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BackgroundEvidence supports a role for the gut-brain axis in Parkinson's disease (PD). Mice overexpressing human wild type α- synuclein (Thy1-haSyn) exhibit slow colonic transit prior to motor deficits, mirroring prodromal constipation in PD. Identifying molecular changes in the gut could provide both biomarkers for early diagnosis and gut-targeted therapies to prevent progression.ObjectiveTo identify early molecular changes in the gut-brain axis in Thy1-haSyn mice through gene expression profiling.MethodsGene expression profiling was performed on gut (colon) and brain (striatal) tissue from Thy1-haSyn and wild-type (WT) mice aged 1 and 3 months using 3' RNA sequencing. Analysis included differential expression, gene set enrichment and weighted gene co-expression network analysis (WGCNA).ResultsAt one month, differential expression (Thy1-haSyn vs. WT) of mitochondrial genes and pathways related to PD was discordant between gut and brain, with negative enrichment in brain (enriched in WT) but positive enrichment in gut. Linear regression of WGCNA modules showed partial independence of gut and brain gene expression changes. Thy1-haSyn-associated WGCNA modules in the gut were enriched for PD risk genes and PD-relevant pathways including inflammation, autophagy, and oxidative stress. Changes in gene expression were modest at 3 months.ConclusionsOverexpression of haSyn acutely disrupts gene expression in the colon. While changes in colon gene expression are highly related to known PD-relevant mechanisms, they are distinct from brain changes, and in some cases, opposite in direction. These findings are in line with the emerging view of PD as a multi-system disease.

G protein-coupled receptors (GPCRs) regulate many pathophysiological processes and are major therapeutic targets. The impact of disease on the subcellular distribution and function of GPCRs is poorly understood. We investigated trafficking and signaling of protease-activated receptor 2 (PAR2) in colitis. To localize PAR2 and assess redistribution during disease, we generated knockin mice expressing PAR2 fused to monomeric ultrastable green fluorescent protein (muGFP). PAR2-muGFP signaled and trafficked normally. PAR2 messenger RNA was detected at similar levels in Par2-mugfp and wild-type mice. Immunostaining with a GFP antibody and RNAScope in situ hybridization using F2rl1 (PAR2) and Gfp probes revealed that PAR2-muGFP was expressed in epithelial cells of the small and large intestine and in subsets of enteric and dorsal root ganglia neurons. In healthy mice, PAR2-muGFP was prominently localized to the basolateral membrane of colonocytes. In mice with colitis, PAR2-muGFP was depleted from the plasma membrane of colonocytes and redistributed to early endosomes, consistent with generation of proinflammatory proteases that activate PAR2 PAR2 agonists stimulated endocytosis of PAR2 and recruitment of Gαq, Gαi, and β-arrestin to early endosomes of T84 colon carcinoma cells. PAR2 agonists increased paracellular permeability of colonic epithelial cells, induced colonic inflammation and hyperalgesia in mice, and stimulated proinflammatory cytokine release from segments of human colon. Knockdown of dynamin-2 (Dnm2), the major colonocyte isoform, and Dnm inhibition attenuated PAR2 endocytosis, signaling complex assembly and colonic inflammation and hyperalgesia. Thus, PAR2 endocytosis sustains protease-evoked inflammation and nociception and PAR2 in endosomes is a potential therapeutic target for colitis.

The original version of this article unfortunately contained a mistake. In the original version of this article, some data in Fig. 6 were presented incorrectly. The online version of this figure has been updated with the correct data. All the results previously reported as statistically significant remain so. There is no associated change to the text of the article. The authors regret this error.

While apoptosis plays a significant role in intestinal homeostasis, it can also be pathogenic if overactive during recovery from inflammation. We recently reported that microRNA-24-3p (miR-24-3p) is elevated in the colonic epithelium of ulcerative colitis patients during active inflammation, and that it reduced apoptosis in vitro. However, its function during intestinal restitution following inflammation had not been examined. In this study, we tested the influence of miR-24-3p on mucosal repair by studying recovery from colitis in both novel miR-24-3p knockout and miR-24-3p-inhibited mice. We observed that knockout mice and mice treated with a miR-24-3p inhibitor had significantly worsened recovery based on weight loss, colon length, and double-blinded histological scoring. In vivo and in vitro analysis of miR-24-3p inhibition in colonic epithelial cells revealed that inhibition promotes apoptosis and increases levels of the pro-apoptotic protein BIM. Further experiments determined that silencing of BIM reversed the pro-apoptotic effects of miR-24-3p inhibition. Taken together, these data suggest that miR-24-3p restrains intestinal epithelial cell apoptosis by targeting BIM, and its loss of function is detrimental to epithelial restitution following intestinal inflammation.

Background & aimsAlterations in microRNA (miRNA) and in the intestinal barrier are putative risk factors for irritable bowel syndrome (IBS). We aimed to identify differentially expressed colonic mucosal miRNAs, their targets in IBS compared to healthy controls (HCs), and putative downstream pathways.MethodsTwenty-nine IBS patients (15 IBS with constipation [IBS-C], 14 IBS with diarrhea [IBS-D]), and 15 age-matched HCs underwent sigmoidoscopy with biopsies. A nCounter array was used to assess biopsy specimen-associated miRNA levels. A false discovery rate (FDR) < 10% was considered significant. Real-time polymerase chain reaction (PCR) was used to validate differentially expressed genes. To assess barrier function, trans-epithelial electrical resistance (TEER) and dextran flux assays were performed on Caco-2 intestinal epithelial cells that were transfected with miRNA-inhibitors or control inhibitors. Protein expression of barrier function associated genes was confirmed using western blots.ResultsFour out of 247 miRNAs tested were differentially expressed in IBS compared to HCs (FDR < 10%). Real-time PCR validation suggested decreased levels of miR-219a-5p and miR-338-3p in IBS (P = .026 and P = .004), and IBS-C (P = .02 and P = .06) vs. HCs as the strongest associations. Inhibition of miR-219a-5p resulted in altered expression of proteasome/barrier function genes. Functionally, miR-219a-5p inhibition enhanced the permeability of intestinal epithelial cells as TEER was reduced (25-50%, P < .05) and dextran flux was increased (P < .01). Additionally, inhibition of miR-338-3p in cells caused alterations in the mitogen-activated protein kinase (MAPK) signaling pathway genes.ConclusionTwo microRNAs that potentially affect permeability and visceral nociception were identified to be altered in IBS patients. MiR-219a-5p and miR-338-3p potentially alter barrier function and visceral hypersensitivity via neuronal and MAPK signaling and could be therapeutic targets in IBS.

Autotaxin (ATX) converts lysophosphatidylcholine and sphingosyl-phosphorylcholine into lysophosphatidic acid and sphingosine 1-phosphate, respectively. Despite the pivotal function of ATX in lipid metabolism, mechanisms by which ATX regulates immune and inflammatory disorders remain elusive. Here, using myeloid cell lineage-restricted Atx knockout mice, we show that Atx deficiency disrupts membrane microdomains and lipid rafts, resulting in the inhibition of Toll-like receptor 4 (TLR4) complex formation and the suppression of adaptor recruitment, thereby inhibiting TLR4-mediated responses in macrophages. Accordingly, TLR4-induced innate immune functions, including phagocytosis and iNOS expression, are attenuated in Atx-deficient macrophages. Consequently, Atx-/- mice exhibit a higher bacterial prevalence in the intestinal mucosa compared to controls. When combined with global Il10-/- mice, which show spontaneous colitis due to the translocation of luminal commensal microbes into the mucosa, myeloid cell lineage-restricted Atx knockout accelerates colitis development compared to control littermates. Collectively, our data reveal that Atx deficiency compromises innate immune responses, thereby promoting microbe-associated gut inflammation.

