Time-course analysis of liver and serum galectin-3 in acute liver injury after alpha-galactosylceramide injection.

Galectin-3 is a beta-galactoside-binding lectin that plays important roles in diverse physiological functions, such as cell proliferation, apoptosis, and mRNA splicing. This protein is expressed on inflammatory cells and acts as a local inflammatory mediator. Recently, galectin-3 has been detected in several diseases, such as chronic liver, heart, and kidney diseases, diabetes, viral infection, autoimmune and neurodegenerative diseases, and tumors, and its role as a biomarker has attracted attention. Alpha-galactosylceramide is an artificially synthesized sphingolipid that can induce acute liver injury via the natural killer T pathway. However, the pathophysiological roles and kinetics of galectin-3 in acute liver injury are not fully understood. This study aimed to elucidate the expression and time course of galectin-3 in liver tissues during acute liver injury following alpha-galactosylceramide injection. Animals were histologically examined on days 1, 2, 4, and 7 after intraperitoneal injection of alpha-galactosylceramide, and the expressions of galectin-3 and ionized calcium-binding adaptor molecule 1 were analyzed. Notably, galectin-3 formed characteristic cluster foci, particularly on day 2 after injection. Cluster formation was not observed in chronic liver disease. Simultaneously, ionized calcium-binding adaptor molecule 1-positive cells were observed in the cluster foci. Serum galectin-3 levels increased on day 2 of treatment and correlated well with the number of galectin-3-positive cell clusters in the liver. Moreover, galectin-3 expression was an important mediator of the early phase of liver injury after alpha-galactosylceramide injection. These results suggest that serum galectin-3 may be a biomarker for the early diagnosis of acute liver injury and that clusters of galectin-3-positive cells may be a specific finding in acute liver injury.

Administration of spermidine attenuates concanavalin A-induced liver injury.

A previous study revealed that treatment with the anticoagulant heparin attenuated concanavalin A (ConA)-induced liver injury. The administration of spermidine (SPD) increased urokinase-type plasminogen activator (uPA) levels in the serum. uPA is clinically used for the treatment of some thrombotic diseases such as cerebral infarction. Therefore, SPD may attenuate ConA-induced liver injury that is exacerbated by blood coagulation. The present study investigated the effect of SPD on liver injury in mice with autoimmune hepatopathy induced by ConA. A model of liver injury was created by intravenous injection of ConA into mice. SPD was administered in free drinking water and was biochemically and pathologically examined over time. The administration of SPD to ConA-treated mice significantly reduced liver injury. However, SPD treatment upregulated the mRNA expression of TNF-α and IFN-ϒ in the livers of ConA-treated mice. In contrast, the mRNA expression of tissue factor in the livers of SPD-treated mice was decreased after ConA injection. The frequency of lymphocytes and lymphocyte activation were not affected by SPD administration in ConA-treated mice. SPD treatment increased uPA levels in the serum and decreased the level of D-dimer in ConA-treated mice. Moreover, SPD decreased fibrin in the livers of ConA-treated mice. These results indicated that SPD treatment increased anticoagulant ability by increasing of uPA and attenuated ConA-induced liver injury.

d-mannose administration improves autoimmune hepatitis by upregulating regulatory T cells.

A recent study revealed that d-mannose suppressed immunopathology in mouse models of autoimmune diabetes and airway inflammation and increased the proportion of regulatory T cells (Tregs) in mice. We investigated the effect of d-mannose on liver injury in murine autoimmune hepatitis (AIH) models induced by concanavalin A (ConA) and α-galactosylceramide (GalCer). Mouse models of AIH were created by intraperitoneal injection of GalCer or intravenous injection of ConA. Drinking water was supplemented with d-mannose and biochemically and pathologically examined over time. The administration of d-mannose to AIH model mice significantly reduced liver injury and reduced inflammatory cytokine expression. In addition, Tregs among splenocytes and intrahepatic lymphocytes were significantly increased by the administration of d-mannose. These results indicate that treatment with d-mannose reduced the inflammatory response in the liver and suppressed liver damage by increasing Tregs.

Systemic and topical administration of spermidine accelerates skin wound healing.

