Targeting pathogenic CD8+ tissue-resident T cells with chimeric antigen receptor therapy in murine autoimmune cholangitis.

Primary biliary cholangitis (PBC) is a cholestatic autoimmune liver disease characterized by autoreactive T cell response against intrahepatic small bile ducts. Here, we use Il12b-/-Il2ra-/- mice (DKO mice) as a model of autoimmune cholangitis and demonstrate that Cd8a knockout or treatment with an anti-CD8α antibody prevents/reduces biliary immunopathology. Using single-cell RNA sequencing analysis, we identified CD8+ tissue-resident memory T (Trm) cells in the livers of DKO mice, which highly express activation- and cytotoxicity-associated markers and induce apoptosis of bile duct epithelial cells. Liver CD8+ Trm cells also upregulate the expression of several immune checkpoint molecules, including PD-1. We describe the development of a chimeric antigen receptor to target PD-1-expressing CD8+ Trm cells. Treatment of DKO mice with PD-1-targeting CAR-T cells selectively depleted liver CD8+ Trm cells and alleviated autoimmune cholangitis. Our work highlights the pathogenic role of CD8+ Trm cells and the potential therapeutic usage of PD-1-targeting CAR-T cells.

Impacts of liver macrophages, gut microbiota, and bile acid metabolism on the differences in iHFC diet-induced MASH progression between TSNO and TSOD mice.

BACKGROUND: Tsumura-Suzuki non-obese (TSNO) mice exhibit a severe form of metabolic dysfunction-associated steatohepatitis (MASH) with advanced liver fibrosis upon feeding a high-fat/cholesterol/cholate-based (iHFC) diet. Another ddY strain, Tsumura-Suzuki diabetes obese (TSOD) mice, are impaired in the progression of iHFC diet-induced MASH. AIM: To elucidate the underlying mechanisms contributing to the differences in MASH progression between TSNO and TSOD mice. METHODS: We analyzed differences in the immune system, gut microbiota, and bile acid metabolism in TSNO and TSOD mice fed with a normal diet (ND) or an iHFC diet. RESULTS: TSOD mice had more anti-inflammatory macrophages in the liver than TSNO mice under ND feeding, and were impaired in the iHFC diet-induced accumulation of fibrosis-associated macrophages and formation of histological hepatic crown-like structures in the liver. The gut microbiota of TSOD mice also exhibited a distinct community composition with lower diversity and higher abundance of Akkermansia muciniphila compared with that in TSNO mice. Finally, TSOD mice had lower levels of bile acids linked to intestinal barrier disruption under iHFC feeding. CONCLUSIONS: The dynamics of liver macrophage subsets, and the compositions of the gut microbiota and bile acids at steady state and post-onset of MASH, had major impacts on MASH development.

Spatial transcriptomics reveals prognosis-associated cellular heterogeneity in the papillary thyroid carcinoma microenvironment.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the most common malignant endocrine tumour, and its incidence and prevalence are increasing considerably. Cellular heterogeneity in the tumour microenvironment is important for PTC prognosis. Spatial transcriptomics is a powerful technique for cellular heterogeneity study. METHODS: In conjunction with a clinical pathologist identification method, spatial transcriptomics was employed to characterise the spatial location and RNA profiles of PTC-associated cells within the tissue sections. The spatial RNA-clinical signature genes for each cell type were extracted and applied to outlining the distribution regions of specific cells on the entire section. The cellular heterogeneity of each cell type was further revealed by ContourPlot analysis, monocle analysis, trajectory analysis, ligand-receptor analysis and Gene Ontology enrichment analysis. RESULTS: The spatial distribution region of tumour cells, typical and atypical follicular cells (FCs and AFCs) and immune cells were accurately and comprehensively identified in all five PTC tissue sections. AFCs were identified as a transitional state between FCs and tumour cells, exhibiting a higher resemblance to the latter. Three tumour foci were shared among all patients out of the 13 observed. Notably, tumour foci No. 2 displayed elevated expression levels of genes associated with lower relapse-free survival in PTC patients. We discovered key ligand-receptor interactions, including LAMB3-ITGA2, FN1-ITGA3 and FN1-SDC4, involved in the transition of PTC cells from FCs to AFCs and eventually to tumour cells. High expression of these patterns correlated with reduced relapse-free survival. In the tumour immune microenvironment, reduced interaction between myeloid-derived TGFB1 and TGFBR1 in tumour focus No. 2 contributed to tumourigenesis and increased heterogeneity. The spatial RNA-clinical analysis method developed here revealed prognosis-associated cellular heterogeneity in the PTC microenvironment. CONCLUSIONS: The occurrence of tumour foci No. 2 and three enhanced ligand-receptor interactions in the AFC area/tumour foci reduced the relapse-free survival of PTC patients, potentially leading to improved prognostic strategies and targeted therapies for PTC patients.