BackgroundClostridium difficile infection (CDI) causes diarrhea and colitis. We aimed to find a common pathogenic pathway in CDI among humans and mice by comparing toxin-mediated effects in human and mouse colonic tissues.MethodUsing multiplex enzyme-linked immunosorbent assay, we determined the cytokine secretion of toxin A- and B-treated human and mouse colonic explants.ResultsToxin A and toxin B exposure to fresh human and mouse colonic explants caused different patterns of cytokine secretion. Toxin A induced macrophage inflammatory protein (MIP) 1α secretion in both human and mouse explants. Toxin A reduced the expression of chloride anion exchanger SLC26A3 expression in mouse colonic explants and human colonic epithelial cells. Patients with CDI had increased colonic MIP-1 α expression and reduced colonic SLC26A3 (solute carrier family 26, member 3) compared with controls. Anti-MIP-1 α neutralizing antibody prevented death, ameliorated colonic injury, reduced colonic interleukin 1β (IL-1β) messenger RNA expression, and restored colonic SLC26a3 expression in C. difficile-infected mice. The anti-MIP-1 α neutralizing antibody prevented CDI recurrence. SLC26a3 inhibition augmented colonic IL-1 β messenger RNA expression and abolished the protective effect of anti-MIP-1 α neutralizing antibody in mice with CDI.ConclusionMIP-1 α is a common toxin A-dependent chemokine in human and mouse colon. MIP-1 α mediates detrimental effects by reducing SLC26a3 and enhancing IL-1 β expression in the colon.

The inflammatory bowel diseases (IBD) are a complex set of chronic gastrointestinal inflammatory conditions arising from the interplay of genetic and environmental factors. This study focuses on noncoding RNA transcripts as potential mediators of IBD pathophysiology. One particular gene, interferon γ-antisense 1 (IFNG-AS1), has been consistently observed to be elevated in the intestinal mucosa of patients with actively inflamed IBD versus healthy controls. This study builds on these observations, demonstrating that the second splice variant is specifically altered, and this alteration even stratifies within inflamed patients. With the use of a CRISPR-based overexpression system, IFNG-AS1 was selectively overexpressed directly from its genomic loci in T cells. An unbiased mRNA array on these cells identified a large increase in many inflammatory cytokines and a decrease in anti-inflammatory cytokines after IFNG-AS1 overexpression. Media from T cells overexpressing IFNG-AS1 elicited an inflammatory signaling cascade in primary human peripheral blood mononuclear cells, suggesting the potential functional importance of IFNG-AS1 in IBD pathophysiology. The significance of these results is amplified by studies suggesting that a single-nucleotide polymorphism in IFNG-AS1, rs7134599, was associated with both subtypes of patients with IBD independently of race.NEW & NOTEWORTHY Long noncoding RNAs are an emerging field of inflammatory bowel disease (IBD) research. This study mechanistically analyzes the role of a commonly upregulated gene in IBD and shows IFNG-AS1 as a mediator of an inflammatory signaling cascade.

Chronic stress is thought to be involved in the occurrence and progression of multiple diseases, via mechanisms that still remain largely unknown. Interestingly, key regulators of the stress response, such as members of the corticotropin-releasing-hormone (CRH) family of neuropeptides and receptors, are now known to be implicated in the regulation of chronic inflammation, one of the predisposing factors for oncogenesis and disease progression. However, an interrelationship between stress, inflammation, and malignancy, at least at the molecular level, still remains unclear. Here, we attempt to summarize the current knowledge that supports the inseparable link between chronic stress, inflammation, and colorectal cancer (CRC), by modulation of a cascade of molecular signaling pathways, which are under the regulation of CRH-family members expressed in the brain and periphery. The understanding of the molecular basis of the link among these processes may provide a step forward towards personalized medicine in terms of CRC diagnosis, prognosis and therapeutic targeting.

Inflammatory bowel disease is characterized by high levels of inflammation and loss of barrier integrity in the colon. The intestinal barrier is a dynamic network of proteins that encircle intestinal epithelial cells. miRNAs regulate protein-coding genes. In this study, miR-24 was found to be elevated in colonic biopsies and blood samples from ulcerative colitis (UC) patients compared with healthy controls. In the colon of UC patients, miR-24 is localized to intestinal epithelial cells, which prompted an investigation of intestinal epithelial barrier function. Two intestinal epithelial cell lines were used to study the effect of miR-24 overexpression on barrier integrity. Overexpression of miR-24 in both cell lines led to diminished transepithelial electrical resistance and increased dextran flux, suggesting an effect on barrier integrity. Overexpression of miR-24 did not induce apoptosis or affect cell proliferation, suggesting that the effect of miR-24 on barrier function was due to an effect on cell-cell junctions. Although the tight junctions in cells overexpressing miR-24 appeared normal, miR-24 overexpression led to a decrease in the tight junction-associated protein cingulin. Loss of cingulin compromised barrier formation; cingulin levels negatively correlated with disease severity in UC patients. Together, these data suggest that miR-24 is a significant regulator of intestinal barrier that may be important in the pathogenesis of UC.

AimsThe role of long non-coding RNA's (lncRNA) in the biology of ulcerative colitis (UC) is not well understood. We have previously detected changes in lncRNA's associated with UC. This study aims to characterize one specific lncRNA, CDKN2B-AS1 whose expression was downregulated in UC patients.Main methodsUC biopsies were used to determine the levels of linear and circular CDKN2B-AS1 relative to healthy controls. In situ hybridization was used to determine the localization of CKDN2B-AS1 in the colon. The intestinal epithelial cell line, Caco-2, was used to study the effects of shRNA mediated loss of CDKN2B-AS1. Transepithelial electrical resistance was used to measure barrier function. An RT-PCR array, immunoblots and immunohistochemistry were used to determine tight junction proteins that CDKN2B-AS1 regulates.Key findingsCDKN2B-AS1 is transcribed into not only linear transcripts but also as circular RNA through back-splicing and both forms are decreased in IBD. CDKN2B-AS1 is expressed mainly in colonic epithelial cells. Cells with down-regulated CDKN2B-AS1 exhibited increased proliferation and no alterations in apoptosis. Targeting both the linear and circular transcripts of CDKN2B-AS1 with short hairpin RNAs enhanced barrier function. We subsequently determined that Claudin-2, a "leaky Claudin" known to decrease barrier function, was decreased in CDKN2B-AS1 knockdown cells.SignificanceThis study identifies a novel lncRNA with both linear and circular transcripts affecting UC biology.

Clostridium difficile infection (CDI) is mediated by two major exotoxins, toxin A (TcdA) and toxin B (TcdB), that damage the colonic epithelial barrier and induce inflammatory responses. The function of the colonic vascular barrier during CDI has been relatively understudied. Here we report increased colonic vascular permeability in CDI mice and elevated vascular endothelial growth factor A (VEGF-A), which was induced in vivo by infection with TcdA- and/or TcdB-producing C. difficile strains but not with a TcdA-TcdB- isogenic mutant. TcdA or TcdB also induced the expression of VEGF-A in human colonic mucosal biopsies. Hypoxia-inducible factor signalling appeared to mediate toxin-induced VEGF production in colonocytes, which can further stimulate human intestinal microvascular endothelial cells. Both neutralization of VEGF-A and inhibition of its signalling pathway attenuated CDI in vivo. Compared to healthy controls, CDI patients had significantly higher serum VEGF-A that subsequently decreased after treatment. Our findings indicate critical roles for toxin-induced VEGF-A and colonic vascular permeability in CDI pathogenesis and may also point to the pathophysiological significance of the gut vascular barrier in response to virulence factors of enteric pathogens. As an alternative to pathogen-targeted therapy, this study may enable new host-directed therapeutic approaches for severe, refractory CDI.

