Ryan Ichikawa

Cathelicidin Signaling via the Toll-like Receptor Protects Against Colitis in Mice

BACKGROUND & AIMS Cathelicidin (encoded by Camp) is an antimicrobial peptide in the innate immune system. We examined whether macrophages express cathelicidin in colons of mice with experimental colitis and patients with inflammatory bowel disease, and we investigated its signaling mechanisms. METHODS Quantitative, real-time, reverse-transcription polymerase chain reaction (PCR), bacterial 16S PCR, immunofluorescence, and small interfering RNA (siRNA) analyses were performed. Colitis was induced in mice using dextran sulfate sodium (DSS); levels of cathelicidin were measured in human primary monocytes. RESULTS Expression of cathelicidin increased in the inflamed colonic mucosa of mice with DSS-induced colitis compared with controls. Cathelicidin expression localized to mucosal macrophages in inflamed colon tissues of patients and mice. Exposure of human primary monocytes to Escherichia coli DNA induced expression of Camp messenger RNA, which required signaling by extracellular signal-regulated kinase (ERK); expression was reduced by siRNAs against Toll-like receptor (TLR)9 and MyD88. Intracolonic administration of bacterial DNA to wild-type mice induced expression of cathelicidin in colons of control mice and mice with DSS-induced colitis. Colon expression of cathelicidin was significantly reduced in TLR9(-/-) mice with DSS-induced colitis. Compared with wild-type mice, Camp(-/-) mice developed a more severe form of DSS-induced colitis, particularly after intracolonic administration of E coli DNA. Expression of cathelicidin from bone marrow-derived immune cells regulated DSS induction of colitis in transplantation studies in mice. CONCLUSIONS Cathelicidin protects against induction of colitis in mice. Increased expression of cathelicidin in monocytes and experimental models of colitis involves activation of TLR9-ERK signaling by bacterial DNA. This pathway might be involved in the pathogenesis of ulcerative colitis.

The antimicrobial peptide cathelicidin modulates Clostridium difficile-associated colitis and toxin A-mediated enteritis in mice

Background Clostridium difficile mediates intestinal inflammation by releasing toxin A (TxA), a potent enterotoxin. Cathelicidins (Camp as gene name, LL-37 peptide in humans and mCRAMP peptide in mice) are antibacterial peptides that also posses anti-inflammatory properties. Objectives To determine the role of cathelicidins in models of Clostridium difficile infection and TxA-mediated ileal inflammation and cultured human primary monocytes. Design Wild-type (WT) and mCRAMP-deficient (Camp−/−) mice were treated with an antibiotic mixture and infected orally with C difficile. Some mice were intracolonically given mCRAMP daily for 3 days. Ileal loops were also prepared in WT mice and treated with either saline or TxA and incubated for 4 h, while some TxA-treated loops were injected with mCRAMP. Results Intracolonic mCRAMP administration to C difficile-infected WT mice showed significantly reduced colonic histology damage, apoptosis, tissue myeloperoxidase (MPO) and tumour necrosis factor (TNF)α levels. Ileal mCRAMP treatment also significantly reduced histology damage, tissue apoptosis, MPO and TNFα levels in TxA-exposed ileal loops. WT and Camp−/− mice exhibited similar intestinal responses in both models, implying that C difficile/TxA-induced endogenous cathelicidin may be insufficient to modulate C difficile/TxA-mediated intestinal inflammation. Both LL-37 and mCRAMP also significantly reduced TxA-induced TNFα secretion via inhibition of NF-κB phosphorylation. Endogenous cathelicidin failed to control C difficile and/or toxin A-mediated inflammation and even intestinal cathelicidin expression was increased in humans and mice. Conclusion Exogenous cathelicidin modulates C difficile colitis by inhibiting TxA-associated intestinal inflammation. Cathelicidin administration may be a new anti-inflammatory treatment for C difficile toxin-associated disease.

