Indoleamine 2,3-dioxygenase-1 (Ido), which catalyzes the first and limiting step of tryptophan catabolism, has been implicated in immune tolerance. However, the roles of Ido in systemic bacterial infection are complicated and remain controversial. To explore this issue, we examined the roles of Ido in bacterial peritonitis and sepsis after cecal ligation and puncture (CLP) in mice by using the Ido inhibitor 1-methyl-d,l-tryptophan (1-MT), by comparing Ido(+/+) and Ido(-/-) mice, or by using chimeric mice in which Ido in the bone marrow-derived cells was deficient. Ido expression in the peritoneal CD11b(+) cells and its metabolite l-kynurenine in the serum were increased after CLP. 1-MT treatment or Ido deficiency, especially in bone marrow-derived cells, reduced mortality after CLP. Compared to Ido(+/+) mice, Ido(-/-) mice showed increased recruitment of neutrophils and mononuclear cells into the peritoneal cavity and a decreased bacterial count in the blood accompanied by increased CXCL-2 and CXCL-1 mRNA in the peritoneal cells. Ido has an inhibitory effect on LPS-induced CXCL-2 and CXCL-1 production in cultured peritoneal cells. These findings indicate that inhibition of Ido reduces mortality from peritonitis and sepsis after CLP via recruitment of neutrophils and mononuclear cells by chemokine production in peritoneal CD11b(+) cells. Thus, blockade of Ido plays a beneficial role in host protection during bacterial peritonitis and sepsis.
Various hydroxycinnamoyl ?-d-xylopyranosides were efficiently prepared from 2,3,4-tri-O-acetyl-?-d-xylopyranosyl bromide (TAXB) with amine by amine-promoted glycosylation. The resulted acetylated hydroxycinnamoyl ?-d-xylopyranosides with acetoxy groups at C-2, C-3, and C-4 were regioselectively deacetylated at C-4 position with Novozym 435. Antioxidant activities of free hydroxycinnamic acids and the respective ?-d-xylopyranosides were evaluated by DPPH radical scavenging activity as well as their inhibitory effect on autoxidation of bulk methyl linoleate. The radical scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) decreased in the order ferulic acid>caffeic acid?caffeoyl ?-d-xylopyranosides?sinapinic acid>sinapoyl ?-d-xylopyranosides?feruloyl ?-d-xylopyranosides>p-coumaric acid>p-coumaroyl ?-d-xylopyranosides. In bulk methyl linoleate, the antioxidant activity order against autoxidation was almost consistent with the scavenging activity order. The results showed that caffeoyl ?-d-xylopyranosides and sinapoyl ?-d-xylopyranosides were as effective as free caffeic acid, sinapinic acid, and ferulic acid.
Acid sphingomyelinase (ASM) regulates the homeostasis of sphingolipids, including ceramides and sphingosine-1-phosphate (S1P). These sphingolipids regulate carcinogenesis and proliferation, survival, and apoptosis of cancer cells. However, the role of ASM in host defense against liver metastasis remains unclear. In this study, the involvement of ASM in liver metastasis of colon cancer was examined using Asm-/- and Asm+/+ mice that were inoculated with SL4 colon cancer cells to produce metastatic liver tumors. Asm-/- mice demonstrated enhanced tumor growth and reduced macrophage accumulation in the tumor, accompanied by decreased numbers of hepatic myofibroblasts (hMFs), which express tissue inhibitor of metalloproteinase 1 (TIMP1), around the tumor margin. Tumor growth was increased by macrophage depletion or by Timp1 deficiency, but was decreased by hepatocyte-specific ASM overexpression, which was associated with increased S1P production. S1P stimulated macrophage migration and TIMP1 expression in hMFs in vitro. These findings indicate that ASM in the liver inhibits tumor growth through cytotoxic macrophage accumulation and TIMP1 production by hMFs in response to S1P. Targeting ASM may represent a new therapeutic strategy for treating liver metastasis of colon cancer.