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since 2007. These studies have confirmed that lncRNAs are altered in almost all diseases. However, studying the functional roles for lncRNAs in the context of disease remains difficult due to the lack of protein products, tissue-specific expression, low expression levels, complexities in splice forms, and lack of conservation among species. Given the species-specific expression, lncRNA studies are often restricted to human research contexts when studying disease processes. Since lncRNAs function at the molecular level, one way to dissect lncRNA biology is to either remove the lncRNA or overexpress the lncRNA and measure cellular effects. In this article, a written and visualized protocol to overexpress lncRNAs in vitro is presented. As a representative experiment, an lncRNA associated with inflammatory bowel disease, Interferon Gamma Antisense 1 (IFNG-AS1), is shown to be overexpressed in a Jurkat T-cell model. To accomplish this, the activating clustered regularly interspaced short palindromic repeats (CRISPR) technique is used to enable overexpression at the endogenous genomic loci. The activating CRISPR technique targets a set of transcription factors to the transcriptional start site of a gene, enabling a robust overexpression of multiple lncRNA splice forms. This procedure will be broken down into three steps, namely (i) guide RNA (gRNA) design and vector construction, (ii) virus generation and transduction, and (iii) colony screening for overexpression. For this representative experiment, a greater than 20-fold enhancement in IFNG-AS1 in Jurkat T cells was observed.

Inflammatory bowel disease (IBD) is a complex disorder that is associated with significant morbidity. While many recent advances have been made with new diagnostic and therapeutic tools, a deeper understanding of its basic pathophysiology is needed to continue this trend toward improving treatments. By utilizing an unbiased, high-throughput transcriptomic analysis of two well-established mouse models of colitis, we set out to uncover novel coding and noncoding RNAs that are differentially expressed in the setting of colonic inflammation. RNA-seq analysis was performed using colonic tissue from two mouse models of colitis, a dextran sodium sulfate-induced model and a genetic-induced model in mice lacking IL-10. We identified 81 coding RNAs that were commonly altered in both experimental models. Of these coding RNAs, 12 of the human orthologs were differentially expressed in a transcriptomic analysis of IBD patients. Interestingly, 5 of the 12 of human differentially expressed genes have not been previously identified as IBD-associated genes, including ubiquitin D. Our analysis also identified 15 noncoding RNAs that were differentially expressed in either mouse model. Surprisingly, only three noncoding RNAs were commonly dysregulated in both of these models. The discovery of these new coding and noncoding RNAs expands our transcriptional knowledge of mouse models of IBD and offers additional targets to deepen our understanding of the pathophysiology of IBD. NEW & NOTEWORTHY Much of the genome is transcribed as non-protein-coding RNAs; however, their role in inflammatory bowel disease is largely unknown. This study represents the first of its kind to analyze the expression of long noncoding RNAs in two mouse models of inflammatory bowel disease and correlate them to human clinical samples. Using high-throughput RNA-seq analysis, we identified new coding and noncoding RNAs that were differentially expressed such as ubiquitin D and 5730437C11Rik.

BackgroundPhosphatase and tensin homolog (Pten) is capable of mediating microbe-induced immune responses in the gut. Thus, Pten deficiency in the intestine accelerates colitis development in Il10-/- mice. As some ambient pollutants inhibit Pten function and exposure to ambient pollutants may increase inflammatory bowel disease (IBD) incidence, it is of interest to examine how Pten inhibition could affect colitis development in genetically susceptible hosts.MethodsWith human colonic mucosa biopsies from pediatric ulcerative colitis and non-IBD control subjects, we assessed the mRNA levels of the PTEN gene and the gene involved in IL10 responses. The data from the human tissues were corroborated by treating Il10-/-, Il10rb-/-, and wild-type C57BL/6 mice with Pten-specific inhibitor VO-OHpic. We evaluated the severity of mouse colitis by investigating the tissue histology and cytokine production. The gut microbiome was investigated by analyzing the 16S ribosomal RNA gene sequence with mouse fecal samples.ResultsPTEN and IL10RB mRNA levels were reduced in the human colonic mucosa of pediatric ulcerative colitis compared with non-IBD subjects. Intracolonic treatment of the Pten inhibitor induced colitis in Il10-/- mice, characterized by reduced body weight, marked colonic damage, and increased production of inflammatory cytokines, whereas Il10rb-/- and wild-type C57BL/6 mice treated with the inhibitor did not develop colitis. Pten inhibitor treatment changed the fecal microbiome, with increased abundance of colitogenic bacteria Bacteroides and Akkermansia in Il10-/- mice.ConclusionsLoss of Pten function increases the levels of colitogenic bacteria in the gut, thereby inducing deleterious colitis in an Il10-deficient condition.

Peripheral factors likely play a role in at least a subset of irritable bowel syndrome (IBS) patients. Few studies have investigated mucosal gene expression using an unbiased approach. Here, we performed mucosal gene profiling in a sex-balanced sample to identify relevant signaling pathways and gene networks and compare with publicly available profiling data from additional cohorts. Twenty Rome III+ IBS patients [10 IBS with constipation (IBS-C), 10 IBS with diarrhea (IBS-D), 5 men/women each), and 10 age-/sex-matched healthy controls (HCs)] underwent sigmoidoscopy with biopsy for gene microarray analysis, including differential expression, weighted gene coexpression network analysis (WGCNA), gene set enrichment analysis, and comparison with publicly available data. Expression levels of 67 genes were validated in an expanded cohort, including the above samples and 18 additional participants (6 each of IBS-C, IBS-D, HCs) using NanoString nCounter technology. There were 1,270 differentially expressed genes (FDR < 0.05) in IBS-C vs. HCs but none in IBS or IBS-D vs. HCs. WGNCA analysis identified activation of the cAMP/protein kinase A signaling pathway. Nine of 67 genes were validated by the NanoString nCounter technology (FDR < 0.05) in the expanded sample. Comparison with publicly available microarray data from the Mayo Clinic and University of Nottingham supports the reproducibility of 17 genes from the microarray analysis and three of nine genes validated by nCounter in IBS-C vs. HCs. This study supports the involvement of peripheral mechanisms in IBS-C, particularly pathways mediating neuronal signaling. NEW & NOTEWORTHY Peripheral factors play a role in the pathophysiology of irritable bowel syndrome (IBS), which, to date, has been mostly evident in IBS with diarrhea. Here, we show that sigmoid colon mucosal gene expression profiles differentiate IBS with constipation from healthy controls. These profiling data and analysis of additional cohorts also support the concept that peripheral neuronal pathways contribute to IBS pathophysiology.

