Core fucose is essential glycosylation for CD14-dependent Toll-like receptor 4 and Toll-like receptor 2 signalling in macrophages.

Core fucosylation, catalysed by α-1, 6 fucosyltransferase (FUT8), regulates growth factor receptors in immune function. Although core fucose regulates many immune cell types, few reports confront the association between core fucose activity and an innate immune reaction. Here, we have investigated the function of core fucose in macrophages in vivo and in vitro using Fut8-deficient mice and cells. Following lipopolysaccharide (LPS) stimulation, inflammatory cytokine production in Fut8-deficient (Fut8-/-) macrophages was suppressed in both in vivo and in vitro experiments. Because LPS is recognized by Toll-like receptor 4 (TLR4), which induces the signalling cascade, TLR4 signalling was assumed to be impaired in Fut8-/- cells. Flow cytometry analyses revealed, however, that a lack of core fucose reduced the expression of, not TLR4, but CD14, which is necessary for TLR4 endocytosis. Because CD14 is necessary for TLR2 signalling, the immune response of TLR2 was also impaired in Fut8-/- macrophages. Moreover, in the dextran sodium sulphate (DSS)-induced murine colitis model, the mice grafted with Fut8-/- bone marrow cells exhibited higher resistance to inflammation than those grafted with Fut8+/+ bone marrow cells. These findings indicate that core fucose is essential for CD14-dependent TLR4 and TLR2 signalling in murine macrophage activity, leading to DSS-induced experimental colitis.

Tissue Drug Concentrations of Anti-tumor Necrosis Factor Agents Are Associated with the Long-term Outcome of Patients with Crohn's Disease.

BACKGROUND: Many reports indicate that a high-serum trough level of anti-tumor necrosis factor (TNF) agents is required for sustained remission in patients with Crohn's disease The pharmacokinetics of anti-TNF agents in inflamed intestinal tissue, however, is not well investigated. We investigated the association between the tissue concentration of anti-TNF agents and long-term disease outcome. METHODS: This was a prospective single-center study that enrolled 25 patients with Crohn's disease who were administered infliximab or adalimumab. All participants underwent endoscopy 2 weeks after administration of the anti-TNF agents, and biopsy samples were obtained from both inflamed and noninflamed intestinal tissue. Tissue concentrations of anti-TNF agents were evaluated and the correlation with serum trough levels was compared. The relation between the tissue drug concentration and clinical course over 24 months was also investigated. RESULTS: Concentrations of anti-TNF agents were significantly higher in inflamed tissue than in noninflamed tissue. Patients with high-serum trough concentrations of anti-TNF agents had significantly higher drug levels in the noninflamed tissue than those with low-serum trough concentrations, but no difference in the levels was detected in the inflamed tissue. Patients with high-drug levels in the noninflamed tissue had a significantly higher sustained response rate than patients with low-drug levels. CONCLUSIONS: Concentrations of anti-TNF agents in the noninflamed tissue can reflect sustained remission and may be a useful biomarker for monitoring therapeutic intensity in patients with Crohn's disease treated with anti-TNF agents (see Video Abstract, Supplemental Digital Content 1, http://links.lww.com/IBD/B623).

A novel pathogenesis of inflammatory bowel disease from the perspective of glyco-immunology.

Oligosaccharide modifications play an essential role in various inflammatory diseases and cancers, but their pathophysiologic roles, especially in inflammation, are not clear. Inflammatory bowel disease (IBD) is an intractable chronic inflammatory disorder with an unknown aetiology, and the number of patients with IBD is increasing throughout the world. Certain types of immunosuppressant drugs, such as corticosteroids, are effective for IBD, suggesting that immune function is closely associated with the pathophysiology of IBD. Recent progress in the analysis of oligosaccharides revealed a role for oligosaccharides in intestinal inflammation based on both experimental models and human samples from IBD patients. Moreover, changes in the oligosaccharide structures on glycoproteins in the sera and tissue samples may serve as biomarkers of IBD. Here, we present current studies of IBD with regard to the immunologic aspects of glycobiology, suggesting a novel concept for IBD pathogenesis and the function of oligosaccharides on immune cells, termed "glyco-immunology".