Two Ectopic Liver Lobes Discovered Incidentally at an Autopsy: A Case Report.

The ectopic liver lobe is a rare anomaly and is most frequently reported as a solitary mass. Herein, we report a case of multiple (two) ectopic liver lobes detected at an autopsy. A Japanese man in his 70s died of an infectious disease associated with acquired immunodeficiency syndrome (AIDS). Autopsy revealed the incidental finding of two 1-cm masses, located anterior to the inferior vena cava. Both masses were composed of liver tissue and had internal microscopic structures resembling the porta hepatis, consisting of an outflow bile duct and blood vessels. The outflow bile duct appeared to be continuous with the common bile duct, but the connection point of the outflow vessel was unclear. The liver tissue showed fibrous thickening of the central veins and portal venopathy, including fibrosis in the portal area as well as narrowing and loss of the portal veins. There was no evidence of congestion, fibrosis, biliary stasis, or neoplasm. The incidence of hepatocellular carcinoma is higher in the ectopic liver lobe than in the proper liver, presumably due to the abnormal circulation and bile excretion pathways. The patient also presented with portal venopathy; this suggests the presence of abnormal circulatory dynamics.

Quantification of collagen fiber properties in alcoholic liver fibrosis using polarization-resolved second harmonic generation microscopy.

Liver fibrosis is assessed mainly by conventional staining or second harmonic generation (SHG) microscopy, which can only provide collagen content in fibrotic area. We propose to use polarization-resolved SHG (PR-SHG) microscopy to quantify liver fibrosis in terms of collagen fiber orientation and crystallization. Liver samples obtained from autopsy cases with fibrosis stage of F0-F4 were evaluated with an SHG microscope, and 12 consecutive PR-SHG images were acquired while changing the polarization azimuth angle of the irradiated laser from 0° to 165° in 15° increments using polarizer. The fiber orientation angle (φ) and degree (ρ) of collagen were estimated from the images. The SHG-positive area increased as the fibrosis stage progressed, which was well consistent with Sirius Red staining. The value of φ was random regardless of fibrosis stage. The mean value of ρ (ρ-mean), which represents collagen fiber crystallinity, varied more as fibrosis progressed to stage F3, and converged to a significantly higher value in F4 than in other stages. Spatial dispersion of ρ (ρ-entropy) also showed increased variation in the stage F3 and decreased variation in the stage F4. It was shown that PR-SHG could provide new information on the properties of collagen fibers in human liver fibrosis.

The Effects of Overnight Fasting Duration on Glucose and Lipid Metabolism in a Sprague-Dawley Rat Model of Nonalcoholic Steatohepatitis with Advanced Fibrosis.