Inhibition of a novel fibrogenic factor Tl1a reverses established colonic fibrosis

Intestinal fibrostenosis is among the hallmarks of severe Crohn’s disease. Patients with certain TNFSF15 (gene name for TL1A) variants over-express TL1A and have a higher risk of developing strictures in the small intestine. In addition, sustained Tl1a expression in mice leads to small and large intestinal fibrostenosis under colitogenic conditions. The aim of this study was to determine whether established murine colonic fibrosis could be reversed with Tl1a antibody (Ab). Treatment with neutralizing Tl1a Ab reversed colonic fibrosis back to the original pre-inflamed levels, potentially as a result of lowered expression of connective tissue growth factor, Il31Ra, transforming growth factor β1 and insulin-like growth factor-1. In addition, blocking Tl1a function by either neutralizing Tl1a Ab or deletion of death domain receptor 3 (Dr3) reduced the number of fibroblasts and myofibroblasts, the primary cell types that mediate tissue fibrosis. Primary intestinal myofibroblasts expressed Dr3 and functionally responded to direct Tl1a signaling by increasing collagen and Il31Ra expression. These data demonstrated a direct role for TL1A–DR3 signaling in tissue fibrosis and that modulation of TL1A–DR3 signaling could inhibit gut fibrosis.

Antifibrogenic Effects of the Antimicrobial Peptide Cathelicidin in Murine Colitis-Associated Fibrosis

Background & Aims Cathelicidin (LL-37 in human and mCRAMP in mice) represents a family of endogenous antimicrobial peptides with anti-inflammatory effects. LL-37 also suppresses collagen synthesis, an important fibrotic response, in dermal fibroblasts. Here, we determined whether exogenous cathelicidin administration modulates intestinal fibrosis in two animal models of intestinal inflammation and in human colonic fibroblasts. Methods C57BL/6J mice (n = 6 per group) were administered intracolonically with a trinitrobenzene sulphonic acid (TNBS) enema to induce chronic (6–7 weeks) colitis with fibrosis. We administered mCRAMP peptide (5 mg/kg every 3 day, week 5–7) or cathelicidin gene (Camp)-expressing lentivirus (107 infectious units week 4) intracolonically or intravenously, respectively. We then infected 129Sv/J mice with Salmonella typhimurium orally to induce cecal inflammation with fibrosis. Camp-expressing lentivirus (107 infectious units day 11) was administered intravenously. Results TNBS-induced chronic colitis was associated with increased colonic collagen (col1a2) mRNA expression. Intracolonic cathelicidin (mCRAMP peptide) administration or intravenous delivery of lentivirus-overexpressing cathelicidin gene significantly reduced colonic col1a2 mRNA expression in TNBS-exposed mice compared with vehicle administration. Salmonella infection also caused increased cecal inflammation associated with collagen (col1a2) mRNA expression that was prevented by intravenous delivery of Camp-expressing lentivirus. Exposure of human primary intestinal fibroblasts and human colonic CCD-18Co fibroblasts to transforming growth factor-β1 (TGF-β1) and/or insulin-like growth factor 1 induced collagen protein and mRNA expression, which was reduced by LL-37 (3–5 μM) through a MAP kinase-dependent mechanism. Conclusions Cathelicidin can reverse intestinal fibrosis by directly inhibiting collagen synthesis in colonic fibroblasts.

Cathelicidin suppresses colon cancer development by inhibition of cancer associated fibroblasts

Background Cathelicidin (LL-37 in humans and mCRAMP in mice) represents a family of endogenous antimicrobial and anti-inflammatory peptides. Cancer-associated fibroblasts can promote the proliferation of colon cancer cells and growth of colon cancer tumors. Methods We examined the role of cathelicidin in the development of colon cancer, using subcutaneous human HT-29 colon-cancer-cell-derived tumor model in nude mice and azoxymethane- and dextran sulfate-mediated colon cancer model in C57BL/6 mice. We also determined the indirect antitumoral mechanism of cathelicidin via the inhibition of epithelial–mesenchymal transition (EMT) of colon cancer cells and fibroblast-supported colon cancer cell proliferation. Results Intravenous administration of cathelicidin expressing adeno-associated virus significantly reduced the size of tumors, tumor-derived collagen expression, and tumor-derived fibroblast expression in HT-29-derived subcutaneous tumors in nude mice. Enema administration of the mouse cathelicidin peptide significantly reduced the size and number of colonic tumors in azoxymethane- and dextran sulfate-treated mice without inducing apoptosis in tumors and the adjacent normal colonic tissues. Cathelicidin inhibited the collagen expression and vimentin-positive fibroblast expression in colonic tumors. Cathelicidin did not directly affect HT-29 cell viability, but did significantly reduce tumor growth factor-β1-induced EMT of colon cancer cells. Media conditioned by the human colonic CCD-18Co fibroblasts promoted human colon cancer HT-29 cell proliferation. Cathelicidin pretreatment inhibited colon cancer cell proliferation mediated by media conditioned by human colonic CCD-18Co fibroblasts. Cathelicidin disrupted tubulin distribution in colonic fibroblasts. Disruption of tubulin in fibroblasts reduced fibroblast-supported colon cancer cell proliferation. Conclusion Cathelicidin effectively inhibits colon cancer development by interfering with EMT and fibroblast-supported colon cancer cell proliferation.