BACKGROUND: The skin wound healing process is regulated by various cytokines, chemokines, and growth factors. Recent reports have demonstrated that spermine/spermidine (SPD) promote wound healing through urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) signaling in vitro. Here, we investigated whether the systemic and topical administration of SPD would accelerate the skin wound-repair process in vivo. METHODS: A skin wound repair model was established using C57BL/6 J mice. SPD was mixed with white petrolatum for topical administration. For systemic administration, SPD mixed with drinking water was orally administered. Changes in wound size over time were calculated using digital photography. RESULTS: Systemic and topical SPD treatment significantly accelerated skin wound healing. The administration of SPD promoted the uPA/uPAR pathway in wound sites. Moreover, topical treatment with SPD enhanced the expression of IL-6 and TNF-α in wound sites. Scratch and cell proliferation assays revealed that SPD administration accelerated scratch wound closure and cell proliferation in vitro. CONCLUSION: These results indicate that treatment with SPD promotes skin wound healing through activation of the uPA/uPAR pathway and induction of the inflammatory response in wound sites. The administration of SPD might contribute to new effective treatments to accelerate skin wound healing. Video Abstract.

Novel hepatitis B virus infection mouse model using herpes simplex virus type 1 thymidine kinase transgenic mice.

BACKGROUND AND AIM: The chronicity of hepatitis B virus (HBV) infection is the result of impaired HBV-specific immune responses that cannot eliminate or cure the infected hepatocytes efficiently. Previous studies have used immunodeficient mice such as herpes simplex virus type 1 thymidine kinase NOD/Scid/IL2Rrnull (HSV-TK-NOG) mice. However, it is difficult to analyze the immune response in the previous models. In the present study, we established a novel HBV infection model using herpes simplex virus type 1 thymidine kinase (HSV-TK) mice in which the host immune system was not impaired. METHODS: Herpes simplex virus type 1 thymidine kinase mice were injected intraperitoneally with ganciclovir (GCV). Seven days after GCV injection, GCV-treated mice were transplanted with 1 × 106 hepatocytes from HBV-transgenic (HBV-Tg) mice. RESULTS: Serum alanine aminotransferase levels in HSV-TK mice increased 1 and 2 weeks after GCV injection. The number and viability of hepatocytes from the whole liver of HBV-Tg mice significantly increased using digestion medium containing liberase. Hepatitis B surface antigen (HBsAg)-positive areas in the liver tissue were observed for at least 20 weeks after HBsAg-positive hepatocyte transplantation. In addition, we measured HBsAg in the serum after transplantation. HBsAg levels in HBV-Tg hepatocyte-replaced mice increased 4 weeks after transplantation. Furthermore, we examined the immune response in HSV-TK mice. The increase in hepatitis B surface antibody levels in replaced mice was maintained for 20 weeks. Also, interferon-γ-producing cells were increased in non-replaced mice. CONCLUSIONS: A novel HBV infection mouse model will help to understand the mechanisms of HBV tolerance similar to human chronic HBV-infected patients and can be used to develop a new strategy to treat chronic HBV infection.

Short-term high-fat diet intake leads to exacerbation of concanavalin A-induced liver injury through the induction of procoagulation state.

Obesity and high-fat diet (HFD) are known to cause proinflammatory and procoagulation states and suggested to become a risk of developing thromboembolic diseases. Non-alcoholic fatty liver disease (NAFLD) is usually associated with obesity and HFD, and a part of NAFLD is known to progress to nonalcoholic steatohepatitis (NASH), the pathogenesis of which has not been fully elucidated. In the current study, we examined the influence of short-term HFD on hepatic expression of the molecules related to inflammation, coagulation, metabolism, and cellular stresses from the perspective that HFD itself can be a risk for the development to NASH. In the analysis in short-term (4 days to 14 days) HFD-fed mice, we found out that HFD increased hepatic expression of IFN-γ, TNF-α, IL-10, monocyte chemotactic protein-1 (MCP-1), tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1) mRNAs, and fibrin/fibrinogen deposition in the liver tissues. And it was suggested that metabolic alterations and endoplasmic reticulum (ER) stresses induced by the HFD intake were associated with this proinflammatory and procoagulation states. When we administered concanavalin A (Con A) to these HFD-fed mice, the extent of liver injury was dramatically exacerbated in HFD-fed mice. Heparin treatment to Con A-administered, HFD-fed mice (for 4 days) profoundly ameliorated the extent of liver injury. These suggest that even short-term of HFD intake induces proinflammatory and procoagulation states in the liver and thereby increases the susceptibility of the liver to circulating inflammatory stimuli. We think that it may explain a part of NASH pathogenesis.