Tumor necrosis factor (TNF)-?, which is a mediator of hepatotoxicity, has been implicated in liver fibrosis. However, the roles of TNF-? on hepatic stellate cell (HSC) activation and liver fibrosis are complicated and remain controversial. To explore this issue, the role of TNF-? in cholestasis-induced liver fibrosis was examined by comparing between TNF-?(-/-) mice and TNF-?(+/+) mice after bile duct ligation (BDL). Serum TNF-? levels in mice were increased by common BDL combined with cystic duct ligation (CBDL+CDL). TNF-? deficiency reduced liver fibrosis without affecting liver injury, inflammatory cell infiltration, and liver regeneration after CBDL+CDL. Increased expression levels of collagen ?1(I) mRNA, transforming growth factor (TGF)-? mRNA, and ?-smooth muscle actin (?SMA) protein by CBDL+CDL in the livers of TNF-?(-/-) mice were comparable to those in TNF-?(+/+) mice. Exogenous administration of TNF-? decreased collagen ?1(I) mRNA expression in isolated rat HSCs. These results suggest that the reduced fibrosis in TNF-?(-/-) mice is regulated in post-transcriptional level. Tissue inhibitor of metalloproteinase (TIMP)-1 plays a crucial role in the pathogenesis of liver fibrosis. TIMP-1 expression in HSCs in the liver was increased by CBDL+CDL, and the induction was lower in TNF-?(-/-) mice than in TNF-?(+/+) mice. Fibrosis in the lobe of TIMP-1(-/-) mice with partial BDL was also reduced. These findings indicate that TNF-? produced by cholestasis can promote liver fibrosis via TIMP-1 production from HSCs. Thus, targeting TNF-? and TIMP-1 may become a new therapeutic strategy for treating liver fibrosis in cholestatic liver injury.
Nonalcoholic fatty liver disease is one of the most common liver diseases. L-tryptophan and its metabolite serotonin are involved in hepatic lipid metabolism and inflammation. However, it is unclear whether L-tryptophan promotes hepatic steatosis. To explore this issue, we examined the role of L-tryptophan in mouse hepatic steatosis by using a high fat and high fructose diet (HFHFD) model. L-tryptophan treatment in combination with an HFHFD exacerbated hepatic steatosis, expression of HNE-modified proteins, hydroxyproline content, and serum alanine aminotransaminase levels, whereas L-tryptophan alone did not result in these effects. We also found that L-tryptophan treatment increases serum serotonin levels. The introduction of adenoviral aromatic amino acid decarboxylase, which stimulates the serotonin synthesis from L-tryptophan, aggravated hepatic steatosis induced by the HFHFD. The fatty acid-induced accumulation of lipid was further increased by serotonin treatment in cultured hepatocytes. These results suggest that L-tryptophan increases the sensitivity to hepatic steatosis through serotonin production. Furthermore, L-tryptophan treatment, adenoviral AADC introduction, and serotonin treatment induced phosphorylation of the mammalian target of rapamycin (mTOR), and a potent mTOR inhibitor rapamycin attenuated hepatocyte lipid accumulation induced by fatty acid with serotonin. These results suggest the importance of mTOR activation for the exacerbation of hepatic steatosis. In conclusion, L-tryptophan exacerbates hepatic steatosis induced by HFHFD through serotonin-mediated activation of mTOR.
The tumor microenvironment (TME) is critical for tumor growth and progression. However, the formation of the TME is largely unknown. This report demonstrates a color-coded imaging model in which the development of the TME can be visualized. In order to image the TME, a green fluorescent protein (GFP)-expressing mouse was used as the host which expresses GFP in all organs but not the parenchymal cells of the liver. Non-colored HCT-116 human colon cancer cells were injected in the spleen of GFP nude mice which led to the formation of experimental liver metastasis. TME formation resulting from the liver metastasis was observed using the Olympus OV100 small animal fluorescence imaging system. HCT-116 cells formed tumor colonies in the liver 28 days after cell transplantation to the spleen. GFP-expressing host cells were recruited by the metastatic tumors as visualized by fluorescence imaging. A desmin positive area increased around and within the liver metastasis over time, suggesting cancer-associated fibroblasts (CAFs) were recruited by the liver metastasis which have a role in tumor progression. The color-coded model of the TME enables its formation to be visualized at the cellular level in vivo, in real-time. This imaging model of the TME should lead to new visual targets in the TME.
