colitis patients.

#### **4.4 TLR5**

Since deficiency of TLR5 results in spontaneous development of colitis, the targeting strategy of TLR5 has been focused on stimulating TLR5 signaling for treatment of colitis. There are conflicting data on the use of the TLR5 agonist flagellin as a therapeutic agent of acute DSS-induced colitis. Intraperitoneal injection of purified flagellin has been reported to be protective in acute DSS-induced colitis (Vijay-Kumar *et al.*, 2008). By contrast, rectal administration of flagellin has been reported to aggravate DSS-induced colitis (Rhee *et al.*, 2005). The discrepancy of the flagellin effect may be due to administration route (systemic vs. topical), but a recent report has suggested that flagellin-mediated aggravation of colitis is independent of TLR5 signaling as the exacerbation of DSS colitis by flagellin is also observed in TLR5-deficient mice (Ivison *et al.*, 2010). TLR5 agonist has been under preclinical stage, but further screening of its efficacy and adverse effects using different mouse models of colitis may be required in addition to clarifying the effector component of flagellin and optimization of their administration route.

#### **4.5 TLR9**

Augmentation and suppression of TLR9 signaling have been challenged in multiple mouse models of colitis. Unmethylated cytosin-guanosin dinucleotide (CpG) dinucleotides, the immunostimulatory components of bacterial DNA, are known to be a TLR9 ligand (Bauer and Wagner, 2002; Krieg *et al.*, 1995). Administration of CpG oligodeoxynucleotides (CpG-ODNs) has been shown to prevent the induction of DSS-induced colitis, but this treatment aggravates colitis when CpG-ODNs are administered after the onset of DSS colitis (Obermeier *et al.*, 2002; Obermeier *et al.*, 2003; Rachmilewitz *et al.*, 2002). The mechanism of these opposing effects of CpG-ODNs is associated with an anti-apoptotic effect and immunostimulatory effect of CpG-ODNs, respectively. Since abundant bacterial ODNs naturally exist in the colorectal lumen that play a part of inflammatory stimuli in the setting of mucosal damage, pretreatment with CpG-ODNs has been suggested to induce host immune tolerance against endogenous bacterial ODNs (Obermeier *et al.*, 2003). Preventive effect of CpG-ODNs on induction of colitis has also been shown in other models such as 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis and can be achieved by oral administration, making this strategy further relevant (Rachmilewitz *et al.*, 2002). Although CpG-ODNs are generally thought as immunostimulative, CpG-ODN stimulation suppresses TNF- and IL-1 expression in *ex vivo* colonic mucosa taken from active UC patients (Rachmilewitz *et al.*, 2006). A subset of patients with ulcerative colitis responds to Type I INF therapy through a yet unknown mechanism (Jacobs *et al.*, 2000; Madsen *et al.*, 2001; Nikolaus *et al.*, 2003). Since TLR9 signaling may induce Type I interferons which may act as immunomodulatory property during intestinal inflammation, emphasizing this aspect of TLR9 signaling may be another therapeutic target in the management of IBD (Katakura *et al.*, 2005).

Suppression of TLR9 signaling has also been beneficial for treating chronic murine colitis. Intraperitoneal as well as oral administration of adenoviral-ODN (AV-ODN) known to block the effect of bacterial CpG-ODN effectively suppressed intestinal inflammation during chronic DSS colitis (Obermeier *et al.*, 2005). The protective effect of AV-ODN was also observed in other chronic colitis models such as IL-10-/- and T cell transfer colitis models (Obermeier *et al.*, 2005). The protective effect of blocking TLR9 signaling during chronic colitis is consistent with the fact that TLR9-deficient mice have reduced severity of chronic colitis (Obermeier *et al.*, 2005). These results indicate that bacterial CpG-ODN abundantly existing in colorectal lumen is one of the indispensable factors inducing intestinal inflammation during chronic colitis.

#### **4.6 NOD2**

186 Colitis

development of acute (DSS-induced) and chronic (MDR1-/-) colitis (Fort *et al.*, 2005). We have further detailed the effect of a specific TLR4 antagonist monoclonal antibody on induction and resolution of acute DSS-induced colitis (Ungaro *et al.*, 2009). Consistent with the former report, our TLR4 antagonist antibody suppressed induction of acute inflammatory infiltrate by blocking the expression of several chemokines in the large intestine when administered prior to DSS treatment (Ungaro *et al.*, 2009). However, blocking TLR4 signaling after colitis was established delayed mucosal healing from the DSS-induced injury (Ungaro *et al.*, 2009). These results indicate that there are multiple roles of TLR4 signaling in the pathogenesis of DSS-induced colitis; it is responsible for acute inflammatory infiltrate through induction of chemokines during induction of colitis, but also contributes to mucosal repair during resolution of colitis. The mechanism underlying the contribution of TLR4 to mucosal repair is associated with TLR4-dependent induction of cyclooxygenase 2 and following production of prostaglandin E2 in response to mucosal damage (Fukata *et al.*, 2006). In addition, the therapeutic effect of the TLR4 antagonist antibody was not found in chronic model of T cell transfer colitis (Ungaro *et al.*, 2009). Therefore, blocking TLR4 may be beneficial in interfering with a particular aspect of colitis pathogenesis i.e., acute inflammatory infiltration, and thus combination therapies with cytoprotective agents may be required to proceed to the clinical stage using this

