IL-17-Producing Vγ4+ γδ T Cells Require Sphingosine 1-Phosphate Receptor 1 for Their Egress from the Lymph Nodes under Homeostatic and Inflammatory Conditions.

Conventional αß T cells require sphingosine 1-phosphate (S1P) receptor 1 (S1P1) for circulation through the lymph nodes (LN); however, it is unclear whether γδ T cells use similar mechanisms. In this study, we found that treatment with fingolimod (FTY720, 1 mg/kg, orally) markedly reduced not only conventional CD4 T cells but also circulating γδ T cells (Vγ4(+) and Vγ4(-) subsets) in the blood of mice. In contrast, IL-17(+)Vγ4(+), IL-17(+)Vγ4(-), and IL-17(-)Vγ4(-) subsets were significantly accumulated in the LN after 6 h of FTY720 treatment. By skin application of a synthetic TLR7/8 agonist, Vγ4(+) γδ T cells (IL-17(+) and IL-17(-) subsets) were accumulated and expanded in the draining LN (DLN), whereas the IL-17(+) subset predominantly migrated to the inflamed skin. FTY720 induced a marked sequestration of IL-17-producing Vγ4(+) γδ T cells in the DLN and inhibited their infiltration into the inflamed skin. Similarly, FTY720 inhibited infiltration of Vγ4(+) γδ T cells into the CNS by their sequestration into the DLN in experimental autoimmune encephalomyelitis. Vγ4(+) γδ T cells expressed a significant level of S1P1 and showed a migratory response toward S1P. FTY720 treatment induced almost complete downregulation of S1P1 expression and S1P responsiveness in Vγ4(+) γδ T cells. Our findings strongly suggest that IL-17-producing Vγ4(+) γδ T cells require S1P1 for their egress from the LN under homeostatic and inflammatory conditions. Consequently, inhibition of S1P1-dependent egress of pathogenic IL-17-producing Vγ4(+) γδ T cells from the DLN may partly contribute the clinical therapeutic effects of FTY720 in relapsing multiple sclerosis.

Pathological characterization and morphometric analysis of hepatic lesions in SHRSP5/Dmcr, an experimental non-alcoholic steatohepatitis model, induced by high-fat and high-cholesterol diet.

SHRSP5/Dmcr is a newly established substrain of stroke-prone spontaneously hypertensive rat (SHRSP). Recently, high-fat and high-cholesterol (HFC) diet-fed SHRSP5/Dmcr has been reported as a novel rat model of developing hepatic lesions similar to human non-alcoholic steatohepatitis (NASH). The aim of this study was to investigate the detailed pathological conditions induced by HFC diet in SHRSP5/Dmcr rats using molecular biological methods and morphometric analysis. SHRSP5/Dmcr rats at 6 weeks of age were fed on either HFC diet or stroke-prone (SP) diet for 2, 4, 6, 8 and 16 weeks and histopathological changes in the liver, blood chemistry and mRNA expression levels in the liver were investigated. As evidenced by the histopathological examination of the liver of the SHRSP5/Dmcr rats, hepatic steatosis and lobular inflammation were present, with gradual increasing severity from 2 weeks after the introduction of the HFC diet. Partial hepatic fibrosis was detected at 6 weeks and spread over the entire region of the liver with more severe bridging formation by 16 weeks. The degrees of NASH-like hepatic lesions such as steatosis (the size distribution of lipid droplets), inflammation and fibrosis were quantified by morphometric analysis. Eosinophilic inclusion bodies encountered in the hepatocytes had immunoreactivity with Cox-4 and double-membrane walls, identified as mega-mitochondria. Serum ALT and bilirubins, and the mRNA expression levels related to fibrosis were closely correlated with hepatic histopathological changes. The clear feeding time-dependent progression of NASH-like hepatic lesion in HFC diet-fed SHRSP5/Dmcr rats reinforced the conclusion that this strain might be a useful model of NASH and of inflammatory fibrotic liver disease.