Establishment of mouse Mac-2 binding protein enzyme-linked immunosorbent assay and its application for mouse chronic liver disease models.

AIM: We identified Mac-2 (galectin-3) binding protein (Mac-2bp) as a novel diagnostic and liver fibrosis predicting biomarker for nonalcoholic steatohepatitis in humans. In mouse models, there are no serum biomarkers predicting liver disease severity. In this study, we developed a mouse Mac-2bp enzyme-linked immunosorbent assay (ELISA) system and determined its efficacy for predicting the severity of liver disease in mouse models, especially in non-alcoholic fatty liver disease (NAFLD) models. METHODS: We established several rat monoclonal antibodies against mouse Mac-2bp, selected two clones for the ELISA, and checked the accuracy and reproducibility of the ELISA, especially for NAFLD models and liver fibrosis models. We also investigated the relationships between serum levels and hepatic gene expression of Mac-2bp in mouse models. RESULTS: Our ELISA system had high accuracy and reproducibility (R2 = 0.999). The intra-assay and inter-assay results for the coefficient of variation were 2.0-3.7% and 1.7-6.9%, respectively. The levels of bilirubin, hemoglobin, and chyle did not affect the Mac-2bp serum levels detected by our ELISA kit. In the mouse models, serum Mac-2bp levels increased with liver disease progression (F0/F1/F2/F3, 239.1 ± 36.7 / 259.1 ± 43.0 / 457.5 ± 162.0 / 643.7 ± 116.0 ng/mL; P < 0.0001), and were significantly correlated with hepatic gene expression of Mac-2bp (R = 0.42, P < 0.0001). CONCLUSION: Our mouse Mac-2bp ELISA system effectively predicts severity of NAFLD and liver fibrosis in mouse models.

Hepatic aberrant glycosylation by N-acetylglucosaminyltransferase V accelerates HDL assembly.

Glycosylation is involved in various pathophysiological conditions. N-Acetylglucosaminyltransferase V (GnT-V), catalyzing ß1-6 branching in asparagine-linked oligosaccharides, is one of the most important glycosyltransferases involved in cancer and the immune system. Recent findings indicate that aberrant N-glycan structure can modify lipid metabolism. In this study, we investigated the effects of aberrant glycosylation by GnT-V on high-density lipoprotein cholesterol (HDL) assembly. We used GnT-V transgenic (Tg) mice and GnT-V Hep3B cell (human hepatoma cell line) transfectants. The study also included 96 patients who underwent medical health check-ups. Total serum cholesterol levels, particularly HDL-cholesterol (HDL-C) levels, were significantly increased in Tg vs. wild-type (WT) mice. Hepatic expression of apolipoprotein AI (ApoAI) and ATP-binding cassette subfamily A member 1 (ABCA1), two important factors in HDL assembly, were higher in Tg mice compared with WT mice. ApoAI and ABCA1 were also significantly elevated in GnT-V transfectants compared with mock-transfected cells. Moreover, ApoAI protein in the cultured media of GnT-V transfectants was significantly increased. Finally, we found a strong correlation between serum GnT-V activity and HDL-C concentration in human subjects. Multivariate logistic analyses demonstrated that GnT-V activity was an independent and significant determinant for serum HDL-C levels even adjusted with age and gender differences. Further analyses represented that serum GnT-V activity had strong correlation especially with the large-size HDL particle concentration. These findings indicate that enhanced hepatic GnT-V activity accelerated HDL assembly and could be a novel mechanism for HDL synthesis.

Serum Mac-2 binding protein is a novel biomarker for chronic pancreatitis.

