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The expression levels of aftiphilin (AFTPH) are significantly lower in inflamed colonic tissues from patients with ulcerative colitis (UC) and mice with experimental colitis. During colonic inflammation, the selective permeability of the colonic epithelium is compromised largely due to dysregulation of proteins associated with either the tight junction (TJ) complex and actin-myosin contraction rings. Here, we hypothesized that inflammation-associated reduction in AFTPH levels might cause an increase in the selective permeability of the colonic epithelium. In this study, we measured the transepithelial electric resistance (TEER), sodium (Na+) ion flux and dextran permeability in polarized colonic epithelial cells after manipulation of AFTPH. Silencing of AFTPH reduced TEER, increased Na+ ion flow and dextran permeability. Examination of mRNA and protein levels of multiple TJ proteins and Na+ ion transporters suggested that AFTPH deficiency did not significantly change expression of most of these transmembrane proteins. While the gross structure of the TJs in AFTPH gene-silenced cells appeared normal, elevated levels of junctional Occludin were observed. Most notably we observed that AFTPH co-localized with myosin light chain kinase (MLCK) and attenuated cellular MLCK activity as observed by phospho-myosin light chain 2 (pMLC2) western blots. Importantly, inhibition of MLCK activity reversed the reduction of TEER in AFTPH-deficient monolayers. Lastly, examination on transmission electron microcopy on microvilli and immunofluorescent microscopy on actin filament arrangement showed that AFTPH deficiency also affected filament arrangement in colonic epithelial cells. Taken together, these results suggest that AFTPH regulates intestinal epithelial permeability and actin polymerization in colonic epithelium through interfering MLCK/MLC interactions.

Colorectal cancer (CRC) is the third most common malignancy in both American men and women. Most of the deaths attributed to CRC are a result of metastatic spread to the liver. In this study, colon cancer cells that highly metastasized to liver in vivo were compared to less metastatic parental cells to investigate the role for long non-coding RNAs (lncRNAs) in CRC metastasis. The highly metastatic daughter cells (LS-3B) were found to be 63-fold more metastatic than the parental cell line (LS-PAR) in vivo. A lncRNA microarray comparing LS-PAR and LS-3B cells revealed that 104 lncRNAs had fold changes > 2.0 and an FDR < 0.05. Real time PCR mediated validation revealed many lncRNAs exhibited high fold changes such as a 60-fold increase in LOC101448202, a 20-fold increase in MRPL23-AS1 and 50-fold decreases in GNAS-AS1 and LOC101928131. In vivo metastasis differences could be recapitulated in vitro as LS-3B cells closed wounds faster than their parental LS-PAR cells. However, intestinal epithelial cancer cells with robust downregulation of MRPL23-AS1, C1QTNF1-AS1, GNAS-AS1, LINCR-0002 and LOC101448202 failed to display differences in comparison to controls in in vitro migration assays. Three of the five lncRNAs with microarray probes for currently available GEO-datasets were significantly altered in liver CRC-associated tumor biopsies as compared to the primary tumor of non-metastatic CRC. Further studies on the lncRNAs identified will better define their roles in metastasis and how they might be useful if targeted therapeutically.

Background Parkinson Disease (PD) is increasingly being recognized as a disease of the gut-brain axis. Overexpression of human wild type α–synuclein under the neuron-specific Thy1 promoter (Thy1-haSyn) is a well-characterized mouse model of PD. In addition to a progressive motor phenotype, Thy1-haSyn mice have delayed colonic transit at three months of age mirroring prodromal constipation common in human PD. Identification of initial molecular changes in the gastrointestinal tract in PD could lead to early detection of PD and/or gut-targeted therapies to prevent motor symptoms in PD. Methods Colon and striatum gene expression profiling was performed in Thy1-haSyn and wild type (WT) mice aged 1 month and 3 months using 3′ RNA sequencing. Analysis of transcriptomic data included identification of differentially expressed genes, gene set enrichment analysis and weighted gene co-expression network analysis (WGCNA). WGCNA results were further analyzed by linear regression to identify modules most strongly associated with Thy1-haSyn in the colon and striatum, as well as in a combined multivariate model. Module annotation included enrichment for gene ontology terms, cell type signatures and risk genes. Results Differential gene expression in Thy1-haSyn vs WT was greater at one vs three months in both the colon and striatum. There was an opposite pattern of enrichment of mitochondrial genes related to PD (KEGG PD pathway) in striatum (enriched in WT) and colon (enriched in Thy1-haSyn). Colon Thy1-haSyn-associated WGCNA modules were enriched for PD risk genes and PD-relevant pathways including inflammation, autophagy, and oxidative stress. When combined with striatum modules, colon modules remained independent predictors of Thy1-haSyn genotype and increased the model fit (p = 0.048). Conclusions Overexpression of alpha synuclein 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.