S1P lyase in thymic perivascular spaces promotes egress of mature thymocytes via up-regulation of S1P receptor 1.

Sphingosine 1-phosphate (S1P) and S1P receptor 1 (S1P1) play an important role in the egress of mature CD4 or CD8 single-positive (SP) thymocytes from the thymus. Fingolimod hydrochloride (FTY720), an S1P1 functional antagonist, induced significant accumulation of CD62L(high)CD69(low) mature SP thymocytes in the thymic medulla. Immunohistochemical staining using anti-S1P1 antibody revealed that S1P1 is predominantly expressed on thymocytes in the thymic medulla and is strongly down-regulated even at 3h after FTY720 administration. 2-Acetyl-4-tetrahydroxybutylimidazole (THI), an S1P lyase inhibitor, also induced accumulation of mature SP thymocytes in the thymic medulla with an enlargement of the perivascular spaces (PVS). At 6h after THI administration, S1P1-expressing thymocytes reduced partially as if to form clusters and hardly existed in the proximity of CD31-expressing blood vessels in the thymic medulla, suggesting S1P lyase expression in the cells constructing thymic medullary PVS. To determine the cells expressing S1P lyase in the thymus, we newly established a mAb (YK19-2) specific for mouse S1P lyase. Immunohistochemical staining with YK19-2 revealed that S1P lyase is predominantly expressed in non-lymphoid thymic stromal cells in the thymic medulla. In the thymic medullary PVS, S1P lyase was expressed in ER-TR7-positive cells (reticular fibroblasts and pericytes) and CD31-positive vascular endothelial cells. Our findings suggest that S1P lyase expressed in the thymic medullary PVS keeps the tissue S1P concentration low around the vessels and promotes thymic egress via up-regulation of S1P1.

Discovery and preclinical profile of teneligliptin (3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-ylcarbonyl]thiazolidine): a highly potent, selective, long-lasting and orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes.

Dipeptidyl peptidase IV (DPP-4) inhibition is suitable mechanism for once daily oral dosing regimen because of its low risk of hypoglycemia. We explored linked bicyclic heteroarylpiperazines substituted at the γ-position of the proline structure in the course of the investigation of l-prolylthiazolidines. The efforts led to the discovery of a highly potent, selective, long-lasting and orally active DPP-4 inhibitor, 3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-ylcarbonyl]thiazolidine (8 g), which has a unique structure characterized by five consecutive rings. An X-ray co-crystal structure of 8 g in DPP-4 demonstrated that the key interaction between the phenyl ring on the pyrazole and the S(2) extensive subsite of DPP-4 not only boosted potency, but also increased selectivity. Compound 8 g, at 0.03 mg/kg or higher doses, significantly inhibited the increase of plasma glucose levels after an oral glucose load in Zucker fatty rats. Compound 8 g (teneligliptin) has been approved for the treatment of type 2 diabetes in Japan.

Amiselimod (MT-1303), a novel sphingosine 1-phosphate receptor-1 functional antagonist, inhibits progress of chronic colitis induced by transfer of CD4+CD45RBhigh T cells.

Amiselimod (MT-1303) is a novel sphingosine 1-phosphate receptor-1 (S1P1 receptor) modulator with a more favorable cardiac safety profile than other S1P1 receptor modulators. MT-1303 phosphate (MT-1303-P), an active metabolite of MT-1303, exhibits S1P1 receptor agonism at a lower EC50 value than other S1P1 receptor modulators currently being developed. We aimed to evaluate the efficacy of MT-1303 and its mode of action in chronic colitis using an inflammatory bowel disease (IBD) model. Oral administration of MT-1303 (0.3 mg/kg) once daily for 3 days to mice almost completely abolished S1P1 receptor expression on CD4+ T cells from mesenteric lymph nodes, which corresponded to a marked decrease in CD4+ T cell count in peripheral blood, indicating that MT-1303-P acts as a functional antagonist of the S1P1 receptor. The potential benefit of MT-1303 for IBD was assessed using immunodeficient SCID mice with chronic colitis induced by adoptive transfer of CD4+CD45RBhigh T cells from BALB/c mice. An oral dose of 0.1 and 0.3 mg/kg MT-1303 administered daily one week after the cell transfer inhibited the development of chronic colitis with an efficacy comparable to that of an anti-mTNF-α mAb (250 µg/mouse). In addition, MT-1303 administration significantly reduced the number of infiltrating Th1 and Th17 cells into the lamina propria of the colon in colitis mice. Our results suggest that MT-1303 acts as a functional antagonist of the S1P1 receptor on lymphocytes, regulates lymphocyte trafficking, and inhibits infiltration of colitogenic Th1 and Th17 cells into the colon to inhibit the development of chronic colitis.