Colorectal cancer (CRC) responds poorly to immuno-mediated cytotoxicity. Underexpression of corticotropin-releasing-hormone-receptor-2 (CRHR2) in CRC, promotes tumor survival, growth and Epithelial to Mesenchymal Transition (EMT), in vitro and in vivo. We explored the role of CRHR2 downregulation in CRC cell resistance to Fas/FasL-mediated apoptosis and the underlying molecular mechanism. CRC cell sensitivity to CH11-induced apoptosis was compared between Urocortin-2 (Ucn2)-stimulated parental and CRHR2-overexpressing CRC cell lines and targets of CRHR2/Ucn2 signaling were identified through in vitro and ex vivo analyses. Induced CRHR2/Ucn2 signaling in SW620 and DLD1 cells increased specifically their sensitivity to CH11-mediated apoptosis, via Fas mRNA and protein upregulation. CRC compared to control tissues had reduced Fas expression that was associated with lost CRHR2 mRNA, poor tumor differentiation and high risk for distant metastasis. YY1 silencing increased Fas promoter activity in SW620 and re-sensitized them to CH11-apoptosis, thus suggesting YY1 as a putative transcriptional repressor of Fas in CRC. An inverse correlation between Fas and YY1 expression was confirmed in CRC tissue arrays, while elevated YY1 mRNA was clinically relevant with advanced CRC grade and higher risk for distant metastasis. CRHR2/Ucn2 signaling downregulated specifically YY1 expression through miR-7 elevation, while miR-7 modulation in miR-7high SW620-CRHR2+ and miR-7low HCT116 cells, had opposite effects on YY1 and Fas expressions and cell sensitivity to CH11-killing. CRHR2/Ucn2 signaling is a negative regulator of CRC cell resistance to Fas/FasL-apoptosis via targeting the miR-7/YY1/Fas circuitry. CRHR2 restoration might prove effective in managing CRC response to immune-mediated apoptotic stimuli.

Mesenteric adipose tissue hyperplasia is a hallmark of Crohn's disease (CD). Recently, we showed that mesenteric adipose-derived stromal cells (ADSCs) from CD, ulcerative colitis, and control patients synthesize and release adipokines in a disease-dependent manner. Here we examined the expression profiles of CD and control patient-derived mesenteric ADSCs and studied the effects of their extracellular mediators on colonocyte signaling in vitro and experimental colitis in vivo. ADSCs were isolated from mesenteric fat of control and CD patients. Microarray profiling and network analysis were performed in ADSCs and human colonocytes treated with conditioned media from cultured ADSCs. Mice with acute colitis received daily injections of conditioned media from patient-derived ADSCs, vehicle, or apolactoferrin. Proliferative responses were evaluated in conditioned media-treated colonocytes and mouse colonic epithelium. Total protein was isolated from cultured colonocytes after treatment with apolactoferrin for Western blot analysis of phosphorylated intracellular signaling kinases. Microarray profiling revealed differential mRNA expression in CD patient-derived ADSCs compared with controls, including lactoferrin. Administration of CD patient-derived medium or apolactoferrin increased colonocyte proliferation compared with controls. Conditioned media from CD patient-derived ADSCs or apolactoferrin attenuated colitis severity in mice and enhanced colonocyte proliferation in vivo. ADSCs from control and CD patients show disease-dependent inflammatory responses and alter colonic epithelial cell signaling in vitro and in vivo. Furthermore, we demonstrate lactoferrin production by adipose tissue, specifically mesenteric ADSCs. We suggest that mesenteric ADSC-derived lactoferrin may mediate protective effects and participate in the pathophysiology of CD by promoting colonocyte proliferation and the resolution of inflammation.

Intestinal wound healing is a new therapeutic goal for inflammatory bowel disease (IBD) as complete healing of the mucosa is the key element of clinical remission in IBD. Previous studies showed that termination of inflammation can be achieved by adding pro-resolving lipids like DHA and EPA exogenously. However, the roles of these lipids in mucosal healing have not been investigated. To recapitulate intestinal healing process, mice were received dextran sodium sulfate (DSS) for 7 days in the drinking water followed by regular tap water for 5 additional days. DSS-induced intestinal inflammation featuring body weight loss, histological tissue damage, increased cytokine production and infiltration of inflammatory cells was gradually reduced upon switching to water. To investigate whether endogenous lipids play a role in mucosal healing, the lipidomics analysis of mouse serum was performed. Reduced levels of arachidonic acid, the biosynthetic precursor of prostaglandin F (PGF)2α, 19H-PGF1α, the metabolite of prostacyclin, and 20H-PGF2α, the metabolite of PGF2α, suggest subsiding inflammation. In contrast, increased levels of an active metabolite of resolvin D1 along with decreased levels of its precursor DHA as well as decreased levels of the precursor of resolvin E, 18-hydroxy-eicosapentaenoic acid, suggest inauguration of mucosal healing by endogenous lipids. Furthermore, exogenously supplied fish oil enhanced the process even further. These results suggest the presence of mucosal healing regulated by endogenous pro-healing lipids and also indicate that the remission state of IBD could be prolonged by enhancing the levels of these lipids.

Substance P is a peptide mainly secreted by neurons and is involved in many biological processes, including nociception and inflammation. Animal models have provided insights into the biology of this peptide and offered compelling evidence for the importance of substance P in cell-to-cell communication by either paracrine or endocrine signaling. Substance P mediates interactions between neurons and immune cells, with nerve-derived substance P modulating immune cell proliferation rates and cytokine production. Intriguingly, some immune cells have also been found to secrete substance P, which hints at an integral role of substance P in the immune response. These communications play important functional roles in immunity including mobilization, proliferation and modulation of the activity of immune cells. This review summarizes current knowledge of substance P and its receptors, as well as its physiological and pathological roles. We focus on recent developments in the immunobiology of substance P and discuss the clinical implications of its ability to modulate the immune response.

C. difficile infection (CDI) is a common debilitating nosocomial infection associated with high mortality. Several CDI outbreaks have been attributed to ribotypes 027, 017, and 078. Clinical and experimental evidence indicates that the nonpathogenic yeast Saccharomyces boulardii CNCM I-745 (S.b) is effective for the prevention of CDI. However, there is no current evidence suggesting this probiotic can protect from CDI caused by outbreak-associated strains. We used established hamster models infected with outbreak-associated C. difficile strains to determine whether oral administration of live or heat-inactivated S.b can prevent cecal tissue damage and inflammation. Hamsters infected with C. difficile strain VPI10463 (ribotype 087) and outbreak-associated strains ribotype 017, 027, and 078 developed severe cecal inflammation with mucosal damage, neutrophil infiltration, edema, increased NF-κB phosphorylation, and increased proinflammatory cytokine TNFα protein expression. Oral gavage of live, but not heated, S.b starting 5 days before C. difficile infection significantly reduced cecal tissue damage, NF-κB phosphorylation, and TNFα protein expression caused by infection with all strains. Moreover, S.b-conditioned medium reduced cell rounding caused by filtered supernatants from all C. difficile strains. S.b-conditioned medium also inhibited toxin A- and B-mediated actin cytoskeleton disruption. S.b is effective in preventing C. difficile infection by outbreak-associated via inhibition of the cytotoxic effects of C. difficile toxins.

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.

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.

In inflammatory bowel disease (IBD), compromised restitution of the epithelial barrier contributes to disease severity. Owing to the complexity in the pathogenesis of IBD, a variety of factors have been implicated in its progress. In this study, we report a functional interaction between macroautophagy and Corticotropin Releasing Hormone (Crh) in the gut. For this purpose we used DSS colitis model on Crh -/- or wild-type (wt) with pharmacological inhibition of autophagy. We uncovered sustained basal autophagy in the gut of Crh -/- mice, which persisted over the course of DSS administration. Autophagy inhibition resulted in partial rescue of Crh -/- mice, while it increased the expression of Crh in the wt gut. Similarly, Crh deficiency was associated with sustained activation of base line autophagy. In vitro models of amino acid deprivation- and LPS-induced autophagy confirmed the in vivo findings. Our results indicate a novel role for Crh in the intestinal epithelium that involves regulation of autophagy, while suggesting the complementary action of the two pathways. These data suggest the intriguing possibility that targeting Crh stimulation in the intestine may provide a novel therapeutic approach to support the integrity of the epithelial barrier and to protect from chronic colitis.