Acid sphingomyelinase (ASM) regulates the homeostasis of sphingolipids, including ceramides and sphingosine-1-phosphate (S1P). Because sphingolipids regulate AKT activation, we investigated the role of ASM in hepatic glucose and lipid metabolism. Initially, we overexpressed ASM in the livers of wild-type and diabetic db/db mice by adenovirus vector (Ad5ASM). In these mice, glucose tolerance was improved, and glycogen and lipid accumulation in the liver were increased. Using primary cultured hepatocytes, we confirmed that ASM increased glucose uptake, glycogen deposition, and lipid accumulation through activation of AKT and glycogen synthase kinase-3?. In addition, ASM induced up-regulation of glucose transporter 2 accompanied by suppression of AMP-activated protein kinase (AMPK) phosphorylation. Loss of sphingosine kinase-1 (SphK1) diminished ASM-mediated AKT phosphorylation, but exogenous S1P induced AKT activation in hepatocytes. In contrast, SphK1 deficiency did not affect AMPK activation. These results suggest that the SphK/S1P pathway is required for ASM-mediated AKT activation but not for AMPK inactivation. Finally, we found that treatment with high-dose glucose increased glycogen deposition and lipid accumulation in wild-type hepatocytes but not in ASM(-/-) cells. This result is consistent with glucose intolerance in ASM(-/-) mice. In conclusion, ASM modulates AKT activation and AMPK inactivation, thus regulating glucose and lipid metabolism in the liver.
IDO converts tryptophan to l-kynurenine, and it is noted as a relevant molecule in promoting tolerance and suppressing adaptive immunity. In this study, we examined the effect of IDO in ?-galactosylceramide (?-GalCer)-induced hepatitis. The increase in IDO expression in the liver of wild-type (WT) mice administered ?-GalCer was confirmed by real-time PCR, Western blotting, and IDO immunohistochemical analysis. The serum alanine aminotransferase levels in IDO-knockout (KO) mice after ?-GalCer injection significantly increased compared with those in WT mice. 1-Methyl-D-tryptophan also exacerbated liver injury in this murine hepatitis model. In ?-GalCer-induced hepatitis models, TNF-? is critical in the development of liver injury. The mRNA expression and protein level of TNF-? in the liver from IDO-KO mice were more enhanced compared with those in WT mice. The phenotypes of intrahepatic lymphocytes from WT mice and IDO-KO mice treated with ?-GalCer were analyzed by flow cytometry, and the numbers of CD49b(+) and CD11b(+) cells were found to have increased in IDO-KO mice. Moreover, as a result of the increase in the number of NK cells and macrophages in the liver of IDO-KO mice injected with ?-GalCer, TNF-? secretion in these mice was greater than that in WT mice. Deficiency of IDO exacerbated liver injury in ?-GalCer-induced hepatitis. IDO induced by proinflammatory cytokines may decrease the number of TNF-?-producing immune cells in the liver. Thus, IDO may suppress overactive immune response in the ?-GalCer-induced hepatitis model.
Indoleamine 2,3-dioxygenase, the L-tryptophan-degrading enzyme, plays a key role in the powerful immunomodulatory effects on several different types of cells. Because modulation of IDO activities after viral infection may have great impact on disease progression, we investigated the role of IDO following infection with LP-BM5 murine leukemia virus. We found suppressed BM5 provirus copies and increased type I IFNs in the spleen from IDO knockout (IDO(-/-)) and 1-methyl-D-L-tryptophan-treated mice compared with those from wild-type (WT) mice. Additionally, the number of plasmacytoid dendritic cells in IDO(-/-) mice was higher in the former than in the WT mice. In addition, neutralization of type I IFNs in IDO(-/-) mice resulted in an increase in LP-BM5 viral replication. Moreover, the survival rate of IDO(-/-) mice or 1-methyl-D-L-tryptophan-treated mice infected with LP-BM5 alone or with both Toxoplasma gondii and LP-BM5 was clearly greater than the survival rate of WT mice. To our knowledge, the present study is the first report to observe suppressed virus replication with upregulated type I IFN in IDO(-/-) mice, suggesting that modulation of the IDO pathway may be an effective strategy for treatment of virus infection.
