John H. Kwon

Identification of microRNAs associated with ileal and colonic Crohn's disease.

Background: Crohns disease (CD) and ulcerative colitis (UC) are associated with expression differences in genes involved in immune function, wound healing, and tissue remodeling. MicroRNAs (miRNAs) are small, noncoding RNAs that act as potent negative regulators of gene expression and are differentially expressed in chronic inflammatory diseases, including UC. We examined the expression of miRNAs in tissues from different intestinal regions and in patients with active ileal and colonic CD. Methods: Colonoscopic pinch biopsies were obtained from the terminal ileum, cecum, transverse colon, sigmoid colon, and rectum of normal, healthy adults and from the ileum and sigmoid colon of patients with active ileal and colonic CD. miRNA expression was assessed using miRNA microarray and validated by mature miRNA quantitative reverse‐transcription polymerase chain reaction (RT‐PCR). Results: Ten intestine region‐specific miRNAs were identified. Three miRNAs were increased and one miRNA was decreased in the terminal ileum as compared to the colon. Six other miRNAs expressed varying levels of expression among the colon regions. Five miRNAs were found to be differentially expressed in tissues of patients with active colonic CD, with three increased and two decreased as compared to normal, healthy controls. Similarly, four miRNAs were found to be significantly increased in tissues of patients with active ileal CD. Conclusions: The expression differences between ileal CD, colonic CD, and previously identified UC‐associated miRNAs support the likelihood that miRNAs influence differing inflammation‐related gene expression in each inflammatory bowel disease (IBD) subtype and may form the basis for future diagnostic tests and therapeutic targets for IBD. Inflamm Bowel Dis 2010

Peripheral blood MicroRNAs distinguish active ulcerative colitis and Crohn's disease

Background: Crohns disease (CD) and ulcerative colitis (UC) result from pathophysiologically distinct dysregulated immune responses, as evidenced by the preponderance of differing immune cell mediators and circulating cytokine expression profiles. MicroRNAs (miRNAs) are small, noncoding RNAs that act as negative regulators of gene expression and have an increasingly recognized role in immune regulation. We hypothesized that differences in circulating immune cells in CD and UC patients are reflected by altered miRNA expression and that miRNA expression patterns can distinguish CD and UC from normal healthy individuals. Methods: Peripheral blood was obtained from patients with active CD, inactive CD, active UC, inactive UC, and normal healthy adults. Total RNA was isolated and miRNA expression assessed using miRNA microarray and validated by mature miRNA quantitative reverse‐transcription polymerase chain reaction. Results: Five miRNAs were significantly increased and two miRNAs (149* and miRplus‐F1065) were significantly decreased in the blood of active CD patients as compared to healthy controls. Twelve miRNAs were significantly increased and miRNA‐505* was significantly decreased in the blood of active UC patients as compared to healthy controls. Ten miRNAs were significantly increased and one miRNA was significantly decreased in the blood of active UC patients as compared to active CD patients. Conclusions: Peripheral blood miRNAs can be used to distinguish active CD and UC from healthy controls. The data support the evidence that CD and UC are associated with different circulating immune cells types and that the differential expression of peripheral blood miRNAs may form the basis of future diagnostic tests for inflammatory bowel disease. (Inflamm Bowel Dis 2011;)

The Microbe-Derived Short Chain Fatty Acid Butyrate Targets miRNA-Dependent p21 Gene Expression in Human Colon Cancer

Colonic microbiota ferment non-absorbed dietary fiber to produce prodigious amounts of short chain fatty acids (SCFAs) that benefit the host through a myriad of metabolic, trophic, and chemopreventative effects. The chemopreventative effects of the SCFA butyrate are, in part, mediated through induction of p21 gene expression. In this study, we assessed the role of microRNA(miRNA) in butyrates induction of p21 expression. The expression profiles of miRNAs in HCT-116 cells and in human sporadic colon cancers were assessed by microarray and quantitative PCR. Regulation of p21 gene expression by miR-106b was assessed by 3′ UTR luciferase reporter assays and transfection of specific miRNA mimics. Butyrate changed the expression of 44 miRNAs in HCT-116 cells, many of which were aberrantly expressed in colon cancer tissues. Members of the miR-106b family were decreased in the former and increased in the latter. Butyrate-induced p21 protein expression was dampened by treatment with a miR-106b mimic. Mutated p21 3′UTR-reporter constructs expressed in HCT-116 cells confirmed direct miR-106b targeting. Butyrate decreased HCT-116 proliferation, an effect reversed with the addition of the miR-106b mimic. We conclude that microbe-derived SCFAs regulate host gene expression involved in intestinal homeostasis as well as carcinogenesis through modulation of miRNAs.

MicroRNAs in Inflammatory Bowel Disease

&NA; 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. (Inflamm Bowel Dis 2011;)

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.

Matrilysin-1 (MMP7) cleaves galectin-3 and inhibits wound healing in intestinal epithelial cells

Background: Galectin‐3 is an animal lectin that has been implicated in wound healing and is decreased in inflammatory bowel disease (IBD). Matrix metalloproteinase‐7 (MMP7), also known as matrilysin‐1, a protease shown to cleave extracellular matrix proteins, is highly expressed in IBD tissues, especially at the leading edge of gastrointestinal ulcers. The ability of MMP7 to cleave galectin‐3 and influence wound healing has not been reported previously. The aim was to determine whether MMP7 cleaves galectin‐3 and modulates wound healing in intestinal epithelial cells. Methods: The cleaved fragments of galectin‐3 were identified by N‐terminal sequencing and mass spectrometry. Western blotting was used to detect the cleaved galectin‐3 products in a colonic epithelial cell line (T84 cells). Cell migration was studied by the in vitro scratch method. Results: We demonstrate for the first time that MMP7 cleaves galectin‐3 in vitro, resulting in three cleaved fragments (20.2 kDa, 18.9 kDa, and 15.5 kDa). Exogenous treatment of T84 cells with recombinant MMP7 resulted in the appearance of secreted galectin‐3 cleavage fragments in the supernatant. MMP7 inhibited cell migration and resulted in wound retraction and the addition of MMP7 to galectin‐3 abrogated the wound healing and cell migration induced by galectin‐3. Conclusions: We have demonstrated that galectin‐3 is a substrate for MMP7. Cleavage of galectin‐3 may be one mechanism by which MMP7 inhibits wound healing. This study has significance in understanding delayed wound healing in chronic intestinal diseases like intestinal ulcers and IBD, where MMP7 protein expression is elevated with a decreased galectin‐3 protein expression. (Inflamm Bowel Dis 2011;)

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.

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.

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

BackgroundThe 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.ResultsWe 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.ConclusionsThe 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.

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.