Activation of STING signaling accelerates skin wound healing.

BACKGROUND: The process of repair after skin injury is precisely regulated by a variety of mediators such as cytokines and chemokines. Recent reports demonstrated that cytoplasmic DNA-sensor cyclic GMP-AMP synthase (cGAS) activates the stimulator of interferon genes (STING) via production of cyclic GMP-AMP (cGAMP) and subsequently induces inflammatory cytokines, including type I interferon (IFN). OBJECTIVE: We examined whether activation of the STING pathway by cGAMP affects the process of skin wound repair. METHODS: The skin wound repair model was established using wild-type (WT) mice. Two full-thickness skin biopsies were taken from the right and left subscapular regions. One site was treated with ointment containing cGAMP, and the other was treated with a control ointment. Changes in wound size over time were calculated using photography. RESULTS: Treatment with cGAMP significantly accelerated skin wound healing up to day 6. Biochemical analyses showed that topical treatment with cGAMP on wound sites promoted STING signaling pathway and enhanced the expression of IFN-ß, CXCL10 and CCL2 in the wound sites treated with cGAMP markedly compared with the control. The scratch assay also revealed that cGAMP treatment accelerated wound closure in mouse embryonic fibroblasts. The acceleration of skin wound repair by cGAMP in WT mouse was impaired by administration of anti-IFNR antibody and anti-CXCR3 antibody respectively. CONCLUSION: These results revealed that topical treatment with cGAMP accelerates skin wound healing by inducing type I IFN and CXCL10/CXCR3. Topical administration of cGAMP might contribute to new effective treatments for accelerating skin wound healing.

Induction of humoral and cellular immune response to HBV vaccine can be up-regulated by STING ligand.

A persistent hepatitis B virus (HBV) infection is characterized by a lack of or a weak immune response to HBV. Efficient induction of the HBV-specific immune response leads to the clearance of HBV. Stimulator of interferon (IFN) genes (STING) is a cytoplasmic sensor of intracellular DNA from microbes and host cells. In the present study, we examined the efficacy of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) that is a ligand of the STING pathway as an HBV vaccine adjuvant. Wild-type (WT) mice and HBV-transgenic (HBV-Tg) mice were immunized with hepatitis B surface antigen (HBsAg) and cGAMP. The vaccination with HBsAg and cGAMP significantly enhanced the humoral and cellular immune response to HBsAg in WT and HBV-Tg mice. Cytokine production related to Th1 and Th2 responses and the activation of antigen-presenting cells in lymphoid tissues were induced by cGAMP. Vaccination using cGAMP may overcome tolerance in patients with chronic HBV infection.

Activation of NLRP3 signalling accelerates skin wound healing.

The process of skin wound healing involves the following three steps: inflammation, tissue formation and tissue remodelling. These optimal steps are required for the development of normal wound healing. Recent reports demonstrated that inflammasomes are involved in the innate immune response. In the present study, we examined whether the activation of inflammasomes affects the process of skin wound repair. The skin wound repair model was established using wild-type (WT), NACHT, LRR and PYD domains-containing protein 3 (NALP3) knockout (KO) and ASC-KO mice. The wounds were observed every other day, and changes in wound size over time were calculated using photography. Wound repair in NALP3-KO and ASC-KO mice was significantly impaired compared with WT mice. Isoliquiritigenin, an inhibitor of NALP3, decreased the rate of wound repair in WT mice. mRNA expression of pro-inflammatory cytokines in the wound sites of NALP3-KO mice was markedly decreased compared with WT mice. Treatment with adenosine triphosphate (ATP), a ligand of NALP3, upregulated the mRNA expression of pro-inflammatory cytokines at the wound site and accelerated wound healing in the WT mice. Scratch assay revealed that ATP accelerated wound closure in mouse embryonic fibroblasts from WT mice but not from NALP3-KO mice. In conclusion, the present study demonstrated that NALP3 pathway activation is involved in wound repair, and the topical use of ATP may be useful as an effective treatment for accelerating wound healing.

