Ivy Ka Man Law

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

BACKGROUND & AIMSnCathelicidin (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.nnnMETHODSnQuantitative, 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.nnnRESULTSnExpression 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.nnnCONCLUSIONSnCathelicidin 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 3u2005days. Ileal loops were also prepared in WT mice and treated with either saline or TxA and incubated for 4u2005h, 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.

Neurotensin—regulated miR-133α is involved in proinflammatory signalling in human colonic epithelial cells and in experimental colitis

Objective Neurotensin (NT) mediates colonic inflammation through its receptor neurotensin receptor 1 (NTR1). NT stimulates miR-133α expression in colonic epithelial cells. We investigated the role of miR-133α in NT-associated colonic inflammation in vitro and in vivo. Design miR-133α and aftiphilin (AFTPH) levels were measured by quantitative PCR. Antisense (as)-miR-133α was administrated intracolonicaly prior to induction of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis and dextran sodium sulfate (DSS)-induced colitis. The effect of AFTPH was examined by gene silencing in vitro. Results NT increased miR-133α levels in NCM-460 overexpressing NTR1 (NCM460-NTR1) and HCT-116 cells. NT-induced p38, ERK1/2, c-Jun, and NF-κB activation, as well as IL-6, IL-8 and IL-1β messenger RNA (mRNA) expression in NCM-460-NTR1 cells were reduced in miR-133α-silenced cells, while overexpression of miR-133α reversed these effects. MiR-133α levels were increased in TNBS (2u2005day) and DSS (5u2005day) colitis, while NTR1 deficient DSS-exposed mice had reduced miR-133α levels, compared to wild-type colitic mice. Intracolonic as-miR-133α attenuated several parameters of colitis as well expression of proinflammatory mediators in the colonic mucosa. In silico search coupled with qPCR identified AFTPH as a downstream target of miR-133α, while NT decreased AFTPH expression in NCM-460-NTR1 colonocytes. Gene silencing of AFTPH enhanced NT-induced proinflammatory responses and AFTPH levels were downregulated in experimental colitis. Levels of miR-133α were significantly upregulated, while AFTPH levels were downregulated in colonic biopsies of patients with ulcerative colitis compared to controls. Conclusions NT-associated colitis and inflammatory signalling are regulated by miR-133α-AFTPH interactions. Targeting of miR-133α or AFTPH may represent a novel therapeutic approach in inflammatory bowel disease.

Neurotensin-induced Proinflammatory Signaling in Human Colonocytes Is Regulated by β-Arrestins and Endothelin-converting Enzyme-1-dependent Endocytosis and Resensitization of Neurotensin Receptor 1

Background: Neurotensin induces proinflammatory responses in human colonic epithelial cells. Results: β-Arrestins and endothelin-converting enzyme 1 regulate neurotensin receptor 1-mediated inflammatory signaling in human colonocytes. Conclusion: Neurotensin-induced proinflammatory responses depend on β-arrestins and are regulated by receptor recycling. Significance: This is a previously unrecognized pathway for regulating neurotensin-induced colonic inflammatory responses. The neuropeptide/hormone neurotensin (NT) mediates intestinal inflammation and cell proliferation by binding of its high affinity receptor, neurotensin receptor-1 (NTR1). NT stimulates IL-8 expression in NCM460 human colonic epithelial cells by both MAP kinase- and NF-κB-dependent pathways. Although the mechanism of NTR1 endocytosis has been studied, the relationship between NTR1 intracellular trafficking and inflammatory signaling remains to be elucidated. In the present study, we show that in NCM460 cells exposed to NT, β-arrestin-1 (βARR1), and β-arrestin-2 (βARR2) translocate to early endosomes together with NTR1. Endothelin-converting enzyme-1 (ECE-1) degrades NT in acidic conditions, and its activity is crucial for NTR1 recycling. Pretreatment of NCM460 cells with the ECE-1 inhibitor SM19712 or gene silencing of βARR1 or βARR2 inhibits NT-stimulated ERK1/2 and JNK phosphorylation, NF-κB p65 nuclear translocation and phosphorylation, and IL-8 secretion. Furthermore, NT-induced cell proliferation, but not IL-8 transcription, is attenuated by the JNK inhibitor, JNK(AII). Thus, NTR1 internalization and recycling in human colonic epithelial cells involves βARRs and ECE-1, respectively. Our results also indicate that βARRs and ECE-1-dependent recycling regulate MAP kinase and NF-κB signaling as well as cell proliferation in human colonocytes in response to NT.