Urocortins (Ucns) 1, 2, and 3 and corticotropin-releasing factor receptor 2 (CRF2) mRNA are prominently expressed in various layers of the upper gut. We tested whether Ucns and CRF2 variants are also expressed in the different layers of the rat colon, regulated by LPS (100 μg/kg ip) and play a modulatory role in the colonic immune response to LPS. Transcripts of Ucns and CRF2b, the most common isoform in the periphery, were detected in all laser microdissected layers, including myenteric neurons. LPS increased the mRNA level of Ucn 1, Ucn 2, and Ucn 3 and decreased that of CRF2b in both the colonic mucosa and submucosa + muscle (S+M) layers at 2, 6, and 9 h after injection with a return to basal at 24 h. In addition, CRF2a, another variant more prominent in the brain, and a novel truncated splice variant CRF2a-3 mRNA were detected in all segments of the large intestine. LPS reciprocally regulated the colonic expression of these CRF2 variants by decreasing both CRF2a and CRF2b, while increasing CRF2a-3 in the mucosa and S+M. The CRF2 antagonist astressin2-B further enhanced LPS-induced increase of mRNA level of interleukin (IL)-1β, TNF-α, and inducible nitric oxide synthase in S+M layers and IL-1β in the mucosa and evoked TNF-α expression in the mucosa. These data indicate that Ucns/CRF2 variants are widely expressed in all colonic layers and reciprocally regulated by LPS. CRF2 signaling dampens the CD14/TLR4-mediated acute inflammatory response to Gram-negative bacteria in the colon.

Neurotensin (NT) triggers signaling in human colonic epithelial cells by activating the G protein-coupled receptor, the neurotensin receptor 1 (NTR1). Activated NTR1 traffics from the plasma membrane to early endosomes, and then recycles. Although sustained NT/NTR1 signaling requires efficient NTR1 recycling, little is known about the regulation of NTR1 recycling. We recently showed that NT/NTR1 signaling increases expression of miR-133α. Herein, we studied the mechanism of NT-regulated miR-133α expression and examined the role of miR-133α in intracellular NTR1 trafficking in human NCM460 colonocytes. We found that NT-induced miR-133α upregulation involves the negative transcription regulator, zinc finger E-box binding homeobox 1. Silencing of miR-133α or overexpression of aftiphilin (AFTPH), a binding target of miR-133α, attenuated NTR1 trafficking to plasma membrane in human colonocytes, without affecting NTR1 internalization. We localized AFTPH to early endosomes and the trans-Golgi network (TGN) in unstimulated human colonic epithelial cells. AFTPH overexpression reduced NTR1 localization in early endosomes and increased expression of proteins related to endosomes and the TGN trafficking pathway. AFTPH overexpression and de-acidification of intracellular vesicles increased NTR1 expression. Our results suggest a novel mechanism of GPCR trafficking in human colonic epithelial cells by which a microRNA, miR-133α regulates NTR1 trafficking through its downstream target AFTPH.

The epithelial cells of the gut form a physical barrier against the luminal contents. The collapse of this barrier causes inflammation, and its therapeutic restoration can protect the gut against inflammation. EGF enhances mucosal barrier function and increases colonocyte proliferation, thereby ameliorating inflammatory responses in the gut. Based on our previous finding that the insect peptide CopA3 promotes neuronal growth, we herein tested whether CopA3 could increase the cell proliferation of colonocytes, enhance mucosal barrier function, and ameliorate gut inflammation. Our results revealed that CopA3 significantly increased epithelial cell proliferation in mouse colonic crypts and also enhanced colonic epithelial barrier function. Moreover, CopA3 treatment ameliorated Clostridium difficile toxin As-induced inflammation responses in the mouse small intestine (acute enteritis) and completely blocked inflammatory responses and subsequent lethality in the dextran sulfate sodium-induced mouse model of chronic colitis. The marked CopA3-induced increase of colonocyte proliferation was found to require rapid protein degradation of p21(Cip1/Waf1), and an in vitro ubiquitination assay revealed that CopA3 directly facilitated ubiquitin ligase activity against p21(Cip1/Waf1). Taken together, our findings indicate that the insect peptide CopA3 prevents gut inflammation by increasing epithelial cell proliferation and mucosal barrier function.

Clostridium difficile is being recognized as a growing threat to many health-care systems. Epidemiology data shows that infection rates are soaring and the disease burden is increasing. Despite the efficacy of standard treatments, it is becoming evident that novel therapeutics will be required to tackle this disease. These new treatments aim to enhance the intestinal microbial barrier, activate the immune system and neutralize the toxins that mediate this disease. Many of these therapies are still in the beginning stages of investigation, however, in the next few years, more clinical data will become available to help implement many of these exciting new therapeutic approaches.

The corticotropin-releasing hormone family mediates functional responses in many organs, including the intestine. Activation of corticotropin-releasing hormone receptor 2 (CRHR2) in the colonic mucosa promotes inflammation during acute colitis but inhibits inflammation during chronic colitis. We hypothesized that specific modulation of CRHR2 signaling in the colonic mucosa can promote restoration of the epithelium through stimulation of cell proliferative, migratory, and wound healing responses. Mucosal repair was assessed after dextran sodium sulfate (DSS)-induced colitis in mice receiving intracolonic injections of a CRHR2 antagonist or vehicle and in Crhr2(-/-) mice. Histologic damage, cytokine expression, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, and Ki-67 immunoreactivity were evaluated. Cell viability, proliferation, and migration were compared between parental and CRHR2-overexpressing colonic epithelial cells. Protein lysates were processed for phosphoprotein assays and a wound healing assay performed in vitro. Administration of a CRHR2 antagonist after DSS-induced colitis increased disease activity, delayed healing, and decreased epithelial cell proliferation in vivo. Colons from these mice also showed increased apoptosis and proinflammatory cytokine expression. Compared with controls, Crhr2(-/-) mice showed increased mortality in the DSS healing protocol. CRHR2-overexpressing cells had increased proliferation and migration compared with parental cells. Wound healing and signal transducer and activator of transcription 3 activity were elevated in CRHR2-overexpressing cells after urocortin 2 and IL-6 treatment, suggesting advanced healing progression. Our results suggest that selective CRHR2 activation may provide a targeted approach to enhance mucosal repair pathways after colitis.

Background & aimsChronic inflammation promotes development and progression of colorectal cancer (CRC). We explored the distribution of Corticotropin-Releasing-Hormone (CRH)-family of receptors and ligands in CRC and their contribution in tumor growth and oncogenic EMT.MethodsmRNA expression of CRH-family members was analyzed in CRC (N=56) and control (N=46) samples, 7 CRC cell lines and normal NCM460 cells. Immunohistochemical detection of CRHR2 was performed in 20 CRC and 5 normal tissues. Cell proliferation, migration and invasion were compared between Urocortin-2 (Ucn2)-stimulated parental and CRHR2-overexpressing (CRHR2+) cells in absence or presence of IL-6. CRHR2/Ucn2-targeted effects on tumor growth and EMT were validated in SW620-xenograft mouse models.ResultsCRC tissues and cell lines showed decreased mRNA and protein CRHR2 expression compared to controls and NCM460, 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 where 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.ConclusionsCRHR2 downregulation 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.

