Yasuhiro Takeda

Long-term effect of tacrolimus therapy in patients with refractory ulcerative colitis

Background  Little is known about long‐term outcome of tacrolimus therapy for ulcerative colitis.

Bovine milk exosomes contain microRNA and mRNA and are taken up by human macrophages.

We reported previously that microRNA (miRNA) are present in whey fractions of human breast milk, bovine milk, and rat milk. Moreover, we also confirmed that so many mRNA species are present in rat milk whey. These RNA were resistant to acidic conditions and to RNase, but were degraded by detergent. Thus, these RNA are likely packaged in membrane vesicles such as exosomes. However, functional extracellular circulating RNA in bodily fluids, such as blood miRNA, are present in various forms. In the current study, we used bovine raw milk and total RNA purified from exosomes (prepared by ultracentrifugation) and ultracentrifuged supernatants, and analyzed them using miRNA and mRNA microarrays to clarify which miRNA and mRNA species are present in exosomes, and which species exist in other forms. Microarray analyses revealed that most mRNA in milk whey were present in exosomes, whereas miRNA in milk whey were present in supernatant as well as exosomes. The RNA in exosomes might exert functional effects because of their stability. Therefore, we also investigated whether bovine milk-derived exosomes could affect human cells using THP-1 cells. Flow cytometry and fluorescent microscopy studies revealed that bovine milk exosomes were incorporated into differentiated THP-1 cells. These results suggest that bovine milk exosomes might have effects in human cells by containing RNA.

Blockade of CXCL12/CXCR4 Axis Ameliorates Murine Experimental Colitis

Recent studies indicate that the CXCL12/CXCR4 interaction is involved in several inflammatory conditions. However, it is unclear whether this interaction has a role in the pathophysiology of inflammatory bowel disease (IBD). We investigated the significance of this interaction in patients with IBD and in mice with dextran sulfate sodium (DSS)-induced colitis and the effect of a CXCR4 antagonist on experimental colitis. First, we measured CXCR4 expression on peripheral T cells in patients with IBD. Furthermore, we investigated CXCR4 expression on leukocytes and CXCL12 expression in the colonic tissue of mice with DSS-induced colitis, and we evaluated the effects of a CXCR4 antagonist on DSS-induced colitis and colonic inflammation of interleukin (IL)-10 knockout (KO) mice. Colonic inflammation was assessed both clinically and histologically. Cytokine production from mesenteric lymph node cells was also examined. CXCR4 expression on peripheral T cells was significantly higher in patients with active ulcerative colitis (UC) compared with normal controls, and CXCR4 expression levels of UC patients correlated with disease activity. Both CXCR4 expression on leukocytes and CXCL12 expression in colonic tissue were significantly increased in mice with DSS-induced colitis. Administration of a CXCR4 antagonist ameliorated colonic inflammation in DSS-induced colitis and IL-10 KO mice. CXCR4 antagonist reduced tumor necrosis factor-α and interferon-γ production from mesenteric lymph node cells, whereas it did not affect IL-10 production. The percentage of mesenteric Foxp3+CD25+ T cells in DSS-induced colitis was not affected by CXCR4 antagonist. These results suggest that blockade of this chemokine axis might have potential as a therapeutic target for the treatment of IBD.

Immunosuppressive effects of tacrolimus on macrophages ameliorate experimental colitis

Background: Tacrolimus is a novel immunomodulator for inflammatory bowel diseases. Immunosuppressive effects of tacrolimus on T cells are well known; however, the effects of tacrolimus on macrophages remain unclear. The aim of this study was to investigate the effects of tacrolimus on activated macrophages and to examine its efficacy in murine colitis models. Methods: Proinflammatory cytokine production from lipopolysaccharide (LPS)–stimulated peritoneal macrophages of IL‐10‐knockout (KO) mice with and without tacrolimus was measured. We investigated the effects of tacrolimus on nuclear factor‐&kgr;B (NF‐&kgr;B), mitogen‐activated protein kinase (MAPK), and caspase activation in macrophages and the induction of apoptosis in macrophages in vitro and examined the in vivo apoptotic effect of tacrolimus on colonic macrophages in IL‐10‐KO mice. We evaluated the effect of the rectal administration of tacrolimus on colonic inflammation in IL‐10‐KO mice and dextran sulfate sodium (DSS)–induced colitis in CB.17/SCID mice. Results: Proinflammatory cytokine production from tacrolimus‐treated macrophages was significantly lower than that from untreated cells. Tacrolimus suppressed LPS‐induced activation of both NF‐&kgr;B and MAPK in macrophages and induced apoptosis of macrophages via activation of caspases 3 and 9. Rectal administration of tacrolimus evoked apoptosis of colonic macrophages in IL‐10‐KO mice. Moreover, the rectal administration of tacrolimus ameliorated colitis in IL‐10‐KO mice and DSS‐induced colitis in CB.17/SCID mice. Gene expression of inflammatory cytokines in colonic mucosa was significantly lower in tacrolimus‐treated mice than in untreated mice. Conclusions: Tacrolimus suppresses the function of activated macrophages and promotes their apoptosis, which may lead to the amelioration of colonic inflammation. Inflamm Bowel Dis 2010