In this study, we demonstrate that the differential behavior, including malignancy and chemosensitivity, of cancer stem-like and non-stem cells can be simultaneously distinguished in the same tumor in real time by color-coded imaging. CD133(+) Huh-7 human hepatocellular carcinoma (HCC) cells were considered as cancer stem-like cells (CSCs), and CD133(-) Huh-7 cells were considered as non-stem cancer cells (NSCCs). CD133(+) cells were isolated by magnetic bead sorting after Huh-7 cells were genetically labeled with green fluorescent protein (GFP) or red fluorescent protein (RFP). In this scheme, CD133(+) cells were labeled with GFP and CD133(-) cells were labeled with RFP. CSCs had higher proliferative potential compared to NSCCs in vitro. The same number of GFP CSCs and the RFP NSCCs were mixed and injected subcutaneously or in the spleen of nude mice. CSCs were highly tumorigenic and metastatic as well as highly resistant to chemotherapy in vivo compared to NSCCs. The ability to specifically distinguish stem-like cancer cells in vivo in real time provides a visual target for prevention of metastasis and drug resistance.
In this study, we demonstrated that lipopolysaccharide (LPS) markedly increased nitric oxide (NO) production and indoleamine 2,3-dioxygenase (IDO) activity in mouse peritoneal cells in the presence of activated Valpha14 natural killer T cells. Moreover, LPS-induced NO production in peritoneal cells from IDO-knockout (KO) mice was more increased than that from wild-type mice. However, there was no significant difference in the expression of inducible nitric oxide synthase (iNOS) mRNA and protein between the wild-type and IDO-KO mice. No significant difference was also observed in the ratio of CD3- and DX5-positive cells and F4/80- and TLR4-positive cells in peritoneal cells between the wild-type and IDO-KO mice. Since the IDO activity was enhanced by an NO inhibitor, NO may be post-translationally consumed by inhibiting the IDO activity. IDO is well known to play an important role in immunosuppression during inflammatory disease. Therefore, the inhibition of IDO by NO may exacerbate inflammation in the peritoneal cavity.
Hepatic fibrosis is a major complication of various chronic liver diseases. Activated hepatic stellate cells (HSCs) play a critical role in the development of liver fibrosis and the axis of platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR), a member of receptor tyrosine kinases (RTKs), is closely associated with the activation of HSC. Insulin-like growth factor (IGF)-1 receptor (IGF-1R), which also belongs to RTKs, interacts with the PDGF/PDGFR axis, thereby cooperatively promoting hepatic fibrosis. We herein examined the effects of (-)-epigallocatechin gallate (EGCG), which inhibits the activation of several types of RTKs, on the development of rat liver fibrosis induced by carbon tetrachloride (CCl4). Drinking water with 0.1% EGCG significantly decreased the serum levels of both aspartate aminotransferase and alanine aminotransferase raised by CCl4, thus indicating an improvement of liver injury. In CCl4-injected rats, EGCG markedly attenuated hepatic fibrosis and decreased the amount of hydroxyproline in the experimental liver. The expression of PDGFRbeta and IGF-1R mRNAs in the liver was significantly lowered by the treatment with EGCG. EGCG also decreased the expression of PDGFRbeta and alpha-smooth muscle actin proteins, thus indicating the inhibition of HSC activation. These findings suggest that EGCG can exert, at least in part, an anti-fibrotic effect on the liver by targeting PDGFRbeta and IGF-1R. EGCG might therefore be useful in both the prevention and treatment of hepatic fibrosis.
