Regulation and Role of αE Integrin and Gut Homing Integrins in Migration and Retention of Intestinal Lymphocytes during Inflammatory Bowel Disease.

Targeting interactions between α4ß7 integrin and endothelial adhesion molecule MAdCAM-1 to inhibit lymphocyte migration to the gastrointestinal tract is an effective therapy in inflammatory bowel disease (IBD). Following lymphocyte entry into the mucosa, a subset of these cells expresses αEß7 integrin, which is expressed on proinflammatory lymphocytes, to increase cell retention. The factors governing lymphocyte migration into the intestinal mucosa and αE integrin expression in healthy subjects and IBD patients remain incompletely understood. We evaluated changes in factors involved in lymphocyte migration and differentiation within tissues. Both ileal and colonic tissue from active IBD patients showed upregulation of ICAM-1, VCAM-1, and MAdCAM-1 at the gene and protein levels compared with healthy subjects and/or inactive IBD patients. ß1 and ß7 integrin expression on circulating lymphocytes was similar across groups. TGF-ß1 treatment induced expression of αE on both ß7+ and ß7- T cells, suggesting that cells entering the mucosa independently of MAdCAM-1/α4ß7 can become αEß7+ ITGAE gene polymorphisms did not alter protein induction following TGF-ß1 stimulation. Increased phospho-SMAD3, which is directly downstream of TGF-ß, and increased TGF-ß-responsive gene expression were observed in the colonic mucosa of IBD patients. Finally, in vitro stimulation experiments showed that baseline ß7 expression had little effect on cytokine, chemokine, transcription factor, and effector molecule gene expression in αE+ and αE- T cells. These findings suggest cell migration to the gut mucosa may be altered in IBD and α4ß7-, and α4ß7+ T cells may upregulate αEß7 in response to TGF-ß once within the gut mucosa.

Dual targeting of lymphocyte homing and retention through α4ß7 and αEß7 inhibition in inflammatory bowel disease.

Anti-integrins are therapeutically effective for inflammatory bowel disease, yet the relative contribution of α4ß7 and αEß7 to gut lymphocyte trafficking is not fully elucidated. Here, we evaluate the effect of α4ß7 and αEß7 blockade using a combination of murine models of gut trafficking and longitudinal gene expression analysis in etrolizumab-treated patients with Crohn's disease (CD). Dual blockade of α4ß7 and αEß7 reduces CD8+ T cell accumulation in the gut to a greater extent than blockade of either integrin alone. Anti-αEß7 reduces epithelial:T cell interactions and promotes egress of activated T cells from the mucosa into lymphatics. Inflammatory gene expression is greater in human intestinal αEß7+ T cells. Etrolizumab-treated patients with CD display a treatment-specific reduction in inflammatory and cytotoxic intraepithelial lymphocytes (IEL) genes. Concurrent blockade of α4ß7 and αEß7 promotes reduction of cytotoxic IELs and inflammatory T cells in the gut mucosa through a stepwise inhibition of intestinal tissue entry and retention.

AlphaE Integrin Expression Is Increased in the Ileum Relative to the Colon and Unaffected by Inflammation.

BACKGROUND: Recent findings suggest that αE expression is enriched on effector T cells and that intestinal αE+ T cells have increased expression of inflammatory cytokines. αE integrin expression is a potential predictive biomarker for response to etrolizumab, a monoclonal antibody against ß7 integrin that targets both α4ß7 and αEß7. We evaluated the prevalence and localization of αE+ cells as well as total αE gene expression in healthy and inflammatory bowel disease patients. METHODS: αE+ cells were identified in ileal and colonic biopsies by immunohistochemistry and counted using an automated algorithm. Gene expression was assessed by quantitative reverse-transcriptase polymerase chain reaction. RESULTS: In both healthy and inflammatory bowel disease patients, significantly more αE+ cells were present in the epithelium and lamina propria of ileal compared with colonic biopsies. αE gene expression levels were also significantly higher in ileal compared with colonic biopsies. Paired biopsies from the same patient showed moderate correlation of αE expression between the ileum and colon. Inflammation did not affect αE expression, and neither endoscopy nor histology scores correlated with αE gene expression. αE expression was not different between patients based on concomitant medication use except 5-aminosalicylic acid. CONCLUSION: αE+ cells, which have been shown to have inflammatory potential, are increased in the ileum in comparison with the colon in both Crohn's disease and ulcerative colitis, as well as in healthy subjects. In inflammatory bowel disease patients, αE levels are stable, regardless of inflammatory status or most concomitant medications, which could support its use as a biomarker for etrolizumab.

Inflammatory Bowel Disease Susceptibility Gene C1ORF106 Regulates Intestinal Epithelial Permeability.

Intestinal epithelial cells form a physical barrier that is tightly regulated to control intestinal permeability. Proinflammatory cytokines, such as TNF-α, increase epithelial permeability through disruption of epithelial junctions. The regulation of the epithelial barrier in inflammatory gastrointestinal disease remains to be fully characterized. In this article, we show that the human inflammatory bowel disease genetic susceptibility gene C1ORF106 plays a key role in regulating gut epithelial permeability. C1ORF106 directly interacts with cytohesins to maintain functional epithelial cell junctions. C1orf106-deficient mice are hypersensitive to TNF-α-induced increase in epithelial permeability, and this is associated with increased diarrhea. This study identifies C1ORF106 as an epithelial cell junction protein, and the loss of C1ORF106 augments TNF-α-induced intestinal epithelial leakage and diarrhea that may play a critical role in the development of inflammatory bowel disease.

