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Non-alcoholic fatty liver disease (NAFLD) has been on a global rise. While animal models have rendered valuable insights to the pathogenesis of NAFLD, discrepancy with patient data still exists. Since non-alcoholic steatohepatitis (NASH) involves chronic inflammation, and CD4 + T cell infiltration of the liver is characteristic of NASH patients, we established and characterized a humanized mouse model to identify human-specific immune response(s) associated with NAFLD progression. Immunodeficient mice engrafted with human immune cells (HIL mice) were fed with high fat and high calorie (HFHC) or chow diet for 20 weeks. Liver histology and immune profile of HIL mice were analyzed and compared with patient data. HIL mice on HFHC diet developed steatosis, inflammation and fibrosis of the liver. Human CD4 + central and effector memory T cells increased within the liver and in the peripheral blood of our HIL mice, accompanied by marked up-regulation of pro-inflammatory cytokines (IL-17A and IFNγ). In vivo depletion of human CD4 + T cells in HIL mice reduced liver inflammation and fibrosis, but not steatosis. Our results highlight CD4 + memory T cell subsets as important drivers of NAFLD progression from steatosis to fibrosis and provides a humanized mouse model for pre-clinical evaluation of potential therapeutics., (Copyright © 2020 Her, Tan, Lim, Tan, Chan, Tan, Liu, Yong, Lai, Ceccarello, Zheng, Fan, Chang, Sun, Chang, Chin, Lee, Dan, Chan, Lim, Chan, Chandy and Chen.)

Background: Liver fibrosis is a common health problem worldwide and there is still a lack of effective medicines. The Chinese herbal medicine, Gan Shen Fu Fang (GSFF) is composed of salvianolic acid B and diammonium glycyrrhizinate. In this study, we observed the effects of GSFF on liver fibrosis in vivo and in vitro in an attempt to provide some hope for the treatment., Aim: To observe the effects of GSFF on liver fibrosis in vivo and in vitro and investigate the mechanism from the perspective of the inflammatory response and extracellular signal-regulated kinase (ERK) phosphorylation., Methods: Common bile duct-ligated rats were used for in vivo experiments. Hepatic stellate cells-T6 (HSC-T6) cells were used for in vitro experiments. Hematoxylin and eosin staining and Masson staining, biochemical assays, hydroxyproline (Hyp) assays, enzyme-linked immunoasorbent assay and western blotting were performed to evaluate the degree of liver fibrosis, liver function, the inflammatory response and ERK phosphorylation. The CCK8 assay, immunofluorescence and western blotting were applied to test the effect of GSFF on HSC-T6 cell activation and determine whether GSFF had an effect on ERK phosphorylation in HSC-T6 cells., Results: GSFF improved liver function and inhibited liver fibrosis in common bile duct-ligated rats after 3 wk of treatment, as demonstrated by histological changes, hydroxyproline assays and collagen I concentrations. GSFF alleviated inflammatory cell infiltration and reduced the synthesis of pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α) and interlukin-1β] and NF-κB. In addition, GSFF decreased ERK phosphorylation. In vitro , GSFF inhibited the viability of HSC-T6 cells with and without transforming growth factor β1 (TGF-β1) stimulation and decreased the synthesis of collagen I. GSFF had the greatest effect at a concentration of 0.5 μmol/L. GSFF inhibited the expression of α-smooth muscle actin (α-SMA), a marker of HSC activation, in HSC-T6 cells. Consistent with the in vivo results, GSFF also inhibited the phosphorylation of ERK and downregulated the expression of NF-κB., Conclusion: GSFF inhibited liver fibrosis progression in vivo and HSC-T6 cell activation in vitro . These effects may be related to an alleviated inflammatory response and downregulated ERK phosphorylation., Competing Interests: Conflict-of-interest statement: All other authors have nothing to disclose., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