Amiselimod (MT-1303), a Novel Sphingosine 1-Phosphate Receptor-1 Modulator, Potently Inhibits the Progression of Lupus Nephritis in Two Murine SLE Models.

Amiselimod (MT-1303) is a novel and selective sphingosine 1-phosphate receptor-1 (S1P1) modulator with a more favorable cardiac safety profile than other S1P1 receptor modulators. In this study, we evaluated the effects of MT-1303 on the progression of lupus nephritis in two well-known murine systemic lupus erythematosus (SLE) models, MRL/lpr and NZBWF1 mice, compared with those of FK506. Daily oral doses of 0.1 and 0.3 mg/kg MT-1303 not only inhibited the development of lupus nephritis when administered before onset in MRL/lpr and NZBWF1 mice but also improved symptoms of lupus nephritis when administered after onset in MRL/lpr mice. Its efficacy in these models was more potent or comparable to that of FK506 (1 and 3 mg/kg). In histological analysis, treatment with MT-1303 inhibited infiltration of T cells into the kidneys, mesangial expansion, and glomerular sclerosis. MT-1303 treatment resulted in a marked reduction in T cells and B cells in the peripheral blood and significantly inhibited increases in the number of plasma cells in the spleen and T cells in the kidneys. In addition, administration of MT-1303 suppressed elevations in serum anti-dsDNA antibody levels in MRL/lpr mice, but not in NZBWF1 mice. Our findings show that MT-1303 exhibits marked therapeutic effects on lupus nephritis in two SLE models, likely by reducing the infiltration of autoreactive T cells into the kidneys. These results suggest that MT-1303 has the potential to be used as a therapeutic agent for patients suffering from SLE, including lupus nephritis.

Inhibitory effects of fasudil on renal interstitial fibrosis induced by unilateral ureteral obstruction.

Renal fibrosis is the major cause of chronic kidney disease, and the Rho/Rho-associated coiled-coil kinase (ROCK) signaling cascade is involved in the renal fibrotic processes. Several studies have reported that ROCK inhibitors attenuate renal fibrosis. However, the mechanism of this process remains to be fully elucidated. The present study assessed the inhibitory effect of fasudil, a ROCK inhibitor using immunohistochemistry, reverse transcription-quantitative polymerase chain reaction and western blot analyses, in vivo and in vitro, to elucidate the mechanisms underlying renal interstitial fibrosis. In mice induced with unilateral ureteral obstruction (UUO), collagen accumulation, the expression of fibrosis­associated genes and the content of hydroxyproline in the kidney increased 3, 7, and 14 days following UUO. Fasudil attenuated the histological changes, and the production of collagen and extracellular matrix in the UUO kidney. The expression of α­smooth muscle actin (α­SMA) and the transforming growth factor­ß (TGFß)­Smad signaling pathway, and macrophage infiltration were suppressed by fasudil in the kidneys of the UUO mice. The present study also evaluated the role of intrinsic renal cells and infiltrated macrophages using NRK­52E, NRK­49F and RAW264.7 cells. The mRNA and protein expression levels of collagen I and α­SMA increased in the NRK­52E and NRK­49F cells stimulated by TGF­ß1. Hydroxyfasudil, a bioactive metabolite of fasudil, attenuated the increase in the mRNA and protein expression levles of α­SMA in the two cell types. However, the reduction in the mRNA expression of collagen I was observed in the NRK­49F cells only. Hydroxyfasudil decreased the mRNA expression of monocyte chemoattractant protein­1 (MCP­1) induced by TGF­ß1 in the NRK­52E cells, but not in the NRK­49F cells. In the RAW264.7 cells, the mRNA expression levels of MCP­1, interleukin (IL)­1ß, IL­6 and tumor necrosis factor α were increased significantly following lipopolysaccharide stimulation, and were not suppressed by hydroxyfasudil. These data suggested that the inhibition of ROCK activity by fasudil suppressed the transformation of renal intrinsic cells into the myofibroblast cells, and attenuated the infiltration of macrophages, without inhibiting the expression or the activation of cytokine/chemokines, in the progression of renal interstitial fibrosis.