Nonalcoholic steatohepatitis (NASH) can progress to hepatic fibrosis, and is associated with cardiovascular and liver-related mortality. To understand the pathogenesis of NASH, reliable animal models of the disease are useful. In animal studies, the animals are usually fasted overnight before biospecimens are taken, but little is known about the effects of fasting. Here, we investigated the impact of overnight fasting for approximately 9 to 17 h on glucose and lipid metabolism in a Sprague-Dawley (SD) rat model of diet-induced moderate and advanced NASH in comparison to normal SD rats. Our results revealed that in the moderate NASH model rats, the fasting duration did not affect glucose and lipid metabolism, the histopathological findings, or the hepatic mRNA expression levels of genes related to lipid metabolism, cholesterol metabolism, inflammation, fibrosis, and oxidative stress. In contrast, in the normal rats, significant fasting time-dependent reductions were observed in the epididymal fat pad weight and the hepatic mRNA expression levels of adipose differentiation-related protein and heme oxygenase-1. Moreover, in the advanced NASH model rats, a significant fasting time-dependent reduction and increase were observed in the serum insulin level and mRNA expression level of alpha-smooth muscle actin, respectively. Our present results suggest that the influence of the overnight fasting duration differs among the healthy condition, moderate NASH, and advanced NASH statuses. Further studies are needed in humans to determine the appropriate overnight fasting duration for the accurate evaluation of glucose and lipid metabolism in NASH patients.

Dynamics of Liver Macrophage Subsets in a Novel Mouse Model of Non-Alcoholic Steatohepatitis Using C57BL/6 Mice.

Macrophages are critical for the development of non-alcoholic steatohepatitis (NASH). Our previous findings in TSNO mouse livers showed that an iHFC (high-fat/cholesterol/cholate) diet induced liver fibrosis similar to human NASH and led to the accumulation of distinct subsets of macrophage: CD11c+/Ly6C- and CD11c-/Ly6C+ cells. CD11c+/Ly6C- cells were associated with the promotion of advanced liver fibrosis in NASH. On the other hand, CD11c-/Ly6C+ cells exhibited an anti-inflammatory effect and were involved in tissue remodeling processes. This study aimed to elucidate whether an iHFC diet with reduced cholic acid (iHFC#2 diet) induces NASH in C57BL/6 mice and examine the macrophage subsets accumulating in the liver. Histological and quantitative real-time PCR analyses revealed that the iHFC#2 diet promoted inflammation and fibrosis indicative of NASH in the livers of C57BL/6 mice. Cell numbers of Kupffer cells decreased and recruited macrophages were accumulated in the livers of iHFC#2 diet-fed C57BL/6 mice. Notably, the iHFC#2 diet resulted in the accumulation of three macrophage subsets in the livers of C57BL/6 mice: CD11c+/Ly6C-, CD11c-/Ly6C+, and CD11c+/Ly6C+ cells. However, CD11c+/Ly6C+ cells were not distinct populations in the iHFC-fed TSNO mice. Thus, differences in cholic acid content and mouse strain affect the macrophage subsets that accumulate in the liver.

Brain-derived neurotrophic factor knock-out mice develop non-alcoholic steatohepatitis.

While brain-derived neurotrophic factor (BDNF), which is a growth factor associated with cognitive improvement and the alleviation of depression symptoms, is known to regulate food intake and body weight, the role of BDNF in peripheral disease is not fully understood. Here, we show that reduced BDNF expression is associated with weight gain and the chronic liver disease non-alcoholic steatohepatitis (NASH). At 10 months of age, BDNF-heterozygous (BDNF+/- ) mice developed symptoms of NASH: centrilobular/perivenular steatosis, lobular inflammation with infiltration of neutrophils, ballooning hepatocytes, and fibrosis of the liver. Obesity and higher serum levels of glucose and insulin - major pathologic features in human NASH - were dramatic. Dying adipocytes were surrounded by macrophages in visceral fat, suggesting that chronic inflammation occurs in peripheral organs. RNA sequencing (RNA-seq) studies of the liver revealed that the most significantly enriched Gene Ontology term involved fatty acid metabolic processes and the modulation of neutrophil aggregation, pathologies that well characterise NASH. Gene expression analysis by RNA-seq also support the notion that BDNF+/- mice are under oxidative stress, as indicated by alterations in the expression of the cytochrome P450 family and a reduction in glutathione S-transferase p, an antioxidant enzyme. Histopathologic phenotypes of NASH were also observed in a knock-in mouse (BDNF+/pro ), in which the precursor BDNF is inefficiently converted into the mature form of BDNF. Lastly, as BDNF reduction causes overeating and subsequent obesity, a food restriction study was conducted in BDNF+/pro mice. Pair-fed BDNF+/pro mice developed hepatocellular damage and showed infiltration of inflammatory cells, including neutrophils in the liver, despite having body weights and blood parameters that were comparable to those of controls. This is the first report demonstrating that reduced BDNF expression plays a role in the pathogenic mechanism of NASH, which is a hepatic manifestation of metabolic syndrome. © 2023 The Pathological Society of Great Britain and Ireland.