Background and Aims: Monocyte chemotactic protein-1 (MCP-1) is a potent chemoattractant for monocytes. It is involved in pathogenesis of several inflammatory diseases. Hepatic MCP-1 is a readout of macrophage activation. While inflammation is a major driver of liver disease progression, the origin and role of circulating MCP-1 as a biomarker remains unclear. Methods: Hepatic CC-chemokine ligand 2 ( CCL2 ) expression and F4/80 staining for Kupffer cells were measured and correlated in a mouse model of chronic liver disease (inhalative CCl 4 for 7 weeks). Next, hepatic RNA levels of CCL2 were measured in explanted livers of 39 patients after transplantation and correlated with severity of disease. Changes in MCP-1 were further evaluated in a rat model of experimental cirrhosis and acute-on-chronic liver failure (ACLF). Finally, we analyzed portal and hepatic vein levels of MCP-1 in patients receiving transjugular intrahepatic portosystemic shunt insertion for complications of portal hypertension. Results: In this mouse model of fibrotic hepatitis, hepatic expression of CCL2 ( P = 0.009) and the amount of F4/80 positive cells in the liver ( P < 0.001) significantly increased after induction of hepatitis by CCl 4 compared to control animals. Moreover, strong correlation of hepatic CCL2 expression and F4/80 positive cells were seen ( P = 0.023). Furthermore, in human liver explants, hepatic transcription levels of CCL2 correlated with the MELD score of the patients, and thus disease severity ( P = 0.007). The experimental model of ACLF in rats revealed significantly higher levels of MCP-1 plasma ( P = 0.028) and correlation of hepatic CCL2 expression ( R = 0.69, P = 0.003). Particularly, plasma MCP-1 levels did not correlate with peripheral blood monocyte CCL2 expression. Finally, higher levels of MCP-1 were observed in the hepatic compared to the portal vein ( P = 0.01) in patients receiving TIPS. Similarly, a positive correlation of MCP-1 with Child-Pugh score was observed ( P = 0.018). Further, in the presence of ACLF, portal and hepatic vein levels of MCP-1 were significantly higher compared to patients without ACLF (both P = 0.039). Conclusion: Circulating levels of MCP-1 mainly derive from the injured liver and are associated with severity of liver disease. Therefore, liver macrophages contribute significantly to disease progression. Circulating MCP-1 may reflect the extent of hepatic macrophage activation., (Copyright © 2020 Queck, Bode, Uschner, Brol, Graf, Schulz, Jansen, Praktiknjo, Schierwagen, Klein, Trautwein, Wasmuth, Berres, Trebicka and Lehmann.)

Background & Aims: Liver inflammation has been recognized as a hallmark of hepatocarcinogenesis. Although Forkhead Box M1 (FoxM1) is a well-defined oncogenic transcription factor that is overexpressed in hepatocellular carcinoma (HCC), its role in liver inflammation has never been explored., Methods: We generated hepatocyte-specific FoxM1 conditional transgenic (TG) mice by using the Cre-loxP and Tetracycline (Tet)-on systems to induce FoxM1 expression in a hepatocyte-specific and time-dependent manner., Results: After treatment of Tet-derivatives doxycycline (DOX) to induce FoxM1, TG mice exhibited spontaneous development of hepatocyte death with elevated serum alanine aminotransferase levels and hepatic infiltration of macrophages. The removal of DOX in TG mice completely removed this effect, suggesting that spontaneous inflammation in TG mice occurs in a hepatocyte FoxM1-dependent manner. In addition, liver inflammation in TG mice was associated with increased levels of hepatic and serum chemokine (C-C motif) ligand 2 (CCL2). In vitro transcriptional analysis confirmed that CCL2 is a direct target of FoxM1 in murine hepatocytes. After receiving FoxM1 induction since birth, all TG mice exhibited spontaneous HCC with liver fibrosis at 12 months of age. Hepatic expression of FoxM1 was significantly increased in liver injury models. Finally, pharmacologic inhibition of FoxM1 reduced liver inflammation in models of liver injury., Conclusions: Hepatocyte FoxM1 acts as a crucial regulator to orchestrate liver inflammation linking to hepatocarcinogenesis. Thus, hepatocyte FoxM1 may be a potential target not only for the treatment of liver injury but also for the prevention toward HCC., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Chronic inflammation is the driver of liver injury and results in progressive fibrosis and eventual cirrhosis with consequences including both liver failure and liver cancer. We have previously described increased expression of the highly multifunctional glycoprotein CD147 in liver injury. This work describes a novel role of CD147 in liver inflammation and the importance of leukocyte aggregates in determining the extent of liver injury., Methods: Non-diseased, progressive injury, and cirrhotic liver from humans and mice were examined using a mAb targeting CD147. Inflammatory cell subsets were assessed by multiparameter flow cytometry., Results: In liver injury, we observe abundant, intrahepatic leukocyte clusters defined as ≥5 adjacent CD45+ cells which we have termed "leukocyte aggregates". We have shown that these leukocyte aggregates have a significant effect in determining the extent of liver injury. If CD147 is blocked in vivo, these leukocyte aggregates diminish in size and number, together with a marked significant reduction in liver injury including fibrosis. This is accompanied by no change in overall intrahepatic leukocyte numbers. Further, blocking of aggregation formation occurs prior to an appreciable increase in inflammatory markers or fibrosis. Additionally, there were no observed, "off-target" or unpredicted effects in targeting CD147., Conclusion: CD147 mediates leukocyte aggregation which is associated with the development of liver injury. This is not a secondary effect, but a cause of injury as aggregate formation proceeds other markers of injury. Leukocyte aggregation has been previously described in inflammation dating back over many decades. Here we demonstrate that leukocyte aggregates determine the extent of liver injury., Competing Interests: The authors have declared that no competing interests exist.