AIM: To determine the efficacy of Mac-2 binding protein (Mac-2bp) for diagnosis of chronic pancreatitis. METHODS: Fifty-nine healthy volunteers (HV), 162 patients with chronic pancreatitis (CP), and 94 patients with pancreatic ductal adenocarcinoma (PDAC) were enrolled in this study. We measured serum Mac-2bp using our developed enzyme-linked immunosorbent assay kit. Additional biochemical variables were measured using an automated analyzer (including aminotransferase, alanine aminotransferase, γ-glutamyltransferase, alkaline phosphatase, triglyceride, C-reactive protein, and amylase levels) or chemiluminescent enzyme immunoassay (carbohydrate antigen 19-9 and carcinoembryonic antigen). The ability of Mac-2bp to predict CP diagnosis accurately was assessed using receiver operating characteristic (ROC) analyses. RESULTS: Serum Mac-2bp levels were significantly increased in CP patients compared to HV (P < 0.0001) and PDAC patients (P < 0.0001). Area under the ROC curve values of Mac-2bp for the discrimination of CP from HV and PDAC were 0.727 and 0.784, respectively. Multivariate analyses demonstrated that serum Mac-2bp levels were independent determinants for CP diagnosis from HV and PDAC patients. Immunohistological staining showed that Mac-2bp was expressed faintly in the pancreas tissues of both CP and PDAC patients. Serum aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase, alkaline phosphatase, and triglyceride levels were significantly higher in patients with CP or PDAC. Serum Mac-2bp levels were highly correlated with protein levels of alanine aminotransferase, γ-glutamyltransferase, and C-reactive protein, but not amylase, suggesting that the damaged liver produces Mac-2bp. CONCLUSION: Measurement of serum Mac-2bp may be a novel and useful biomarker for CP diagnosis as well as liver fibrosis in the general population.

Core Fucosylation on T Cells, Required for Activation of T-Cell Receptor Signaling and Induction of Colitis in Mice, Is Increased in Patients With Inflammatory Bowel Disease.

BACKGROUND & AIMS: Attachment of a fucose molecule to the innermost N-glycan in a glycoprotein (core fucosylation) regulates the activity of many growth factor receptors and adhesion molecules. The process is catalyzed by α1-6 fucosyltransferase (FUT8) and required for immune regulation, but it is not clear whether this process is dysregulated during disease pathogenesis. We investigated whether core fucosylation regulates T-cell activation and induction of colitis in mice, and is altered in patients with inflammatory bowel disease (IBD). METHODS: Biopsy samples were collected from inflamed and noninflamed regions of intestine from patients (8 with Crohn's disease, 4 with ulcerative colitis, and 4 without IBD [controls]) at Osaka University Hospital. Colitis was induced in FUT8-deficient (Fut8(-/-)) mice and Fut8(+/+) littermates by administration of trinitrobenzene sulfonic acid. Intestinal tissues were collected and analyzed histologically. Immune cells were collected and analyzed by lectin flow cytometry, immunofluorescence, and reverse-transcription polymerase chain reaction, as well as for production of cytokines and levels of T-cell receptor (TCR) in lipid raft fractions. T-cell function was analyzed by intraperitoneal injection of CD4(+)CD62L(+) naïve T cells into RAG2-deficient mice. RESULTS: Levels of core fucosylation were increased on T cells from mice with colitis, compared with mice without colitis, as well as on inflamed mucosa from patients with IBD, compared with their noninflamed tissues or tissues from control patients. Fut8(-/-) mice developed less-severe colitis than Fut8(+/+) mice, and T cells from Fut8(-/-) mice produced lower levels of T-helper 1 and 2 cytokines. Adoptive transfer of Fut8(-/-) T cells to RAG2-deficient mice reduced the severity of colitis. Compared with CD4(+) T cells from Fut8(+/+) mice, those from Fut8(-/-) mice expressed similar levels of TCR and CD28, but these proteins did not contain core fucosylation. TCR complexes formed on CD4(+) T cells from Fut8(-/-) mice did not signal properly after activation and were not transported to lipid rafts. CONCLUSIONS: Core fucosylation of the TCR is required for T-cell signaling and production of inflammatory cytokines and induction of colitis in mice. Levels of TCR core fucosylation are increased on T cells from intestinal tissues of patients with IBD; this process might be blocked as a therapeutic strategy.

Specific increase in serum core-fucosylated haptoglobin in patients with chronic pancreatitis.

