Patient Perception and Clinical Impact of Direct-to-Consumer Advertising in Inflammatory Bowel Disease.

INTRODUCTION: With an increasing number of available therapies for inflammatory bowel disease (IBD), little is known about patients' attitudes regarding IBD-related direct-to-consumer advertising (IBD-DTCA) and its impact on treatment decisions in clinical practice. METHODS: We administered a 58-item, mailed questionnaire to patients with IBD receiving Gastroenterology subspecialty care at a large academic health system. The survey assessed patient awareness and perception of IBD-DTCA and its effect on IBD treatment discussions and decisions. We used bivariate analysis to evaluate patient-level factors associated with awareness and favorable perception of IBD-DTCA. RESULTS: We achieved a response rate of 15.2% (n = 226 of 1486). Most patients (93.3%) reported awareness of IBD-DTCA, with adalimumab receiving the most exposure. A majority of respondents reported IBD-DTCA made them more aware of treatments they otherwise would not know about (53.6%), provided information in a balanced manner (63.5%), and taught them about new potential risks and side effects (64.5%). Patients without a college degree and those with a household income less than $75 k per year perceived IBD-DTCA more favorably. However, IBD-DTCA rarely changed IBD management, with only 7.6% of respondents having a discussion with their provider about the advertised drug and only two (0.9%) being initiated on the advertised drug. CONCLUSION: IBD patients were aware of IBD-DTCA and perceived it favorably; however, IBD-DTCA rarely led to patient-provider discussions or changes in treatment regimen.

Genome-Wide Association Study Identifies African-Specific Susceptibility Loci in African Americans With Inflammatory Bowel Disease.

BACKGROUND & AIMS: The inflammatory bowel diseases (IBD) ulcerative colitis (UC) and Crohn's disease (CD) cause significant morbidity and are increasing in prevalence among all populations, including African Americans. More than 200 susceptibility loci have been identified in populations of predominantly European ancestry, but few loci have been associated with IBD in other ethnicities. METHODS: We performed 2 high-density, genome-wide scans comprising 2345 cases of African Americans with IBD (1646 with CD, 583 with UC, and 116 inflammatory bowel disease unclassified) and 5002 individuals without IBD (controls, identified from the Health Retirement Study and Kaiser Permanente database). Single-nucleotide polymorphisms (SNPs) associated at P < 5.0 × 10-8 in meta-analysis with a nominal evidence (P < .05) in each scan were considered to have genome-wide significance. RESULTS: We detected SNPs at HLA-DRB1, and African-specific SNPs at ZNF649 and LSAMP, with associations of genome-wide significance for UC. We detected SNPs at USP25 with associations of genome-wide significance for IBD. No associations of genome-wide significance were detected for CD. In addition, 9 genes previously associated with IBD contained SNPs with significant evidence for replication (P < 1.6 × 10-6): ADCY3, CXCR6, HLA-DRB1 to HLA-DQA1 (genome-wide significance on conditioning), IL12B,PTGER4, and TNC for IBD; IL23R, PTGER4, and SNX20 (in strong linkage disequilibrium with NOD2) for CD; and KCNQ2 (near TNFRSF6B) for UC. Several of these genes, such as TNC (near TNFSF15), CXCR6, and genes associated with IBD at the HLA locus, contained SNPs with unique association patterns with African-specific alleles. CONCLUSIONS: We performed a genome-wide association study of African Americans with IBD and identified loci associated with UC in only this population; we also replicated IBD, CD, and UC loci identified in European populations. The detection of variants associated with IBD risk in only people of African descent demonstrates the importance of studying the genetics of IBD and other complex diseases in populations beyond those of European ancestry.