The inhibition of NLRP3 signaling attenuates liver injury in an α-galactosylceramide-induced hepatitis model.

Inflammasomes are involved in innate immune responses. Several NOD-Like receptors (NLRs) participate in the formation of inflammasomes. NACHT, LRR and PYD domains-containing protein 3 (NALP3) belongs to the NLR family and recognizes adenosine triphosphate (ATP), crystals, and Reactive Oxygen Species (ROS). This study examined the effect of inflammasomes on alpha-galactosylceramide (GalCer)-induced liver injury using NALP3-knockout (KO) mice. GalCer administration induced inflammasome activation and IL-1ß-maturation. In NALP3-KO mice treated with GalCer, serum ALT levels were significantly reduced compared with those in GalCer-treated WT mice. Histological examination revealed decreased necrosis in NALP3-KO mice compared with WT mice, consistent with ALT levels. Expression of proinflammatory cytokines (such as IL-6, and TNF-α) and chemokines was also significantly suppressed in NALP3-KO mice. Moreover, flow cytometry analysis revealed fewer infiltrating immune cells in the livers of GalCer-treated NALP3-KO mice. Inportantly, the frequency of MDSCs (CD11b+Gr-1int cells), which suppress the immune response, was significantly increased in GalCer-treated NALP3-KO mice. In conclusion, NALP3 inhibition attenuated liver injury in GalCer-induced hepatitis. The inhibition of NALP3 signaling coused be a therapeutic target in immune-mediated liver injury.

Deficiency of NALP3 Signaling Impairs Liver Regeneration After Partial Hepatectomy.

Inflammatory response is required to proceed the optimal liver regeneration after liver injury. Recent reports demonstrated that inflammasomes are involved in the innate immune response. Several NOD-Like receptors (NLRs) participated in the formation of the inflammasomes. NACHT, LRR, and PYD domain-containing protein 3 (NALP3) belongs to the NLR families and recognizes adenosine triphosphate (ATP), crystals, and reactive oxygen species. The present study examined the effect of inflammasomes on the process of liver regeneration using NALP3 knockout (KO) mice. The activation of inflammasomes in the liver was induced after 70% partial hepatectomy (PHx). The liver-to-body weight ratio was significantly decreased in NALP3-KO mice compared to that in WT mice after PHx. The number of Ki67-positive cells and the expression of Cyclin D1 and E1 after PHx were reduced in NALP3-KO mice compared to WT mice. The expression of pro-inflammatory cytokines (IL-1ß, TNF-α, and IL-6) were decreased in the remnant liver of NALP3-KO mice compared to WT mice. Flow cytometric analysis revealed that the expression of chemokines was decreased and the accumulation of CD11b-positive cells was suppressed in NALP3-KO mice after PHx. The treatment with ATP, which is a ligand to NALP3, increased the liver-to-body weight ratio in WT mice. These results indicate that NALP3 signaling is required for the induction of inflammatory response and the development of liver regeneration after PHx.

The Inhibition of Indoleamine 2,3-Dioxygenase Accelerates Early Liver Regeneration in Mice After Partial Hepatectomy.

BACKGROUND AND AIM: The inflammatory response accelerates early liver regeneration after liver injury and resection. Recent studies have demonstrated that indoleamine 2,3-dioxygenase-1 (IDO1) suppresses the activation of inflammatory cells and induces immune tolerance. In this study, we examined the role of IDO1 in liver regeneration after partial hepatectomy (PHx). METHODS: WT or IDO1-knockout (IDO1-KO) mice received 70% PHx. The liver-body weight ratio after PHx was measured and hepatocyte growth was assessed by immunostaining. The expression of cell cycle genes and pro-inflammatory cytokines in the liver was analyzed by quantitative RT-PCR. In addition, 1-methyl-DL-tryptophan (1-MT), which is an IDO1 inhibitory agent, was given to WT mice and the liver-body weight ratio was measured after PHx. RESULTS: The liver-body weight ratio was significantly increased in IDO1-KO mice compared with that in WT mice after PHx. More Ki-67-positive cells were present in IDO1-KO mice than in WT mice after PHx. The expression of cell cycle genes (cyclin D1, cyclin E) and pro-inflammatory cytokines (IL-1ß, TNF-α and IL-6) was up-regulated in the remnant liver of IDO1-KO mice compared with WT mice. Moreover, treatment with 1-MT promoted liver regeneration. CONCLUSION: IDO1 deficiency promoted early liver regeneration after PHx, indicating that IDO1 suppresses the production of inflammatory cytokines and subsequently inhibits hepatocyte proliferation during liver regeneration.