BACKGROUND/OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all malignancies, and its diagnosis in early stages is the most important prognostic factor. Chronic pancreatitis (CP), a common background of PDAC occurrence, is morphologically defined as progressive pancreatic fibrosis and inflammation accompanied by pancreatic exocrine cell atrophy. We recently found that inflammation and fibrosis are independent characteristic histological changes in noncancerous lesions in PDAC patients despite the absence of a past history of clinical CP. Subclinical CP is an important background for PDAC occurrence. Therefore, there is an urgent need to develop a noninvasive and reliable biomarker for CP diagnosis. METHODS: Fifty-nine healthy volunteers (HV), 159 patients with CP, and 83 patients with PDAC were enrolled in this study. We measured serum total fucosylated haptoglobin (Fuc-Hpt) and core-Fuc-Hpt levels using lectin-antibody enzyme-linked immunosorbent assay kits that we developed. In these kits, total Fuc-Hpt and core-Fuc-Hpt were measured using Aleuria aurantia lectin and Pholiota squarrosa lectin, respectively. RESULTS: Serum Fuc-Hpt levels were significantly increased in CP patients compared to HV (P < 0.0001) and were further increased in PDAC patients (P < 0.0001). Interestingly, serum core-Fuc-Hpt levels were significantly higher in CP patients compared to HV (P < 0.0001) and PDAC patients (P < 0.0001). Multivariate analyses demonstrated that total serum core-Fuc-Hpt was an independent determinant for CP diagnosis, but Fuc-Hpt was not. CONCLUSIONS: A dramatic change in oligosaccharides was observed in serum haptoglobin between CP and PDAC. Serum core-Fuc-Hpt may be a novel and useful biomarker for CP diagnosis.

N-Acetylglucosaminyltransferase V exacerbates murine colitis with macrophage dysfunction and enhances colitic tumorigenesis.

BACKGROUND: Oligosaccharide structures and their alterations have important roles in modulating intestinal inflammation. N-Acetylglucosaminyltransferase V (GnT-V) is involved in the biosynthesis of N-acetylglucosamine (GlcNAc) by ß1,6-branching on N-glycans and is induced in various pathologic processes, such as inflammation and regeneration. GnT-V alters host immune responses by inhibiting the functions of CD4(+) T cells and macrophages. The present study aimed to clarify the role of GnT-V in intestinal inflammation using GnT-V transgenic mice. METHODS: Colitis severity was compared between GnT-V transgenic mice and wild-type mice. ß1,6-GlcNAc levels were investigated by phytohemagglutinin-L4 lectin blotting and flow cytometry. We investigated phagocytosis of macrophages by measuring the number of peritoneal-macrophage-ingested fluorescent latex beads by flow cytometry. Cytokine production in the culture supernatant of mononuclear cells from the spleen, mesenteric lymph nodes, and bone-marrow-derived macrophages was determined by enzyme-linked immunosorbent assay. Clodronate liposomes were intravenously injected to deplete macrophages in vivo. Chronic-colitis-associated tumorigenesis was assessed after 9 months of repeated administration of dextran sodium sulfate (DSS). RESULTS: DSS-induced colitis and colitis induced by trinitrobenzene sulfonic acid were markedly exacerbated in GnT-V transgenic mice compared with wild-type mice. Production of interleukin-10 and phagocytosis of macrophages were significantly impaired in GnT-V transgenic mice compared with wild-type mice. Clodronate liposome treatment to deplete macrophages blocked the exacerbation of DSS-induced colitis and impairment of interleukin-10 production in GnT-V transgenic mice. Chronic-colitis-associated tumorigenesis was significantly increased in GnT-V transgenic mice. CONCLUSIONS: Overexpression of GnT-V exacerbated murine experimental colitis by inducing macrophage dysfunction, thereby enhancing colorectal tumorigenesis.

Vitamin K deficiency leads to exacerbation of murine dextran sulfate sodium-induced colitis.