Characterization of genetic loci that affect susceptibility to inflammatory bowel diseases in African Americans.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) has familial aggregation in African Americans (AAs), but little is known about the molecular genetic susceptibility. Mapping studies using the Immunochip genotyping array expand the number of susceptibility loci for IBD in Caucasians to 163, but the contribution of the 163 loci and European admixture to IBD risk in AAs is unclear. We performed a genetic mapping study using the Immunochip to determine whether IBD susceptibility loci in Caucasians also affect risk in AAs and identify new associated loci. METHODS: We recruited AAs with IBD and without IBD (controls) from 34 IBD centers in the United States; additional controls were collected from 4 other Immunochip studies. Association and admixture loci were mapped for 1088 patients with Crohn's disease, 361 with ulcerative colitis, 62 with IBD type unknown, and 1797 controls; 130,241 autosomal single-nucleotide polymorphisms (SNPs) were analyzed. RESULTS: The strongest associations were observed between ulcerative colitis and HLA rs9271366 (P = 7.5 × 10(-6)), Crohn's disease and 5p13.1 rs4286721 (P = 3.5 × 10(-6)), and IBD and KAT2A rs730086 (P = 2.3 × 10(-6)). Additional suggestive associations (P < 4.2 × 10(-5)) were observed between Crohn's disease and IBD and African-specific SNPs in STAT5A and STAT3; between IBD and SNPs in IL23R, IL12B, and C2orf43; and between ulcerative colitis and SNPs near HDAC11 and near LINC00994. The latter 3 loci have not been previously associated with IBD, but require replication. Established Caucasian associations were replicated in AAs (P < 3.1 × 10(-4)) at NOD2, IL23R, 5p15.3, and IKZF3. Significant admixture (P < 3.9 × 10(-4)) was observed for 17q12-17q21.31 (IZKF3 through STAT3), 10q11.23-10q21.2, 15q22.2-15q23, and 16p12.2-16p12.1. Network analyses showed significant enrichment (false discovery rate <1 × 10(-5)) in genes that encode members of the JAK-STAT, cytokine, and chemokine signaling pathways, as well those involved in pathogenesis of measles. CONCLUSIONS: In a genetic analysis of 3308 AA IBD cases and controls, we found that many variants associated with IBD in Caucasians also showed association evidence with these diseases in AAs; we also found evidence for variants and loci not previously associated with IBD. The complex genetic factors that determine risk for or protection against IBD in different populations require further study.

Variation in treatment of patients with inflammatory bowel diseases at major referral centers in the United States.

We performed a prospective study of patients with inflammatory bowel diseases to examine variations in treatment among medical centers. In a prospective cohort study of 1659 patients with Crohn's disease and 946 patients with ulcerative colitis seen at 7 high-volume referral centers, we collected data on demographics, disease characteristics, and medical and surgical treatments. We used logistic regression to determine differences in treatment among centers, controlling for potential confounders. We found significant variations among centers in the treatment of Crohn's disease with immunomodulators (odds ratio [OR], 3.34; 95% confidence interval [CI], 2.09-5.32) but not anti-tumor necrosis factor agents (OR, 1.64; 95% CI, 0.97-2.77). There was less variation in the treatment of ulcerative colitis; we found no difference in use of immunomodulators (OR, 1.83; 95% CI, 1.00-3.36) or anti-tumor necrosis factor therapy (OR, 0.81; 95% CI, 0.40-1.65). The development and implementation of evidence-based standards of care for inflammatory bowel disease may help reduce variation and improve outcomes.

Translational repression of SLC26A3 by miR-494 in intestinal epithelial cells.