Background & aimsChronic inflammation promotes development and progression of colorectal cancer (CRC). We explored the distribution of Corticotropin-Releasing-Hormone (CRH)-family of receptors and ligands in CRC and their contribution in tumor growth and oncogenic EMT.MethodsmRNA expression of CRH-family members was analyzed in CRC (N=56) and control (N=46) samples, 7 CRC cell lines and normal NCM460 cells. Immunohistochemical detection of CRHR2 was performed in 20 CRC and 5 normal tissues. Cell proliferation, migration and invasion were compared between Urocortin-2 (Ucn2)-stimulated parental and CRHR2-overexpressing (CRHR2+) cells in absence or presence of IL-6. CRHR2/Ucn2-targeted effects on tumor growth and EMT were validated in SW620-xenograft mouse models.ResultsCRC tissues and cell lines showed decreased mRNA and protein CRHR2 expression compared to controls and NCM460, 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 where 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.ConclusionsCRHR2 downregulation 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.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease with increasing incidence and prevalence worldwide. Here we investigated the newly synthesized jasmonate analogue 2-hydroxyethyl 5-chloro-4,5-didehydrojasmonate (J11-Cl) for its anti-inflammatory effects on intestinal inflammation. First, to test whether J11-Cl can activate peroxisome proliferator-activated receptors (PPARs), we performed docking simulations because J11-Cl has a structural similarity with anti-inflammatory 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2), one of the endogenous ligands of PPARγ. J11-Cl bound to the ligand binding domain of PPARγ in the same manner as 15d-PGJ2 and rosiglitazone, and significantly increased transcriptional activity of PPARγ. In animal experiments, colitis was significantly reduced in mice with J11-Cl treatment, determined by analyses of survival rate, body weight changes, clinical symptoms, and histological evaluation. Moreover, J11-Cl decreased production of pro-inflammatory cytokines including IL-6, IL-8, and G-CSF as well as chemokines including chemokine (C-C motif) ligand (CCL)20, chemokine (C-X-C motif) ligand (CXCL)2, CXCL3, and chemokine (C-X3-C motif) ligand 1 (CX3CL1) in colon tissues, and LPS or TNF-α-stimulated macrophages and epithelial cells. In contrast, production of anti-inflammatory cytokines including IL-2 and IL-4 as well as the proliferative factor, GM-CSF, was increased by J11-Cl. Furthermore, inhibition of MAPKs and NF-κB activation by J11-Cl was also observed. J11-Cl reduced intestinal inflammation by increasing the transcriptional activity of PPARγ and modulating inflammatory signaling pathways. Therefore, our study suggests that J11-Cl may serve as a novel therapeutic agent against IBD.

Colonic epithelium is the first line of defense against various pathological offenses in the gut. Previous studies have shown that the peptides of the corticotropin-releasing hormone (CRH) family modulate vascular endothelial growth factor (VEGF)-A production in other cells. Here we sought to investigate whether CRH and urocortin (Ucn) 3 regulate VEGF-A secretion in colonocytes through CRH receptors and to elucidate the underlying mechanism of action. CRH and Ucn 3 significantly increased the expression levels of VEGF-A mRNA and protein through CRH receptor 1 and 2, respectively, in human colonic epithelial cells and primary mouse intestinal epithelial cells. Underlying mechanisms involve activation of adenylyl cyclase with subsequent increase of intracellular cAMP level and increased DNA binding activity of transcription factor CREB on VEGF-A promoter region. Finally, genetic deficiency of CREB decreased intestinal inflammation and VEGF-A expression in a dextran sodium sulfate-induced colitis model. These results show that activation of CRH receptors by CRH ligands stimulates VEGF-A expression in intestinal epithelial cells through the cAMP/CREB pathway. Since VEGF-A boosts inflammatory responses through angiogenesis, these data suggest that CREB may be a key effector of CRH and Ucn 3-dependent inflammatory angiogenesis.

Background & aimsPersistent activation of the inflammatory response contributes to the development of inflammatory bowel diseases, which increase the risk of colorectal cancer. We aimed to identify microRNAs that regulate inflammation during the development of ulcerative colitis (UC) and progression to colitis-associated colon cancer (CAC).MethodsWe performed a quantitative polymerase chain reaction analysis to measure microRNAs in 401 colon specimens from patients with UC, Crohn's disease, irritable bowel syndrome, sporadic colorectal cancer, or CAC, as well as subjects without these disorders (controls); levels were correlated with clinical features and disease activity of patients. Colitis was induced in mice by administration of dextran sodium sulfate (DSS), and carcinogenesis was induced by addition of azoxymethane; some mice also were given an inhibitor of microRNA214 (miR214).ResultsA high-throughput functional screen of the human microRNAome found that miR214 regulated the activity of nuclear factor-κB. Higher levels of miR214 were detected in colon tissues from patients with active UC or CAC than from patients with other disorders or controls and correlated with disease progression. Bioinformatic and genome-wide profile analyses showed that miR214 activates an inflammatory response and is amplified through a feedback loop circuit mediated by phosphatase and tensin homolog (PTEN) and PDZ and LIM domain 2 (PDLIM2). Interleukin-6 induced signal transducer and activator of transcription 3 (STAT3)-mediated transcription of miR214. A miR214 chemical inhibitor blocked this circuit and reduced the severity of DSS-induced colitis in mice, as well as the number and size of tumors that formed in mice given azoxymethane and DSS. In fresh colonic biopsy specimens from patients with active UC, the miR214 inhibitor reduced inflammation by increasing levels of PDLIM2 and PTEN.ConclusionsInterleukin-6 up-regulates STAT3-mediated transcription of miR214 in colon tissues, which reduces levels of PDLIM2 and PTEN, increases phosphorylation of AKT, and activates nuclear factor-κB. The activity of this circuit correlates with disease activity in patients with UC and progression to colorectal cancer.

UnlabelledThe neuropeptide substance P (SP) exhibits cytokine-like properties and exerts different effects in autoimmune inflammation. Various immune cells express SP and its neurokinin-1 receptor (NK1R) isoforms. A role for SP has been demonstrated in a number of autoimmune conditions, including multiple sclerosis (MS). In this work, we studied the role of SP and NK1R in human immune cells with a focus on their relationship with IL-12/IL-23 family cytokines and the associated IFN-γ/IL-17.Aims(1) To determine the role of SP mediated effects on induction of various inflammatory cytokines in peripheral blood mononuclear cells (PBMC); (2) to investigate the expression of SP and its receptor in T cells and the effects of stimulation with IL-12 and IL-23. Quantitative real-time PCR, flow cytometry, ELISA, promoter studies on PBMC and primary T cells from healthy volunteers, and Jurkat cell line. Treatment with SP significantly increased the expression of IL-12/IL-23 subunit p40, IL-23 p19 and IL-12 p35 mRNA in human PBMC. Expression of NK1R and SP in T cells was upregulated by IL-23 but a trend was observed with IL-12. The IL-23 effect likely involves IL-17 production that additionally mediates IL-23 effects. Mutual interactions exist with SP enhancing the cytokines IL-23 and IL-12, and SP and NK1R expression being differentially but potentially synergistically regulated by these cytokines. These findings suggest a proinflammatory role for SP in autoimmune inflammation. We propose a model whereby immunocyte derived SP stimulates Th1 and Th17 autoreactive cells migrating to the central nervous system (CNS), enhances their crossing the blood brain barrier and perpetuates inflammation in the CNS by being released from damaged nerves and activating both resident glia and infiltrating immune cells. SP may be a therapeutic target in MS.

