Low concentrations of human neutrophil peptide ameliorate experimental murine colitis.

Human neutrophil peptides (HNPs) not only have antimicrobial properties, but also exert multiple immunomodulatory effects depending on the concentration used. We have previously demonstrated that the intraperitoneal administration of high-dose HNP-1 (100 µg/day) aggravates murine dextran sulfate sodium (DSS)-induced colitis, suggesting a potential pro-inflammatory role for HNPs at high concentrations. However, the role of low physiological concentrations of HNPs in the intestinal tract remains largely unknown. The aim of this study was to examine the effects of low concentrations of HNPs on intestinal inflammation. We first examined the effects of the mild transgenic overexpression of HNP-1 in DSS-induced colitis. HNP-1 transgenic mice have plasma HNP-1 levels similar to the physiological concentrations in human plasma. Compared to wild-type mice treated with DSS, HNP-1 transgenic mice treated with DSS had significantly lower clinical and histological scores, and lower colonic mRNA levels of pro-inflammatory cytokines, including interleukin (IL)-1ß and tumor necrosis factor (TNF)-α. We then injected low-dose HNP-1 (5 µg/day) or phosphate-buffered saline (PBS) intraperitoneally into C57BL/6N and BALB/c mice administered DSS. The HNP-1-treated mice exhibited significantly milder colitis with reduced expression levels of pro-inflammatory cytokines compared with the PBS-treated mice. Finally, we examined the in vitro effects of HNP-1 on the expression of cytokines associated with macrophage activation. Low physiological concentrations of HNP-1 did not significantly affect the expression levels of IL-1ß, TNF-α, IL-6 or IL-10 in colonic lamina propria mononuclear cells activated with heat-killed Escherichia coli, suggesting that the anti-inflammatory effects of HNP-1 on murine colitis may not be exerted by direct action on intestinal macrophages. Collectively, our data demonstrated a biphasic dose-dependent effect of HNP-1 on DSS-induced colitis: an amelioration at low concentrations and an aggravation at high concentrations. Low concentrations of HNPs may contribute to the maintenance of intestinal homeostasis.

Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development.

Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical ß-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial ß-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation.

[Jejunal duplication in an adult presenting with hematochezia].

A 56-year-old man was admitted to our hospital with appetite loss, palpitations, orthostatic syncope, and hematochezia. Contrast-enhanced abdominal computed tomography (CT) revealed a proximal jejunal diverticulum with contrast extravasation. We immediately performed transoral double balloon enteroscopy (DBE) to treat the bleed in the jejunum, and this revealed a small ulcer with an exposed vessel at the opening of the jejunal diverticulum. Hemostasis was achieved endoscopically with argon plasma coagulation (APC) and hemoclips. During subsequent surgery, the diverticulum was found on the mesenteric side of the jejunum. We performed laparoscopy-assisted partial resection of the jejunum, and pathological examination showed that the diverticulum shared a common proper muscle layer with the jejunum and was covered by jejunal mucosa with no ectopic mucosa. Therefore, we diagnosed jejunal duplication. After hospital discharge, the patient had no recurrence of hematochezia or anemia. We report a rare case of jejunal duplication presenting with hematochezia, which was diagnosed as jejunal diverticular bleeding by CT and DBE before surgery. Pathological analysis confirmed jejunal duplication after surgery. We suggest that intestinal diverticular bleeding, as well as duplication of the gastrointestinal tract, should be considered as part of the differential diagnosis of obscure gastrointestinal bleeding.

Expression of glycoprotein nonmetastatic melanoma protein B in macrophages infiltrating injured mucosa is associated with the severity of experimental colitis in mice.

Glycoprotein nonmetastatic melanoma protein B (Gpnmb) is a transmembrane glycoprotein, which negatively regulates the inflammatory responses of macrophages. However, the role of Gpnmb in intestinal macrophages remains to be fully elucidated. The present study aimed to investigate the expression of Gpnmb and its effects on colonic mucosal injuries associated with dextran sulfate sodium (DSS)­induced colitis in BALB/c mice, DBA/2J (D2) mice lacking Gpnmb and Gpnmb­transgenic DBA/2J mice (D2­gpnmb+). The colonic expression of Gpnmb increased with the severity of DSS­induced colitis in BALB/c mice, and macrophages infiltrating the inflamed mucosa were found to express Gpnmb. The D2 mice lacking Gpnmb exhibited more severe DSS­induced colitis, which was accompanied by higher levels of pro­inflammatory cytokines, including interleukin (IL)­1ß and IL­6, compared with the D2­gpnmb+ mice. Following lipopolysaccharide stimulation, macrophages from the D2 mice expressed higher levels of pro­inflammatory cytokines and lower levels of IL­10, compared with the D2­gpnmb+mice. In addition, in the RAW264.7 murine macrophage cell line, knockdown of Gpnmb by small interfering RNA was associated with increased production of pro­inflammatory cytokines, which were potentially mediated by the extracellular signal­regulated kinase (ERK) and p38 signaling pathways. The results of the present study indicated that macrophages infiltrating injured mucosa express Gpnmb, and that Gpnmb­positive macrophages may ameliorate inflammation in the intestinal mucosa by decreasing pro­inflammatory cytokine production via the ERK and p38 signaling pathways.