SLC26A3 [downregulated in adenoma (DRA)] is a Cl(-)/HCO3(-) exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Altered DRA expression levels are associated with infectious and inflammatory diarrheal diseases. Therefore, it is critical to understand the regulation of DRA expression. MicroRNAs (miRNAs) are endogenous, small RNAs that regulate protein expression via blocking the translation and/or promoting mRNA degradation. To investigate potential modulation of DRA expression by miRNA, five different in silico algorithms were used to predict the miRNAs that target DRA. Of these miRNAs, miR-494 was shown to have a highly conserved putative binding site in the DRA 3'-untranslated region (3'-UTR) compared with other DRA-targeting miRNAs in vertebrates. Transfection with pmirGLO dual luciferase vector containing DRA 3'-UTR (pmirGLO-3'-UTR DRA) resulted in a significant decrease in relative luciferase activity compared with empty vector. Cotransfection of the DRA 3'-UTR luciferase vector with a miR-494 mimic further decreased luciferase activity compared with cells transfected with negative control. The transfection of a miR-494 mimic into Caco-2 and T-84 cells significantly increased the expression of miR-494 and concomitantly decreased the DRA protein expression. Mutation of the seed sequences for miR-494 in 3'-UTR of DRA abrogated the effect of miR-494 on 3'-UTR. These data demonstrate a novel regulatory mechanism of DRA expression via miR-494 and indicate that targeting this microRNA may serve to be a potential therapeutic strategy for diarrheal diseases.

IL-23 receptor regulation by Let-7f in human CD4+ memory T cells.

CD4(+) memory T cells include the Th17 cell population, which has been shown to be implicated in autoimmune and inflammatory diseases. These memory T cells express higher IL-23R and produce more IL-17 compared with their naive counterparts. However, the molecular mechanisms that regulate IL-23R expression in human T cells are not completely understood. MicroRNAs play important roles in a wide range of biological events through posttranscriptional suppression of target mRNAs. In this article, we provide evidence that a specific microRNA, Let-7f, inhibits IL-23R expression in human CD4(+) memory T cells. Endogenous expression of Let-7f in memory T cells is significantly lower when compared with naive T cells, and Let-7f blocks IL-23R expression through its complementary target sequence within 3' untranslated region of target gene. Furthermore, exogenous transfection of a Let-7f mimic into memory T cells results in downregulation of IL-23R and its downstream cytokine, IL-17. Our findings reveal a novel mechanism in regulating the IL-23/IL-23R pathway and subsequent downstream IL-17 production, which may provide novel therapeutics for human inflammatory and autoimmune diseases.

Novel players in inflammatory bowel disease pathogenesis.

Technological and conceptual advances in inflammatory bowel disease research have uncovered new mechanisms that contribute to the pathogenesis of these disorders. It is becoming increasingly clear that the microbiota of the gut and the response of intestinal cells to that microbiota can initiate or contribute to intestinal inflammation. Evidence from genetic studies have identified IBD-associated genes implicated in autophagy and innate sensing of microbes. These genes also play key roles in the homeostasis of a cell type that stands at the interface of host-microbial interaction - the Paneth cell. Here we discuss recent findings that underscore the importance of the microbiome, Paneth cells and autophagy in inflammatory bowel disease.

Intravenous immunoglobulin for the treatment of Clostridium difficile infection: a review.

Clostridium difficile infection (CDI) has increased sharply in incidence, mortality rate, and burden on the healthcare system over the past decade. Therefore, novel treatment modalities have been developed, including intravenous immunoglobulin (IVIG). The level of immune response to Clostridium difficile colonization is the major determinant of the magnitude and duration of clinical manifestations. This effect is mediated predominantly by serum IgG anti-toxin A antibodies. Based on this finding, anti-toxin A and B antibodies were successfully used in multiple in vitro and in vivo experimental settings to passively immunize hamsters in CDI models. In humans, IVIG was used as the source of those antibodies. Fifteen small, mostly retrospective and non-randomized reports documented IVIG's success in the treatment of protracted, recurrent, or severe CDI. Diarrhea resolution rates were higher in the former patient group, but the recurrence rates were similar. IVIG mechanism of action is neutralization of mainly toxin A through IgG anti-toxin A antibodies. Purified anti-toxin A and B antibodies were successfully used to decrease CDI recurrence rates among patients with no or one previous CDI episodes. In conclusion, the efficacy of IVIG for CDI treatment in animal models has been convincingly demonstrated. However, only few small non-randomized, mostly uncontrolled reports have been published on human subjects. A phase II trial results support the use of purified anti-toxin A and B antibodies to decrease CDI recurrence rates. Therefore, IVIG should currently only be used as adjunct therapy until results from large, randomized controlled trials are available.

