Keith J. Breglio

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.

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.

Toll-like receptor 4 differentially regulates epidermal growth factor-related growth factors in response to intestinal mucosal injury

Epiregulin (EPI) and amphiregulin (AR) are epidermal growth factor receptor (EGFR) ligands implicated in mucosal repair and tumorigenesis. We have shown that Toll-like receptor 4 (TLR4) induces intestinal epithelial cell (IEC) proliferation by activating EGFR through AR expression. We examined whether TLR4 differentially regulates expression of EGFR ligands in response to mucosal injury. The human IEC line SW480 was examined expression of EGFR ligands, EGFR phosphorylation, and proliferation in response to lipopolysaccharide (LPS). Small-interfering RNA (siRNA) was used to block TLR4. Neutralizing antibodies to EGFR ligands were used to examine inhibition of LPS-dependent EGFR activation. Acute colitis and recovery were examined in the mice given 2.5% dextran sodium sulfate (DSS). Colonic secretion of EPI and AR was analyzed by enzyme-linked immunosorbent assay. LPS selectively induces EPI and AR but not other EGFR ligands. LPS induced early EPI mRNA expression between 30 min and 24 h. The neutralizing antibodies to EPI and AR prevented activation of EGFR by LPS. LPS induces IEC proliferation (200%, P=0.01) in 24 h but blocking EPI and AR significantly decreased proliferation. In vivo, mucosal EPI and AR expression are significantly decreased in TLR4−/− mice (P=0.02) compared to wild-type mice during acute colitis. EPI and AR exhibit different kinetics in response to mucosal damage: EPI expression is upregulated acutely at day 7 of DSS, but falls during recovery at day 14. By contrast, a sustained upregulation of AR expression is seen during mucosal injury and repair. We show that TLR4 regulates EPI and AR expression and that both these EGFR ligands are necessary for optimal proliferation of IEC. The diverse kinetics of EPI and AR expression suggest that they function in distinct roles with respect to acute injury vs repair. Our results highlight the role of bacterial sensing for IEC homeostasis and may lead to targeted therapy for mucosal healing and prevention of tumorigenesis.

The role of prostaglandin E2 (PGE 2) in toll-like receptor 4 (TLR4)-mediated colitis-associated neoplasia.

BackgroundWe have previously found that TLR4-deficient (TLR4-/-) mice demonstrate decreased expression of mucosal PGE 2 and are protected against colitis-associated neoplasia. However, it is still unclear whether PGE 2 is the central factor downstream of TLR4 signaling that promotes intestinal tumorigenesis. To further elucidate critical downstream pathways involving TLR4-mediated intestinal tumorigenesis, we examined the effects of exogenously administered PGE 2 in TLR4-/- mice to see if PGE 2 bypasses the protection from colitis-associated tumorigenesis.MethodMouse colitis-associated neoplasia was induced by azoxymethane (AOM) injection followed by two cycles of dextran sodium sulfate (DSS) treatment. Two different doses of PGE 2 (high dose group, 200 μg, n = 8; and low dose group, 100 μg, n = 6) were administered daily during recovery period of colitis by gavage feeding. Another group was given PGE 2 during DSS treatment (200 μg, n = 5). Inflammation and dysplasia were assessed histologically. Mucosal Cox-2 and amphiregulin (AR) expression, prostanoid synthesis, and EGFR activation were analyzed.ResultsIn control mice treated with PBS, the average number of tumors was greater in WT mice (n = 13) than in TLR4-/- mice (n = 7). High dose but not low dose PGE 2 treatment caused an increase in epithelial proliferation. 28.6% of PBS-treated TLR4-/- mice developed dysplasia (tumors/animal: 0.4 ± 0.2). By contrast, 75.0% (tumors/animal: 1.5 ± 1.2, P < 0.05) of the high dose group and 33.3% (tumors/animal: 0.3 ± 0.5) of the low dose group developed dysplasia in TLR4-/- mice. Tumor size was also increased by high dose PGE 2 treatment. Endogenous prostanoid synthesis was differentially affected by PGE 2 treatment during acute and recovery phases of colitis. Exogenous administration of PGE 2 increased colitis-associated tumorigenesis but this only occurred during the recovery phase. Lastly, PGE 2 treatment increased mucosal expression of AR and Cox-2, thus inducing EGFR activation and forming a positive feedback mechanism to amplify mucosal Cox-2.ConclusionsThese results highlight the importance of PGE 2 as a central downstream molecule involving TLR4-mediated intestinal tumorigenesis.

