Chronic skin damage induces small intestinal damage via IL-13-induced apoptosis.

The gut-skin axis has recently been widely recognized, and both the gut and skin have been found to affect each other through a bidirectional connection; however, the precise mechanisms remain to be elucidated. Therefore, we aimed to investigate the effects of chronic skin damage on mouse intestines. Following the chronic skin damage (CSD) model, 4 % sodium dodecyl sulfate (SDS) was applied to the back-shaved murine skin six times for 2 weeks after tape stripping. The small and large intestines were analyzed histologically and immunologically, respectively. Intestinal permeability was measured using fluorescein isothiocyanate-conjugated (FITC)-dextran. The role of IL-13 in the ileum was investigated using an anti-IL-13 antibody. Apoptotic intestinal cells were analyzed using TUNEL staining. Villus atrophy was observed in the small intestine in the CSD model, along with increased permeability. Mast cells, but not T cells, eosinophils, nor ILC-2, were increased in the intestinal mucosa. However, no significant changes were observed in the large intestine. mRNA expression of IL-13 was increased only in the ileum of the CSD model. Apoptotic intestinal epithelial cells were significantly increased in the ileum of the CSD model. Administration of an anti-IL-13 antibody ameliorated the intestinal damage caused by CSD, along with decreased apoptotic cells and mast cell infiltration. Skin damage causes morphological changes in the small intestine, accompanied by increased intestinal permeability, possibly through the IL-13-induced apoptosis of mast cells in the epithelium. Surfactant-mediated mechanical skin damage can cause a leaky gut.

Transgenerational impacts of oral probiotic administration in pregnant mice on offspring gut immune cells and colitis susceptibility.

BACKGROUND AND AIM: The study of the impact of environmental factors during pregnancy on fetal development has so far been focused primarily on those negatively affecting human health; however, little is known about the effects of probiotic treatment during pregnancy on inflammatory bowel diseases (IBD). In this study, we investigated whether oral administration of heat-killed probiotics isolated from fermented foods decreased the vulnerability of offspring to IBD. METHODS: Probiotics were administered to the pregnant mice until the birth of pups, after which the parent mice were maintained with autoclaved water. Partial pups were evaluated for dextran sodium sulfate-induced colitis. The influence of CD11c+ CD103+ dendritic cells (DCs) and regulatory T cells (Tregs) in mesenteric lymph nodes of parent mice and their pups was analyzed. RESULTS: Oral administration of heat-killed probiotics to pregnant dams significantly decreased inflammation induced by dextran sodium sulfate in pups. Probiotic treatment increased the number of CD103+ DCs, and the expression of ß8-integrin in CD103+ DCs and Tregs in mesenteric lymph nodes, not only in dams themselves but also in their offspring. CONCLUSIONS: Oral administration of probiotics during gestation induced transgenerational immunomodulatory effects on the gut-associated immune system and resilience to experimental colitis in the offspring. Our results suggest that consumption of fermented foods during pregnancy can be effective in preventing inflammatory diseases such as IBD beyond generation.

Ameliorating Role of Hydrogen-Rich Water Against NSAID-Induced Enteropathy via Reduction of ROS and Production of Short-Chain Fatty Acids.

BACKGROUND: Nonsteroidal anti-inflammatory drug (NSAID)-induced enteropathy, the mechanism of which is involved in oxidative stress, can be lethal due to hemorrhage. Thus, we aimed to investigate the effect of hydrogen-rich water (HRW), in terms of oxidative stress, on intestinal mucosal damage as well as changes in the gut microbiome and the short-chain fatty acids (SCFAs) content in feces. METHODS: Hydrogen-rich water was orally administered for 5 days to investigate the effectiveness of indomethacin-induced enteropathy in mice. Small intestinal damage and luminal reactive oxygen species (ROS) were evaluated to investigate the ameliorating effects of hydrogen. Then, components of the gut microbiome were analyzed; fecal microbiota transplantation (FMT) was performed using the cecal contents obtained from mice drinking HRW. The cecal contents were analyzed for the SCFAs content. Finally, cells from the macrophage cell line RAW264 were co-cultured with the supernatants of cecal contents. RESULTS: Hydrogen-rich water significantly ameliorated IND-induced enteropathy histologically and reduced the expression of IND-induced inflammatory cytokines. Microscopic evaluation revealed that luminal ROS was significantly reduced and that HRW did not change the gut microbiota; however, FMT from HRW-treated animals ameliorated IND-induced enteropathy. The SCFA content in the cecal contents of HRW-treated animals was significantly higher than that in control animals. The supernatant had significantly increased interleukin-10 expression in RAW264 cells in vitro. CONCLUSION: Hydrogen-rich water ameliorated NSAID-induced enteropathy, not only via direct antioxidant effects but also via anti-inflammatory effects by increasing luminal SCFAs. These results suggest that hydrogen may have therapeutic potential in small intestinal inflammatory diseases.