BACKGROUND: Patients with inflammatory bowel disease (IBD) often exhibit vitamin K deficiency. Vitamin K has been shown to inhibit inflammation via interleukin (IL)-6 suppression. This study aimed to evaluate the effect of vitamin K in a murine model of colitis. METHODS: Colitis was induced using dextran sulfate sodium (DSS) in mice fed either a vitamin K-deficient (K-def) or a vitamin K-supplemented (K-sup) diet. The clinical and histological severity of colitis was assessed, and levels of cytokine production from the spleen and colonic lamina propria were measured by enzyme-linked immunosorbent assay and quantitative real-time reverse transcription polymerase chain reaction. Cytokine expression levels in CD4(+), CD11b(+), and CD19(+) cells in the presence and absence of vitamin K [menatetrenone (MK-4)] were measured in vitro and apoptosis was determined by caspase 3/7 activity and Annexin V staining. RESULTS: DSS administration resulted in significantly more severe body weight loss, shorter colon length, and higher histological scores in mice fed a K-def diet than those fed a K-sup diet. IL-6 expression in lamina propria mononuclear cells was significantly higher in the K-def group than in the K-sup group. IL-6 expression was significantly decreased in the presence of MK-4 in CD19(+) cells, but not in the CD4(+) and CD11b(+) subpopulations. Apoptotic cell population in CD19(+) cells was increased in the presence of MK-4 in vitro and in vivo. CONCLUSIONS: Vitamin K exerts a protective effect against DSS colitis; this effect is associated with IL-6 downregulation. Vitamin K could be a potential treatment target for IBD.

Role of aberrant IgG glycosylation in the pathogenesis of inflammatory bowel disease.

The intestine is one of the most important organs associated with the immune system. It is thought that disruption of intestinal immunity causes inflammatory bowel disease (IBD). Recent advances in immune glycobiology have provided novel insights into many human diseases. For example, studies of glycosylation remodeling in mice have underscored the importance of oligosaccharides in the pathogenesis of IBD. Furthermore, aberrant glycosylation of IgG is a good serum marker of IBD activity. In this review, we examine current understanding of the role of aberrant glycosylation in the pathogenesis of IBD in terms of our original data and recent reports.

Ectopic expression of N-acetylglucosaminyltransferase V accelerates hepatic triglyceride synthesis.

AIM: Glycosylation changes induce various types of biological phenomena in human diseases. N-Acetylglucosaminyltransferase V (GnT-V) is one of the most important glycosyltransferases involved in cancer biology. Recently, many researchers have challenged studies of lipid metabolism in cancer. To elucidate the relationships between cancer and lipid metabolism more precisely, we investigated the effects of GnT-V on lipid metabolism. In this study, we investigated the effects of aberrant glycosylation by GnT-V on hepatic triglyceride production. METHODS: We compared lipid metabolism in GnT-V transgenic (Tg) mice with that of wild-type (WT) mice fed with normal chow or a choline-deficient amino acid-defined (CDAA) diet in vivo. HepG2 cells and GnT-V transfectants of Hep3B cells were used in an in vitro study. RESULTS: Serum triglyceride levels and hepatic very low-density lipoprotein (VLDL) secretion in Tg mice were significantly elevated compared with that of WT mice. Hepatic lipogenic genes (Lxrα, Srebp1, Fas and Acc) and VLDL secretion-related gene (Mttp1) were significantly higher in Tg mice. Expression of these genes was also significantly higher in GnT-V transfectants than in mock cells. Knockdown of GnT-V decreased, while both epidermal growth factor and transforming growth factor-ß1 stimulation increased LXRα gene expression in HepG2 cells. Finally, we found that the blockade of VLDL secretion by CDAA diet induced massive hepatic steatosis in Tg mice. CONCLUSION: Our study demonstrates that enhancement of hepatic GnT-V activity accelerates triglyceride synthesis and VLDL secretion. Glycosylation modification by GnT-V regulation could be a novel target for a therapeutic approach to lipid metabolism.

A novel noninvasive diagnostic method for nonalcoholic steatohepatitis using two glycobiomarkers.