Establishment of repeated liver biopsy technique in experimental mice.

Biopsy is a commonly used method for determining pathological diagnoses by directly using human tissues and cells. Biopsies are widely used to determine disease progression and treatment efficacy. Although organs and tissues are usually obtained by sacrifice during animal experiments, it is theoretically possible to use the same biopsy techniques in humans. In the present study, we examined the feasibility of performing four repeated liver biopsies in a spontaneous metabolic syndrome mouse model. Even though a small number of mice died accidently, most mice were able to undergo four liver biopsies without significant adverse events. We also performed three liver biopsies in mouse liver tumor carcinogen models at 4, 8, and 12 weeks of age. In addition to the sample collected at 16 weeks of age during sacrifice, we successfully collected four liver samples from the same mice at different stages of disease progression. The application of this liver biopsy technique might make it possible for direct evaluation of pathological conditions in the same individual over time, thereby reducing the number of experimental animals.

The innate immune receptor RP105 promotes metabolic syndrome by altering gut microbiota composition and intestinal barrier function.

Radioprotective 105 (RP105) plays a key role in the development of high-fat diet (HFD)-induced metabolic disorders; however, the underlying mechanisms remain to be understood. Here, we aimed to uncover whether RP105 affects metabolic syndrome through the modification of gut microbiota. We confirmed that body weight gain and fat accumulation by HFD feeding were suppressed in Rp105-/- mice. Fecal microbiome transplantation from HFD-fed donor Rp105-/- mice into HFD-fed recipient wild-type mice significantly improved various abnormalities associated with metabolic syndrome, including body weight gain, insulin resistance, hepatic steatosis, macrophage infiltration and inflammation in the adipose tissue. In addition, HFD-induced intestinal barrier dysfunction was attenuated by fecal microbiome transplantation from HFD-fed donor Rp105-/- mice. A 16S rRNA sequence analysis indicated that RP105 modified gut microbiota composition and was involved in the maintenance of its diversity. Thus, RP105 promotes metabolic syndrome by altering gut microbiota composition and intestinal barrier function.

Histological and immunohistochemical analysis of epithelial cells in epidermoid cysts in intrapancreatic accessory spleen.

BACKGROUND: Epidermoid cysts in intrapancreatic accessory spleen (ECIPAS) are a rare lesion. Its pathogenesis, including the origin of cystic epithelium, is not well established. We aimed to elucidate new aspects of the pathological features of ECIPAS to clarify its pathogenesis. METHODS: Six cases of ECIPAS were included in this study. As well as histopathological analysis, to elucidate the features and nature of cystic epithelial cells, immunohistochemical analysis including Pbx1 and Tlx1 and imaging mass spectrometry was performed. RESULTS: Histologically, the cysts were covered by either monolayered or multilayered epithelium. Immunohistochemistry revealed that the epithelial cells in multilayered epithelium exhibited different attributes between the basal and superficial layers. Few epithelial cells had abundant clear cytoplasm and were immunohistochemically positive for adipophilin, suggesting lipid-excreting function. The intracystic fluid contained cholesterol clefts and foamy macrophages, and imaging mass spectrometry revealed the accumulation of lipids. Immunohistochemical analysis indicated that the epithelial cells were positive for Pbx1 in some cases. CONCLUSION: Novel histological features of epithelial cells of ECIPAS were indicated. Although more cases need to be evaluated, we propose that the cause of ECIPAS may be different from that of pancreatic ductal origin. J. Med. Invest. 70 : 251-259, February, 2023.