Engelbreth-Holm-Swarm (EHS) gel has been reported to maintain the mature hepatocyte phenotypes in primary cultured hepatocytes. We investigated the effect of EHS gel on the differentiation of fetal liver cells, which contain stem/progenitor cells. The isolated fetal liver cells cultured on EHS gel formed a spherical shape and increased liver-specific gene expressions compared with cells cultured on collagen. The hepatic progenitor cells that were transplanted subcutaneously to BALB/c nude mice could survive and express hepatocyte marker alpha-fetoprotein when the cells were suspended with EHS gel. These findings demonstrate that EHS gel supports cytodifferentiation from immature progenitor cells to hepatocytes and maintain its differentiated phenotypes in vitro and in vivo.
The relationship between sphingosine kinase (SPHK), cellular ceramide concentration and chemosensitivity was investigated in human colon cancer cell lines. Among nine colon cancer cell lines, SPHK1 and SPHK2 activity and protein expression was highest in RKO cells and lowest in HCT116 cells. A viability assay revealed that HCT116 cells were sensitive to the effects of oxaliplatin (l-OHP), whereas RKO cells were resistant to those of l-OHP. Treatment with 5microg/ml l-OHP induced a marked time-dependent increase in various ceramides (C16, C24, C24:1) in HCT116 cells but not in RKO cells, as indicated by liquid chromatography/mass spectrometry. The increase in ceramide and caspase activation induced by l-OHP in the sensitive HCT116 cells was abolished by pretreatment with a neutral sphingomyelinase inhibitor, suggesting that the ceramide formation was due to the activation of neutral, rather than acid, sphingomyelinase. In contrast, in l-OHP-resistant RKO cells, treatment with an SPHK inhibitor or SPHK1 and SPHK2 silencing by RNA interference suppressed cell viability and increased caspase activity and cellular ceramide formation after l-OHP treatment. The elevated ceramide formation induced by SPHK inhibition and l-OHP was inhibited by fumonisin B1 but not myriocin, suggesting that ceramide formation was through the salvage pathway. Endogenous phosphorylated Akt levels were much higher in the resistant RKO cells than in the sensitive HCT116 cells. Either SPHK1 or SPHK2 silencing in RKO cells decreased phosphorylated Akt levels and increased p53 and p21 protein levels as well as poly(ADP-ribose) polymerase cleavage in response to l-OHP treatment. These findings indicate that SPHK isoforms and neutral sphingomyelinase contribute to the regulation of chemosensitivity by controlling ceramide formation and the downstream Akt pathway in human colon cancer cells.
c-Jun N-terminal Kinase (JNK) is a key regulator in tumor necrosis factor (TNF)-mediated liver injury. However, distinct roles for JNK1 and JNK2 in hepatocyte apoptosis are still unresolved. Although myeloid cell leukemia-1 (Mcl-1) has been reported as a substrate of JNK, the role of Mcl-1 and its functional regulation by JNK in TNF-induced hepatocyte apoptosis and liver injury remain to be elucidated.
Cytokine-dependent cell lines have been used to analyze the cytokine-induced cellular signaling and the mechanism of oncogenesis. In the current study, we analyzed MOTN-1 and PLT-2 cell lines established from different stages of a T-cell large granular lymphocyte leukemia patient (Daibata et al. 2004). MOTN-1 is IL-2-dependent derived from the chronic phase, whereas IL-2-independent PLT-2 is from the aggressive and terminal stage. They shared considerable chromosome abnormalities and the pattern of T-cell receptor rearrangement, presuming that the cytokine independence of PLT-2 was due to the additive genetic abnormality. Besides IL-2, IL-15 supported MOTN-1 cell growth, because these receptors share beta- and gamma-subunits. IL-2 activated ERK, AKT and STAT pathway of MOTN-1. STAT3 pathway of PLT-2 was also activated by IL-2, suggesting intact IL-2 induces signal transduction of PLT-2. However, ERK1/2 but not AKT, was continuously activated in PLT-2, consistent with the increased Ras-activity of PLT-2. Sequence analysis revealed KRAS G12A mutation but not NRAS and HRAS mutation of PLT-2 but not MOTN-1. Another signaling molecule affecting Ras-signaling pathway, SHP2, which has been frequently mutated in juvenile myelomonocytic leukemia (JMML), did not show mutation. Moreover, MEK inhibitor, PD98059, as well as farnesylation inhibitor inhibited PLT-2 cell growth. Using NIH3T3 and MOTN-1, ERK activation, increased cell proliferation and survival by KRAS G12A were shown, suggesting the important role of KRAS G12A in IL-2-independent growth of PLT-2. Taken together, KRAS G12A is important for IL-2-independent growth of PLT-2 cells and suggests the possibility of involvement of KRAS mutation with disease progression.