SR-PSOX/CXCL16 plays a critical role in the progression of colonic inflammation

Background and aims Inflammatory bowel disease (IBD) is initiated and perpetuated by a dysregulated immune response to unknown environmental antigens such as luminal bacteria in genetically susceptible hosts. SR-PSOX/CXCL16, a scavenger receptor that binds phosphatidylserine and oxidised lipoprotein, has both phagocytic activity and chemotactic properties. The aim of this study was to investigate the role of SR-PSOX/CXCL16 in patients with IBD and experimental murine colitis. Methods The serum levels of SR-PSOX/CXCL16 were measured in patients with IBD. The roles of SR-PSOX/CXCL16 in phagocytosis of bacterial components and cytokine production by macrophages from wild-type (WT) and SR-PSOX/CXCL16 knockout (KO) mice were assessed. Colitis was induced by administering dextran sulfate sodium (DSS) to WT and SR-PSOX/CXCL16 KO mice. Colonic inflammation was analysed by clinical, histological and immunological parameters. Finally, the effect of a monoclonal antibody (mAb) to SR-PSOX/CXCL16 on DSS-induced colitis and trinitrobenzene sulfonic acid-induced colitis models was evaluated. Results Serum levels of SR-PSOX/CXCL16 correlated significantly with the disease activity of patients with IBD. Ex vivo experiments showed that SR-PSOX/CXCL16 was involved in both phagocytosis of bacterial antigens and the T helper 1 immune response through the production of interleukin 12 and interferon γ. In vivo murine experiments demonstrated the upregulated gene expression of SR-PSOX/CXCL16 in inflamed colonic tissues and the predominant expression of SR-PSOX/CXCL16 on macrophages. SR-PSOX/CXCL16 KO mice were less susceptible to colonic inflammation than were their WT littermates. Administration of SR-PSOX/CXCL16 mAb ameliorated the condition in the two different experimental colitis models. Conclusions SR-PSOX/CXCL16 plays a critical role in colonic inflammation and could be a potential therapeutic target for patients with IBD.

Upregulation of T-bet and tight junction molecules by Bifidobactrium longum improves colonic inflammation of ulcerative colitis