An ingredient was isolated from Acanthus ilicifolius and identified as 4-hydroxy-2(3H)-benzoxazolone (HBOA). Its protective effects and underlying mechanism on liver fibrosis were investigated. Briefly, rats were intragastrically administrated with 50% CCl 4 twice a week for 12 weeks to induce liver fibrosis. Meanwhile, the animals were treated with various medicines from weeks 8 to 12. Then the histological change, serum biochemical index, inflammatory factors and hepatocyte apoptosis were detected. Moreover, the TGF-β1/Smads, NF-κB and ERK signaling pathways were also detected to illustrate the underlying mechanism. The results showed that HBOA significantly ameliorated CCl 4 -induced liver injury and collagen accumulation in rats, as evidenced by the histopathologic improvement. Moreover, HBOA markedly decreased hepatocyte apoptosis by regulating the expression levels of caspase-3, -9 and -12, as well as the Bcl-2 family. The mechanism study showed that HBOA significantly decreased the expressions of α-smooth muscle actin (α-SMA) and collagen and inhibited the generation of excessive extracellular matrix (ECM) components by restoring the balance between matrix metalloproteinases (MMPs) and its inhibitor (TIMPs). HBOA markedly alleviated oxidative stress and inflammatory cytokines through inhibiting the NF-κB pathway. In addition, HBOA significantly down-regulated the levels of TGF-β1, Smad2/3, Smad4 and up-regulated the level of Smad7, inhibiting the TGF-β1/Smads signaling pathway. Moreover, HBOA significantly blocked the ERK signaling pathway, leading to the inactivation of hepatic stellate cells. This study suggests that HBOA exerts a protective effect against liver fibrosis via modulating the TGF-β1/Smads, NF-κB and ERK signaling pathways, which will be developed as a potential agent for the treatment of liver fibrosis., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Aim: To investigate whether Yiguanjian decoction (YGJ) has an anti-liver cirrhotic effect and whether it regulates hepatic stem cell differentiation., Methods: A rat model of liver cirrhosis was established via subcutaneous injection of carbon tetrachloride (CCl 4 ) for 8 wk. From the beginning of the ninth week, the rats received 2-acetylaminofluorene (2-AAF) by oral gavage and a DLK-1 + fetal liver stem/progenitor cell (FLSPC) transplant or an FLSPC transplant in combination with YGJ treatment for 4 wk. In vitro , lipopolysaccharide (LPS)-activated macrophages were co-cultured with WB-F344 cells, and the differentiation of WB-F344 cells was observed in the presence and absence of YGJ treatment., Results: FLSPC transplantation improved liver function and histopathology, and inhibited the activation of the non-canonical Wnt signaling pathway, while activating the canonical Wnt signaling pathway. YGJ enhanced the therapeutic effects of FLSPCs and also promoted the liver regeneration differentiation of FLSPCs into hepatocytes. In vitro , LPS-activated macrophages promoted the differentiation of WB-F344 cells into myofibroblasts, and the canonical Wnt signaling was inhibited while the non-canonical Wnt signaling was activated in WB-F344 cells. YGJ suppressed the activation of macrophages and then inhibited non-canonical Wnt signaling and promoted canonical Wnt signaling., Conclusion: YGJ enhances FLSPC-mediated repair of liver cirrhosis through regulation of macrophage activation state, and YGJ in combination with stem cell transplantation may be a suitable treatment for end-stage liver cirrhosis., Competing Interests: Conflict-of-interest statement: The authors declare that they have no conflicts of interest.