Effect of daikenchuto (TU-100) on carcinogenesis in non-alcoholic steatohepatitis.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is associated with a higher risk of hepatocellular carcinoma (HCC), and the importance of the gut?liver axis has been recognized in NASH-associated HCC. We investigated the effect of TU-100 on the intestinal microbiome and hepatocarcinogenesis in a NASH model. METHODS: Seven-week-old Tsumura Suzuki obese diabetes mice, a model that shows the spontaneous onset of NASH and HCC, were used. They were divided into a TU-100 treated group and a control group. Mice were sacrificed at 24 and 48 weeks to evaluate hepatic steatosis, fibrosis, carcinogenesis, cytokine expression, and microbiome abundance. RESULTS: At 24 weeks, the TU-100 group showed significantly lower expression of IL6, IL1B, and ACTA2 mRNA in the liver (P?

Revisiting Aire and tissue-restricted antigens at single-cell resolution.

The thymus is a highly specialized organ that plays an indispensable role in the establishment of self-tolerance, a process characterized by the "education" of developing T-cells. To provide competent T-cells tolerant to self-antigens, medullary thymic epithelial cells (mTECs) orchestrate negative selection by ectopically expressing a wide range of genes, including various tissue-restricted antigens (TRAs). Notably, recent advancements in the high-throughput single-cell analysis have revealed remarkable heterogeneity in mTECs, giving us important clues for dissecting the mechanisms underlying TRA expression. We overview how recent single-cell studies have furthered our understanding of mTECs, with a focus on the role of Aire in inducing mTEC heterogeneity to encompass TRAs.

Accumulation of α-synuclein in hepatocytes in nonalcoholic steatohepatitis and its usefulness in pathological diagnosis.

BACKGROUNDS: Nonalcoholic steatohepatitis (NASH) is characterized by fat deposition, inflammation, and hepatocellular damage. The diagnosis of NASH is confirmed pathologically, and hepatocyte ballooning is an important finding for definite diagnosis. Recently, α-synuclein deposition in multiple organs was reported in Parkinson's disease. Since it was reported that α-synuclein is taken up by hepatocytes via connexin 32, the expression of α-synuclein in the liver in NASH is of interest. The accumulation of α-synuclein in the liver in NASH was investigated. Immunostaining for p62, ubiquitin, and α-synuclein was performed, and the usefulness of immunostaining in pathological diagnosis was examined. METHODS: Liver biopsy tissue specimens from 20 patients were evaluated. Several antibodies against α-synuclein, as well as antibodies against connexin 32, p62, and ubiquitin were used for immunohistochemical analyses. Staining results were evaluated by several pathologists with varying experience, and the diagnostic accuracy of ballooning was compared. RESULTS: Polyclonal α-synuclein antibody, not the monoclonal antibody, reacted with eosinophilic aggregates in ballooning cells. Expression of connexin 32 in degenerating cells was also demonstrated. Antibodies against p62 and ubiquitin also reacted with some of the ballooning cells. In the pathologists' evaluations, the highest interobserver agreement was obtained with hematoxylin and eosin (H&E)-stained slides, followed by slides immunostained for p62 and α-synuclein, and there were cases with different results between H&E staining and immunostaining CONCLUSION: These results indicate the incorporation of degenerated α-synuclein into ballooning cells, suggesting the involvement of α-synuclein in the pathogenesis of NASH. The combination of immunostaining including polyclonal α-synuclein may contribute to improving the diagnosis of NASH.

Serum/glucose starvation strikingly reduces heterogeneous nuclear ribonucleoprotein A1 protein and its target, cyclin D1.