The neurotensin-HIF-1α-VEGFα axis orchestrates hypoxia, colonic inflammation, and intestinal angiogenesis.

The expression of neurotensin (NT) and its receptor (NTR1) is up-regulated in experimental colitis and inflammatory bowel disease; NT/NTR1 interactions regulate gut inflammation. During active inflammation, metabolic shifts toward hypoxia lead to the activation of hypoxia-inducible factor (HIF)-1, which enhances vascular endothelial growth factor (VEGF) expression, promoting angiogenesis. We hypothesized that NT/NTR1 signaling regulates intestinal manifestations of hypoxia and angiogenesis by promoting HIF-1 transcriptional activity and VEGFα expression in experimental colitis. We studied NTR1 signaling in colitis-associated angiogenesis using 2,4,6-trinitrobenzenesulfonic acid-treated wild-type and NTR1-knockout mice. The effects of NT on HIF-1α and VEGFα were assessed on human colonic epithelial cells overexpressing NTR1 (NCM460-NTR1) and human intestinal microvascular-endothelial cells. NTR1-knockout mice had reduced microvascular density and mucosal integrity score compared with wild-type mice after 2,4,6-trinitrobenzenesulfonic acid treatment. VEGFα mRNA levels were increased in NCM460-NTR1 cells treated with 10(-7) mol/L NT, at 1 and 6 hours post-treatment. NT exposure in NCM460-NTR1 cells caused stabilization, nuclear translocation, and transcriptional activity of HIF-1α in a diacylglycerol kinase-dependent manner. NT did not stimulate tube formation in isolated human intestinal macrovascular endothelial cells but did so in human intestinal macrovascular endothelial cells cocultured with NCM460-NTR1 cells. Our results demonstrate the importance of an NTR1-HIF-1α-VEGFα axis in intestinal angiogenic responses and in the pathophysiology of colitis and inflammatory bowel disease.

A long noncoding RNA signature for ulcerative colitis identifies IFNG-AS1 as an enhancer of inflammation

High-throughput technologies revealed new categories of genes, including the long noncoding RNAs (lncRNAs), involved in the pathogenesis of human disease; however, the role of lncRNAs in the ulcerative colitis (UC) has not been evaluated. Gene expression profiling was used to develop lncRNA signatures in UC samples. Jurkat T cells were activated by PMA/ionomycin subsequently interferon-γ (IFNG) and tumor necrosis factor (TNF)-α protein levels were assessed by ELISA. Anti-sense molecules were designed to block IFNG-AS1 expression. A unique set of lncRNAs was differentially expressed between UC and control samples. Of these, IFNG-AS1 was among the highest statistically significant lncRNAs (fold change: 5.27, P value: 7.07E-06). Bioinformatic analysis showed that IFNG-AS1 was associated with the IBD susceptibility loci SNP rs7134599 and its genomic location is adjacent to the inflammatory cytokine IFNG. In mouse models of colitis, active colitis samples had increased colonic expression of this lncRNA. Utilizing the Jurkat T cell model, we found IFNG-AS1 to positively regulate IFNG expression. Novel lncRNA signatures differentiate UC patients with active disease, patients in remission, and control subjects. A subset of these lncRNAs was found to be associated with the clinically validated IBD susceptibility loci. IFNG-AS1 was one of these differentially expressed lncRNAs in UC patients and found to regulate the key inflammatory cytokine, IFNG, in CD4 T cells. Taking these findings together, our study revealed novel lncRNA signatures deregulated in UC and identified IFNG-AS1 as a novel regulator of IFNG inflammatory responses, suggesting the potential importance of noncoding RNA mechanisms on regulation of inflammatory bowel disease-related inflammatory responses.