MicroRNAs are differentially expressed in ulcerative colitis and alter expression of macrophage inflammatory peptide-2 alpha.

BACKGROUND & AIMS: Chronic inflammatory bowel diseases such as ulcerative colitis (UC) are associated with differential expression of genes involved in inflammation and tissue remodeling. MicroRNAs (miRNAs), which direct mRNA degradation and translational inhibition, influence a number of disease processes. We examined whether miRNAs are differentially expressed in UC tissues and are associated with expression of genes that regulate inflammation. METHODS: miRNA expression was assessed in patients with active UC, inactive UC, Crohn's disease, irritable bowel syndrome, infectious colitis, and microscopic colitis, as well as in healthy subjects by microarray, quantitative reverse transcription-polymerase chain reaction and in situ hybridization analyses. Colonic epithelial cell (HT29) expression of miRNAs was assessed. Regulation of gene expression by miRNAs was assessed by luciferase reporter construct assays and transfection of specific miRNA mimics. RESULTS: Active UC was associated with the differential expression of 11 miRNAs; 3 were significantly decreased and 8 were significantly increased in UC tissues. In situ hybridization analysis indicated that miR-192, an miRNA with decreased expression in active UC, was predominantly localized to colonic epithelial cells. Macrophage inflammatory peptide (MIP)-2 alpha, a chemokine expressed by epithelial cells, was identified as a target of miR-192. In colon epithelial cells, induction of MIP-2 alpha expression by tumor necrosis factor-alpha was accompanied by a concomitant reduction in miR-192 expression and miR-192 was observed to regulate the expression of MIP-2 alpha. CONCLUSIONS: These findings expand the known roles of miRNAs, indicating that tissues from patients with UC, and possibly other chronic inflammatory diseases, have altered miRNA expression patterns. These findings also demonstrate that miRNAs regulate colonic epithelial cell-derived chemokine expression.

miR-193a-3p is a Key Tumor Suppressor in Ulcerative Colitis-Associated Colon Cancer and Promotes Carcinogenesis through Upregulation of IL17RD.

Purpose: Patients with ulcerative colitis are at increased risk for colorectal cancer, although mechanisms underlying neoplastic transformation are poorly understood. We sought to evaluate the role of microRNAs in neoplasia development in this high-risk population.Experimental Design: Tissue from 12 controls, 9 ulcerative colitis patients without neoplasia, and 11 ulcerative colitis patients with neoplasia was analyzed. miRNA array analysis was performed and select miRNAs assayed by real-time PCR on the discovery cohort and a validation cohort. DNA methylation of miR-193a was assessed. Following transfection of miR-193a-3p, proliferation, IL17RD expression, and luciferase activity of the 3'UTR of IL17RD were measured. Tumor growth in xenografts as well as EGFR signaling were assessed in HCT116 cells expressing IL17RD with either a mutant 3' untranslated region (UTR) or wild-type (WT) 3'UTR.Results: miR-31, miR-34a, miR-106b, and miR-193a-3p were significantly dysregulated in ulcerative colitis-neoplasia and adjacent tissue. Significant down-regulation of miR-193a-3p was also seen in an independent cohort of ulcerative colitis cancers. Changes in methylation of miR-193a or expression of pri-miR-193a were not observed in ulcerative colitis cancer. Transfection of miR-193a-3p resulted in decreased proliferation, and identified IL17RD as a direct target of miR-193a-3p. IL17RD expression was increased in ulcerative colitis cancers, and miR-193a-3p treatment decreased growth and EGFR signaling of HCT116 cells in xenografts expressing both IL17RD with WT 3'UTR compared with cells expressing IL17RD with mutant 3'UTR.Conclusions: miR-193a-3p is downregulated in ulcerative colitis neoplasia, and its loss promotes carcinogenesis through upregulation of IL17RD. These findings provide novel insight into inflammation-driven colorectal cancer and could suggest new therapeutic targets in this high-risk population. Clin Cancer Res; 23(17); 5281-91. ©2017 AACR.