783 TL1A Modulates the Differential Effect of IL-17 Blockade on Mucosal Inflammation

Background: IL-17 has been thought to be pathogenic in IBD. However, a recent clinical trial using IL-17 blockade unexpectedly worsened IBD (Hueber, et al. Gut. 2012). A proportion of trial patients who responded to IL-17 blockade were found to carry a risk TL1A IBD polymorphism that predicted elevated expression of TL1A. The mechanism of IL-17 and its interaction with TL1A on mucosal inflammation has not been found. Aim: To determine the mechanism of TL1A mediated differential effects of IL-17 on mucosal inflammation Methods: Naive (CD4+CD45RBhi) T-cells were isolated from WT, Tl1a-Tg, Il-17a-/-, and Tl1a-Tg with deficiency in Il-17a (Tl1a-Tg/Il-17a-/-) mice and adoptively transferred into Rag-/mice. Naive (CD4+CD25loCD44loCD62hi) T-cells were differentiated in vitro into T helper (Th) effector cells. Intestinal inflammation was evaluated by disease activity index (DAI) and histological analyses. Flow cytometry was used to determine CD4+ T-cell infiltration, activation, and cytokine expression in the LPMC from the colon. Results: Consistent with the human IL-17 trial, we found that mice that received Il-17a-/Naive cells had worsened colitis (increased DAI scores and cecal inflammation) as compared to WT. Comparable results were seen with Tl1a-Tg Naive cells. Similar to the human IL-17 trial, Il-17a deficiency under Tl1a driven conditions (Tl1a-Tg/Il-17a-/-) ameliorated colitis (reduced DAI and cecal inflammation). Mucosal CD4+ T-cell infiltration and activation (CD69 and CD44) were increased in both Il-17a-/and Tl1a-Tg, but reduced with Tl1a-Tg/Il-17a-/-. Analysis of Th effector pathways demonstrated a shift towards Th-1 (increased Ifn-γ) and Th-9 (increased Il-9), and reduced regulatory cytokine Il-10 production with both Il-17a-/and Tl1a-Tg. In contrast, Il-17 deficiency under Tl1a driven conditions (Tl1a-Tg/Il-17a-/-) had a reduction in Th1 (reduced Ifn-γ) and Th9 (reduced Il-9) responses, and increased regulatory responses (increased Il-10). To assess whether intrinsic cellular differences exist in Naive T-cells between WT, Il-17a-/-, Tl1a-Tg, and Tl1a-Tg/Il-17a-/-, in vitro differentiation assays were performed. Consistently, in vitro differentiated Naive Il-17a-/and Tl1a-Tg T-cells expressed increased Ifn-γ and Il-9, whereas Tl1a-Tg/Il17a-/T-cells expressed lower Il-9 but higher Il-10 levels. Conclusion: IL-17 blockade induces mucosal inflammation by enhancing Th1 and Th9 effector pathways and reducing regulatory response. However under TL1A driven conditions, inhibiting IL-17 may be beneficial by reducing Th1 and Th9 effector responses and enhancing regulatory responses. This may be one mechanism of why the subset of IBD patients with TL1A polymorphisms improved with IL-17 blockade, while most trial patients did not. This highlights the importance of understanding pathway-pathway interactions in designing clinical trials. Table 1: Inflammatory Markers

Erratum: Sustained TL1A (TNFSF15) expression on both lymphoid and myeloid cells leads to mild spontaneous intestinal inflammation and fibrosis (DOI: 10.1556/EuJMI.3.2013.1.2).

After the publication of this article (EurJ Microbiol Immunol (Bp) 2013;3: , 13 Mar), we discovered that the version of Figure 3b included in the article is incorrect due to it being done for a separate project. Please see the correct Figure 3b file here. This change does not affect the summary data for Figure 3c, statistical analysis, or figure legend. Fig. 3b. Sustained Tl1a expression led to an increased expression of activation and gut homing marker on Tl1a-Tg T cells.