Proinflammatory role of basophils in oxazolone-induced chronic intestinal inflammation.

BACKGROUND AND AIM: The functions of basophils have not been elucidated until recently because of their rarity. However, with recent developments in basophil-specific antibodies and basophil-deficient animals, the roles of basophils in various diseases related to chronic inflammation have been clarified. In this study, we aimed to investigate the roles of basophils in human ulcerative colitis (UC) and oxazolone (OXA) colitis using genetically engineered Mcpt8DTR mice. METHODS: Immunohistochemical staining of human colon specimens was performed to examine the involvement of basophils in the pathogenesis of UC. We examined the correlation between the number of infiltrating basophils and the UC endoscopic index of severity (UCEIS), Mayo score, and Matts score. We also examined the correlation between eosinophil count and basophil infiltration. In murine experiments, we examined whether basophil infiltration was involved in OXA-induced colitis and whether basophil depletion improved inflammation in Mcpt8DTR mice. RESULTS: Colonic basophil infiltration was significantly increased in patients with UC. There were significant correlations between UCEIS, Mayo score, Matts score, and the number of infiltrating basophils. In murine OXA-induced colitis, a significant increase in basophil infiltration was observed. When basophils were depleted by diphtheria toxin in Mcpt8DTR mice, inflammation improved significantly and mRNA expression of some proinflammatory cytokines, including Tnf-α and Ifn-γ decreased significantly. CONCLUSION: Basophil infiltration correlated with endoscopic, clinical, and pathological scores in human UC independently of eosinophil infiltration, and depletion of basophils ameliorated mucosal inflammation in murine OXA-induced colitis, collectively suggesting that basophils exert a proinflammatory role in chronic intestinal inflammation such as UC.

Protective Effect of Luminal Uric Acid Against Indomethacin-Induced Enteropathy: Role of Antioxidant Effect and Gut Microbiota.

BACKGROUND: Uric acid (UA) has anti- and pro-inflammatory properties. We previously revealed that elevated serum UA levels provide protection against murine small intestinal injury probably via luminal UA secreted in the small intestine. Luminal UA may act as an antioxidant, preventing microbiota vulnerability to oxidative stress. However, whether luminal UA is increased under hyperuricemia and plays a protective role in a dose-dependent manner as well as the mechanism by which luminal UA exerts its protective effects on enteropathy remains unknown. METHODS: Inosinic acid (IMP) (1000 mg/kg, i.p.) was administered to obtain high serum UA (HUA) and moderate serum UA (500 mg/kg IMP, i.p.) mice. UA concentrations and levels of oxidative stress markers in the serum and intestine were measured. Mice received indomethacin (20 mg/kg, i.p.) to evaluate the effects of UA on indomethacin-induced enteropathy. Reactive oxygen species (ROS) on the ileal mucosa were analyzed. The fecal microbiota of HUA mice was transplanted to investigate its effect on indomethacin-induced enteropathy. RESULTS: IMP increased luminal UA dose-dependently, with higher levels of luminal antioxidant markers. Indomethacin-induced enteropathy was significantly ameliorated in both UA-elevated groups, with decreased indomethacin-induced luminal ROS. The microbiota of HUA mice showed a significant increase in α-diversity and a significant difference in ß-diversity from the control. Fecal microbiota transplantation from HUA mice ameliorated indomethacin-induced enteropathy. CONCLUSIONS: The protective role of luminal UA in intestinal injury is likely exerted via oxidative stress elimination and microbiota composition modulation, preferably for gut immunity. Therefore, enhancing anaerobic conditions using antioxidants is a potential therapeutic target.

Intestinal inflammations increase efflux of innate lymphoid cells from the intestinal mucosa to the mesenteric lymph nodes through lymph-collecting ducts.