The protection role of Atg16l1 in CD11c+dendritic cells in murine colitis.

The autophagy-related 16-like 1 gene (Atg16l1) is associated with inflammatory bowel disease (IBD) and has been shown to play an essential role in paneth cell function and intestinal homeostasis. However, the functional consequences of Atg16l1 deficiency in myeloid cells, particularly in dendritic cells (DCs), are not fully characterized. The aim of this study is to investigate the functional consequence of Atg16l1 in CD11c+DCs in murine colitis. We generated mice deficient in Atg16l1 in CD11c+DCs. Dextran Sulfate Sodium (DSS) and S. typhimurium infection induced colitis was used to assess the role of DCs specific Atg16l1 deficiency in vivo in murine colitis. Bone marrow derived dendritic cells (BMDC) were isolated and autophagy function was assessed with microtubule-associated protein 1 light chain 3ß (Map1lc3b or LC3) by western blot. Uptake of Salmonella enteric serovar typhimurium (S. typhimurium) was assessed by flow cytometry and transmission electron microscopy (TEM). The production of reactive oxygen species (ROS) and intracellular S. typhimurium killing in BMDCs were assessed. We showed worsened colonic inflammation in Atg16l1 deficiency mice in DSS induced murine colitis with increased proinflammatory cytokines of IL-1ß and TNF-α. Mechanistic studies performed in primary murine BMDCs showed that Atg16l1 deficiency increased ROS production, reduced microbial killing and impaired antigen processing for altered intracellular trafficking. Together, these results indicate impaired CD11c+DCs function with Atg16l1 deficiency contributes to the severity of murine colitis.

Myeloid ATG16L1 Facilitates Host-Bacteria Interactions in Maintaining Intestinal Homeostasis.

Intact ATG16L1 plays an essential role in Paneth cell function and intestinal homeostasis. However, the functional consequences of ATG16L1 deficiency in myeloid cells, particularly macrophages, are not fully characterized. We generated mice with Atg16l1 deficiency in myeloid and dendritic cells and showed that mice with myeloid Atg16l1 deficiency had exacerbated colitis in two acute and one chronic model of colitis with increased proinflammatory to anti-inflammatory macrophage ratios, production of proinflammatory cytokines, and numbers of IgA-coated intestinal microbes. Mechanistic analyses using primary murine macrophages showed that Atg16l1 deficiency led to increased reactive oxygen species production, impaired mitophagy, reduced microbial killing, impaired processing of MHC class II Ags, and altered intracellular trafficking to the lysosomal compartments. Increased production of reactive oxygen species and reduced microbial killing may be general features of the myeloid compartment, as they were also observed in Atg16l1-deficient primary murine neutrophils. A missense polymorphism (Thr300Ala) in the essential autophagy gene ATG16L1 is associated with Crohn disease (CD). Previous studies showed that this polymorphism leads to enhanced cleavage of ATG16L1 T300A protein and thus reduced autophagy. Similar findings were shown in primary human macrophages from controls and a population of CD patients carrying the Atg16l1 T300A risk variant and who were controlled for NOD2 CD-associated variants. This study revealed that ATG16L1 deficiency led to alterations in macrophage function that contribute to the severity of CD.

Anti-fibrogenic effects of the anti-microbial peptide cathelicidin in murine colitis-associated fibrosis.

BACKGROUND AND 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. mCRAMP peptide (5 mg/kg every 3 day, week 5-7) or cathelicidin gene (Camp)-expressing lentivirus (107 infectious units week 4) were administered intracolonically or intravenously, respectively. 129Sv/J mice were infected 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 to 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-beta1 (TGF-beta1) and/or insulin-like growth factor 1 induced collagen protein and mRNA expression, that was reduced by LL-37 (3-5 µM) through a MAP kinase-dependent mechanism. CONCLUSION: 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-ß1-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.

SUSTAINED TL1A (TNFSF15) EXPRESSION ON BOTH LYMPHOID AND MYELOID CELLS LEADS TO MILD SPONTANEOUS INTESTINAL INFLAMMATION AND FIBROSIS.

TL1A is a member of the TNF superfamily, and its expression is increased in the mucosa of inflammatory bowel disease patients. Moreover, patients with certain TNFSF15 variants over-express TL1A and have a higher risk of developing strictures in the small intestine. Consistently, mice with sustained Tl1a expression in either lymphoid or myeloid cells develop spontaneous ileitis and increased intestinal collagen deposition. Transgenic (Tg) mice with constitutive Tl1a expression in both lymphoid and myeloid cells were generated to assess their in vivo consequence. Constitutive expression of Tl1a in both lymphoid and myeloid cells showed increased spontaneous ileitis and collagen deposition than WT mice. T cells with constitutive expression of Tl1a in both lymphoid and myeloid cells were found to have a more activated phenotype, increased gut homing marker CCR9 expression, and enhanced Th1 and Th17 cytokine activity than WT mice. Although no differences in T cell activation marker, Th1 or Th17 cytokine activity, ileitis, or collagen deposition were found between constitutive Tl1a expression in lymphoid only, myeloid only, or combined lymphoid and myeloid cells. Double hemizygous Tl1a-Tg mice appeared to have worsened ileitis and intestinal fibrosis. Our findings confirm that TL1A-DR3 interaction is involved in T cell-dependent ileitis and fibrosis.

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.

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.

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.