Masayuki Fukata

TLR Signaling in the Gut in Health and Disease

The human intestine has evolved in the presence of diverse enteric microflora. TLRs convert the recognition of pathogen-associated molecules in the gut into signals for anti-microbial peptide expression, barrier fortification, and proliferation of epithelial cells. Healing of injured intestinal epithelium and clearance of intramucosal bacteria require the presence of intact TLR signaling. Nucleotide oligomerization domain (Nod)1 and Nod2 are additional pattern recognition receptors that are required for defense against invasive enteric pathogens. Through spatial and functional localization of TLR and Nod molecules, the normal gut maintains a state of controlled inflammation. By contrast, patients with inflammatory bowel disease demonstrate inflammation in response to the normal flora. A subset of these patients carry polymorphisms in TLR and CARD15/NOD2 genes. A better understanding of the delicate regulation of TLR and Nod molecules in the gut may lead to improved treatment for enteric infections and idiopathic inflammatory bowel diseases.

β-Defensin-2 Expression Is Regulated by TLR Signaling in Intestinal Epithelial Cells

The intestinal epithelium serves as a barrier to the intestinal flora. In response to pathogens, intestinal epithelial cells (IEC) secrete proinflammatory cytokines. To aid in defense against bacteria, IEC also secrete antimicrobial peptides, termed defensins. The aim of our studies was to understand the role of TLR signaling in regulation of β-defensin expression by IEC. The effect of LPS and peptidoglycan on β-defensin-2 expression was examined in IEC lines constitutively or transgenically expressing TLRs. Regulation of β-defensin-2 was assessed using promoter-reporter constructs of the human β-defensin-2 gene. LPS and peptidoglycan stimulated β-defensin-2 promoter activation in a TLR4- and TLR2-dependent manner, respectively. A mutation in the NF-κB or AP-1 site within the β-defensin-2 promoter abrogated this response. In addition, inhibition of Jun kinase prevents up-regulation of β-defensin-2 protein expression in response to LPS. IEC respond to pathogen-associated molecular patterns with expression of the antimicrobial peptide β-defensin-2. This mechanism may protect the intestinal epithelium from pathogen invasion and from potential invaders among the commensal flora.

Toll-like receptors (TLRs) and Nod-like receptors (NLRs) in inflammatory disorders

Toll-like receptors (TLRs) and Nod-like receptors (NLRs) are two major forms of innate immune sensors, which provide immediate responses against pathogenic invasion or tissue injury. Activation of these sensors induces the recruitment of innate immune cells such as macrophages and neutrophils, initiates tissue repair processes, and results in adaptive immune activation. Abnormalities in any of these innate sensor-mediated processes may cause excessive inflammation due to either hyper responsive innate immune signaling or sustained compensatory adaptive immune activation. Recent gene association studies appear to reveal strong associations of NLR gene mutations and development of several idiopathic inflammatory disorders. In contrast, TLR polymorphisms are less often associated with inflammatory disorders. Nevertheless, TLRs are up-regulated in the affected tissue of most inflammatory disorders, suggesting TLR signaling is involved in the pathogenesis of chronic and/or idiopathic inflammatory disorders. NLR signaling results in the formation of a molecular scaffold complex (termed an inflammasome) and orchestrates with TLRs to induce IL-1beta and IL-18, both of which are important mediators in the majority of inflammatory disorders. Therefore, understanding the roles of TLRs and NLRs in the pathogenesis of chronic and idiopathic inflammatory disorders may provide novel targets for the prevention and/or treatment of many common and uncommon diseases involving inflammation.

Toll-Like Receptor Signaling in Small Intestinal Epithelium Promotes B-Cell Recruitment and IgA Production in Lamina Propria

BACKGROUND & AIMS Several lines of evidence support a role for Toll-like receptor (TLR) signaling to protect the intestine from pathogenic infection. We hypothesized that TLR signaling at the level of the intestinal epithelium is critical for mucosal immune responses. METHODS We generated transgenic mice that express a constitutively active form of TLR4 in the intestinal epithelium (V-TLR4 mice). Lamina propria cellularity was evaluated by immunostaining and flow cytometry. Immunoglobulin (Ig) A levels in the stool and serum were measured by enzyme-linked immunosorbent assay. Chemokine and cytokine expression were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS V-TLR4 transgenic mice reproduced normally and had a normal life span. Constitutive activity of TLR4 in the intestinal epithelium promoted recruitment of B cells and an increase in fecal IgA levels. Intestinal epithelial cells of V-TLR4 mice expressed higher levels of CCL20 and CCL28, chemokines known to be involved in B-cell recruitment, and of a proliferation-inducing ligand (APRIL), a cytokine that promotes T-cell-independent class switching of B cells to IgA. The changes in B-cell numbers and IgA levels were blocked by simultaneous expression in intestinal epithelial cells of M3, a herpes virus protein that binds and inhibits multiple chemokines. CONCLUSIONS TLR signaling in the intestinal epithelial cells significantly elevated the production of IgA in the intestine. This effect was mediated by TLR-induced expression of a specific set of chemokines and cytokines that promoted both recruitment of B cells into the lamina propria and IgA class switching of B cells.