INTRODUCTION: Innate lymphoid cells (ILCs) are abundant in the intestinal mucosa, forming boundaries externally. Herein, ILCs were directly obtained from intestinal lymph using a lymph fistula rat model and analyzed under physiological and pathological conditions. METHODS: Thoracic duct (TD) lymphocytes were collected by cannulation with/without preceded mesenteric lymphadenectomy, which were comparable to lymphocytes flowing through mesenteric lymphatic vessels (MLVs) or TD, respectively. The collected ILCs were classified according to gene transcription factors and analyzed by flow cytometry. The effect of IL-25 or indomethacin was studied. RESULTS: The proportion of total ILCs in the MLVs (MLV-ILCs) was significantly higher than that in TD (TD-ILCs, 0.01% vs. 0.003%, respectively). Physiologically, there were several significant differences in the MLV-ILCs compared with TD-ILCs, including the proportion of ILC2 (42.3% vs. 70.9%) and ILC3 (33.3% vs. 13.8%), and the proportion of α4-integrin-positive cells (36.8% vs. 0.3%). IL-25 significantly increased the proportion of MLV-ILC2 after 3 days. Indomethacin-induced intestinal injury increased the proportion of MLV-ILC3 in the early phase within 12 h. CONCLUSION: Intestinal ILCs were found to migrate through MLVs. The altered mobilization of MLV-ILCs after stimuli suggests that ILCs play an important role in regulating the immune responses at the secondary lymph nodes.

Angiopoietin-like protein 4 deficiency augments liver fibrosis in liver diseases such as nonalcoholic steatohepatitis in mice through enhanced free cholesterol accumulation in hepatic stellate cells.

AIM: We recently reported that lipoprotein lipase (LPL)-mediated free cholesterol (FC) accumulation in hepatic stellate cells (HSCs) augmented liver fibrosis in non-alcoholic steatohepatitis (NASH). The aim of the present study was to explore the role of angiopoietin-like protein 4 (Angptl4), an LPL inhibitor, in the pathogenesis of liver fibrosis in NASH. METHODS: Angptl4-deficient or wild-type mice were used to investigate the role of Angptl4 in the pathogenesis of NASH induced by feeding a methionine- and choline-deficient diet. We also examined the effect of Angptl4 on FC accumulation in HSCs, and the subsequent activation of HSCs, using Angptl4-deficient HSCs. RESULTS: In the NASH model, Angptl4-deficient mice had significantly aggravated liver fibrosis and activated HSCs without enhancement of hepatocellular injury, liver inflammation, or liver angiogenesis. FC levels were significantly higher in HSCs from Angptl4-deficient mice than in those from wild-type mice. Treatment with Angptl4 reversed low-density lipoprotein-induced FC accumulation in HSCs through the inhibition of LPL. The Angptl4 deficiency-induced FC accumulation in HSCs suppressed HSC expression of the transforming growth factor-ß (TGF-ß) pseudoreceptor, bone morphogenetic protein, and activin membrane-bound inhibitor, and sensitized HSCs to TGF-ß-induced activation in vivo and in vitro. CONCLUSIONS: Angptl4 plays an important role in the pathogenesis of FC accumulation in HSCs. In addition, regulation of FC levels in HSCs by Angptl4 plays a critical role in the pathogenesis of liver fibrosis in NASH. Thus, Angptl4 could represent a novel therapeutic option for NASH.

Acesulfame potassium induces dysbiosis and intestinal injury with enhanced lymphocyte migration to intestinal mucosa.

BACKGROUND AND AIM: The artificial sweetener acesulfame potassium (ACK) is officially approved as safe for intake and has been used in processed foods. However, ACKs have been reported to induce metabolic syndrome, along with alteration of the gut microbiota in mice. In recent years, studies have suggested that this artificial sweetener promotes myeloperoxidase reactivity in Crohn's disease-like ileitis. We aimed to investigate the effect of ACK on the intestinal mucosa and gut microbiota of normal mice. METHODS: Acesulfame potassium was administered to C57BL/6J mice (8 weeks old) via free drinking. Intestinal damage was evaluated histologically, and messenger RNA (mRNA) levels of TNF-α, IFN-γ, IL1-ß, MAdCAM-1, GLP1R, and GLP2R were determined with quantitative reverse transcription polymerase chain reaction (qRT-PCR). Immunohistochemistry was performed to examine the expression of MAdCAM-1 in the small intestine. The composition of gut microbiota was assessed using high-throughput sequencing. We performed intravital microscopic observation to examine if ACK altered lymphocyte migration to the intestinal microvessels. RESULTS: Acesulfame potassium increased the expression of proinflammatory cytokines, decreased the expression of GLP-1R and GLP-2R, and induced small intestinal injury with an increase in intestinal permeability, and ACK treatment induced microbial changes, but the transfer of feces alone from ACK mice did not reproduce intestinal damage in recipient mice. ACK treatment significantly increased the migration of lymphocytes to intestinal microvessels. CONCLUSION: Acesulfame potassium induces dysbiosis and intestinal injury with enhanced lymphocyte migration to intestinal mucosa. Massive use of non-caloric artificial sweeteners may not be as safe as we think.

Deoxycholic acid enhancement of lymphocyte migration through direct interaction with the intestinal vascular endothelium.