Human neutrophil peptides induce interleukin-8 in intestinal epithelial cells through the P2 receptor and ERK1/2 signaling pathways.

Human neutrophil peptides (HNPs) are antimicrobial peptides produced predominantly by neutrophils. We have previously reported that HNP 1-3 levels are increased in the sera and plasma of patients with active ulcerative colitis. The increased expression of interleukin-8 (IL-8) has also been demonstrated in the colonic mucosa of patients with active ulcerative colitis. HNPs induce IL-8 in lung epithelial cells and monocytes through the P2Y6 signaling pathway. However, the association between HNPs and IL-8 in the intestinal mucosa has not yet been investigated. In the present study, we investigated the effects of HNP-1 on the production of IL-8 by human intestinal epithelial cells and the underlying signaling mechanisms. We observed a significant increase in IL-8 expression in the human colon carcinoma cell line, Caco-2, following treatment with HNP-1. The non-selective P2 receptor antagonists, suramin and pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) tetrasodium salt hydrate (PPADS), significantly blocked the HNP-1-induced expression of IL-8 in the Caco-2 cells. The P2Y6-specific antagonist, MRS2578, led to a significant but partial decrease in IL-8 expression, suggesting that P2 receptors in addition to P2Y6 are involved in the HNP-1-induced production of IL-8 by Caco-2 cells. In agreement with this finding, HNP-1 also significantly increased IL-8 production in the P2Y6-negative human colon cancer cell line, HT-29, and this increase was blocked by treatment with suramin and PPADS. HNP-1 significantly increased the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) in the HT-29 cells. However, the HNP-1-induced production of IL-8 was suppressed by the ERK1/2 inhibitor, U0126, but not by the p38 MAPK inhibitor, SB203580. In conclusion, our data demonstrate that HNP-1 induces IL-8 production not only through P2Y6, but also through additional P2 receptors via an ERK1/2-dependent mechanism in intestinal epithelial cells.

Hepatocyte growth factor stimulates the migration of gastric epithelial cells by altering the subcellular localization of the tight junction protein ZO-1.

BACKGROUND: Hepatocyte growth factor (HGF) is essential for epithelial restitution, a process in which epithelial cells rapidly migrate to cover desquamated epithelium after mucosal injury in the gastrointestinal tract. In this study, we aimed to elucidate the molecular mechanisms of the HGF-mediated reconstitution of gastric epithelial structures by analyzing the expression and subcellular dynamics of tight junction proteins. METHODS: We treated human gastric epithelial MKN74 cells with HGF, and examined the effects of HGF on cell migration and proliferation, and the expression and subcellular dynamics of tight junction proteins; as well, we investigated the effect of HGF on paracellular permeability to macromolecules (using fluorescein isothiocyanate [FITC]-dextran). RESULTS: HGF significantly stimulated the migration of MKN74 cells, but not their proliferation, in a dose-dependent manner. HGF did not affect the expression of tight junction proteins, including claudin-1, -3, -4 and -7; occludin; and zonula occludens (ZO)-1. However, fluorescence immunostaining revealed that, in the cell membrane, the levels of ZO-1, but not those of occludin or claudin-4, were transiently decreased 1 h after HGF treatment. The results were further confirmed by western blotting: HGF reduced the amount of ZO-1 protein in the cell membrane fraction concomitantly with an increase in cytoplasmic ZO-1. Furthermore, HGF reduced the interaction between ZO-1 and occludin, and induced the tyrosine phosphorylation of occludin, whereas the phosphorylation status of ZO-1 was not affected by exposure to HGF. Despite a decrease in the ZO-1/occludin interaction, HGF did not affect paracellular permeability to macromolecules. CONCLUSIONS: HGF alters the subcellular localization of ZO-1, probably through the tyrosine phosphorylation of occludin, which may induce cell dispersion during epithelial restitution.

[Diagnostic biomarkers in inflammatory bowel disease].