Our investigation to explore cellular alterations related to undernutrition in cancer cells revealed that the protein level of heterogenous nuclear ribonucleoprotein A1 (hnRNP A1) is drastically decreased by serum/glucose starvation. Its loss was reversible, serum/glucose starvation-specific and universal throughout cell types and species. The hnRNP A1 mRNA level and hnRNP A1 mRNA/protein stability were not altered under this condition. CCND1 mRNA, which we newly identified as the binding target of hnRNP A1, was decreased by serum/glucose starvation. Under similar conditions, CCND1 protein was reduced in vitro and in vivo, whereas hnRNP A1 mRNA level and CCND1 mRNA level revealed no correlation in most clinical samples. Functional analyses revealed that CCND1 mRNA stability is certainly dependent on hnRNP A1 protein level and that RNA recognition motif-1 (RRM1) in hnRNP A1 plays a central role in maintaining CCND1 mRNA stability and subsequent protein expression. The injection of RRM1-deleted hnRNP A1-expressing cancer cells in the mouse xenograft model did not form any tumours, and that of hnRNP A1-expressing cancer cells retained CCND1 expression at the lesion adjacent to necrosis with a slight increase in tumour volume. Furthermore, RRM1 deletion caused growth suppression with the induction of apoptosis and autophagy, whereas CCND1 restoration completely recovered it. Our results indicate that serum/glucose starvation triggers entire hnRNP A1 protein loss, and its loss may play a role in CCND1 mRNA destabilization and CCND1-mediated cellular event inhibition, i.e. growth promotion, apoptosis induction and autophagosome formation.

Cooperative but Distinct Role of Medullary Thymic Epithelial Cells and Dendritic Cells in the Production of Regulatory T Cells in the Thymus.

Regulatory T cells (Tregs) are produced in the thymus to establish self-tolerance, and agonistic stimuli by self-Ags play a pivotal role in this process. Although two types of APCs, medullary thymic epithelial cells (mTECs) and dendritic cells (DCs), are responsible for presenting self-Ags together with costimulatory/cytokine signals, the distinct role of each APC in producing Tregs remains enigmatic. We have approached this issue by depleting the mTECs and DCs using mice expressing diphtheria toxin receptors driven by Aire and CD11c promoters, respectively. Depletion of mTECs showed an effect on Treg production quantitatively and qualitatively more profound than that of DCs followed by the development of distinct organ-specific autoimmune lesions in the hosts. Because self-Ags produced by mTECs are transferable to DCs through a process known as Ag transfer, we monitored the process of Ag transfer using mice expressing GFP from TECs. Although GFP expressed from total TECs was effectively transferred to DCs, GFP expressed from cortical TECs was not, suggesting that mTECs are the predominant source of self-Ags. We also found that GFP expressed not only from mature mTECs but also from immature mTECs was transferred to DCs, suggesting that a broad spectrum of molecules were subjected to Ag transfer during mTEC development. Interestingly, the numbers of recirculating non-Tregs producing IL-2, an important source for Treg expansion in the thymus, were reduced only in the mTEC-depleted mice. These results suggested the cooperative but distinct role of mTECs and DCs in the production of Tregs to avoid autoimmunity.

AIRE illuminates the feature of medullary thymic epithelial cells in thymic carcinoma.

Despite the clear distinction between cortical (cTECs) and medullary thymic epithelial cells (mTECs) in physiology, the cell of origin of thymic carcinomas (TCs) and other thymic epithelial tumors remained enigmatic. We addressed this issue by focusing on AIRE, an mTEC-specific transcriptional regulator that is required for immunological self-tolerance. We found that a large proportion of TCs expressed AIRE with typical nuclear dot morphology by immunohistochemistry. AIRE expression in TCs was supported by the RNA-seq data in the TCGA-THYM database. Furthermore, our bioinformatics approach to the recent single-cell RNA-seq data on human thymi has revealed that TCs hold molecular characteristics of multiple mTEC subpopulations. In contrast, TCs lacked the gene signatures for cTECs. We propose that TCs are tumors derived from mTECs.