Various glycosyl ferulates were efficiently synthesized from 2,4,6-tetra-O-acetyl-?-D-glucopyranosyl bromide (TAGB) with amine by amine-promoted glycosylation without using heavy metal. The resulted acetylated glycosyl ferulates with acetoxyl groups at C-2, C-3 and C-4 were regioselectively deacetylated at C-4 and C-6 positions with Novozym 435. Antioxidant abilities of free ferulic acids and its synthetic glycosyl ferulates were evaluated by inhibitory effect on autoxidation of bulk methyl linoleate as well as their radical scavenging activity. The radical scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH·) decreased in the order ferulic acid>sinapinic acid ? glycosyl sinapinates ? glycosyl ferulates>p-coumaric acid>glycosyl p-coumarates. In bulk methyl linoleate, the antioxidant activity order against autoxidation was very consistent with the scavenging activity order. The results showed that glycosyl ferulates and sinapinates were effective as well as free carboxylic acid forms.
Secondary bacterial infection in humans is one of the pathological conditions requiring clinical attention. In this study, we examined the effect of lipopolysaccharide (LPS) on encephalomyocarditis virus (EMCV) infected mice. All mice inoculated with EMCV at 5 days before LPS challenge died within 24?h. LPS-induced TNF-? mRNA expression was significantly increased in the brain and heart at 5 days after EMCV infection. CD11b(+)/TLR4(+) cell population in the heart was remarkably elevated at 5 days after EMCV infection, and sorted CD11b(+) cells at 5 days after EMCV infection produced a large amount of TNF-? on LPS stimulation in vivo and in vitro. In conclusion, we found that the infiltration of CD11b(+) cells into infected organs is involved in the subsequent LPS-induced lethal shock in viral encephalomyocarditis. This new experimental model can help define the mechanism by which secondary bacterial infection causes a lethal shock in viral encephalomyocarditis.
The activity of IDO that catalyzes the degradation of tryptophan (Trp) into kynurenine (Kyn) increases after diseases caused by different infectious agents. Previously, we demonstrated that IDO has an important immunomodulatory function in immune-related diseases. However, the pathophysiological role of IDO following acute viral infection is not fully understood. To investigate the role of IDO in the l-Trp-Kyn pathway during acute viral myocarditis, mice were infected with encephalomyocarditis virus, which induces acute myocarditis. We used IDO-deficient (IDO(-/-)) mice and mice treated with 1-methyl-d,l-Trp (1-MT), an inhibitor of IDO, to study the importance of Trp-Kyn pathway metabolites. Postinfection with encephalomyocarditis virus infection, the serum levels of Kyn increased, whereas those of Trp decreased, and IDO activity increased in the spleen and heart. The survival rate of IDO(-/-) or 1-MT-treated mice was significantly greater than that of IDO(+/+) mice. Indeed, the viral load was suppressed in the IDO(-/-) or 1-MT-treated mice. Furthermore, the levels of type I IFNs in IDO(-/-) mice and IDO(-/-) bone marrow-transplanted IDO(+/+) mice were significantly higher than those in IDO(+/+) mice, and treatment of IDO(-/-) mice with Kyn metabolites eliminated the effects of IDO(-/-) on the improved survival rates. These results suggest that IDO has an important role in acute viral myocarditis. Specifically, IDO increases the accumulation of Kyn pathway metabolites, which suppress type I IFNs production and enhance viral replication. We concluded that inhibition of the Trp-Kyn pathway ameliorates acute viral myocarditis.
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