Inflammatory bowel diseases (IBD), which includes ulcerative colitis and Crohn's disease, are chronic intestinal disorders of unknown etiology. The diagnosis of IBD is based upon clinical history, endoscopic, and histological findings; however, accurate diagnosis can still be difficult with these current methodologies. Recent advances in molecular techniques in proteomics and genetic analysis have driven the discovery of novel IBD biomarkers and genetic susceptibility factors that may facilitate the diagnosis of IBD. In the future, biomarkers will play a key role in the diagnosis of IBD and the development of individual courses of treatment.

Human neutrophil peptide-1 aggravates dextran sulfate sodium-induced colitis.

BACKGROUND: Human neutrophil peptide (HNP)-1, HNP-2, and HNP-3 (HNP-1-3) are useful biomarkers for ulcerative colitis (UC). The precise roles of these peptides in UC are poorly understood, however. The aim of this study was to determine whether HNP-1 affects disease activity in mice with experimental colitis. METHODS: Experimental colitis was induced in BALB/c or severe combined immunodeficiency (SCID) mice using dextran sulfate sodium (DSS). Mice were subsequently treated intraperitoneally with HNP-1 (100 µg/day) or phosphate-buffered saline (PBS) from day 4 to day 6. The severity of colitis was evaluated based on a disease activity index, histologic score, and cytokine expression. RESULTS: Body weight and colon length significantly decreased and the disease activity index score, histologic score, and myeloperoxidase activity significantly increased in HNP-1-treated BALB/c mice compared with PBS-treated mice. Interferon-γ and tumor necrosis factor-α levels in colon culture supernatants-derived HNP-1-treated mice were also significantly higher, and interleukin (IL)-1ß levels tended to increase in response to HNP-1. In addition, treating SCID mice with HNP-1 aggravated DSS-induced colitis and IL-1ß levels in colon culture supernatants from these mice were significantly higher than in cultures obtained from control mice. Furthermore, in both BALB/c and SCID mice increased recruitment of F4/80-positive macrophages was observed in the inflamed colonic mucosa following HNP-1 injections. CONCLUSIONS: High concentrations of HNP-1 aggravate DSS-induced colitis, including upregulated expression of such macrophage-derived cytokines as IL-1ß. These results indicate that high concentrations of HNP-1-3 in patients with UC may exacerbate disease activity via increased cytokine production.

Repeated enemas with hepatocyte growth factor selectively stimulate epithelial cell proliferation of injured mucosa in rats with experimental colitis.

AIMS: Hepatocyte growth factor (HGF) modulates intestinal epithelial cell proliferation and migration. We previously reported that systemic administration of recombinant human HGF (rh-HGF) ameliorated experimental colitis. However, an increase in serum HGF concentrations may induce undesired systemic effects, limiting the use of rh-HGF. To avoid possible side effects, we investigated the safety and efficacy of rectally administered rh-HGF as a treatment for experimental colitis. MAIN METHODS: We measured serum human HGF concentration following a single rectal enema of rh-HGF. Rats with 2,4,6-trinitrobenzene sulfonic acid (TNBS)- or dextran sulfate sodium (DSS)-induced colitis were treated with rectal enemas of rh-HGF once a day for seven days. The degree of mucosal injuries and the proliferative activity of the colon epithelium were examined. KEY FINDINGS: Rats administered a rectal enema of rh-HGF at a dose of 0.1 mg/ml or less had no detectable rh-HGF in the serum. Repeated enemas of rh-HGF at this dose significantly reduced mucosal injuries, both with respect to lesion size and inflammatory cell infiltration. This regimen also stimulated proliferation of epithelial cells surrounding injured mucosa; however, the cell proliferation of uninjured mucosa was not affected by this local treatment. SIGNIFICANCE: Rectally administered rh-HGF selectively accelerates the repair of injured mucosa in rat experimental colitis without systemic exposure to HGF. Rectal enemas of HGF are thus a potential novel and safe therapy for IBD.

Hepatocyte growth factor ameliorates mucosal injuries leading to inhibition of colon cancer development in mice.