Recent advances in the understanding of celiac disease: therapeutic implications for the management of pediatric patients.

Celiac disease (CD) is an autoimmune condition occurring in genetically susceptible individuals characterized by inflammatory injury to the mucosa of the small intestine after the ingestion of wheat glutens or related barley and rye products. Originally thought to be highly prevalent only in Northern European populations, growing evidence indicates a much higher prevalence in many other regions, including the US as well as South America, North Africa, and Asia. The growing awareness that pediatric patients may present with quite diverse and protean manifestations and the significant impact of CD on childhood development has prompted efforts to increase CD awareness for the early diagnosis and treatment of this disease. The current diagnostic criteria for CD requires characteristic histologic findings in small bowel biopsies and clinical remission when placed on a gluten-free diet. Serologic testing for CD can provide additional support for the diagnosis of CD or a means to assess efficacy and adherence to a gluten-free diet. The mainstay of treatment remains the institution of a gluten-free diet. However, patients with refractory CD may require treatment with immunosuppressant medications. With the increased identification of specific gluten epitopes and understanding of the pathogenesis of CD, future therapies may rely on genetically altering gluten proteins, immunization techniques, or therapies focused on either the development of specific immune tolerance or regulation of mucosal inflammation.

Ulcerative Colitis-Associated Long Noncoding RNA, BC012900, Regulates Intestinal Epithelial Cell Apoptosis.

BACKGROUND: Long noncoding RNAs (lncRNAs) were recently found to be key regulators of biological functions and associated with human diseases. Thus far, the roles of lncRNAs in inflammatory bowel disease (IBD) remain unknown. We aimed to determine whether lncRNAs are associated with IBD and regulate epithelial cell physiology. METHODS: lncRNAs microarray and quantitative RT-PCR were performed on 60 sigmoid colon biopsies from patients with active ulcerative colitis (UC) and relevant controls. Localization of lncRNAs was detected by in situ hybridization and on subcellular RNA. The boundaries of BC012900 were assessed by 5' and 3'-rapid amplification of cDNA ends. Apoptosis and proliferation assays were performed on BC012900-expressing construct or siRNA-transfected cells. RESULTS: We identified 329 lncRNAs with increased and 126 lncRNAs with decreased expression in active UC tissues compared with normal control tissues, including the most significantly upregulated (BC012900, AK001903, and AK023330) and downregulated (BC029135, CDKN2B-AS1, and BC062296) transcripts. We found that most of the lncRNAs are localized to the nucleus. In particular, BC012900 expression was significantly increased in active UC and stimulated by cytokines and pathogenic molecules. Furthermore, BC012900 overexpression in epithelial cells results in a significant inhibition of cell proliferation and an increased susceptibility to apoptosis, which differ from its adjacent gene DUSP4. CONCLUSIONS: Multiple lncRNAs are differentially expressed in IBD and play a role in regulating cellular physiology. Our results indicate that lncRNAs may be integral modulators of intestinal inflammation associated with IBD and represent novel targets for future therapeutics and diagnostic marker development.

The risk of lymphoma in the treatment of inflammatory bowel disease with immunosuppressive agents.

Immunosuppressive agents have become an established part of the therapeutic armamentarium for inflammatory bowel disease (IBD). However, when used in transplant recipients or for other indications, agents that suppress or modulate the immune system (immunomodulators) have been associated with an increased risk of lymphoma. Fortunately, in part because of the lower doses used in IBD patients, the risk of lymphoma in IBD patients appears to be significantly less than that associated with renal and hepatic transplant-related immunosuppression. Whether the risk of azathioprine or 6-mercaptopurine associated lymphoma in IBD is real or relates to the underlying disease remains unclear. The results of several recent large well designed population-based studies suggest that the lymphoma risk associated with azathioprine and 6-mercaptopurine therapy is likely to be of minimal clinical significance compared to the established and more frequent risks of myelosuppression and infection, and is far outweighed by the clinical benefit of immunomodulator therapy in IBD. While the issue of lymphoma risk is likely to become more relevant with the growing number of biologic and immunomodulators being tested in clinical trials for IBD, early post-marketing surveillance data on infliximab suggests that the lymphoma risk may not be any greater than that associated with azathioprine and 6-mercaptopurine.