To the Editor: Manipulation of the mucosal microbiota to reduce the inflammatory potential of colonizing bacteria is an attractive therapy for patients with ulcerative colitis (UC). Therefore, probiotics treatments have been focused on the point of improving its intestinal microbial balance. We read with a great interest the review by Isaacs and Herfarth entitled “Role of probiotic therapy in IBD.”1 In that review, the authors described the effect of 4 weeks of symbiotic treatment (Bifidobactrium longum with Synergy 1) on patients with active UC reported by Furrie et al.2 Bifidobactrium longum (hereinafter BB536) was isolated from feces of a healthy baby in 1969.3 Thereafter, many physiological effects of BB536 have been reported and it has been used commercially for various food applications in several countries. The size of this study with BB536 was small, but there was a significant reduction of proinflammatory cytokines in mucosal biopsies in patients treated with the active therapy as compared to placebo. This finding was very promising for treatment with UC, but the exact mechanism of BB536 on patients with UC was not elucidated. Here we report data on an openlabel trial of BB536 for Japanese patients with UC and demonstrate its mechanism of reducing intestinal inflammation in patients with UC. Human study: From 2005 to 2007, 14 patients with UC (5 male, 9 female; mean age 43 5 years) who were refractory to more than 2250 mg of 5-aminosalicylate (5-ASA) were enrolled in this open-label study. The median disease duration prior to BB536 was 10 3.3 years. Six patients of these patients (42.8%) were afflicted with pancolitis and 3 (21.4%) had left-sided colitis; 5 (35.7%) had proctitis. Clinical activity was assessed with the Clinical Activity Index (CAI). The mean CAI score was 7 1.5 before BB536 administration. Patients who scored higher than 5 points in CAI were regarded as active phase and were treated with 2–3 1011 freeze-dried viable BB536 for 24 weeks. The clinical response was assessed at 24 weeks after administration of BB536. Clinical remission was defined as a decrease in the CAI to 4 or less. CAI was reduced in 12 of 14 patients at 24 weeks after starting BB536 therapy. Of note, 10 (67%) of 14 patients achieved clinical remission (2 had pancolitis; 3 had left-sided, and 5 had proctitis). The mean CAI significantly decreased at 8, 12, and 24 weeks compared to that before administration of BB536 (Fig. 1). Next we investigated the molecular mechanism of BB536 on cytokine production and expression of molecules related to mucosal barrier function. 1) Splenocytes of T cell receptorknockout (KO) mice resembling cytokine phenotype of UC (Th2 dominant) were stimulated with CD3/CD28 after pretreatment of heat-inactivated BB536. The production of IL-12, IFN, IL-4, and IL-13 in supernatants was measured by enzyme-linked immunosorbent assay (ELISA). T-bet and GATA-3 mRNA expression was evaluated by reversetranscriptase polymerase chain reaction (RT-PCR). 2) After inoculation of BB536 (1.5 1010CFU/mouse) to wildtype and Toll like receptor 2 KO mice, the gene expression of tight junction molecule (Claudin-1 and ZO-1) in colonic mucosa were analyzed by RTPCR. Stimulation with BB536 induced the production of both IL-12 and IFNfrom splenocytes and inhibited the production of IL-4 and IL-13. Stimulation with BB536 resulted in induction of Tbet mRNA expression in splenocytes, while it reduced GATA-3 mRNA expression. Moreover, gene expressions of claudin-1 and ZO-1 was upregulated in colonic tissue of wildtype mice after stimulation with BB536, while these upregulations were not observed in that of TLR-2 KO mice (Fig. 2).

Role of heat shock protein 47 in intestinal fibrosis of experimental colitis.

BACKGROUND AND AIMS Intestinal fibrosis is a clinically important issue of inflammatory bowel disease (IBD). It is unclear whether or not heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, plays a critical role in intestinal fibrosis. The aim of this study is to investigate the role of HSP47 in intestinal fibrosis of murine colitis. METHODS HSP47 expression and localization were evaluated in interleukin-10 knockout (IL-10KO) and wild-type (WT, C57BL/6) mice by immunohistochemistry. Expression of HSP47 and transforming growth factor-β1 (TGF-β1) in colonic tissue was measured. In vitro studies were conducted in NIH/3T3 cells and primary culture of myofibroblasts separated from colonic tissue of IL-10KO (PMF KO) and WT mice (PMF WT) with stimulation of several cytokines. We evaluated the inhibitory effect of administration of small interfering RNA (siRNA) targeting HSP47 on intestinal fibrosis in IL-10KO mice in vivo. RESULTS Immunohistochemistry revealed HSP47 positive cells were observed in the mesenchymal and submucosal area of both WT and IL-10 KO mice. Gene expressions of HSP47 and TGF-β1 were significantly higher in IL-10KO mice than in WT mice and correlated with the severity of inflammation. In vitro experiments with NIH3T3 cells, TGF-β1 only induced HSP47 gene expression. There was a significant difference of HSP47 gene expression between PMF KO and PMF WT. Administration of siRNA targeting HSP47 remarkably reduced collagen deposition in colonic tissue of IL-10KO mice. CONCLUSIONS Our results indicate that HSP47 plays an essential role in intestinal fibrosis of IL-10KO mice, and may be a potential target for intestinal fibrosis associated with IBD.