Hepatocyte growth factor (HGF), which facilitates the repair of injured mucosa, has the potential to be a new therapeutic agent for inflammatory bowel disease (IBD). However, given that the incidence of colorectal cancer increases continuously with disease duration in patients with IBD, the fact that HGF is a potent mitogen for intestinal epithelial cells may further heighten the risk of bowel cancer in this patient population. In this study, we examined the effects of recombinant HGF on colorectal cancer development in mice with or without experimentally induced colitis. Although HGF stimulated proliferation of colonic epithelial cells in normal mucosa, the development of colorectal cancer induced by repeated injection of azoxymethane (AOM) was significantly inhibited by HGF treatment. In a mouse model of colitis-associated cancer, colorectal cancer frequently developed despite only a single injection of AOM prior to three cycles of dextran sulfate sodium administration. However, HGF treatment significantly facilitated the repair of injured mucosa, leading to inhibition of colorectal cancer development in a dose-dependent manner. Thus, HGF-induced repair of injured mucosa inhibits rather than accelerates the development of colorectal cancer, and these results also suggest the importance of blocking the cycles of mucosal injury and repair to prevent colitis-associated colorectal cancer.

Polyamines mediate glutamine-dependent induction of the intestinal epithelial heat shock response.

Heat shock proteins (Hsps) are highly conserved proteins that play a role in cytoprotection and maintaining intestinal homeostasis. Glutamine is essential for the optimal induction of intestinal epithelial Hsp expression, but its mechanisms of action are incompletely understood. Glutamine is a substrate for polyamine synthesis and stimulates the activity of ornithine decarboxylase (ODC), a key enzyme for polyamine synthesis, in intestinal epithelial cells. Thus we investigated whether polyamines (putrescine, spermidine, or spermine) and their precursor ornithine mediate the induction of Hsp expression in IEC-18 rat intestinal epithelial cells. As previously observed, glutamine was required for heat stress induction of Hsp70 and Hsp25, although it had little effect under basal conditions. Under conditions of glutamine depletion, supplementation of ornithine or polyamines restored the heat-induced expression of Hsp70 and Hsp25. When ODC was inhibited by α-difluoromethylornithine (DFMO), an irreversible ODC inhibitor, the heat stress induction of Hsp70 and Hsp25 was decreased significantly, even in the presence of glutamine. Ornithine, polyamines, and DFMO did not modify the nuclear localization of heat shock transcription factor 1 (HSF-1). However, DFMO dramatically reduced glutamine-dependent HSF-1 binding to an oligonucleotide with heat shock elements (HSE), which was increased by glutamine. In addition, exogenous polyamines recovered the DNA-binding activity. These results indicate that polyamines play a critical role in the glutamine-dependent induction of the intestinal epithelial heat shock response through facilitation of HSF-1 binding to HSE.

Liver regeneration after partial hepatectomy in rat is more impaired in a steatotic liver induced by dietary fructose compared to dietary fat.

Hepatic steatosis (HS) has a negative effect on liver regeneration, but different pathophysiologies of HS may lead to different outcomes. Male Sprague-Dawley rats were fed a high fructose (66% fructose; H-fruc), high fat (54% fat; H-fat), or control chow diet for 4 weeks. Based on hepatic triglyceride content and oil red O staining, HS developed in the H-fruc group, but was less severe compared to the H-fat group. Hepatic mRNA expression levels of fatty acid synthase and fructokinase were increased and those of carnitine palmitoyltransferase-1 and peroxisome proliferator-activated receptor-α were decreased in the H-fruc group compared to the H-fat group. Liver regeneration after 70% partial hepatectomy (PHx) was evaluated by measuring the increase in postoperative liver mass and PCNA-positive hepatocytes, and was impaired in the H-fruc group compared to the H-fat and control groups on days 3 and 7. Serum levels of tumor necrosis factor-α, interleukin-6 and hepatocyte growth factor did not change significantly after PHx. In contrast, serum TGF-ß1 levels were slightly but significantly lower in the control group on day 1 and in the H-fat group on day 3 compared to the level in each group on day 0, and then gradually increased. However, the serum TGF-ß1 level did not change after PHx in the H-fruc group. These results indicate that impairment of liver regeneration after PHx in HS is related to the cause, rather than the degree, of steatosis. This difference may result from altered metabolic gene expression profiles and potential dysregulation of TGF-ß1 expression.

Impact of cigarette smoking on onset of nonalcoholic fatty liver disease over a 10-year period.