TET-catalyzed 5-hydroxymethylcytosine regulates gene expression in differentiating colonocytes and colon cancer.

The formation of differentiated cell types from pluripotent progenitors involves epigenetic regulation of gene expression. DNA hydroxymethylation results from the enzymatic oxidation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) by the ten-eleven translocation (TET) 5-mC dioxygenase enzymes. Previous work has mapped changes in 5-mC during differentiation of intestinal stem cells. However, whether or not 5-hmC regulates colonocyte differentiation is unknown. Here we show that 5-hmC regulates gene expression during colonocyte differentiation and controls gene expression in human colon cancers. Genome-wide profiling of 5-hmC during in vitro colonic differentiation demonstrated that 5-hmC is gained at highly expressed and induced genes and is associated with intestinal transcription factor binding sites, including those for HNF4A and CDX2. TET1 induction occurred during differentiation, and TET1 knockdown altered gene expression and inhibited barrier formation of colonocytes. We find that the 5-hmC distribution in primary human colonocytes parallels the distribution found in differentiated cells in vitro, and that gene-specific 5-hmC changes in human colon cancers are directly correlated with changes in gene expression. Our results support a model in which 5-hmC regulates differentiation of adult human intestine and 5-hmC alterations contribute to the disrupted gene expression in colon cancer.

Divergent influence of microRNA-21 deletion on murine colitis phenotypes.

BACKGROUND: MicroRNAs (miRNAs) acting as negative regulators of gene expression are differentially expressed in intestinal tissues of patients with inflammatory bowel disease (IBD). Assessing the functions of miRNAs in murine colitis facilitates elucidating the role of specific miRNAs in human IBD. We aimed to determine the miRNA signature of several models of colitis and to assess the influence of miR-21 on intestinal inflammation. METHODS: miR-21-deficient mouse and littermate controls were assessed in the standard DSS, TNBS, and CD4+ T-cell transfer models of colitis. RNAs of mouse colon and CD4+CD45RB High cells were analyzed by miRNA and mRNA microarray, and quantitative reverse transcription polymerase chain reaction. T helper (Th) cell polarization was accessed by flow cytometry and enzyme-linked immunosorbent assay. RESULTS: Alterations in miRNA expression were identified for acute and chronic DSS and TNBS colitis, respectively. The expression of miRs-21, -142-3p, and -223 were distinct between DSS and TNBS models while overlap of numerous miRNAs was found. Importantly, miRs-19b, -192, and -215, that are decreased in IBD, were decreased in these models of colitis. miR-21, which is increased in IBD, was increased in TNBS colitis. Furthermore, the deletion of miR-21 resulted in the exacerbation of both the TNBS and T-cell transfer models of colitis. CD4+CD45RB High T cells from miR-21 mice were prone to Th1 polarization. CONCLUSIONS: MiRNAs are differentially expressed in both human IBD and murine colitis, with overlap of several IBD-associated miRNAs. The demonstration that miR-21-/- deletion exacerbated CD4+ T-cell-mediated models of colitis provide further evidence that miRNAs play significant roles in the pathogenesis of IBD.

NOD2 expression is regulated by microRNAs in colonic epithelial HCT116 cells.