Quantitative analysis of markers of podocyte injury in the rat puromycin aminonucleoside nephropathy model.

Podocytes are an essential component of the renal glomerular filtration barrier, their injury playing an early and important role in progressive renal dysfunction. This makes quantification of podocyte marker immunoreactivity important for early detection of glomerular histopathological changes. Here we have specifically applied a state-of-the-art automated computational method of glomerulus recognition, which we have recently developed, to study quantitatively podocyte markers in a model with selective podocyte injury, namely the rat puromycin aminonucleoside (PAN) nephropathy model. We also retrospectively investigated mRNA expression levels of these markers in glomeruli which were isolated from the same formalin-fixed, paraffin-embedded kidney samples by laser microdissection. Among the examined podocyte markers, the immunopositive area and mRNA expression level of both podoplanin and synaptopodin were decreased in PAN glomeruli. The immunopositive area of podocin showed a slight decrease in PAN glomeruli, while its mRNA level showed no change. We have also identified a novel podocyte injury marker ß-enolase, which was increased exclusively by podocytes in PAN glomeruli, similarly to another widely used marker, desmin. Thus, we have shown the specific application of a state-of-the-art computational method and retrospective mRNA expression analysis to quantitatively study the changes of various podocyte markers. The proposed methods will open new avenues for quantitative elucidation of renal glomerular histopathology.

One-step RT-PCR without initial RNA isolation sStep for laser-microdissected tissue sample.

One-step RT-PCR procedure without initial RNA extraction step is tested for laser microdissected tissue sample. Unfixed cryosections of liver and kidney tissue of male SD rats were cut using laser microdissection system and directly used as templates for RT-PCR study. To check the sensitivity, 5, 25, 125, and 625 hepatocytes were cut and put in PCR-tube. After DNase treatment and cDNA synthesis with pd(N)6 random primer, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs were amplified by 60 thermal cycles. GAPDH-specific bands were observed at as few as 25 hepatocytes. Specificity of this procedure was tested for hepatocytes, renal tubular epithelium and glomerular tissue using albumin PCR primers. Approximately 250 cells were cut and albumin cDNA was amplified as described above. Albumin specific band was observed only in hepatocytes sample. To apply this approach to quantitative PCR, various numbers of hepatocytes were cut and put in 0.2 mL PCR tube. After reverse transcription and 10 cycles of GAPDH cDNA amplification by regular thermal-cycler, PCR solution was transferred to 96-well plate designed for real-time PCR system, and further 40 cycles were performed. As a result, GAPDH cDNAs were successfully amplified with a good correlation between the number of template hepatocytes and the intensity of PCR signal. From these results, we concluded this approach would be very useful for the expression analysis of microdissected pathology samples.

The novel dipeptidyl peptidase-4 inhibitor teneligliptin prevents high-fat diet-induced obesity accompanied with increased energy expenditure in mice.