Since deficiency of TLR5 results in spontaneous development of colitis, the targeting strategy of TLR5 has been focused on stimulating TLR5 signaling for treatment of colitis. There are conflicting data on the use of the TLR5 agonist flagellin as a therapeutic agent of acute DSS-induced colitis. Intraperitoneal injection of purified flagellin has been reported to be protective in acute DSS-induced colitis (Vijay-Kumar *et al.*, 2008). By contrast, rectal administration of flagellin has been reported to aggravate DSS-induced colitis (Rhee *et al.*, 2005). The discrepancy of the flagellin effect may be due to administration route (systemic vs. topical), but a recent report has suggested that flagellin-mediated aggravation of colitis is independent of TLR5 signaling as the exacerbation of DSS colitis by flagellin is also observed in TLR5-deficient mice (Ivison *et al.*, 2010). TLR5 agonist has been under preclinical stage, but further screening of its efficacy and adverse effects using different mouse models of colitis may be required in addition to clarifying the effector component of

Augmentation and suppression of TLR9 signaling have been challenged in multiple mouse models of colitis. Unmethylated cytosin-guanosin dinucleotide (CpG) dinucleotides, the immunostimulatory components of bacterial DNA, are known to be a TLR9 ligand (Bauer and Wagner, 2002; Krieg *et al.*, 1995). Administration of CpG oligodeoxynucleotides (CpG-ODNs) has been shown to prevent the induction of DSS-induced colitis, but this treatment aggravates colitis when CpG-ODNs are administered after the onset of DSS colitis (Obermeier *et al.*, 2002; Obermeier *et al.*, 2003; Rachmilewitz *et al.*, 2002). The mechanism of these opposing effects of CpG-ODNs is associated with an anti-apoptotic effect and

strategy to treat colitis patients.

flagellin and optimization of their administration route.

**4.4 TLR5**

**4.5 TLR9**

NOD2 signaling is a plausible therapeutic target as NOD2 mutations are associated with susceptibility to Crohn's disease. The mutations are known to be loss-of-function type mutations and thus the backup of this signaling is a reasonable idea. The use of NOD2 knockout models is consistent with the human disease setting. NOD2 transgenic mice are resistant to TNBS colitis (Yang *et al.*, 2007). In addition, intraperitoneal injection of a NOD2 ligand MDP protected mice from induction of acute colitis in DSS and TNBS models (Watanabe *et al.*, 2008). The mechanism underlying MDP-mediated protection is associated with down regulation of multiple TLR responses by NOD2 stimulation, as pretreatment of dendritic cells with MDP has been shown to inhibit cytokine responses induced by TLR stimulation. One concern is that the NOD2-deficient mice introduced NOD2 construct with a CARD15 frame-shift mutation were not protected from colitis by MDP (Watanabe *et al.*, 2008). Nevertheless, development of NOD2-stimulation strategy is advantageous because MDP has already been applied to several clinical trials of cancer treatments (Meyers *et al.*, 2008).

#### **5. Conclusion and future direction**

We discussed the unique aspects of innate immunity in the context of intestinal homeostasis and the pathogenesis of colitis. It is important to understand intestinal-specific innate

Innate Immunity: A Potent Target for Management of Inflammatory Bowel Disease 189

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## *Edited by Masayuki Fukata*

Inflammation of the colon is collectively called "Colitis". Since a variety of conditions may cause colitis and its manifestations are similar among the causes, selection of the right treatment based on the correct diagnosis is important in the management of this group of illnesses. Over the last few decades, a major shift has been observed in the clinical attention to the pathogenesis of colitis from infectious to idiopathic inflammatory bowel diseases. Colitis cases that are associated with chemical therapeutics and specific pathogens such as amoeba, have become prominent in hospitalized individuals and immune deficient patients, respectively. In addition, a great deal of progress has been made in colitis research triggering the need for updating our knowledge about colitis. This book Colitis provides comprehensive information on the pathogenesis, mechanism of resolution, and treatment strategies of colitis.