Background & aimsSubstance P (SP), a neuropeptide member of the tachykinin family, plays a critical role in colitis. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression. However, whether SP modulates expression of microRNAs in human colonic epithelial cells remains unknown.MethodsWe 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 TNBS and DSS models of colitis.ResultsSP 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, while overexpression of miR-221-5p reduced IL-6R expression. NF-κB and JNK inhibition decreased SP-induced miR-221-5p expression. MiR-221-5p expression was increased in both TNBS- and DSS-induced colitis and colonic biopsies from Ulcerative Colitis, but not Crohn's Disease subjects, compared to controls. In mice, intracolonic administration of a miR-221-5p chemical inhibitor, exacerbated TNBS-and DSS-induced colitis, and increased colonic TNF-α, Cxcl10, and Col2 α 1 mRNA expression. In situ hybridization in TNBS-and DSS-exposed colons revealed increased miR-221-5p expression primarily in colonocytes.ConclusionsOur 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.

Substance P (SP), a neuropeptide member of the tachykinin family, plays a critical role in colitis. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression. However, whether SP modulates expression of microRNAs in human colonic epithelial cells remains unknown.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 TNBS and DSS models of colitis.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, while overexpression of miR-221-5p reduced IL-6R expression. NF-κB and JNK inhibition decreased SP-induced miR-221-5p expression. MiR-221-5p expression was increased in both TNBS- and DSS-induced colitis and colonic biopsies from Ulcerative Colitis, but not Crohn's Disease subjects, compared to controls. In mice, intracolonic administration of a miR-221-5p chemical inhibitor, exacerbated TNBS-and DSS-induced colitis, and increased colonic TNF-α, Cxcl10, and Col2 α 1 mRNA expression. In situ hybridization in TNBS-and DSS-exposed colons revealed increased miR-221-5p expression primarily in colonocytes.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.

ObjectiveNeurotensin (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.DesignmiR-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.ResultsNT 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 (2 day) and DSS (5 day) 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.ConclusionsNT-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.

Background & aimsSubstance P (SP), neurokinin-1 receptors (NK-1Rs) are expressed in mesenteric preadipocytes and SP binding activates proinflammatory signalling in these cells. We evaluated the expression levels of SP (Tac-1), NK-1R (Tacr-1), and NK-2R (Tacr-2) mRNA in preadipocytes isolated from patients with Inflammatory Bowel Disease (IBD) and examined their responsiveness to SP compared to control human mesenteric preadipocytes. The Aim of our study is to investigate the effects of the neuropeptide SP on cytokine expression in preadipocytes of IBD vs control patients and evaluate the potential effects of these cells on IBD pathophysiology via SP-NK-R interactions.MethodsMesenteric fat was collected from control, Ulcerative colitis (UC) and Crohn's disease (CD) patients (n=10-11 per group). Preadipocytes were isolated, expanded in culture and exposed to substance P. Colon biopsies were obtained from control and IBD patients.ResultsTacr-1 and -2 mRNA were increased in IBD preadipocytes compared to controls, while Tac-1 mRNA was increased only in UC preadipocytes. SP differentially regulated the expression of inflammatory mediators in IBD preadipocytes compared to controls. Disease-dependent responses to SP were also observed between UC and CD preadipocytes. IL-17A mRNA expression and release increased after SP treatment in both CD and UC preadipocytes, while IL-17RA mRNA increased in colon biopsies from IBD patients.ConclusionsPreadipocyte SP-NK-1R interactions during IBD may participate in IBD pathophysiology. The ability of human preadipocytes to release IL-17A in response to SP together with increased IL-17A receptor in IBD colon opens the possibility of a fat-colonic mucosa inflammatory loop that may be active during IBD.

Background & aimsSubstance P (SP), neurokinin-1 receptors (NK-1Rs) are expressed in mesenteric preadipocytes and SP binding activates proinflammatory signalling in these cells. We evaluated the expression levels of SP (Tac-1), NK-1R (Tacr-1), and NK-2R (Tacr-2) mRNA in preadipocytes isolated from patients with Inflammatory Bowel Disease (IBD) and examined their responsiveness to SP compared to control human mesenteric preadipocytes. The Aim of our study is to investigate the effects of the neuropeptide SP on cytokine expression in preadipocytes of IBD vs control patients and evaluate the potential effects of these cells on IBD pathophysiology via SP-NK-R interactions.MethodsMesenteric fat was collected from control, Ulcerative colitis (UC) and Crohn's disease (CD) patients (n=10-11 per group). Preadipocytes were isolated, expanded in culture and exposed to substance P. Colon biopsies were obtained from control and IBD patients.ResultsTacr-1 and -2 mRNA were increased in IBD preadipocytes compared to controls, while Tac-1 mRNA was increased only in UC preadipocytes. SP differentially regulated the expression of inflammatory mediators in IBD preadipocytes compared to controls. Disease-dependent responses to SP were also observed between UC and CD preadipocytes. IL-17A mRNA expression and release increased after SP treatment in both CD and UC preadipocytes, while IL-17RA mRNA increased in colon biopsies from IBD patients.ConclusionsPreadipocyte SP-NK-1R interactions during IBD may participate in IBD pathophysiology. The ability of human preadipocytes to release IL-17A in response to SP together with increased IL-17A receptor in IBD colon opens the possibility of a fat-colonic mucosa inflammatory loop that may be active during IBD.

BackgroundTwenty to 25% of the patients with inflammatory bowel disease (IBD) present the disease before the age of 18 to 20, with worse extent and severity, compared with adult-onset IBD. We sought to identify the differential expression of microRNAs in pediatric ulcerative colitis (UC) and their association with different clinical phenotypes.MethodsMicroRNA expression analysis was performed in colonic tissues derived from pediatric patients with UC and controls without IBD. MiR-4284 levels were verified by real-time quantitative polymerase chain reaction in 2 additional cohorts of pediatric patients with UC. Bioinformatics analysis was performed to predict the targets of miR-4284. In vitro experiments using luciferase reporter assays and real-time polymerase chain reaction evaluated the direct effect of miR-4284 on CXCL5 mRNA. In vivo experiments were performed in 2 mouse models of experimental colitis.ResultsA 24-microRNA signature was identified in colonic tissues derived from pediatric patients with UC. The most downregulated microRNA in the tissue of pediatric patients UC, relative to non-IBD controls, was miR-4284. In situ hybridization revealed that miR-4284 is present in colonic epithelial cells, and its levels correlate with the disease activity. Furthermore, we found that miR-4284 regulates CXCL5 mRNA expression through binding to its 3'UTR. CXCL5 had increased mRNA levels in colonic tissue from pediatric patients with UC and correlated with disease activity. Furthermore, we found an inverse correlation between miR-4284 and CXCL5 levels in the colonic pediatric UC tissues and in 2 mouse models of experimental colitis.ConclusionsOur data reveal a novel microRNA pediatric UC signature and provide evidence that miR-4284 directly regulates CXCL5 and correlates with the disease activity.