Diminished Expression of Corticotropin-Releasing Hormone Receptor 2 in Human Colon Cancer Promotes Tumor Growth and Epithelial-to-Mesenchymal Transition via Persistent Interleukin-6/Stat3 Signaling

Background & Aims Chronic inflammation promotes development and progression of colorectal cancer (CRC). We explored the distribution of the corticotropin-releasing-hormone (CRH) family of receptors and ligands in CRC and their contribution in tumor growth and oncogenic epithelial-to-mesenchymal transition (EMT). Methods The mRNA expression of CRH-family members was analyzed in CRC (n = 56) and control (n = 46) samples, seven CRC cell lines, and normal NCM460 cells. Immunohistochemical detection of CRHR2 was performed in 20 CRC and five normal tissues. Cell proliferation, migration, and invasion were compared between urocortin-2 (Ucn2)-stimulated parental and CRHR2-overexpressing (CRHR2+) cells in the absence or presence of interleukin-6 (IL-6). CRHR2/Ucn2-targeted effects on tumor growth and EMT were validated in SW620-xenograft mouse models. Results CRC tissues and cell lines showed decreased mRNA and protein CRHR2 expression compared with controls and NCM460 cells, respectively. The opposite trend was shown for Ucn2. CRHR2/Ucn2 signaling inhibited cell proliferation, migration, invasion, and colony formation in CRC-CRHR2+ cells. In vivo, SW620-CRHR2+ xenografts showed decreased growth, reduced expression of EMT-inducers, and elevated levels of EMT-suppressors. IL-1b, IL-6, and IL-6R mRNAs were diminished in CRC-CRHR2+ cells, while CRHR2/Ucn2 signaling inhibited IL-6-mediated Stat3 activation, invasion, migration, and expression of downstream targets acting as cell cycle– and EMT-inducers. Expression of cell cycle– and EMT-suppressors was augmented in IL-6/Ucn2-stimulated CRHR2+ cells. In patients, CRHR2 mRNA expression was inversely correlated with IL-6R and vimentin levels and metastasis occurrence, while positively associated with E-cadherin expression and overall survival. Conclusions CRHR2 down-regulation in CRC supports tumor expansion and spread through maintaining persistent inflammation and constitutive Stat3 activation. CRHR2low CRC phenotypes are associated with higher risk for distant metastases and poor clinical outcomes.

Neurotensin promotes the development of colitis and intestinal angiogenesis via Hif-1α-MIR-210 signaling

Neurotensin (NT) via its receptor 1 (NTR1) modulates the development of colitis, decreases HIF-1α/PHD2 interaction, stabilizes and increases HIF-1α transcriptional activity, and promotes intestinal angiogenesis. HIF-1α induces miR-210 expression, whereas miR-210 is strongly upregulated in response to NT in NCM460 human colonic epithelial cells overexpressing NTR1 (NCM460-NTR1). In this study, we examined whether NT activates a NTR1-HIF-1α-miR-210 cascade using in vitro (NCM460-NTR1 cells) and in vivo (transgenic mice overexpressing [HIF-1α-OE] or lacking HIF-1α [HIF-1α-knockout (KO)] in intestinal epithelial cells and mice lacking NTR1 [NTR1-KO]) models. Pretreatment of NCM460-NTR1 cells with the HIF-1α inhibitor PX-478 or silencing of HIF-1α (small interfering HIF-1α) attenuated miR-210 expression in response to NT. Intracolonic 2,4,6-trinitrobenzenesulfonic acid (TNBS) administration (2-d model) increased colonic miR-210 expression that was significantly reduced in NTR1-KO, HIF-1α-KO mice, and wild-type mice pretreated intracolonically with locked nucleic acid anti–miR-210. In contrast, HIF-1α-OE mice showed increased miR-210 expression at baseline that was further increased following TNBS administration. HIF-1α-OE mice had also exacerbated TNBS-induced neovascularization compared with TNBS-exposed wild-type mice. TNBS-induced neovascularization was attenuated in HIF-1α-KO mice, or mice pretreated intracolonically with anti–miR-210. Intracolonic anti–miR-210 also reduced colitis in response to TNBS (2 d). Importantly, miR-210 expression was increased in tissue samples from ulcerative colitis patients. We conclude that NT exerts its proinflammatory and proangiogenic effects during acute colitis via a NTR1-prolyl hydroxylase 2/HIF-1α-miR-210 signaling pathway. Our results also demonstrate that miR-210 plays a proinflammatory role in the development of colitis.