UNLABELLED: Nonalcoholic fatty liver disease (NAFLD) is a growing medical problem; thus, discriminating nonalcoholic steatohepatitis (NASH) from NAFLD is of great clinical significance. For the diagnosis of NASH, liver biopsy-proven histological examination is the current gold standard, and noninvasive and reliable biomarkers are greatly needed. Recently, we found that two glycobiomarkers, fucosylated haptoglobin (Fuc-Hpt) and Mac-2 binding protein (Mac2bp), are useful independently for NASH diagnosis. In this study, we confirmed that serum Fuc-Hpt is suitable for the prediction of ballooning hepatocytes and that serum Mac2bp is suitable for the prediction of liver fibrosis severity in 124 biopsy-proven NAFLD patients (training cohort). In addition, we found that the combination of serum Fuc-Hpt and Mac2bp levels was an excellent tool for NASH diagnosis. Using receiver operating characteristic analyses, the area under the receiver operating characteristic curve, sensitivity, and specificity of the combination of these two glycobiomarkers were 0.854, 81.1%, and 79.3%, respectively. We established a prediction model for NASH diagnosis using logistic regression analysis: logit (p)=-2.700+0.00242×Fuc-Hpt+1.225×Mac2bp. To validate the prediction model, another 382 biopsy-proven NAFLD patients were enrolled (validation cohort). In the validation cohort, the area under the receiver operating characteristic curve of this model for NASH diagnosis was 0.844, with 71.4% and 82.3% sensitivity and specificity, respectively. In addition, we investigated the significance of our developed NASH diagnosis model in ultrasound-diagnosed NAFLD subjects who received medical health checkups (n = 803). Our model also could predict NAFLD disease severity in this larger population. CONCLUSION: The combination of serum Fuc-Hpt and Mac2bp can distinguish NASH from NAFLD patients. Our noninvasive model using two serum glycobiomarkers contributes to a novel NASH diagnostic methodology that could replace liver biopsy.

Fetuin-A negatively correlates with liver and vascular fibrosis in nonalcoholic fatty liver disease subjects.

BACKGROUND & AIMS: Fetuin-A (α2HS-glycoprotein), a liver secretory glycoprotein, is known as a transforming growth factor (TGF)-ß1 signalling inhibitor. Serum fetuin-A concentration is associated with nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease. However, the usefulness of serum fetuin-A as a predictive fibrosis biomarker in NAFLD patients remains unclear. In this study, we investigated the relationship between circulating fetuin-A levels and fibrosis-related markers [platelet count, NAFLD fibrosis score and carotid intima media thickness (IMT)] in subjects with NAFLD. METHODS: A total of 295 subjects (male, 164; female, 131) who received medical health check-ups were enrolled in this study. NAFLD was diagnosed using abdominal ultrasonography. Serum fetuin-A was measured by ELISA. IMT was assessed using a high-resolution ultrasound scanner. Using recombinant human fetuin-A, we investigated the effects of fetuin-A on hepatic stellate cells, which play a pivotal role in the process of hepatic fibrosis. RESULTS: Serum fetuin-A concentration was significantly correlated with platelet count (R = 0.19, P < 0.01), NAFLD fibrosis score (R = -0.25, P < 0.01) and mean IMT (R = -0.22, P < 0.01). Multivariate analyses revealed that the fetuin-A concentration is a significant and independent determinant of platelet count, NAFLD fibrosis score and mean IMT. Recombinant fetuin-A suppressed TGF-ß1 signalling and fibrosis-related gene expression and increased the expression of TGF-ß1 pseudoreceptor bone morphogenic protein and activin membrane-bound inhibitor (BAMBI). CONCLUSIONS: Serum fetuin-A level is associated with liver/vessel fibrosis-related markers in NAFLD patients. Circulating fetuin-A could be a useful serum biomarker for predicting liver and vascular fibrosis progression in NAFLD patients.

N-acetylglucosaminyltransferase V exacerbates concanavalin A-induced hepatitis in mice.