In inflammatory bowel disease (IBD), obesity is associated with worsening of the course of disease. Here, we examined the role of obesity in the development of colitis and studied mesenteric fat-epithelial cell interactions in patients with IBD. We combined the diet-induce obesity with the trinitrobenzene sulfonic acid (TNBS) colitis mouse model to create groups with obesity, colitis, and their combination. Changes in the mesenteric fat and intestine were assessed by histology, myeloperoxidase assay, and cytokine mRNA expression by real-time PCR. Medium from human mesenteric fat and cultured preadipocytes was obtained from obese patients and those with IBD. Histological analysis showed inflammatory cell infiltrate and increased histological damage in the intestine and mesenteric fat of obese mice with colitis compared with all other groups. Obesity also increased the expression of proinflammatory cytokines including IL-1β, TNF-α, monocyte chemoattractant protein 1, and keratinocyte-derived chemokine, while it decreased the TNBS-induced increases in IL-2 and IFN-γ in mesenteric adipose and intestinal tissues. Human mesenteric fat isolated from obese patients and those with and IBD demonstrated differential release of adipokines and growth factors compared with controls. Fat-conditioned media reduced adiponectin receptor 1 (AdipoR1) expression in human NCM460 colonic epithelial cells. AdipoR1 intracolonic silencing in mice exacerbated TNBS-induced colitis. In conclusion, obesity worsens the outcome of experimental colitis, and obesity- and IBD-associated changes in adipose tissue promote differential mediator release in mesenteric fat that modulates colonocyte responses and may affect the course of colitis. Our results also suggest an important role for AdipoR1 for the fat-intestinal axis in the regulation of inflammation during colitis.

Ulcerative colitis (UC) and Crohn's Disease (CD) are the two most common forms of Inflammatory Bowel Diseases (IBD) marked by chronic and persistent inflammation. Neurotensin (NT), together with its receptor, NT receptor 1 (NTR1), are important mediators in intestinal inflammation and their expression is upregulated in the intestine of experimental colitis models and UC colonic biopsies. MicroRNAs (miRNAs) are short, non-coding RNA molecules which act as transcription repressors. We have previously shown that NT exposure upregulates miR-133α expression in human colonocytes NCM460 cells overexpressing NTR1 (NCM460-NTR1). Recently, miR-133α was further examined forits role in NT-associated proinflammatory signaling cascades and acute colitis in vivo. Our study shows that NT-induced miR-133α upregulation modulates NF-κB phosphorylation and promotes proinflammatory cytokine production. In addition, intracolonicinjection of antisense-miR-133α before colitis induction improves histological scores and proinflammatory cytokine transcription. More importantly, dysregulation of miR-133α levels and aftiphilin (AFTPH), a newly-identified miR-133α downstream target, is found only in UC patients, but not in patients with CD. Taken together, we identified NTR1/miR-133α/aftiphilin as a novel regulatory axis involved in NT-associated colonic inflammation in human colonocytes, acute colitis mouse model and in colonic biopsies from UC patients. Our results also provide evidence that colonic levels of NTR1, miR-133α and aftiphilin may also serve as potential biomarkers in UC.

Background and aimsCathelicidin (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.MethodsC57BL/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.ResultsTNBS-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.ConclusionCathelicidin can reverse intestinal fibrosis by directly inhibiting collagen synthesis in colonic fibroblasts.

IntroductionAnimal studies have shown that stress could induce epigenetic and transcriptomic alterations essential in determining the balance between adaptive or maladaptive responses to stress. We tested the hypothesis that chronic stress in rats deregulates coding and non-coding gene expression in the spinal cord, which may underline neuroinflammation and nociceptive changes previously observed in this model.MethodsMale Wistar rats were exposed to daily stress or handled, for 10 days. At day 11, lumbar spinal segments were collected and processed for mRNA/miRNA isolation followed by expression profiling using Agilent SurePrint Rat Exon and Rat miRNA Microarray platforms. Differentially expressed gene lists were generated using the dChip program. Microarrays were analyzed using the Ingenuity Pathways Analysis (IPA) tool from Ingenuity Systems. Multiple methods were used for the analysis of miRNA-mRNA functional modules. Quantitative real time RT-PCR for Interleukin 6 signal transducer (gp130), the Signal Transducer And Activator Of Transcription 3 (STAT3), glial fibrillary acidic protein and mir-17-5p were performed to confirm levels of expression.ResultsGene network analysis revealed that stress deregulated different inflammatory (IL-6, JAK/STAT, TNF) and metabolic (PI3K/AKT) signaling pathways. MicroRNA array analysis revealed a signature of 39 deregulated microRNAs in stressed rats. MicroRNA-gene network analysis showed that microRNAs are regulators of two gene networks relevant to inflammatory processes. Specifically, our analysis of miRNA-mRNA functional modules identified miR-17-5p as an important regulator in our model. We verified miR-17-5p increased expression in stress using qPCR and in situ hybridization. In addition, we observed changes in the expression of gp130 and STAT3 (involved in intracellular signaling cascades in response to gp130 activation), both predicted targets for miR-17-5p. A modulatory role of spinal mir17-5p in the modulation of visceral sensitivity was confirmed in vivo.ConclusionUsing an integrative high throughput approach, our findings suggest a link between miR-17-5p increased expression and gp130/STAT3 activation providing new insight into the possible mechanisms mediating the effect of chronic stress on neuroinflammation in the spinal cord.

Background and aimsCathelicidin (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.MethodsC57BL/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.ResultsTNBS-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.ConclusionCathelicidin can reverse intestinal fibrosis by directly inhibiting collagen synthesis in colonic fibroblasts.

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.

Background & aimsEarly-onset ulcerative colitis, which is considered severe colonic inflammation that develops in infants and young children, can be caused by alterations in interleukin (IL)-10 signaling, although other factors are involved in its pathogenesis. We investigated whether loss of phosphatase and tensin homologue (PTEN), which regulates many important cell functions such as cell proliferation, cell survival, and Toll-like receptor (TLR) signaling pathways, contributes to the development of colitis in Il10(-/-) mice.MethodsWe generated Il10(-/-) mice (in C57BL/6 and C3H/HeJBir background strains) with disruption of Pten in the intestinal epithelium (Ints(ΔPten/ΔPten);Il10(-/-) mice) and Ints(ΔCont);Il10(-/-) (control) mice. Colon tissues were collected and histological, transmission electron microscopy, and gene expression analysis were performed. Fecal microbiota samples were analyzed by sequencing of 16S ribosomal RNA genes. We disrupted Tlr4 in Ints(ΔPten/ΔPten);Il10(-/-) mice. Lipopolysaccharide signaling via TLR4 was blocked by treating mice with polymyxin B.ResultsIl10(-/-) mice developed colitis when they were 6 to 7 months old, whereas Ints(ΔPten/ΔPten);Il10(-/-) mice developed severe colitis and colon tumors by the time they were 36 days old. Within 3 months of birth, 80% of Ints(ΔPten/ΔPten);Il10(-/-) mice developed severe colitis and colonic malignancy, whereas none of the Ints(ΔCont);Il10(-/-) mice had these phenotypes. Ints(ΔPten/ΔPten);Il10(-/-) mice had alterations in fecal microbiota compared with controls, such as increased proportions of Bacteroides species, which are gram negative. Disruption of Tlr4 or treating Ints(ΔPten/ΔPten);Il10(-/-) mice with polymyxin B delayed the development of colitis and reduced disease severity.ConclusionsDisruption of Pten in the intestinal epithelium of Il10(-/-) mice speeds the onset and increases the severity of colitis. Fecal microbiota from Ints(ΔPten/ΔPten);Il10(-/-) mice have increased proportions of Bacteroides species. Development of colitis is delayed and reduced by blocking TLR4 signaling. Ints(ΔPten/ΔPten);Il10(-/-) mice may be studied as a model for early-onset ulcerative colitis and used to identify new therapeutic targets.