Background & AimsMicroRNAs (miRNAs) are transported in body fluids within exosomes, and exosomal-miRNA sorting is highly selective. In colonic epithelial cells (CECs), Substance P-neurokinin-1-receptor (SP/NK1R) signaling regulates miR-21 sorting into secreted exosomes. Here, we studied the molecular mechanisms driving miR-21 sorting into colonic epithelial exosomes (CEEs) under SP-stimulation.MethodsWe performed studies with human colonic epithelial NCM460 cells overexpressing neurokinin-1-receptor (NCM460-NK1R) and in intestinal-epithelial-specific NK1R knockout mice. SP-regulated gene targets were validated by real-time polymerase chain reaction (RT-PCR) and immunoblotting. Small non-coding RNAs (sncRNAs) were isolated from NCM460-NK1R cells and secreted exosomes, and 3′-end adenylated and 3′-end uridylated fractions were separated. Cellular and exosomal sncRNA fractions were processed for miR-21 and miR-1307-3p expression and 3′-end adenylation to uridylation (A/U) ratios using RT-PCR. Pharmacological inhibition studies in NCM460-NK1R cells were performed in the presence of the Adenosine A2B receptor (ADORA2B) antagonist, PSB-1115, followed by RT-PCR and immunoblotting. Mass spectrometry validated in silico interacting partners of miR-21 in CECs.ResultsIn CECs, miR-21 is predominantly polyuridylated under SP-stimulation and preferentially sorted to CEEs. SP/NK1R signaling activation upregulates extracellular adenosine (ADO) signaling via ADORA2B concomitant with reduced ADO uptake via epithelial-specific equilibrative nucleoside transporter-2 (ENT-2). Mass spectrometry and immunoblotting revealed upregulation of TUT7 in SP-stimulated CEEs. Knockdown of TUT7 decreased both TUT7 and miR-21 content in these exosomes. Pharmacological inhibition of ADORA2B in CECs resulted in decreased TUT7 and miR-21 in CEEs, regardless of SP stimulation.ConclusionsSP/NK1R coupling in CECs activates ADORA2B and its downstream signaling cascade which mediates TUT7/miR-21 interaction and subsequent miR-21 polyuridylation and exosomal export.SynopsisSubstance P-neurokinin-1 receptor signaling regulates terminal uridyl transferase7-mediated predominant 3’-end polyuridylation and exosomal recruitment of miR-21 in human colonic epithelial NCM460 cells overexpressing NK-1R via activation of Adenosine A2B receptor and its downstream signaling cascade. Because Adenosine A2B receptor signaling is involved in IBD pathogenesis, it could therefore be exploited as a pharmacological target for the therapeutic benefits in IBD.

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.

Background Clostridium 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. Method Using multiplex enzyme-linked immunosorbent assay, we determined the cytokine secretion of toxin A– and B–treated human and mouse colonic explants. Results Toxin 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. Conclusion MIP-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.

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/barrierfunction genes. Functionally, miR-219a-5p inhibition enhanced the permeability of intestinal epithelial cells as TEER was reduced (25-50%, P < .05) and dextran flux wasincreased (P < .01). Additionally, inhibition of miR-338-3p incells caused alterations in the mitogen-activated protein kinase (MAPK) signaling pathway genes.ConclusionTwomicroRNAs 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.

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.

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.

BACKGROUND: Inflammatory bowel disease is associated with an increased risk of Parkinson’s Disease (PD) in epidemiological studies. (PMIDs: 29788069, 29785965, 29459115) The mechanism underlying this association is unknown.AIMS: 1) To test the hypothesis that PD-related genes are dysregulated in ulcerative colitis (UC) and experimental murine colitis using gene set enrichment analysis (GSEA) of microarray gene expression profiling data. 2) To test the association of PD-related genes with UC status controlling for age and sex.METHODS: The UC analysis used previously published gene expression profiling data (Padua et al PMID: 27492330) including surgical samples (n, %women, mean age) from controls (7, 43%, 45) and UC (12, 67%, 39). Data for the experimental colitis analysis, obtained from GEO (GSE22307, Fang et al PMID:20923862), included colon tissues from male C57BL/6J mice aged 12–14 weeks treated with 3% dextran sodium sulfate (DSS) in in the drinking water for 6 days (n=6) and untreated mice (n=5). The GSEA software was used to determine enrichment of the KEGG Parkinson’s Disease gene set in UC/control and DSS/untreated. The GSEA recommended threshold of false discovery rate (FDR) of 0.25 was used to determine statistical significance. The effect of UC vs control on expression of PD-genes present in the array (n=78) controlling for age and sex was determined using linear models and a Benjamini-Hochberg adjusted p-value threshold of 0.05 was used to determine statistical significance.RESULTS: PD genes were enriched in controls vs UC (FDR=0.19) and in untreated vs DSS-treated mice (FDR=0.14). Pathway representation of genes enriched in controls (downregulated in UC/DSS, Figure 1) reveals that most down-regulated genes are associated with mitochondrial function. Controlling for age and sex, four genes (COX5B, NDUFV1, NDUFB1, CYCS, Table 1), all with roles in cellular respiration, were down-regulated in UC vs controls. CONCLUSIONS: Genes related to PD pathogenesis are down-regulated in UC and experimental colitis. The predicted impaired mitochondrial function associated with these changes is in concordance with putative pathophysiologic mechanisms of PD. We hypothesize that increased oxidative stress associated with inflammation (perhaps in combination with genetic and environmental risk factors) may contribute to the observed association between IBD and PD.