N­Acetylglucosaminyltransferase V (GnT­V) catalyzes ß1­6 branching in asparagine­linked oligosaccharides and is one of the most important glycosyltransferases involved in carcinogenesis, cancer metastasis and immunity. To investigate the biological functions of GnT­V, the present study developed GnT­V transgenic (Tg) mice and the role of GnT­V in experimental immune­mediated hepatitis, induced by concanavalin A (ConA), were investigated. It was found that the aberrant expression of GnT­V exacerbated ConA­induced hepatitis in the Tg mice compared with the wild­type (WT) mice. The survival rate of the ConA­induced hepatitis at a high­dose of ConA was significantly lower in the Tg mice. Intravenously injected ConA is known to initially bind predominantly to the mannose gland of the liver sinusoidal endothelial cell (LSEC) surface and to leads to the activation of various immune cells. In the present study, the binding affinity of ConA to the LSECs did not differ between the WT and Tg mice. In addition, T cell receptor stimulation by anti­cluster of differentiation (CD)3/CD28 antibodies produced lower levels of T helper (Th)1 cytokine (interferon­Î³) and higher levels of Th2 cytokine (interleukin­10) in the Tg mouse splenic lymphocytes compared with WT mice. The composition of the hepatic mononuclear cells revealed that CD11b­positive cells were significantly increased in the GnT­V Tg mice. In addition, F4/80­positive cells were significantly increased in the Tg mouse liver and the depletion of macrophages reduced the difference in the severity of ConA­induced hepatitis between the WT and Tg mice. In conclusion, the present findings indicated that the aberrant expression of GnT­V led to an increase in hepatic macrophage infiltration and enhanced ConA­induced hepatitis. Modulation of glycosylation may be a novel therapeutic target for immunity­associated acute hepatitis.

Peyer's patches play a protective role in nonsteroidal anti-inflammatory drug-induced enteropathy in mice.

BACKGROUND: Peyer's patches (PPs) play a major role in mucosal immunity. However, their roles in nonsteroidal anti-inflammatory drug-induced enteropathy are poorly understood. METHODS: Wild-type (WT) and PP-null mice were injected with indomethacin. Twenty-four hours later, the cellular profiles and cytokine levels in the PPs, mesenteric lymph nodes (MLNs), and lamina propria (LP) of the small intestine were measured. WT and PP-null mice were given antibiotics before indomethacin treatment to evaluate enteropathy. Naive CD4 T cells were co-cultured with CD103 or CD103 dendritic cells (DCs) to analyze the interleukin (IL)-10 expression levels. Finally, WT mice adoptively transferred with CD103 or CD103 DCs were injected with indomethacin. RESULTS: The proportion of CD103 DCs in PPs and MLNs and IL-10-expressing CD4 T cells of PPs and the LP increased after indomethacin treatment. The PP-null mice showed greater indomethacin-induced enteropathy, fewer CD103 DCs in their MLNs, and lower proportion of IL-10-expressing CD4 T cells of their LP than WT mice, regardless of commensal bacteria. Naive splenic CD4 T cells co-cultured with CD103 DCs isolated from the MLNs of indomethacin-injected WT mice produced a higher amount of IL-10 compared with those co-cultured with CD103 DCs. Moreover, WT mice that received CD103 DCs showed milder enteropathy than those that received CD103 DCs. CONCLUSIONS: PPs play a protective role in nonsteroidal anti-inflammatory drug-induced enteropathy, and this protection is associated with an increase in CD103 DCs and IL-10-producing CD4 T cells in the intestine, independent of the commensal bacteria.

Regulation of anergy-related ubiquitin E3 ligase, GRAIL, in murine models of colitis and patients with Crohn's disease.

BACKGROUND: Abrogating tolerance is a critical step in the pathogenesis of Crohn's disease (CD). T cell-anergy is one of the main mechanisms of tolerance and is regulated by the gene related to anergy in lymphocytes (GRAIL). This study investigated the expressions and regulation of GRAIL in CD and murine colitis models. METHODS: Expressions of GRAIL mRNA and protein in CD4+ T cells were investigated in the peripheral blood and mucosal tissues of patients with CD, mice with dextran sodium salt (DSS)-induced colitis, and Il-10-deficient mice. MicroRNAs responsible for the regulation of GRAIL were examined by miRNA microarray. GRAIL-overexpressing T cells were intravenously injected in mice with DSS-induced colitis. RESULTS: The GRAIL expression was higher in the lamina propria (LP) CD4+ T cells of CD patients than of the control subjects, while it was lower in the peripheral blood CD4+ T cells of the CD patients than of the control subjects. The GRAIL mRNA expression was lower, but the GRAIL protein expression was higher in the LP of colitic mice than that of non-colitic mice. The miRNA microarray identified miR-290-5p as an miRNA that inhibits expression of the GRAIL protein and that is highly expressed in the LP of non-colitic mice. GRAIL-expressing T cells expressed regulatory T cell markers and showed suppressive effects in murine DSS-induced colitis. CONCLUSIONS: Our results show that expression of GRAIL is uniquely regulated by the specific miRNA in the intestinal mucosa, and suggest that GRAIL may associate with the pathophysiology of CD.