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

271 Cathelicidin Inhibits Colitis Associated Colon Cancer Development by Inhibition of Epithelial-Mesenchymal Transition and Cancer Associated Fibroblasts

Oxyntic atrophy and intestinal metaplasia are considered critical precursors for the development of gastric adenocarcinoma. Autoimmune atrophic gastritis (AAG) is characterized by immune-mediated destruction of gastric parietal cells, chronic inflammation, atrophic gastritis, and pernicious anemia. While AAG is associated with increased risk of carcinoids, the risk for adenocarcinoma in the absence of H. pylori infection is controversial. Here, we investigated the differences between AAG and chronic atrophic gastritis (CAG) with intestinal metaplasia (IM). Methods: Tissue from 6 patients with CAG were obtained at the time of resection for gastric adenocarcinomna and all patients showed IM. 20 AAG patients were diagnosed by upper endoscopy, oxyntic atrophy, and the presence of anti-parietal cell antibodies. 20/20 AAG patients were negative for H. pylori. 19/20 AAG patient showed marked elevations in both serum gastrin and serum chromogranin. In the body mucosa, we evaluated the presence of spasmolytic-polypeptide expressing metaplasia (SPEM) with TFF2 staining, IM with MUC2, macrophages with CD68, neutrophils with myeloperoxidase (MPO) and proliferative cells with Ki67. Results: 20/20 AAG patients showed profound parietal cell loss with SPEM and 15 demonstrated IM (75%). AAG patients had significantly fewer MPO positive neutrophils (6.4/1000 cells) compared to CAG patients (21.9/1000 cells) (p<0.01). The gastric biopsies from AAG patients also showed significantly fewer CD68 positive macrophages (10.1/1000 cells) compared to CAG patients (17.4/1000 cells, p= 0.01). Ki67 positive cells were markedly decreased in AAG patients (0.2/1000 cells) compared to CAG patients (14.5/1000 cells) (p<0.01). Conclusions: The metaplasia observed in AAG patients exhibited a low proliferative rate compared with the metaplasia samples from CAG patients. This lower proliferative rate was associated with smaller macrophage and neutrophil numbers in AAG patients compared to those in CAG patients. The lower proliferative rate in metaplasia and reduced macrophage and neutrophil infiltrates may explain in part the lower tendency of metaplasia in H. pylori-negative AAG patients to evolve into gastric adenocarcinoma compared with individuals with CAG.

Su1182 Intestinal Cathelicidin Levels Predict Prognosis of Ulcerative Colitis Patients

Introduction: Faecal Calprotectin (FC) is a cytosolic protein belonging to the S-100 family of calcium binding proteins found in neutrophils. It is excreted in the intestinal lumen in inflammatory conditions of the gut and can be used to distinguish irritable bowel syndrome (IBS) from other inflammatory bowel conditions such as colitis, diverticulosis, etc. Pointof-contact qualitative FC tests are now available and can be used in primary care to aid decision making for referrals to gastroenterologists for young patients presenting with chronic diarrhoea. Aims: To assess the feasibility and cost effectiveness of a primary care Pathway using a point-of-contact FC Test (Caldetect®) to aid decision making for referrals to gastroenterology in young patients presenting to their primary physicians with chronic diarrhoea. Methods: Primary Care data indicated that approximately 253 referrals are made annually to gastroenterologists from Primary Care to assess patients ,60years presenting with diarrhoea, costing approx. £119,000 for investigations and consultations. Using a Caldetect® (Preventis, GmbH) point of contact FC test, it was estimated that a saving of £89,000 could be achieved. A pathway for investigating chronic diarrhoea using Caldetect® was designed and implemented in the community (population 150,000) between September 2011-March 2012. (this will be presented). FC results were categorised using manufacturer cut-offs of ,15ug/ g, 15-60ug/g and .60ug/g. Patients with FC results of 15-60 and .60 were deemed to have an inflammatory process and referred to Gastroenterology Clinics. Cost analysis was carried out using the 2010-11 tariffs for the NHS. Results: 142 Caldetect® tests were carried out in Primary Care during this pilot phase. Of these, a negative result ( ,15ug/g) was present in 89, with 36 tests being .60ug/g. 3 tests were at the intermediate level and 14 tests could not be accurately reported. Negative results were managed in primary care as IBS. A monthly cost savings of £6100 was calculated taking consultation and endoscopy tariffs into account. Conclusion: This pilot study demonstrates the feasibility and cost effectiveness of a Pathway for decision making and a point-of-care faecal calprotectin test in rationalising referrals to Gastroenterologists for chronic diarrhoea.