Role of Toll-like receptors in gastrointestinal malignancies

Mounting evidence supports the tenet that innate immune responses to luminal microbes participate in the development of gastrointestinal malignancies. The gastrointestinal tract is relatively unique in that it has evolved in the presence of diverse enteric microflora. Intestinal flora is required to develop a normal adaptive immune response in the periphery. With the characterization of the innate immune system, we have begun to understand the adaptations the intestine has made to the microbiota. The interaction between the microbiota and the intestinal mucosa through Toll-like receptors (TLRs) is required to maintain intestinal homeostasis. In particular, intestinal epithelial cells and lamina propria mononuclear cells such as antigen-presenting cells and T cells must respond to breaches in the mucosal barrier by activating TLR-dependent pathways that result in increased epithelial proliferation, wound healing and recruitment of acute inflammatory cells. In the setting of chronic inflammation such as Helicobacter pylori (H. pylori) infection in the stomach or idiopathic inflammatory bowel disease, the process of repair may eventually result in carcinogenesis. The following review highlights human and animal data that support a role for innate immune responses and TLRs specifically in promoting gastrointestinal malignancies. Candidate pathways linking TLRs to gastrointestinal malignancies include activation of nuclear factor-κB and cyclooxygenase-2. Studying the link between innate immune signaling and gastrointestinal malignancies offers the possibility to identify novel ways to both prevent and treat gastrointestinal cancer.

A novel Toll-like receptor 4 antagonist antibody ameliorates inflammation but impairs mucosal healing in murine colitis

Dysregulated innate immune responses to commensal bacteria contribute to the development of inflammatory bowel disease (IBD). TLR4 is overexpressed in the intestinal mucosa of IBD patients and may contribute to uncontrolled inflammation. However, TLR4 is also an important mediator of intestinal repair. The aim of this study is to examine the effect of a TLR4 antagonist on inflammation and intestinal repair in two murine models of IBD. Colitis was induced in C57BL/6J mice with dextran sodium sulfate (DSS) or by transferring CD45Rb(hi) T cells into RAG1-/- mice. An antibody (Ab) against the TLR4/MD-2 complex or isotype control Ab was administered intraperitoneally during DSS treatment, recovery from DSS colitis, or induction of colitis in RAG1-/- mice. Colitis severity was assessed by disease activity index (DAI) and histology. The effect of the Ab on the inflammatory infiltrate was determined by cell isolation and immunohistochemistry. Mucosal expression of inflammatory mediators was analyzed by real-time PCR and ELISA. Blocking TLR4 at the beginning of DSS administration delayed the development of colitis with significantly lower DAI scores. Anti-TLR4 Ab treatment decreased macrophage and dendritic cell infiltrate and reduced mucosal expression of CCL2, CCL20, TNF-alpha, and IL-6. Anti-TLR4 Ab treatment during recovery from DSS colitis resulted in defective mucosal healing with lower expression of COX-2, PGE(2), and amphiregulin. In contrast, TLR4 blockade had minimal efficacy in ameliorating inflammation in the adoptive transfer model of chronic colitis. Our findings suggest that anti-TLR4 therapy may decrease inflammation in IBD but may also interfere with colonic mucosal healing.

The myeloid differentiation factor 88 (MyD88) is required for CD4 + T cell effector function in a murine model of inflammatory bowel disease

Abnormal T cell responses to commensal bacteria are involved in the pathogenesis of inflammatory bowel disease. MyD88 is an essential signal transducer for TLRs in response to the microflora. We hypothesized that TLR signaling via MyD88 was important for effector T cell responses in the intestine. TLR expression on murine T cells was examined by flow cytometry. CD4+CD45Rbhigh T cells and/or CD4+CD45RblowCD25+ regulatory T cells were isolated and adoptively transferred to RAG1−/− mice. Colitis was assessed by changes in body weight and histology score. Cytokine production was assessed by ELISA. In vitro proliferation of T cells was assessed by [3H]thymidine assay. In vivo proliferation of T cells was assessed by BrdU and CFSE labeling. CD4+CD45Rbhigh T cells expressed TLR2, TLR4, TLR9, and TLR3, and TLR ligands could act as costimulatory molecules. MyD88−/− CD4+ T cells showed decreased proliferation compared with WT CD4+ T cells both in vivo and in vitro. CD4+CD45Rbhigh T cells from MyD88−/− mice did not induce wasting disease when transferred into RAG1−/− recipients. Lamina propria CD4+ T cell expression of IL-2 and IL-17 and colonic expression of IL-6 and IL-23 were significantly lower in mice receiving MyD88−/− cells than mice receiving WT cells. In vitro, MyD88−/− T cells were blunted in their ability to secrete IL-17 but not IFN-γ. Absence of MyD88 in CD4+CD45Rbhigh cells results in defective T cell function, especially Th17 differentiation. These results suggest a role for TLR signaling by T cells in the development of inflammatory bowel disease.