Role of invariant NKT cells in lipopolysaccharide-induced lethal shock during encephalomyocarditis virus infection.

Viral infections can give rise to secondary bacterial infections. In the present study, we examined the role of invariant natural killer T (iNKT) cells in lipopolysaccharide (LPS)-induced lethal shock during encephalomyocarditis virus (EMCV) infection. Wild-type (WT) mice and Jα18 gene knockout (Jα18 KO) mice were inoculated with EMCV, 5days prior to challenging with LPS. The survival rate of Jα18 KO mice subjected to EMCV and LPS was significantly higher than that of WT mice. TNF-α and nitric oxide (NO) production were increased in WT mice, than that in Jα18 KO mice, after the administration of EMCV and LPS. EMCV infection increased the number of iNKT cells and IFN-γ production by iNKT cells in WT mice. Moreover, EMCV infection enhanced the expression of Toll-like receptor 4 (TLR4) in the lung and spleen. IFN-γ also increased the expression of TLR4 in splenocytes. These findings indicated that EMCV infection activated iNKT cells, and IFN-γ secreted from the iNKT cells up-regulated the expression of TLR4 in various tissues. As a result, EMCV-infected mice were susceptible to LPS and easily developed the lethal shock. In conclusion, iNKT cells were involved in the development of LPS-induced lethal shock during EMCV infection.

The Deficiency of Indoleamine 2,3-Dioxygenase Aggravates the CCl4-Induced Liver Fibrosis in Mice.

In the present study, we examined the role of indoleamine 2,3-dioxygenase (IDO) in the development of CCl4-induced hepatic fibrosis. The liver fibrosis induced by repetitive administration with CCl4 was aggravated in IDO-KO mice compared to WT mice. In IDO-KO mice treated with CCl4, the number of several inflammatory cells and the expression of pro-inflammatory cytokines increased in the liver. In the results, activated hepatic stellate cells (HSCs) and fibrogenic factors on HSCs increased after repetitive CCl4 administration in IDO-KO mice compared to WT mice. Moreover, the treatment with l-tryptophan aggravated the CCl4-induced hepatic fibrosis in WT mice. Our findings demonstrated that the IDO deficiency enhanced the inflammation in the liver and aggravated liver fibrosis in repetitive CCl4-treated mice.

[Clinical significance of hyaline casts in the new CKD risk classification (KDIGO 2009)].

Chronic kidney disease (CKD) significantly contributes to the increased number of dialysis patients with end stage renal disease. A new CKD risk classification (KDIGO 2009) established in 2011, which is defined by albuminuria and estimated glomerular filtration rate (eGFR) values, demonstrates the relative risks of CKD in great detail. In this study, we evaluated the clinical significance of urinary casts by categorizing a risk Group 1 to 5 according to the KDIGO 2009 classification. In the high risk CKD group (risk group 3 and over), we found a significantly higher number of patients who had > 100 hyaline casts/whole field (WF) in their urine than those that had < 100 hyaline casts/WF. Further, we determined the diagnostic accuracy for the high risk CKD group when the cutoff value for the number of hyaline casts was set at > or = 100 hyaline casts/WF (sensitivity: 44.7%, specificity: 96.5%). The eGFR value was significantly lower in the group with > or = 100 hyaline casts/WF, particularly in hypertensive patients, than that in the group with < 100 hyaline casts/ WF. Of interest is that the eGFR value was significantly lower in patients with 100-999 hyaline casts/WF and > or = 1,000 hyaline casts/WF than that in patients with < 100 hyaline casts/WF in A1 stage. Thus, our present study suggests that the presence of > or = 100 hyaline casts/WF indicates decreased eGFR, and the urinary casts counting may be important and useful for the screening and early detection of high-risk CKD.