Serum Mac-2 binding protein levels as a novel diagnostic biomarker for prediction of disease severity and nonalcoholic steatohepatitis.

PURPOSE: Mac-2 binding protein (Mac-2 bp) is one of the major fucosylated glycoproteins, which we identified with glycol-proteomic analyses. We previously reported that fucosylated glycoproteins are secreted into bile, but scarcely secreted into sera in normal liver and hypothesized that the fucosylation-based sorting machinery would be disrupted in ballooning hepatocytes due to the loss of cellular polarity. In the present study, we investigated the availability of Mac-2 bp for differential diagnosis of nonalcoholic steatohepatitis (NASH) from nonalcoholic fatty liver disease (NAFLD) as a biomarker. EXPERIMENTAL DESIGN: Serum Mac-2 bp levels were determined with our developed ELISA kit. Our cohort of 127 patients with NAFLD had liver biopsy to make a histological diagnosis of NASH and simple fatty liver. RESULTS: Mac-2 bp levels were significantly elevated in NASH patients compared with non-NASH (simple steatosis) patients (2.132 ± 1.237 vs. 1.103 ± 0.500 µg/mL, p < 0.01). The area under the receiver-operating characteristic curve for predicting NASH by Mac-2 bp was 0.816. Moreover, multivariate logistic regression analyses showed Mac-2 bp levels could predict the fibrosis stage and the presence of ballooning hepatocytes in NAFLD patients. CONCLUSIONS AND CLINICAL RELEVANCE: These results support the potential usefulness of measuring Mac-2 bp levels in clinical practice as a biomarker for NASH.

Serum Fucosylated Haptoglobin as a Novel Diagnostic Biomarker for Predicting Hepatocyte Ballooning and Nonalcoholic Steatohepatitis.

UNLABELLED: Nonalcoholic fatty liver disease (NAFLD) is a growing medical problem around the world. NAFLD patients with nonalcoholic steatohepatitis (NASH) can develop cirrhosis and hepatocellular carcinoma. The ability to distinguish NASH from simple steatosis would be of great clinical significance. Ballooning hepatocytes are characteristic of typical pathological NASH; here, the polarized secretion of proteins is disrupted due to destruction of the cytoskeleton. We previously reported that fucosylated glycoproteins are secreted into bile, but not into sera in normal liver. Therefore, we hypothesized that the fucosylation-based sorting machinery would be disrupted in ballooning hepatocytes, and serum fucosylated glycoproteins would increase in NASH patients. To confirm our hypothesis, we evaluated serum fucosylated haptoglobin (Fuc-Hpt) levels in biopsy-proven NAFLD patients (n = 126) using a lectin-antibody ELISA kit. Fuc-Hpt levels were significantly increased in NASH patients compared with non-NASH (NAFLD patients without NASH) patients. Interestingly, Fuc-Hpt levels showed a significant stepwise increase with increasing hepatocyte ballooning scores. Multiple logistic regression analysis showed that Fuc-Hpt levels were independent and significant determinants of the presence of ballooning hepatocytes. Moreover, Fuc-Hpt levels were useful in monitoring liver fibrosis staging. Next, to investigate the significance of serum Fuc-Hpt in a larger population, we measured Fuc-Hpt levels in ultrasound-diagnosed NAFLD subjects (n = 870) who received a medical health checkup. To evaluate NAFLD disease severity, we used the FIB-4 index (based on age, serum AST and ALT levels, and platelet counts). Fuc-Hpt levels increased stepwise with increasing FIB-4 index. CONCLUSION: Measurement of serum Fuc-Hpt levels can distinguish NASH from non-NASH patients, and predict the presence of ballooning hepatocytes in NAFLD patients with sufficient accuracy. These results support the potential usefulness of measuring Fuc-Hpt levels in clinical practice.