Background: Evidence supports that Parkinson’s Disease (PD) is a disease of the gut-brain axis. Constipation is prevalent in patients with PD, and mice overexpressing human wild type alpha–synuclein (ASO) have delayed colonic transit in comparison to wild-type (WT) mice. Alpha synuclein overexpression in ASO mice begins at about 1 month of age, and changes in colonic transit are present at 3 months. Improved understanding of initial molecular changes in the gastrointestinal tract in PD could lead to early detection of PD and/or gut-targeted therapies to prevent motor symptoms in PD.Aim: To perform gene expression profiling of the distal colon and brain in ASO vs. WT mice at 1 and 3 months.Methods: RNA was extracted from descending colon and brain tissues of male mice at 1 month (n=9 ASO, n= 7 WT) and 3 months (n=9 ASO, n=9WT) of age. RNA sequencing was performed in the UCLA Neuroscience Genomics Core Laboratory using Lexogen QuantSeq 3′ FWD cDNA library synthesis. Results: The number of differentially expressed genes (DEGs) is shown in Table 1. Glial cell line derived neurotrophic factor (GDNF) family receptor alpha 4 (Gfra4), and SRY (sex determining region Y)-box 10 (Sox10) were among up-regulated DEGs in the colons of ASO vs. WT mice at 1 month (fold change (FC), adjusted p-value for Gfra4 and Sox10: 4.4, 0.006; 5.8, 0.02). Sox10 was marginally increased at 3 months (FC=2.3, unadjusted p=0.03).Conclusions: Both the brain and colon of ASO vs. WT mice shown altered gene expression as early as 1 month. Early colonic gene expression is notable for Sox10, a marker of enteric glial cells (EGCs). Increased activation of EGCs has been found in colonic biopsies from patients with PD, which adds support to the use of ASO mice to study the gastrointestinal tract in PD.

BACKGROUND: Identification of alpha-synuclein (aSyn) aggregates, a pathological hallmark of Parkinson’s Disease (PD), in the colon of PD patients, supports PD as a disease of the gut-brain axis. Molecular mechanisms of the gut-brain axis, particularly related to effects of colonic aSyn are poorly understood. RNA-sequencing data from post-mortem human tissue samples is available (GTEx). Consensus weighted gene correlation network analysis (WGCNA) can identify modules of highly correlated genes in both brain and colon. AIMS: 1. To perform WGCNA of sigmoid colon (SC) and cerebral cortex (CC) from the same individuals to identify gut-brain consensus modules. 2. To compare connectivity of aSyn-containing networks within brain and colon. METHODS: GTEx data in the form of gene-level counts was obtained from the recount2 database. Counts were scaled, prepared for linear modeling using variance stabilization and quantile normalized using the recount, DESeq2 and limma packages in R. Donors with data from both CC and SC were included (n=29 after removal of 5 outliers). The WGCNA package (R) was used to generate signed networks in CC and SC and consensus modules (similar clustering in both networks). Overrepresented gene ontology (GO) terms were determined with the hypergeometric function in the GOstats package (R). RESULTS: There were 15 consensus modules. Overall, there was increased intermodular connectivity in the CC, likely related to increased homogeneity of cell types. While CC and SC intermodular connectivity was quite different, some relationships were conserved. Interestingly, the module containing the gene for aSyn (SNCA) was differentially associated in SC (positively) vs CC (negatively) with a module enriched for the GO term "Astrocyte differentiation". Hub genes in this module included SRY-box 10 (SOX10) and S100 calcium binding protein B (S100B), markers of enteric glial cells (EGCs) and astrocytes. Pairwise correlations between SNCA (gene encoding aSyn) and these markers mirrored module relationships. Glial fibrillary acidic protein (GFAP) and proteolipid protein 1 (PLP1) are also EGC markers. GFAP, which was not in the consensus modules (did not cluster similarly in brain and colon) was correlated with aSyn in brain but not colon. PLP1 on the X chromosome was excluded from clustering. CONCLUSIONS: The GTEx data set is a valuable resource to explore gene networks relevant to the gut-brain axis. EGCs may function differently than astrocytes in response to aSyn and/or the tissue response to aSyn may differ in CC vs SC. The interaction of aSyn and EGCs in the colon merits further investigation in disease and animal models.