BACKGROUND AND AIM: The small intestine plays a central role in gut immunity, and enhanced lymphocyte migration is involved in the pathophysiology of various enteropathy. Bile acid (BA) is closely related to lipid metabolism and gut microbiota and essential for gut homeostasis. However, the effects of BA on gut immunity have not been studied in detail, especially on the small intestine and lymphocyte migration. Therefore, we aimed to investigate the effect of BA on small intestinal lymphocyte microcirculation. METHODS: The effect of deoxycholic acid (DCA), taurocholic acid (tCA), or cholic acid (CA) on the indomethacin (IND)-induced small intestinal enteropathy in mice was investigated. Lymphocyte movements were evaluated after exposure to BA using intravital microscopy. The effects of BA on surface expression of adhesion molecules on the vascular endothelium and lymphocytes through BA receptors were examined in vitro. RESULTS: IND-induced small intestinal enteropathy was histologically aggravated by DCA treatment alone. The expression of adhesion molecules ICAM-1 and VCAM-1 was significantly enhanced by DCA. Exposure to DCA increased lymphocyte adhesion in the microvessels of the ileum, which was partially blocked by anti-α4ß1 integrin antibody in vivo. The expression of ICAM-1 and VCAM-1 was significantly enhanced by DCA in vitro, which was partially suppressed by the sphingosine-1-phosphate receptor 2 (S1PR2) antagonist. The S1PR2 antagonist significantly ameliorated IND-induced and DCA-exaggerated small intestinal injury. CONCLUSION: DCA exacerbated IND-induced small intestinal enteropathy. DCA directly acts on the vascular endothelium and enhances the expression levels of adhesion molecules partially via S1PR2, leading to enhanced small intestinal lymphocyte migration.

Dietary emulsifier polysorbate-80-induced small-intestinal vulnerability to indomethacin-induced lesions via dysbiosis.

BACKGROUND AND AIM: Dietary emulsifiers are widely used in processed foods and officially approved as safe for intake. However, recent studies have demonstrated that some emulsifiers alter the colonic microbiota, leading to colonic low-grade inflammation, in mice. The effect of dietary emulsifiers on small-intestinal microbiota, which is important for gut immunity, has not been studied. We aimed to investigate the effect of a representative dietary emulsifier, polysorbate-80 (P80), on the small-intestinal microbiota in normal mice. METHODS: Some mice were pretreated with P80 for 8 weeks with or without indomethacin administration on the last 2 days, and intestinal damage was evaluated histologically. The ileal and colonic microbiota composition was assessed using 16S rRNA polymerase chain reaction. RESULTS: Polysorbate-80 increased the Gammaproteobacteria abundance and decreased the α-diversity in the small intestine. No decrease in α-diversity was observed in the colon. P80 pretreatment exacerbated the indomethacin-induced small-intestinal lesions and significantly increased the interleukin-1ß expression. Culture of ileal content on deoxycholate hydrogen sulfide lactose agar showed that P80 significantly increased the colonies of the sulfide-producing bacteria Proteus spp. (genetically identified as Proteus mirabilis). Antibiotic pretreatment abolished the P80-induced aggravation of indomethacin-induced ileitis. Motility assay in semisolid agar showed that adding 0.02% P80 to the agar significantly increased the diameter of P. mirabilis colonies but not that of Escherichia coli colonies. CONCLUSIONS: Polysorbate-80 enhances the vulnerability of the small intestine to indomethacin-induced injury by inducing ileal dysbiosis. Direct enhancement of the motility of specific flagellated microbiota by P80 might be related to dysbiosis and intestinal injury.

Chitinase 3-like 1 deficiency ameliorates liver fibrosis by promoting hepatic macrophage apoptosis.

AIM: Chitinase 3-like 1 (CHI3L1), an 18-glycosyl hydrolase-related molecule, is a member of the enzymatically inactive chitinase-like protein family. Serum levels of CHI3L1 are strongly correlated with hepatic fibrosis progression during many liver diseases. Therefore, this protein could be involved in the development of hepatic fibrosis pathology; however, its role has not been elucidated. We aimed to elucidate its role in the pathophysiology of liver fibrosis. METHODS: Chitinase 3-like 1-deficient (Chi3l1-/- ) mice were given carbon tetrachloride twice per week for 4 weeks or fed a methionine choline-deficient diet for 12 weeks to generate mouse liver fibrosis models. Human fibrotic liver tissues were also examined immunohistochemically. RESULTS: In human and mouse fibrotic livers, CHI3L1 expression was mainly localized to hepatic macrophages, and the intrahepatic accumulation of CHI3L1+ macrophages was significantly enhanced compared to that in control livers. In the two mouse models, hepatic fibrosis was significantly ameliorated in Chi3l1-/- mice compared to that in wild-type mice, which was dependent on hepatic macrophages. The accumulation and activation of hepatic macrophages was also significantly suppressed in Chi3l1-/- mice compared to that in wild-type mice. Furthermore, apoptotic hepatic macrophages were significantly increased in Chi3l1-/- mice. Chitinase 3-like 1 was found to inhibit hepatic macrophage apoptosis by suppressing Fas expression and activating Akt signaling in an autocrine manner, which resulted in hepatic macrophage accumulation and activation, exaggerating liver fibrosis. CONCLUSIONS: Chitinase 3-like 1 exacerbates liver fibrosis progression by suppressing apoptosis in hepatic macrophages. Therefore, this might be a potential therapeutic target for the treatment of liver fibrosis.