BACKGROUND: Crohn's disease (CD) is associated with defective sensing of pathogens in genetically susceptible individuals. Nucleotide-binding oligomerization domain containing 2 (NOD2) mutations in coding regions are strongly linked to CD pathogenesis. Our laboratory has reported that microRNAs (miRNAs) are differentially expressed in CD. However, miRNA regulation of NOD2 remains unknown. This study was designed to determine whether miRNAs regulate NOD2 expression as well as downstream nuclear factor kappaB activation and inflammatory responses in colonic epithelial HCT116 cells. METHODS: NOD2 and miRNA expression in stimulated HCT116 cells were assessed by quantitative reverse transcription-polymerase chain reaction. Regulation of NOD2 expression by miRNAs was determined by luciferase reporter construct assays and transfection of specific miRNA mimics. Regulation of NOD2 signaling and immune response by miRNAs was assessed by transfection of mimics followed by muramyl dipeptide stimulation. RESULTS: Muramyl dipeptide-induced increases in NOD2, interleukin-8, and CXCL3 expression were inversely associated with miRNA expression. Overexpression of miR-192, miR-495, miR-512, and miR-671 suppressed NOD2 expression, muramyl dipeptide-mediated NF-κB activation, and messenger RNA expressions of interleukin-8 and CXCL3 in HCT116 cells. A single-nucleotide polymorphism (rs3135500) located in the NOD2 3'-untranslated region significantly reduced miR-192 effects on NOD2 gene expression. CONCLUSIONS: To our knowledge, this is the first report demonstrating that miRNAs regulate NOD2 and its signaling pathway. Four miRNAs downregulate NOD2 expression, suppress NF-κB activity, and inhibit interleukin-8 and CXCL3 messenger RNA expression. Treatment of CD with miRNAs may represent a potential anti-inflammatory therapeutic strategy in CD patients with and without NOD2 gene mutations.

Human autophagy gene ATG16L1 is post-transcriptionally regulated by MIR142-3p.

Multiple genetic studies have implicated the autophagy-related gene, ATG16L1, in the pathogenesis of Crohn disease (CD). While CD-related research on ATG16L1 has focused on the functional significance of ATG16L1 genetic variations, the mechanisms underlying the regulation of ATG16L1 expression are unclear. Our laboratory has described that microRNAs (miRNAs), key regulators of gene expression, are dysregulated in CD. Here, we report miRNA-mediated regulation of ATG16L1 in colonic epithelial cells as well as Jurkat T cells. Dual luciferase reporter assays following the transfection of vectors containing the ATG16L1 3'-untranslated region (3'UTR) or truncated 3'UTR fragments suggest that the first half of ATG16L1 3'UTR in the 5' end is more functional for miRNA targeting. Of 5 tested miRNAs with putative binding sites within the region, MIR142-3p, upon transient overexpression in the cells, resulted in decreased ATG16L1 mRNA and protein levels. Further observation demonstrated that the luciferase reporter vector with a mutant MIR142-3p binding sequence in the 3'UTR was unresponsive to the inhibitory effect of MIR142-3p, suggesting ATG16L1 is a gene target of MIR142-3p. Moreover, the regulation of ATG16L1 expression by a MIR142-3p mimic blunted starvation- and L18-MDP-induced autophagic activity in HCT116 cells. Additionally, we found that a MIR142-3p inhibitor enhanced starvation-induced autophagy in Jurkat T cells. Our study reveals MIR142-3p as a new autophagy-regulating small molecule by targeting ATG16L1, implying a role of this miRNA in intestinal inflammation and CD.

miR-106b fine tunes ATG16L1 expression and autophagic activity in intestinal epithelial HCT116 cells.