Breach of tolerance versus burden of bile acids: Resolving the conundrum in the immunopathogenesis and natural history of primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a classic autoimmune disease due to the loss of tolerance to self-antigens. Bile acids (BA) reportedly play a major role in biliary inflammation and/or in the modulation of dysregulated immune responses in PBC. Several murine models have indicated that molecular mimicry plays a role in autoimmune cholangitis; however, they have all been limited by the relative failure to develop hepatic fibrosis. We hypothesized that species-specific differences in the BA composition between mice and humans were the primary reason for this limited pathology. Here, we aimed to study the impact of human-like hydrophobic BA composition on the development of autoimmune cholangitis and hepatic fibrosis. We took advantage of a unique construct, Cyp2c70/Cyp2a12 double knockout (DKO) mice, which have human-like BA composition, and immunized them with a well-defined mimic of the major mitochondrial autoantigen of PBC, namely 2-octynoic acid (2OA). 2OA-treated DKO mice were significantly exacerbated portal inflammation and bile duct damage with increased Th1 cytokines/chemokines at 8 weeks post-initial immunization. Most importantly, there was clear progression of hepatic fibrosis and increased expression of hepatic fibrosis-related genes. Interestingly, these mice demonstrated increased serum BA concentrations and decreased biliary BA concentrations; hepatic BA levels did not increase because of the upregulation of transporters responsible for the basolateral efflux of BA. Furthermore, cholangitis and hepatic fibrosis were more advanced at 24 weeks post-initial immunization. These results indicate that both the loss of tolerance and the effect of hydrophobic BA are essential for the progression of PBC.

Urokinase-type plasminogen activator blockade ameliorates experimental colitis in mice.

Although several angiogenesis-related factors are reportedly involved in the pathogenesis of ulcerative colitis (UC), the mechanisms by which they contribute to disease are unclear. We first examined the expression of angiogenesis-related factors in inflamed colorectal tissue of UC patients using antibody array, and identified the 5 factors with highest expression, which included matrix metalloproteinase-8, urokinase-type plasminogen activator (uPA), angiostatin/plasminogen, hepatocyte growth factor and endoglin. Subsequent real-time PCR experiments using additional colorectal tissues revealed that uPA mRNA levels were significantly higher in inflamed tissues than in non-inflamed tissues, and significantly correlated with the severity of UC. Mirror section immunohistochemistry revealed that uPA was expressed in the neutrophils of inflamed colorectal tissues. We administered dextran sulfate sodium (DSS) in drinking water to uPA knockout (uPA-/-) mice, and found that the disease activity index in uPA-/- mice was marginally lower and the histological score in uPA-/- mice was significantly lower than those in wild-type mice, suggesting the importance of uPA in colitis. When an uPA-selective inhibitor, UK122, was administered to DSS-treated C57BL6J mice, the disease activity index and histological score in those mice were significantly lower compared with control mice. Multiple cytokine/chemokine assay using colorectal tissues from uPA-/- and UK122-treated mice revealed significantly lowered level of RANTES. In conclusion, uPA was highly expressed in neutrophils of the inflamed mucosa of UC patients, and the expression level correlated with the severity of UC. Genetic uPA deletion or pharmacological uPA blockade significantly ameliorated colitis in mice, concomitant with downregulation of RANTES.

Impact of Vancomycin Treatment and Gut Microbiota on Bile Acid Metabolism and the Development of Non-Alcoholic Steatohepatitis in Mice.

The potential roles of the gut microbiota in the pathogenesis of non-alcoholic fatty liver disease, including non-alcoholic steatohepatitis (NASH), have attracted increased interest. We have investigated the links between gut microbiota and NASH development in Tsumura-Suzuki non-obese mice fed a high-fat/cholesterol/cholate-based (iHFC) diet that exhibit advanced liver fibrosis using antibiotic treatments. The administration of vancomycin, which targets Gram-positive organisms, exacerbated the progression of liver damage, steatohepatitis, and fibrosis in iHFC-fed mice, but not in mice fed a normal diet. F4/80+-recruited macrophages were more abundant in the liver of vancomycin-treated iHFC-fed mice. The infiltration of CD11c+-recruited macrophages into the liver, forming hepatic crown-like structures, was enhanced by vancomycin treatment. The co-localization of this macrophage subset with collagen was greatly augmented in the liver of vancomycin-treated iHFC-fed mice. These changes were rarely seen with the administration of metronidazole, which targets anaerobic organisms, in iHFC-fed mice. Finally, the vancomycin treatment dramatically modulated the level and composition of bile acid in iHFC-fed mice. Thus, our data demonstrate that changes in inflammation and fibrosis in the liver by the iHFC diet can be modified by antibiotic-induced changes in gut microbiota and shed light on their roles in the pathogenesis of advanced liver fibrosis.