Vitamin A-coupled liposome system targeting free cholesterol accumulation in hepatic stellate cells offers a beneficial therapeutic strategy for liver fibrosis.

AIM: Liver fibrosis is a life-threatening disorder for which no approved therapy is available. Recently, we reported that mouse hepatic stellate cell (HSC) activation increased free cholesterol (FC) accumulation, partly by enhancing signaling through sterol regulatory element-binding protein 2 (SREBP2) and microRNA-33a (miR-33a), which resulted in HSC sensitization to transforming growth factor-ß (TGFß)-induced activation in a "vicious cycle" of liver fibrosis. METHODS: Human HSCs were isolated from surgical liver specimens from control patients and patients with liver fibrosis. C57BL/6 mice were treated with carbon tetrachloride for 4 weeks and concurrently given SREBP2-siRNA- or anti-miR-33a-bearing vitamin A-coupled liposomes. RESULTS: In human activated HSCs obtained from patients with liver fibrosis, FC accumulation was enhanced independently of serum cholesterol levels through increased signaling by both SREBP2 and miR-33a. This increased FC accumulation enhanced Toll-like receptor 4 (TLR4) protein levels and lowered the TGFß-pseudoreceptor Bambi (bone morphogenetic protein and activin membrane-bound inhibitor) mRNA levels in HSCs. Notably, in a mouse liver fibrosis model, reduction of FC accumulation, specifically in activated HSCs by suppression of SREBP2 or miR-33a expression using SREBP2-siRNA- or anti-miR-33a-bearing vitamin A-coupled liposomes, downregulated TLR4 signaling, increased Bambi expression, and consequently ameliorated liver fibrosis. CONCLUSIONS: Our results suggest that FC accumulation in HSCs, as an intracellular mediator promoting HSC activation, contributes to a vicious cycle of HSC activation in human and mouse liver fibrosis independent of serum cholesterol levels. Targeting FC accumulation-related molecules in HSCs through a vitamin A-coupled liposomal system represents a favorable therapeutic strategy for liver fibrosis.

Amelioration of colitis through blocking lymphocytes entry to Peyer's patches by sphingosine-1-phosphate lyase inhibitor.

BACKGROUND AND AIM: Sphingosine-1-phosphate (S1P) receptor 1, a therapeutic target of the S1P1 agonist FTY720, plays a crucial role in lymphocyte migration and is expressed in several cells including naïve T lymphocytes and endothelial cells. 2-Acetyl-4-tetrahydroxybutyl imidazole (THI), an inhibitor of S1P lyase, exhibits immunomodulatory activity through increasing the S1P concentration in the secondary lymphoid organs, but its effects on colitis remain unclear. This study aimed to clarify how THI affects colitis and migration of naïve T lymphocytes in Peyer's patches (PPs). METHODS: The effect of THI on gut immunity was investigated by analyzing the dextran sulfate sodium (DSS)-induced murine colitis model, lymphocyte components in thoracic duct lymphocytes (TDLs), and microscopic movement of TDLs in PPs. RESULTS: 2-Acetyl-4-tetrahydroxybutyl imidazole ameliorated DSS-induced colitis histologically by causing a significant decrease in colonic lymphocyte infiltration and expression of mucosal pro-inflammatory cytokines. THI suppressed the inflow of naïve T lymphocytes into the thoracic duct. Microscopic observation of PPs in control animals revealed that many TDLs egressed to the stroma and migrated to lymph capillaries after attaching to the high endothelial venules (HEVs). THI or FTY720 treatment in recipient animals blocked lymphocyte egression from the HEVs to the stroma. CONCLUSIONS: This is the first study to clarify the ameliorating effects of THI on DSS-induced colitis. Microscopic observations demonstrated the involvement of HEVs in the egression of S1P-dependent gut-tropic T lymphocytes to lymph capillaries. This S1P lyase inhibitor might become a novel immunosuppressant for inflammatory bowel disease therapy by blocking infiltration of lymphocytes through HEVs into the stroma in PPs.