Photo by selvanegra / iStock

Colitis

Colitis

*Edited by Masayuki Fukata*


3.3.4. Silage Yield (Maximum FBA) Forecasting in Area by MOD11A1-LST Values

The shapes of the FBA and RFBA curves depicted above suggest that the silage yield should be near the peak of the curve to maximize profits. Therefore, the areal silage yield could be simulated by the validated Equation (6) in 2019 with *T*LST from MOD11A1 as a substitute for *T*canopy in combination with the field FBA observations on three different days at different growth stages (Figure 16). The differences in spatial distribution were captured. Similarly, the predicted FBA increased and got closer to the maximum output when the field observation date was near harvest.

**Figure 16.** Predicting silage yield values using the NR-logistic model calibrated in 2019 based on the field observations in three different days: (**a**) 2017/7/16; (**b**) 2017/8/10; (**c**) 2017/8/31.

According to the summarized data in Table 6, the model simulation showed only a slight difference on the observation day (31 August) in the experimental station. This is not surprising given that the measured silage yield was recorded on the same date. The results on 10 August still produced a satisfying accuracy, which indicates that it might be an appropriate harvest date for silage yield in view of various factors. There were no measurements of silage yield in the three subareas, so the comparison could not be analyzed and displayed between observations and predictions in the region.

**Table 6.** Comparisons between the forecasted silage yields (maximum FBAs) based on the field observation 1 in different days and experimental station.

1 Sample numbers in experimental station are 13 and 30. 2 The data were measured on 2017/8/31.

#### *3.4. Verification in Jiefangzha Sub-Irrigation District*

As mentioned in Section 2.3, an LST map (30 m) of Jiefangzha showed the results fused from Landsat 8 and MOD11A1 images by the ESTARFM algorithm. Scatters of the fused LSTs and Landsat-LSTs were evenly distributed on both sides of the 1:1 line, showing the fused LSTs were relatively reliable (Figure 17a). The fused LSTs were also compared with the Tc recorded at 11:30 a.m., in which the values of *R*2 (0.547), *RMSE* (3.96◦C), and *d* (0.79) indicate good consistency between the observed values and the fused ones (Figure 17b). The grain yield forecasting map was constructed by employing the N-Logistic model described previously with the fused LSTs (Figure 18).

**Figure 17.** Regressions for accuracy evaluation of the fused LST: (**a**) the fused LST vs. the inversed values from Landsat 8; (**b**) the fused LST vs. the observed Tc in experimental station in 2016.

**Figure 18.** Forecasting results of grain yield in the Jiefangzha sub-irrigation district using the 2019 calibrated model based on the field observations in four different days: (**a**) 2016/7/4; (**b**)2016/7/21; (**c**) 2016/8/4; (**d**) 2016/8/26.

Figure 18 shows the spatial grain yield estimates retrieved using DBA measured on four acquisition dates that include (a) 4 July, (b) 21 July, (c) 4 August, and (d) 26 August in 2016 based on the model calibrated in 2019. The predicted yield showed a significant upward trend as the acquisition date of DBA approached harvest. Meanwhile, the comparisons between the predicted and measured yield in situ were conducted with statistical parameters as evaluation metrics of accuracy (Table 7). The predicted yields coincide with the measured ones with *RE* values, ranging from −16.14% to 9.84%, which confirms that assimilating once-measured data into the model can produce a great estimate of grain yield. Comparatively speaking, DBA assimilation closer to the harvest date allows for a more accurate prediction of yield except for the results based on the data on 21 July, which may be caused by the irrigation measures or the model parameters. Assimilating DBA measured on 4 July also provides a reliable result, albeit with a slightly lower *R*2. Such a result proves the feasibility of the approach for early yield forecasting in other large areas though the optimal date of sampling still needs to be explored and determined further.

**Table 7.** Comparisons between the forecasted grain yields in the Jiefangzha sub-irrigation district based on field observation in different days and the measurements 1.

1. The data were measured on 2016//9/15.

#### **4. Discussion**

According to results reported above, our data on maize biomass show that the Tc should be included as a valuable proxy of other independent variables in models because the Tc can be recognized as LST in large regions covered by maize that can be derived from remote sensing data [50,51].