BACKGROUND: The microRNAs (miRNAs) regulate gene expression at the posttranscriptional level. ATG16L1, an essential component for autophagy and a risk gene for Crohn's disease, contains two binding sites in the 3'UTR for miR-17 family, including miRs-20a, -93, -106a, and -106b. The purpose of this study was to assess the effects of these miRNAs on ATG16L1 expression and autophagic activity in HCT116 cells. METHODS: The functional binding sites in the ATG16L1 3'UTR were evaluated by transfection of pMIR-GLO vectors bearing the wild type or mutant 3'UTR into cells for luciferase reporter assay. The miRNA regulation of ATG16L1 expression was determined by quantitative real-time polymerase chain reaction and Western blot. The miRNA regulation of autophagic activity was evaluated by examining LC3II formation using Western blot and confocal imaging. RESULTS: Both miR-106a and miR-106b mimics inhibited starvation-induced autophagy. The miR-106b mimic reduced ATG16L1 protein expression. Luciferase reporter assays showed that mutating the binding sequence at the positions 1036 to 1042 abrogated miR-106b regulation of ATG16L1 3'UTR luciferase activity. In addition, miR-106a and miR-106b overexpression inhibited the expression of several other autophagy genes, including ATG12. CONCLUSIONS: miR-106b targets ATG16L1 and modulates autophagy, partially through the binding site at the 3' end of ATG16L1 3'UTR. miR-106a regulates autophagy, possibly irrelevant to ATG16L1 regulation. Both miR-106a and miR-106b regulate multiple autophagy genes so that they may play an integral role in fine-tuning autophagy.

Multiphasic analysis of the temporal development of the distal gut microbiota in patients following ileal pouch anal anastomosis.

BACKGROUND: The indigenous gut microbiota are thought to play a crucial role in the development and maintenance of the abnormal inflammatory responses that are the hallmark of inflammatory bowel disease. Direct tests of the role of the gut microbiome in these disorders are typically limited by the fact that sampling of the microbiota generally occurs once disease has become manifest. This limitation could potentially be circumvented by studying patients who undergo total proctocolectomy with ileal pouch anal anastomosis (IPAA) for the definitive treatment of ulcerative colitis. A subset of patients who undergo IPAA develops an inflammatory condition known as pouchitis, which is thought to mirror the pathogenesis of ulcerative colitis. Following the development of the microbiome of the pouch would allow characterization of the microbial community that predates the development of overt disease. RESULTS: We monitored the development of the pouch microbiota in four patients who underwent IPAA. Mucosal and luminal samples were obtained prior to takedown of the diverting ileostomy and compared to samples obtained 2, 4 and 8 weeks after intestinal continuity had been restored. Through the combined analysis of 16S rRNA-encoding gene amplicons, targeted 16S amplification and microbial cultivation, we observed major changes in structure and function of the pouch microbiota following ileostomy. There is a relative increase in anaerobic microorganisms with the capacity for fermentation of complex carbohydrates, which corresponds to the physical stasis of intestinal contents in the ileal pouch. Compared to the microbiome structure encountered in the colonic mucosa of healthy individuals, the pouch microbial community in three of the four individuals was quite distinct. In the fourth patient, a community that was much like that seen in a healthy colon was established, and this patient also had the most benign clinical course of the four patients, without the development of pouchitis 2 years after IPAA. CONCLUSIONS: The microbiota that inhabit the ileal-anal pouch of patients who undergo IPAA for treatment of ulcerative colitis demonstrate significant structural and functional changes related to the restoration of fecal flow. Our preliminary results suggest once the pouch has assumed the physiologic role previously played by the intact colon, the precise structure and function of the pouch microbiome, relative to a normal colonic microbiota, will determine if there is establishment of a stable, healthy mucosal environment or the reinitiation of the pathogenic cascade that results in intestinal inflammation.

MicroRNAs in inflammatory bowel disease.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene and protein expression. miRNAs are critical to a normal immune response and have altered expression in multiple immune-mediated disorders. This emerging role of miRNAs in the pathogenesis of multiple disease states has led to investigations into miRNA expression profiles in inflammatory bowel disease (IBD). The discovery of miRNAs in IBD is likely to contribute to our understanding of IBD pathogenesis and lead to clinical advances in IBD. This review focuses on miRNA expression in inflammation, autoimmune disorders, and inflammation-associated cancer, as well as their function in the biology and management of IBD.