Constitutive activation of epithelial TLR4 augments inflammatory responses to mucosal injury and drives colitis-associated tumorigenesis

Background: Chronic intestinal inflammation culminates in cancer and a link to Toll‐like receptor‐4 (TLR4) has been suggested by our observation that TLR4 deficiency prevents colitis‐associated neoplasia. In the current study we address the effect of the aberrant activation of epithelial TLR4 on induction of colitis and colitis‐associated tumor development. We take a translational approach to address the consequences of increased TLR signaling in the intestinal mucosa. Methods: Mice transgenic for a constitutively active TLR4 under the intestine‐specific villin promoter (villin‐TLR4 mice) were treated with dextran sodium sulfate (DSS) for acute colitis and azoxymethane (AOM)‐DSS TLR4 expression was analyzed by immunohistochemistry in colonic tissue from patients with ulcerative colitis (UC) and UC‐associated cancer. The effect of an antagonist TLR4 antibody (Ab) was tested in prevention of colitis‐associated neoplasia in the AOM‐DSS model. Results: Villin‐TLR4 mice were highly susceptible to both acute colitis and colitis‐associated neoplasia. Villin‐TLR4 mice had increased epithelial expression of COX‐2 and mucosal PGE2 production at baseline. Increased severity of colitis in villin‐TLR4 mice was characterized by enhanced expression of inflammatory mediators and increased neutrophilic infiltration. In human UC samples, TLR4 expression was upregulated in almost all colitis‐associated cancer and progressively increased with grade of dysplasia. As a proof of principle, a TLR4/MD‐2 antagonist antibody inhibited colitis‐associated neoplasia in the mouse model. Conclusions: Our results show that regulation of TLRs can affect the outcome of both acute colitis and its consequences, cancer. Targeting TLR4 and other TLRs may ultimately play a role in prevention or treatment of colitis‐associated cancer. (Inflamm Bowel Dis 2010;)

Innate immunity in the small intestine

Purpose of review In this article, we provide an update of the latest findings related to the innate immunity in the small intestine. In particular, we will focus on innate immune receptors and antimicrobial strategies that keep luminal bacteria and viral pathogens under control to avoid mucosal damage. These strategies include IgA secretion and antimicrobial peptides produced by Paneth cells, and downregulation or anergy of the innate immune receptors themselves. Recent findings Pattern-recognition receptors are the main target in the study of innate immunity in the intestinal mucosa due to their involvement in the regulation of host–commensal interactions. It has been shown that TLR5-deficient mice develop metabolic syndrome and have altered intestinal microbiota. On the contrary, NOD2 has been associated with the activation of autophagy and the inhibition of TLR4. Moreover, NOD2 has been described to be essential to keep a feedback loop in the host–commensal homeostasis, through the kinase Rip-2. Summary Innate immunity in the small intestine is mainly characterized by IgA secretion and Paneth cell antimicrobial function. In both cases pattern-recognition receptors, Toll-like receptors and nucleotide-binding and oligomerization domain-like receptors, are involved. A better understanding of the innate immunity in the small intestine would provide valuable information to develop vaccines against pathogens.

Innate immune signaling by Toll-like receptor-4 (TLR4) shapes the inflammatory microenvironment in colitis-associated tumors

Background: Patients with ulcerative colitis are at increased risk for developing colorectal cancer. We have shown that Toll‐like receptor‐4 (TLR4) is overexpressed in human colitis‐associated cancer (CAC) and that mice deficient in TLR4 are markedly protected against colitis‐associated neoplasia. We wished to elucidate the specific contributions of TLR4 signaling by myeloid cells and colonic epithelial cells (CEC) in colitis‐associated tumorigenesis. Methods: TLR4‐deficient mice or wildtype littermates (WT) were transplanted with bone marrow (BM) cells: TLR4−/− BM→WT mice (TLR4‐expressing CEC) and WT BM→TLR4−/− mice (TLR4‐expressing myeloid cells). Colitis‐associated neoplasia was induced by azoxymethane (AOM 7.3 mg/kg) injection and 2 cycles of dextran sodium sulfate (DSS) treatment. Results: The number and size of dysplastic lesions were greater in TLR4−/− BM→WT mice than in WT BM→TLR4−/− mice (P < 0.005). Histologically, TLR4−/− BM→WT mice had greater numbers of mucosal neutrophils and macrophages compared to WT BM→TLR4−/− mice. The chemokines KC and CCL2, important in recruitment of neutrophils and macrophages, respectively, were induced in mice expressing TLR4 in CEC rather than the myeloid compartment. The lamina propria infiltrate of mice expressing TLR4 in CEC was characterized by macrophages expressing Cox‐2. Moreover, mice expressing TLR4 in CEC rather than the myeloid compartment had increased production of amphiregulin and EGFR activation. Conclusions: These findings indicate that TLR4 signaling on CEC is necessary for recruitment and activation of Cox‐2‐expressing macrophages and increasing the number and size of dysplastic lesions. Our results implicate innate immune signaling on CEC as a key regulator of a tumor‐promoting microenvironment.