BACKGROUND: Metabolic syndrome, which includes obesity, hyperglycemia, dyslipidemia, and hypertension, is a major risk factor for the development of nonalcoholic fatty liver disease (NAFLD). Cigarette smoking is a well-known risk factor for metabolic syndrome, but the epidemiological impact of cigarette smoking on development of NAFLD is unclear. METHODS: In this retrospective study, 2,029 subjects underwent a complete medical health checkup in 1998 and again in 2008. Those who were positive for hepatitis B surface antigen or hepatitis C virus antibody, or had an alcohol intake of > 20 g/day as assessed by questionnaire, were excluded. Fatty liver was diagnosed by abdominal ultrasonography. Independent risk factors associated with the development of NAFLD were determined by multiple logistic regression analysis. Smoking status was expressed using the Brinkman index (BI), which was calculated as the number of cigarettes smoked per day multiplied by the number of years of smoking. RESULTS: Of 1,560 subjects without NAFLD in 1998, 266 (17.1%) were newly diagnosed with NAFLD in 2008. Multiple logistic analysis identified age [adjusted odds ratio (AOR) 0.95, 95% confidence interval (95% CI) 0.94-0.97], male sex (AOR 1.46, 95% CI 1.01-2.10), body mass index ≥ 25 (AOR 3.08, 95% CI 2.20-4.32), dyslipidemia (AOR 1.79, 95% CI 1.25-2.58) and cigarette smoking (AOR 1.91, 95% CI 1.34-2.72) as risk factors associated with the development of NAFLD. Smoking status at baseline was also associated with the development of NAFLD (BI 1-399: AOR 1.77, 95% CI 1.02-3.07, BI ≥ 400: AOR 2.04, 95% CI 1.37-3.03). CONCLUSION: Cigarette smoking is an independent risk factor for onset of NAFLD.

Glutamine increases autophagy under Basal and stressed conditions in intestinal epithelial cells.

BACKGROUND & AIMS: Glutamine plays a protective role in intestinal cells during physiologic stress; however, the protection mechanisms are not fully understood. Autophagy functions in bulk degradation of cellular components, but has been recognized recently as an important mechanism for cell survival under conditions of stress. We therefore sought to see if glutamine's actions involve the induction of autophagy in intestinal cells and, if so, the mechanisms that underlie this action. METHODS: Formation of microtubule-associated protein light chain 3 (LC3)-phospholipid conjugates (LC3-II) in rat intestinal epithelial IEC-18 cells and human colonic epithelial Caco-2(BBE) cells was determined by Western blotting and localized by confocal microscopy. Activation of mammalian target of rapamycin (mTOR) pathway, mitogen-activated protein (MAP) kinases, caspase-3, and poly (ADP-ribose) polymerase were monitored by Western blotting. RESULTS: Glutamine increased LC3-II as well as the number of autophagosomes. Glutamine-induced LC3-II formation was paralleled by inactivation of mTOR and p38 MAP kinase pathways, and inhibition of mTOR and p38 MAP kinase allowed LC3-II induction in glutamine-deprived cells. Under glutamine starvation, LC3-II recovery after heat stress or the increase under oxidative stress was blunted significantly. Glutamine depletion increased caspase-3 and poly (ADP-ribose) polymerase activity after heat stress, which was inhibited by treatment with inhibitors of mTOR and p38 MAP kinase. CONCLUSIONS: Glutamine induces autophagy under basal and stressed conditions, and prevents apoptosis under heat stress through its regulation of the mTOR and p38 MAP kinase pathways. We propose that glutamine contributes to cell survival during physiologic stress by induction of autophagy.

Autoantibodies against ubiquitination factor E4A (UBE4A) are associated with severity of Crohn's disease.

BACKGROUND: Identification of a disease- and organ-specific autoantigen can potentially contribute to understanding molecular mechanisms involved in Crohn's disease (CD) and lead to development of clinically useful markers. The aim was to identify potential intestinal autoantigens specific to patients with CD and evaluate their diagnostic value. METHODS: A cDNA expression library from normal terminal ileum was created and screened with pooled sera from 3 randomly selected patients with CD. For evaluation of the diagnostic value of antibody screening, serum samples obtained from 39 patients with CD, 28 patients with ulcerative colitis (UC), and 60 healthy controls were examined for IgG against the strongest clone. RESULTS: We identified an intestinal cDNA clone encoding ubiquitination factor E4A (UBE4A), a U-box-type ubiquitin-protein ligase. The prevalence of anti-UBE4A IgG in patients with CD was significantly higher than that in patients with UC or healthy controls (46.2% versus respectively 7.1%, P = 0.0006; 3.3%, P < 0.0001). Anti-UBE4A-positive patients with CD were more likely to require surgery (P = 0.0013). The level of anti-UBE4A IgG was correlated with disease activity (r = 0.777, P < 0.0001) and associated with stricturing or penetrating disease (P = 0.0028). Immunohistochemical studies showed upregulation of UBE4A in enteroendocrine cells of inflamed ileal mucosa with CD. CONCLUSIONS: Anti-UBE4A antibodies are potentially useful markers for detection and prediction of clinical activity and outcome in patients with CD.