Dipeptidyl peptidase-4 (DPP-4)-deficient mice exhibit prevention of obesity with increased energy expenditure, whereas currently available DPP-4 inhibitors do not induce similar changes. We investigated the impact of the novel DPP-4 inhibitor teneligliptin on body weight, energy expenditure, and obesity-related manifestations in diet-induced obese mice. Six-weeks-old C57BL/6N mice were fed a high-fat diet (60%kcal fat) ad libitum and administered teneligliptin (30 or 60mg/kg) via drinking water for 10 weeks. Mice fed a high-fat diet showed accelerated body weight gain. In contrast, compared with the vehicle group, the administration of teneligliptin reduced body weight to 88% and 71% at dose of 30mg/kg/day and 60mg/kg/day, respectively. Although there was no change in locomotor activity, indirect calorimetry studies showed that teneligliptin (60mg/kg) increased oxygen consumption by 22%. Adipocyte hypertrophy and hepatic steatosis induced by a high-fat diet were suppressed by teneligliptin. The mean adipocyte size in the 60-mg/kg treatment group was 44% and hepatic triglyceride levels were 34% of the levels in the vehicle group. Furthermore, treatment with teneligliptin (60mg/kg) reduced plasma levels of insulin to 40% and increased the glucose infusion rate to 39%, as measured in the euglycemic clamp study, indicating its beneficial effect on insulin resistance. We showed for the first time that the DPP-4 inhibitor prevents obesity and obesity-related manifestations with increased energy expenditure. Our findings suggest the potential utility of teneligliptin for the treatment of a broad spectrum of metabolic disorders related to obesity beyond glycemic control.

Automated image analysis of a glomerular injury marker desmin in spontaneously diabetic Torii rats treated with losartan.

Diabetic nephropathy is a major complication in diabetes and a leading cause of end-stage renal failure. Glomerular podocytes are functionally and structurally injured early in diabetic nephropathy. A non-obese type 2 diabetes model, the spontaneously diabetic Torii (SDT) rat, is of increasing preclinical interest because of its pathophysiological similarities to human type 2 diabetic complications including diabetic nephropathy. However, podocyte injury in SDT rat glomeruli and the effect of angiotensin II receptor blocker treatment in the early stage have not been reported in detail. Therefore, we have evaluated early stages of glomerular podocyte damage and the beneficial effect of early treatment with losartan in SDT rats using desmin as a sensitive podocyte injury marker. Moreover, we have developed an automated, computational glomerulus recognition method and illustrated its specific application for quantitatively studying glomerular desmin immunoreactivity. This state-of-the-art method enabled automatic recognition and quantification of glomerular desmin-positive areas, eliminating the need to laboriously trace glomerulus borders by hand. The image analysis method not only enabled assessment of a large number of glomeruli, but also clearly demonstrated that glomerular injury was more severe in the juxtamedullary region than in the superficial cortex region. This applied not only in SDT rat diabetic nephropathy but also in puromycin aminonucleoside-induced nephropathy, which was also studied. The proposed glomerulus image analysis method combined with desmin immunohistochemistry should facilitate evaluations in preclinical drug efficacy studies as well as elucidation of the pathophysiology of diabetic nephropathy.

Automated recognition and quantification of pancreatic islets in Zucker diabetic fatty rats treated with exendin-4.

Type 2 diabetes is characterized by impaired insulin secretion from pancreatic ß-cells. Quantification of the islet area in addition to the insulin-positive area is important for detailed understanding of pancreatic islet histopathology. Here we show computerized automatic recognition of the islets of Langerhans as a novel high-throughput method to quantify islet histopathology. We utilized state-of-the-art tissue pattern recognition software to enable automatic recognition of islets, eliminating the need to laboriously trace islet borders by hand. After training by a histologist, the software successfully recognized even irregularly shaped islets with depleted insulin immunostaining, which were quite difficult to automatically recognize. The results from automated image analysis were highly correlated with those from manual image analysis. To establish whether this automated, rapid, and objective determination of islet area will facilitate studies of islet histopathology, we showed the beneficial effect of chronic exendin-4, a glucagon-like peptide-1 analog, treatment on islet histopathology in Zucker diabetic fatty (ZDF) rats. Automated image analysis provided qualitative and quantitative evidence that exendin-4 treatment ameliorated the loss of pancreatic insulin content and gave rise to islet hypertrophy. We also showed that glucagon-positive α-cell area was decreased significantly in ZDF rat islets with disorganized structure. This study is the first to demonstrate the utility of automatic quantification of digital images to study pancreatic islet histopathology. The proposed method will facilitate evaluations in preclinical drug efficacy studies as well as elucidation of the pathophysiology of diabetes.