Free cholesterol accumulation in liver sinusoidal endothelial cells exacerbates acetaminophen hepatotoxicity via TLR9 signaling.

BACKGROUND & AIMS: Although obesity is a risk factor for acute liver failure, the pathogenic mechanisms are not yet fully understood. High cholesterol (HC) intake, which often underlies obesity, is suggested to play a role in the mechanism. We aimed to elucidate the effect of a HC diet on acetaminophen-induced acute liver injury, the most frequent cause of acute liver failure in the USA. METHODS: C57BL/6 Toll-like receptor 9 (TLR9) knockout (Tlr9-/-) mice and their Tlr9+/+ littermates were fed an HC diet for fourweeks and then treated with acetaminophen. Liver sinusoidal endothelial cells (LSECs) were isolated from the mice for in vivo and in vitro analyses. RESULTS: The HC diet exacerbated acetaminophen-induced acute liver injury in a TLR9/inflammasome pathway-dependent manner. LSECs played a major role in the cholesterol loading-induced exacerbation. The accumulation of free cholesterol in the endolysosomes in LSECs enhanced TLR9-mediated signaling, thereby exacerbating the pathology of acetaminophen-induced liver injury through the activation of the TLR9/inflammasome pathway. The accumulation of free cholesterol in LSEC endolysosomes induced a dysfunction of the Rab7 membrane trafficking recycling mechanism, thus disrupting the transport of TLR9 from late endosomes to the lysosomes. Consequently, the level of active TLR9 in the late endosomes increased, thereby enhancing TLR9 signaling in LSECs. CONCLUSIONS: HC intake exaggerated acetaminophen-induced acute liver injury via free cholesterol accumulation in LSECs, demonstrating a novel role of free cholesterol as a metabolic factor in TLR9 signal regulation and pathologies of acetaminophen-induced liver injury. Therapeutic approaches may target this pathway. Lay summary: High cholesterol intake exacerbated acetaminophen-induced acute liver injury via the accumulation of free cholesterol in the endolysosomes of liver sinusoidal endothelial cells. This accumulation enhanced Toll-like receptor 9 signaling via impairment of its membrane trafficking mechanism. Thus, free cholesterol accumulation, as an underlying metabolic factor, exacerbated the pathology of acetaminophen-induced liver injury through activation of the TLR9/inflammasome pathway.

Uric acid ameliorates indomethacin-induced enteropathy in mice through its antioxidant activity.

BACKGROUND AND AIM: Uric acid is excreted from blood into the intestinal lumen, yet the roles of uric acid in intestinal diseases remain to be elucidated. The study aimed to determine whether uric acid could reduce end points associated with nonsteroidal anti-inflammatory drug (NSAID)-induced enteropathy. METHODS: A mouse model of NSAID-induced enteropathy was generated by administering indomethacin intraperitoneally to 8-week-old male C57BL/6 mice, and then vehicle or uric acid was administered orally. A group of mice treated with indomethacin was also concurrently administered inosinic acid, a uric acid precursor, and potassium oxonate, an inhibitor of uric acid metabolism, intraperitoneally. For in vitro analysis, Caco-2 cells treated with indomethacin were incubated in the presence or absence of uric acid. RESULTS: Oral administration of uric acid ameliorated NSAID-induced enteropathy in mice even though serum uric acid levels did not increase. Intraperitoneal administration of inosinic acid and potassium oxonate significantly elevated serum uric acid levels and ameliorated NSAID-induced enteropathy in mice. Both oral uric acid treatment and intraperitoneal treatment with inosinic acid and potassium oxonate significantly decreased lipid peroxidation in the ileum of mice with NSAID-induced enteropathy. Treatment with uric acid protected Caco-2 cells from indomethacin-induced oxidative stress, lipid peroxidation, and cytotoxicity. CONCLUSIONS: Uric acid within the intestinal lumen and in serum had a protective effect against NSAID-induced enteropathy in mice, through its antioxidant activity. Uric acid could be a promising therapeutic target for NSAID-induced enteropathy.

Platelet interaction with lymphatics aggravates intestinal inflammation by suppressing lymphangiogenesis.