This study demonstrated the benefits of integrating remote sensing LST into crop growth models in combination with once-observed values (DBA or FBA) to enhance yield prediction. The introduction of remote sensing LST to the logistic models offers effective information about regional crop status, overcoming the limitation of model in regional application. Moreover, the ground crop truth was profitably considered by using oncemeasured observations as the drive of the yield forecasting model. Another point worth emphasizing is that the subsequent consequences, after undertaking necessary agronomic measures based on the yield forecasting results, can be assessed with just one observation as input again because the effect on the crop can be reflected by LST and measured biomass. All in all, LST as a key factor for characterizing field drought [52] provides an intuitive basis to determine the water shortage of crops in large regions. This offers a solid theoretical basis for the synergistic prediction of future crop drought and yield evaluation in combination using remote sensing technology.

In addition, to obtain a more robust estimate of maize yield, data fusion technology may be recommended to improve the spatio-temporal resolution of LST [40]. Unfortunately, this method is unavailable due to the scarce Landsat images caused by the cloud cover on most days in maize growing season in Changchun. Our attempt in the Jiefangzha sub-irrigation district indicated that the data fusion technology improved the performance of spatial variation of grain yield forecasting under available conditions of suitable Landsat images. It follows that high-resolution satellite imagery such as Sentinel-2 might be a further exploration tool for improving yield forecasting precision.

The values of *t*m in models of RDBA and RFBA are key input data, which can be obtained for different hydrological years by analysis of the local yearly rainfall. When the hydrological year of the current growing season is estimated, the N-logistic model and the NR-logistic model can be utilized with the corresponding value of *t*m. To obtain the values of *y*Dm and *y*Fm, the data of DBA and FBA should be collected in field at least once during the maize growing period. Furthermore, the growth patterns of DBA and FBA can be simulated when the models are set up and calibrated. HI, an empirical value describing the relationship between DBA and grain yield, offers an opportunity to evaluate grain yield. By combining the growth curves for DBA and FBA with the future market demands, maize can be flexibly harvested as silage or grain during the growing season.

Apparently, the early yield forecasting accuracy varies depending on the growth stages, which may be caused by many factors. Firstly, the retrieval of *t*m is pivotal, as mentioned above. Secondly, the quality of remote sensing images obtained over time varies slightly, which can introduce some uncertainty into the yield forecasting results. Thirdly, the date of acquiring DBA or FBA is particularly critical for yield forecasting accuracy. As shown in Tables 3 and 7, the optimal predicting date is clearly different in different areas. In Changchun, the optimal date at which the DBA provides more accurate yield forecasting is approximately 38 days (10 August) ahead of harvest. As for the Jiefangzha sub-irrigation district, the results on 26 August produced a better prediction of yield, relatively speaking. Furthermore, the latter is better than the former in statistical parameters. This phenomenon may be caused by a variety of factors. For instance, a rare downpour amounting to more than 160 mm of rain occurred in Changchun on 21 July 2017, which may destroy plants and influence yield forecasting results. In addition, the area of Changchun is larger than the Jiefangzha sub-irrigation district, which may have an impact on the results. Taking the three results into account (Tables 3, 6 and 7), it is suggested to forecast yield by using the field data (DBA or FBA) measured at the middle growth period (early August). Furthermore, it is necessary to determine the optimal date by taking into consideration multiple factors.

Another noteworthy point about the results presented here is that the field soil water content was sufficient or at least was not in water deficit in the research area. The FBA, RFBA, and silage yield may be impacted by the crop and soil water conditions. Future efforts to prove model generality should include examination of changes in the model parameters for different levels of plant and field soil moisture.


**Daniela Pantusa 1,\*, Felice D'Alessandro 1, Luigia Riefolo 2, Francesca Principato 3 and Giuseppe Roberto Tomasicchio 1**

Received: 4 July 2018; Accepted: 5 September 2018; Published: 10 September 2018

**Abstract:** The coastal vulnerability index (CVI) is a popular index in literature to assess the coastal vulnerability of climate change. The present paper proposes a CVI formulation to make it suitable for the Mediterranean coasts; the formulation considers ten variables divided into three typological groups: geological; physical process and vegetation. In particular, the geological variables are: geomorphology; shoreline erosion/accretion rates; coastal slope; emerged beach width and dune width. The physical process variables are relative sea-level change; mean significant wave height and mean tide range. The vegetation variables are width of vegetation behind the beach and posidonia oceanica. The first application of the proposed index was carried out for a stretch of the Apulia region coast, in the south of Italy; this application allowed to (i) identify the transects most vulnerable to sea level rise, storm surges and waves action and (ii) consider the usefulness of the index as a tool for orientation in planning strategies. For the case study presented in this work, the most influential variables in determining CVI are dune width and geomorphology. The transects that present a very high vulnerability are characterized by sandy and narrow beaches (without dunes and vegetation) and by the absence of Posidonia oceanica.

**Keywords:** coastal vulnerability index; climate change; sea level rise; storm surges; waves action; Mediterranean coasts