Health maintenance and vaccination strategies in pediatric inflammatory bowel disease.

Abstract: The development of inflammatory bowel disease during childhood and adolescence is not uncommon. As advances in medical therapies continue to emerge, so does the recognition that treatment goals must include achieving and maintaining childhood wellness. Preservation of normal linear growth, development and psychosocial wellbeing along with appropriate vaccination and preventative care strategies are elements critical to assuring the complete health of the pediatric patient who is affected by inflammatory bowel disease.

574 Transgenic Epithelial Expression of TLR4 Results in Duodenal Adenoma and Colitis-Associated Neoplasia

Background & Aims: Recently, the role of stromal cells such as inflammatory cells in tumor development has received much attention. Nuclear factor kappa B (NF-κB) is an important transcription factor in various biological processes and is involved in carcinogenesis. The exact mechanism how NF-κB activation contributes to inflammation-associated carcinogenesis is not clear.We conducted this study to investigate how colon cancer cells induced inflammatory response to link tumor growth. Methods: Mouse bone marrow macrophages (BMDMs) (wildtype, IKKβ-/-, TLR2-/-, TLR4-/and MyD88-/-), J774.1 and THP-1 cells were stimulated with the culture supernatant of several colon cancer cell lines. NF-κB activation was evaluated by EMSA, immunoblot of phosphorylated-IκBα and IκBα. Macrophage invasion assay were performed using chemotaxis chamber. In tumors of subcutaneously transplantation of the cells into nude mice, macrophage infiltration and IL-6 expression which is regulated NFκB activation were evaluated by immunostaining . Results: Six out of 10 (60%) of the supernatants prepared from colon cancer cells activated NF-κB in BMDMs and THP-1 cells (NF-κB activating colon cancer cells). This activation was observed in macrophages from wild-type and TLR4-/-, but not in TLR2-/-, MyD88-/and IKKβ-/-, suggesting that the activation is dependent on TLR2, MyD88 and IKKβ. Supernatant from NF-κB activating colon cancer cells also induces chemotaxis. In transplanted tumor tissue, macrophages accumulation and IL-6 expression were markedly increased in tumors of NF-κB activating colon cancer cells compared with non-activating ones. In addition, NF-κB activating colon cancer cells grewmore rapidly and aggressively than non-activating ones inmice. Surprisingly, NF-κB activating colon cancer cells had constitutive NF-κB activity. Conclusions: Colon cancer-derived certain factors induce accumulation of macrophages and activate NF-κB in macrophages through TLR2 and MyD88, eventually link tumor growth.

S1649 The Role of Prostaglandin E2 (PGE2) in Toll-Like Receptor-4 (TLR4)-Mediated Colitis-Associated Neoplasia

Background We have previously found that TLR4-deficient (TLR4-/-) mice demonstrate decreased expression of mucosal PGE 2 and are protected against colitis-associated neoplasia. However, it is still unclear whether PGE 2 is the central factor downstream of TLR4 signaling that promotes intestinal tumorigenesis. To further elucidate critical downstream pathways involving TLR4-mediated intestinal tumorigenesis, we examined the effects of exogenously administered PGE 2 in TLR4-/- mice to see if PGE 2 bypasses the protection from colitis-associated tumorigenesis.