Lymphatic failure is a histopathological feature of inflammatory bowel disease (IBD). Recent studies show that interaction between platelets and podoplanin on lymphatic endothelial cells (LECs) suppresses lymphangiogenesis. We aimed to investigate the role of platelets in the inflammatory process of colitis, which is likely to be through modulation of lymphangiogenesis. Lymphangiogenesis in colonic mucosal specimens from patients with IBD was investigated by studying mRNA expression of lymphangiogenic factors and histologically by examining lymphatic vessel (LV) densities. Involvement of lymphangiogenesis in intestinal inflammation was studied by administering VEGF-receptor 3 (VEGF-R3) inhibitors to the mouse model of colitis using dextran sulfate sodium and evaluating platelet migration to LVs. The inhibitory effect of platelets on lymphangiogenesis was investigated in vivo by administering antiplatelet antibody to the colitis mouse model and in vitro by coculturing platelets with lymphatic endothelial cells. Although mRNA expressions of lymphangiogenic factors such as VEGF-R3 and podoplanin were significantly increased in the inflamed mucosa of patients with IBD compared with those with quiescent mucosa, there was no difference in LV density between them. In the colitis model, VEGF-R3 inhibition resulted in aggravated colitis, decreased lymphatic density, and increased platelet migration to LVs. Administration of an antiplatelet antibody increased LV densities and significantly ameliorated colitis. Coculture with platelets inhibited proliferation of LECs in vitro. Our data suggest that despite elevated lymphangiogenic factors during colonic inflammation, platelet migration to LVs resulted in suppressed lymphangiogenesis, leading to aggravation of colitis by blocking the clearance of inflammatory cells. Modulating the interaction between platelets and LVs could be a new therapeutic means for treating IBD.

Free cholesterol accumulation in hepatic stellate cells: mechanism of liver fibrosis aggravation in nonalcoholic steatohepatitis in mice.

UNLABELLED: Although nonalcoholic steatohepatitis (NASH) is associated with hypercholesterolemia, the underlying mechanisms of this association have not been clarified. We aimed to elucidate the precise role of cholesterol in the pathophysiology of NASH. C57BL/6 mice were fed a control, high-cholesterol (HC), methionine-choline-deficient (MCD), or MCD+HC diet for 12 weeks or a control, HC, high-fat (HF), or HF+HC diet for 24 weeks. Increased cholesterol intake accelerated liver fibrosis in both the mouse models without affecting the degree of hepatocellular injury or Kupffer cell activation. The major causes of the accelerated liver fibrosis involved free cholesterol (FC) accumulation in hepatic stellate cells (HSCs), which increased Toll-like receptor 4 protein (TLR4) levels through suppression of the endosomal-lysosomal degradation pathway of TLR4, and thereby sensitized the cells to transforming growth factor (TGF)ß-induced activation by down-regulating the expression of bone morphogenetic protein and activin membrane-bound inhibitor. Mammalian-cell cholesterol levels are regulated by way of a feedback mechanism mediated by sterol regulatory element-binding protein 2 (SREBP2), maintaining cellular cholesterol homeostasis. Nevertheless, HSCs were sensitive to FC accumulation because the high intracellular expression ratio of SREBP cleavage-activating protein (Scap) to insulin-induced gene (Insig) disrupted the SREBP2-mediated feedback regulation of cholesterol homeostasis in these cells. HSC activation subsequently enhanced the disruption of the feedback system by Insig-1 down-regulation. In addition, the suppression of peroxisome proliferator-activated receptor γ signaling accompanying HSC activation enhanced both SREBP2 and microRNA-33a signaling. Consequently, FC accumulation in HSCs increased and further sensitized these cells to TGFß-induced activation in a vicious cycle, leading to exaggerated liver fibrosis in NASH. CONCLUSION: These characteristic mechanisms of FC accumulation in HSCs are potential targets to treat liver fibrosis in liver diseases including NASH.

Toll-like receptor (TLR) 2 agonists ameliorate indomethacin-induced murine ileitis by suppressing the TLR4 signaling.