Identification of a Novel Substance P-Neurokinin-1 Receptor MicroRNA-221-5p Inflammatory Network in Human Colonic Epithelial Cells

Background & Aims Substance P (SP), a neuropeptide member of the tachykinin family, plays a critical role in colitis. MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression. We examined whether SP modulates expression of microRNAs in human colonic epithelial cells. Methods We performed microRNA profiling analysis of SP-stimulated human colonic epithelial NCM460 cells overexpressing neurokinin-1 receptor (NCM460-NK-1R). Targets of SP-regulated microRNAs were validated by real-time polymerase chain reaction (RT-PCR). Functions of miRNAs were tested in NCM460-NK-1R cells and the trinitrobenzene sulfonic acid (TNBS) and dextran sulfate sodium (DSS) models of colitis. Results SP stimulated differential expression of 29 microRNAs, including miR-221-5p, the highest up-regulated miR (by 12.6-fold) upon SP stimulation. Bioinformatic and luciferase reporter analyses identified interleukin-6 receptor (IL-6R) mRNA as a direct target of miR-221-5p in NCM460 cells. Accordingly, SP exposure of NCM460-NK-1R cells increased IL-6R mRNA expression, and overexpression of miR-221-5p reduced IL-6R expression. Nuclear factor κB and c-Jun N-terminal kinase inhibition decreased SP-induced miR-221-5p expression. MiR-221-5p expression was increased in both TNBS- and DSS-induced colitis and in colonic biopsy samples from ulcerative colitis but not Crohn’s disease patients compared with controls. In mice, intracolonic administration of a miR-221-5p chemical inhibitor exacerbated TNBS- and DSS-induced colitis and increased colonic tumor necrosis factor-α, C-X-C motif chemokine 10 (Cxcl10), and collagen, type II, α 1 (Col2α1) mRNA expression. In situ hybridization in TNBS- and DSS-exposed colons revealed increased miR-221-5p expression primarily in colonocytes. Conclusions Our results reveal a novel NK-1R-miR-221-5p-IL-6R network that protects from colitis. The use of miR-221-5p mimics may be a promising approach for colitis treatment.

The Roles of Antimicrobial Peptides in the Regulation of Gastrointestinal Microbiota and Innate Immunity

Abstract Numerous antimicrobial peptides (AMPs) are expressed in the mucosa of the gastrointestinal tract where they are able to modulate innate immune responses and gut microbiota. Interestingly, the antimicrobial functions of AMPs are not necessarily the most important mechanism in the modulation of gastrointestinal diseases. Instead, AMPs may promote certain protective microbial species and modulate innate immune responses. The interactions of AMPs with innate immunity and gut microbiota reveal interesting drug targets. AMPs may serve as novel therapeutic approaches against gastrointestinal infection and inflammation, but the application of AMPs and their derivatives in treating gastrointestinal diseases remains at an early stage. AMPs may even directly modulate obesity and metabolic diseases. Additionally, AMPs may serve as biomarkers of gastrointestinal diseases, as expression of AMPs is often altered during the development of gastrointestinal diseases. This report summarizes the latest development of AMP-related gastrointestinal research with emphasis on innate immunity and gut microbiota.