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.

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.

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.