Modulation of the Th1/Th2 balance by infliximab improves hyperthyroidism associated with a flare-up of ulcerative colitis

To the Editor: Several reports have suggested an association between inflammatory bowel disease and autoimmune thyroid disease.1,2 Hyperthyroidism in patients with ulcerative colitis (UC) is very rare, with an incidence reported to be 0.82%3 to 3.7%.2 However, chronic thyroiditis is well known as an extramanifestation of UC. The cause of the association between UC and Graves’ disease remains unclear because the colon and thyroid do not have a common trigger antigen for embryological origins. Alteration in T-cell function might be a possible mechanism, accounting for their association. We report a case of a patient with both UC and hyperthyroidism successfully treated with infliximab. Intracellular cytokine assay demonstrated that infliximab modulated the Th1/Th2 balance and finally led to the improvement of both diseases.

Combinational effects of prebiotic oligosaccharides on bifidobacterial growth and host gene expression in a simplified mixed culture model and neonatal mice.

It is important to provide formula-fed infants with a bifidobacteria-enriched gut microbiota similar to those of breastfed infants to ensure intestinal health. Prebiotics, such as certain oligosaccharides, are a useful solution to this problem, but the combinational benefits of these oligosaccharides have not been evaluated. This study investigated the benefits of oligosaccharide combinations and screened for an optimal combination of oligosaccharides to promote healthy gut microbiota of formula-fed infants. In vitro and in vivo experiments were performed to assess the bifidogenic effects of lactulose (LAC) alone and LAC combined with raffinose (RAF) and/or galacto-oligosaccharide (GOS), using a mixed culture model and neonatal mice orally administered with these oligosaccharides and Bifidobacterium breve. In the in vitro culture model, the combination of the three oligosaccharides (LAC-RAF-GOS) significantly increased cell numbers of B. breve and Bifidobacterium longum (P<0·05) compared with either LAC alone or the combination of two oligosaccharides, and resulted in the production of SCFA under anaerobic conditions. In the in vivo experiment, the LAC-RAF-GOS combination significantly increased cell numbers of B. breve and Bacteroidetes in the large intestinal content (P<0·05) and increased acetate concentrations in the caecal content and serum of neonatal mice. Genes related to metabolism and immune responses were differentially expressed in the liver and large intestine of mice administered with LAC-RAF-GOS. These results indicate a synergistic effect of the LAC-RAF-GOS combination on the growth of bifidobacteria and reveal possible benefits of this combination to the gut microbiota and health of infants.

Decreased expression of CD200R3 on mouse basophils as a novel marker for IgG1-mediated anaphylaxis

IgE‐mediated mast cell activation is the trigger of anaphylaxis in humans, whereas it is known that not only IgE but also IgG can induce anaphylaxis in mice. In our preliminary experiments, the expression of a murine basophil identification marker, CD200R3, on antigen‐sensitized basophils decreased following specific antigen challenge. Interestingly, this decrease did not always correspond with increased expression of the IgE‐mediated basophil activation marker CD200R1. Since IgG as well as IgE plays a role in mouse anaphylaxis, we hypothesized that the observed decrease in CD200R3 on basophils was caused by IgG‐mediated cell activation. We attempted to establish whether CD200R3 is a marker of IgG‐mediated basophil activation and if its expression is correlated with anaphylaxis in a mouse model. Mouse basophils were stimulated via FcϵRs and/or FcγRs, and levels of CD200R1 and CD200R3 were analyzed by flow cytometry. Basophils derived from naive mice were challenged with a natural antigen, β‐lactoglobulin, after passive sensitization with anti‐β‐LG serum or IgG/IgG subclass‐depleted antiserum. Systemic anaphylaxis was induced by i.v. injection of anti‐FcγRIII/II monoclonal antibody, and CD200R3 expression on peripheral basophils was assessed. Stimulation via FcϵRs induced a significant increase in CD200R1 expression but had only a small effect on that of CD200R3. However, anti‐FcγRIII/II stimulation reduced CD200R3 expression markedly. In passive sensitization experiments, down‐regulation of CD200R3 induced by antigen challenge was strongly negated by the depletion of IgG or IgG1 from antiserum. Intravenous injection of anti‐FcγRIII/II induced CD200R3 down‐regulation on peripheral basophils, together with a drop in rectal temperature. Lowered CD200R3 expression on basophils is induced by IgG‐mediated stimulation via FcγRs. Use of CD200R1 and CD200R3 as activation markers enables the evaluation of murine basophil activation mediated by IgE and IgG, respectively.