Expression of vascular endothelial growth factor (VEGF) associated with histopathological changes in rodent models of osteoarthritis.

Vascular endothelial growth factor (VEGF) and its receptors have recently reported to be expressed in human osteoarthritis (OA), suggesting that VEGF could be implicated in the pathogenesis of this disease. In the present study, expression of VEGF in the articular cartilage was determined in three different OA models: medial meniscectomy and monoiodoacetate (MIA) injection in rats and age-associated spontaneous joint cartilage destruction in guinea pigs. VEGF was detected by immunohistochemical analysis in the regenerative and hypertrophic chondrocytes, perichondrium and osteophyte areas and chondrocyte clones. Stain intensity of VEGF immunoreactivity increased simultaneously with the degree of cartilage destruction and reparation. These results suggest that VEGF is a key factor in the articular cartilage in human OA and animal OA models.

Evaluation of gene expression related to hepatic cell maturation and differentiation in a chemically induced mouse hepatoblastoma cell line.

The MHB-2 cell line, established from a mouse hepatoblastoma (HB), was subjected to the reverse transcriptase-polymerase chain reaction (RT-PCR) for evaluation of gene expression related to cell differentiation. RNAs for c-kit, CD34, thy-1, albumin, cytokeratin (CK) 8, 18 and 19 could be detected, but expression of alpha-fetoprotein, glucose-6-phosphatase, tyrosine aminotransferase and CK7 was not observed. MHB-2 cells were positive for CK8/18 but negative for c-kit, CD34, thy-1 and albumin on protein level. Immunohistochemical staining of the HB in vivo revealed diffusely expressed c-kit. Thy-1-positive HB cells were sparsely observed, but the tumor was negative for CD34 and rarely positive for CK8/18. By in situ hybridization, the HB was positive for CK18 but negative for CK19. Slight expression of albumin, but the lack of immature hepatocytic marker suggested some heterogeneous hepatocyte or an undifferentiated cell from other origin. Furthermore, positive expression of CK19 as well as CK8 and CK18 in culture strongly suggested the differentiation into a biliary lineage or the bidirectional state. In conclusion, the present study indicated the mouse HB to have de-differentiated, bipotent, or biliary-like cell characteristics, and considering the histological difference between HB and biliary tumors, it suggests the mouse HB cells are closely like some sort of hepatic undifferentiated cells.

Barrier function of microvessels and roles of glial cell line-derived neurotrophic factor in the rat testis.

The expression and localization of glial cell line-derived neurotrophic factor (GDNF) and its receptor GFR alpha1 in the testis were examined, because the blood-testis barrier is a well-known tissue barrier and we previously reported that GDNF reduced the endothelial permeability of the blood-brain barrier (BBB). Five minutes after intravenous injection of Evans blue (molecular weight, 960.6) or fluorescent dextran (molecular weight 10000 and 70000), Evans blue was observed outside microvessels of the testis, whereas the fluorescent dextran was not. Immunohistochemically, GDNF was detected in alpha-smooth muscle actin-positive cells around the seminiferous tubules and in microvessels. On the other hand, GFR alpha1 was detected in endothelial cells in the interstitial space, as well as in spermatocytes. Although occludin was positive in Sertoli cells and endothelium, claudin-5 was localized only in the endothelium of the microvessels. Thus, it became very clear that the microvessels in the testis possessed relatively impermeable tight junctions, and that the alpha-smooth muscle actin-positive cells secreted GDNF, which receptor was expressed in endothelial cells. Because this relation between GDNF and GFR alpha1 is similar to that observed in the BBB, we hypothesize that GDNF is a general regulator of tight junctions of the endothelium forming a blood-tissue barrier in a paracrine fashion.