BACKGROUND AND AIM: Few drugs have been found satisfactory in the treatment of nonsteroidal anti-inflammatory drugs (NSAIDs)-induced enteropathy. Toll-like receptor (TLR) 4 and aberrant leukocyte migration to the intestinal mucosa are reported to be involved in the pathology of intestinal enteropathy and TLR2 agonists have been found to evoke hyposensitivity to TLR4 stimulation in vitro. In this study, we investigated whether and how lipoarabinomannan (LAM) or lipoteichoic acid (LTA), TLR2 agonists, attenuated indomethacin (IND)-induced intestinal damage. METHODS: LAM (0.5 mg/kg) or LTA (15 mg/kg) was administered intraperitoneally to mice before IND (10 mg/kg) administration. Disease activity was evaluated macroscopically and histologically. In the migration analysis, fluorescence-labeled leukocyte movement in the intestinal microvessels was observed by intravital microscopy. Expression of P-selectin, MAdCAM-1, TLR2, TLR4, and F4/80 was observed immunohistochemically. In the in vitro analysis, RAW264.7 macrophage cells were preincubated with LAM and stimulated with lipopolysaccharide (LPS), and the mRNA expression levels of TLR4, tumor necrosis factor-α, and interleukin-12p40 were measured. RESULTS: Pretreatment with LAM or LTA significantly decreased IND-induced injury as well as decreased leukocyte infiltration. Pretreatment with LAM decreased IND-induced TLR4 expression on F4/80(+) macrophages, the level of P-selectin expression, and leukocyte migration in the small intestinal vessels. In the in vitro study, a single administration of LAM decreased TLR4 mRNA expression and inhibited the increase in mRNA expression of inflammatory cytokines by LPS in a dose-dependent manner. CONCLUSION: TLR2 agonists attenuated IND-induced small intestinal lesions and leukocyte infiltration probably by suppressing the TLR4 signaling pathway in tissue macrophages.

Recombinant Thrombomodulin Modulates Murine Colitis Possibly via High-Mobility Group Box 1 Protein Inhibition.

BACKGROUND AND AIM: Thrombomodulin (TM) is an anticoagulant cofactor protein. We hypothesized that its recombinant soluble TM (rhTM) form, widely used to treat disseminated intravascular coagulation, might have anti-inflammatory action in inflammatory bowel disease (IBD), possibly through its inhibition of high-mobility group box 1 protein (HMGB1). METHODS: We investigated inflammatory effects of HMGB1 and anti-inflammatory effect of rhTM in dextran sulfate sodium (DSS)-treated mice, some cell lines and ulcerative colitis (UC) patients, particularly focusing on changes of vascular endothelial adhesion molecules. RESULTS: Treatments with rhTM significantly attenuated DSS-treated mice clinically and histologically. The mRNA levels of proinflammatory cytokines and adhesion molecules were decreased by rhTM. Increased inflammatory cells in the colonic mucosa strongly expressed HMGB1 in the cytoplasm in the DSS-treated mice and UC patients' colonic mucosa, which were significantly decreased by rhTM in mice. In in vitro experiments, rhTM significantly decreased the mRNA levels of tumor necrosis factor-alpha (TNF-α) and adhesion molecules increased by endotoxin exposures in RAW 264.7 (macrophage cell line) and bEND.3 cells (endothelial cell line), suggesting the proinflammatory role of HMGB1 in TNF-α production from macrophages. CONCLUSIONS: These findings suggest that rhTM may be useful for the treatment of IBD by attenuating inflammatory cytokine production and adhesion molecule expression, partly because of its inhibition of HMGB1.

Acyl-CoA:cholesterol acyltransferase 1 mediates liver fibrosis by regulating free cholesterol accumulation in hepatic stellate cells.

BACKGROUND & AIMS: Acyl-coenzyme A: cholesterol acyltransferase (ACAT) catalyzes the conversion of free cholesterol (FC) to cholesterol ester, which prevents excess accumulation of FC. We recently found that FC accumulation in hepatic stellate cells (HSCs) plays a role in progression of liver fibrosis, but the effect of ACAT1 on liver fibrosis has not been clarified. In this study, we aimed to define the role of ACAT1 in the pathogenesis of liver fibrosis. METHODS: ACAT1-deficient and wild-type mice, or Toll-like receptor 4 (TLR4)(-/-)ACAT1(+/+) and TLR4(-/-)ACAT1(-/-) mice were subjected to bile duct ligation (BDL) for 3 weeks or were given carbon tetrachloride (CCl4) for 4 weeks to induce liver fibrosis. RESULTS: ACAT1 was the major isozyme in mice and human primary HSCs, and ACAT2 was the major isozyme in mouse primary hepatocytes and Kupffer cells. ACAT1 deficiency significantly exaggerated liver fibrosis in the mouse models of liver fibrosis, without affecting the degree of hepatocellular injury or liver inflammation, including hepatocyte apoptosis or Kupffer cell activation. ACAT1 deficiency significantly increased FC levels in HSCs, augmenting TLR4 protein and downregulating expression of transforming growth factor-ß (TGFß) pseudoreceptor Bambi (bone morphogenetic protein and activin membrane-bound inhibitor), leading to sensitization of HSCs to TGFß activation. Exacerbation of liver fibrosis by ACAT1 deficiency was dependent on FC accumulation-induced enhancement of TLR4 signaling. CONCLUSIONS: ACAT1 deficiency exaggerates liver fibrosis mainly through enhanced FC accumulation in HSCs. Regulation of ACAT1 activities in HSCs could be a target for treatment of liver fibrosis.