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.

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.

Exosomes are cellular vesicles involved in intercellular communication via their specialized molecular cargo, such as miRNAs. Substance P (SP), a neuropeptide/hormone, and its high-affinity receptor, NK-1R, are highly expressed during colonic inflammation. Our previous studies show that SP/NK-1R signaling stimulates differential miRNA expression and promotes colonic epithelial cell proliferation. In this study, we examined whether SP/NK-1R signaling regulates exosome biogenesis and exosome-miRNA cargo sorting. Moreover, we examined the role of SP/NK-1R signaling in exosome-regulated cell proliferation and migration. Exosomes produced by human colonic NCM460 epithelial cells overexpressing NK-1R (NCM460-NK1R) were isolated from culture media. Exosome abundance and uptake were assessed by Western blot analysis (abundance) and Exo-Green fluorescence microscopy (abundance and uptake). Cargo-miRNA levels were assessed by RT-PCR. Cell proliferation and migration were assessed using xCELLigence technology. Colonic epithelial exosomes were isolated from mice pretreated with SP for 3 days. Cell proliferation in vivo was assessed by Ki-67 staining. SP/NK-1R signaling in human colonic epithelial cells (in vitro) and mouse colons (in vivo) increased 1) exosome production, 2) the level of fluorescence in NCM460s treated with Exo-Green-labeled exosomes, and 3) the level of miR-21 in exosome cargo. Moreover, our results showed that SP/NK-1R-induced cell proliferation and migration are at least in part dependent on intercellular communication via exosomal miR-21 in vitro and in vivo. Our results demonstrate that SP/NK-1R signaling regulates exosome biogenesis and induces its miR-21 cargo sorting. Moreover, exosomal miR-21 promotes proliferation and migration of target cells. NEW & NOTEWORTHY Substance P signaling regulates exosome production in human colonic epithelial cells and colonic crypts in wild-type mice. MiR-21 is selectively sorted into exosomes induced by Substance P stimulation and promotes cell proliferation and migration in human colonocytes and mouse colonic crypts.

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.

Aims The 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 methods UC 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 findings CDKN2B-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. Significance This study identifies a novel lncRNA with both linear and circular transcripts affecting UC biology.

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.

A variety of biological processes are regulated by posttranslational modifications. Posttranslational modifications including phosphorylation, ubiquitination, glycosylation, and proteolytic cleavage, control diverse physiological functions in the gastrointestinal tract. Therefore, a better understanding of their implications in intestinal diseases, including inflammatory bowel disease, irritable bowel syndrome, celiac disease, and colorectal cancer would provide a basis for the identification of novel biomarkers as well as attractive therapeutic targets. Posttranslational modifications can be common denominators, as well as distinct biomarkers, characterizing pathological differences of various intestinal diseases. This review provides experimental evidence that identifies changes in posttranslational modifications from patient samples, primary cells, or cell lines in intestinal disorders, and a summary of carefully selected information on the use of pharmacological modulators of protein modifications as therapeutic options.

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.

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.

The neuroendocrine circuit of the corticotropin-releasing hormone (CRH) family peptides, via their cognate receptors CRHR1 and CRHR2, copes with psychological stress. However, peripheral effects of the CRH system in colon cancer remains elusive. Thus, we investigate the role of CRHR1 and CRHR2 in colon cancer. Human colon cancer biopsies were used to measure the mRNA levels of the CRH family by quantitative real-time PCR. Two animal models of colon cancer were used: Apcmin/+ mice and azoxymethane (AOM)/dextran sulfate sodium (DSS)-treated mice. The mRNA levels of CRHR2 and UCN III are reduced in human colon cancer tissues compared to those of normal tissues. Crhr1 deletion suppresses the tumor development and growth in Apcmin/+ mice, while Crhr2 deficiency exacerbates the tumorigenicity. Crhr1 deficiency not only inhibits the expression of tumor-promoting cyclooxygenase 2, but also upregulates tumor-suppressing phospholipase A2 in Apcmin/+ mice, however, Crhr2 deficiency does not change these expressions. In the AOM/DSS model, Crhr2 deficiency worsens the tumorigenesis. In conclusion, Crhr1 deficiency confers tumor-suppressing effects in Apcmin/+ mice, but Crhr2 deficiency worsens the tumorigenicity in both Apcmin/+ and AOM/DSS-treated mice. Therefore, pharmacological inhibitors of CRHR1 or activators of CRHR2 could be of significance as anti-colon cancer drugs.