Yu Gan Long (YGL) is a Chinese traditional herbal medicine that has been used in the treatment of liver fibrosis for many years in clinical practice. However, its anti-hepatofibrotic mechanism has not been studied yet. In this study, the effect and mechanism of YGL in reducing liver fibrosis was demonstrated in vivo. Our results showed that liver fibrosis biomarkers collagen IV (Col IV), type III precollagen (PCIII), hyaluronuc acid (HA) and laminin (LN), were increased after CCl 4 treatment and decreased by YGL. Among the liver fibrosis indicators, α-smooth muscle actin (α-SMA) was decreased by YGL in the CCl 4 -treated rats, while MMP2 and MMP9 was upregulated followed by TIMP1 downregulation. Proteins involved in liver fibrosis such as p-Smad2, p-Smad3 and Smad4 were down-regulated, while Smad7 protein was up-regulated by YGL after CCl 4 -induced liver damage. YGL also suppressed the increase of TGF-β1, TNF-α, IL-1β, IL-6, IL-4 and IL-17 A induced by CCl 4 treatment, while promoted IFN-γ expression. Finally, the transcription factors ROR-γt and GATA3 were decreased, while T-bet was increased after YGL treatment. These results suggested that YGL attenuated CCl 4 -induced hepatic fibrosis by accelerating the extracellular matrix degradation, blocking the TGF-β1/Smad signaling pathway and modulating the balance among IL-4, IL-17 A and IFN-γ, demonstrating YGL protective effect and its potential mechanisms in treating liver fibrosis., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Aim: To reveal the protective mechanism of the combined use of vitamin D and puerarin in the progression of hepatic fibrosis induced by carbon tetrachloride (CCl 4 )., Methods: Eight-week-old male Wistar rats were randomly divided into a normal control group (C group), a CCl 4 group (CCl 4 group), a vitamin D group (V group), a puerarin group (P group), and a combined group of vitamin D and puerarin (V + P group), each of which contained ten rats. In this way, we built a rat model of CCl 4 -induced hepatic fibrosis with intervention by vitamin D, puerarin, or a combination of the two. After eight weeks, the mice were sacrificed to collect serum and liver specimens. Blood was collected to detect the hyaluronic acid (HA). We also measured hydroxyproline (Hyp) and prepared paraffin sections of liver. After Sirius red staining, the liver specimens were observed under a microscope. RT-PCR and western blot analysis were adopted to detect the mRNA and the protein levels of Collagen I, Collagen III, Wnt1, and β-catenin in the liver tissues, respectively., Results: Hepatic fibrosis was observed in the CCl 4 group. In comparison, hepatic fibrosis was attenuated in the V, P, and V + P groups: the HA level in blood and the Hyp level in liver were reduced, and the mRNA levels of Collagen I, Collagen III, Wnt, and β-catenin in liver were also decreased, as well as the protein levels of Wnt1 and β-catenin. Among these groups, the V + P group demonstrated the greatest amelioration of hepatic fibrosis., Conclusion: The combined application of vitamin D and puerarin is capable of alleviating CCl 4 -induced hepatic fibrosis of rats. As to the mechanism, it is probably because the combined use is able to silence the Wnt1/β-catenin pathway, suppress the activation of hepatic stellate cells, and reduce the secretion of collagen fibers, therefore improving the anti-hepatic fibrosis effect., Competing Interests: Conflict-of-interest statement: All authors have no conflict of interest.

Patients with liver cirrhosis present an increased incidence of infections. The main cause has been founded in alterations of the enteric flora and of the intestinal barrier probably due to portal hypertension, in addition to a reticulo-endothelial system dysfunction. Furthermore, those living with cirrhosis can report a high predisposition to sepsis and septic shock, due to the excessive response of pro-inflammatory cytokines and a complessive hemodynamic derangement. By the analysis in the experimental model of the cirrhotic rat, it was demonstrated that radio-labelled Escherichia coli given by the oral route resulted in the location of the bacteria in the gut, the ascitic fluid and mesenteric lymph nodes, a phenomenon known as bacterial translocation. Bacteria encountered with the highest frequency are those colonizing the intestinal tract, such as E. coli, Klebsiella pneumoniae and Enterobacteriaceae, intracellular bacteria and parasites are reported with a lower frequency. Multi-drug resistant bacteria are cultured with the highest frequency in those with frequent hospitalisations and report both high septic shock and mortality rates. Spontaneous bacterial peritonitis (SBP) is the commonest infection in cirrhotic, estimated to occur in 10-30% of the cases with ascites. A practical approach may include administration of a protected penicillin, III generation cephalosporin or quinolones in uncomplicated cases. Instead, in complicated cases and in nosocomial SBP, administration of cephalosporin or quinolones can be burned by the high resistance rate and drugs active against ESBL-producing bacteria and multi-drug resistant Gram positive bacteria have to be considered as empiric therapy, until cultures are available. When cultures are not readily available and patients fail to improve a repeated diagnostic paracentesis should be performed. Current investigations suggest that norfloxacin 400 mg/day orally has been reported to successfully prevent SBP in patients with low-protein ascites and patients with prior SBP.

Interleukin-17A has been identified as a driver of hepatic stellate cell activation and plays a critical role in the pathogenesis of hepatic fibrosis. However, the underlining fibrosis-promoting mechanism of IL-17A is far from understood. Here we aimed to define whether hepatocytes directly respond to IL-17A stimulation and are associated with the development of hepatic fibrosis. The functional significance of IL-17A was evaluated in bile duct ligation (BDL) or thioacetamide (TAA) injection-induced mouse models of hepatic fibrosis. Human cirrhosis and control tissues were obtained from the patients with cirrhosis who received an open surgical repair process. Neutralizing IL-17A promoted the resolution of BDL or TAA-induced acute or chronic inflammation and fibrosis, resulted in a shift of the suppressive immune response in fibrotic liver toward a Th1-type immune response, and restored autophagy activity in both cholestatic and hepatotoxic liver injury induced fibrotic liver tissues, which was accompanied by a significant inhibition of STAT3 phosphorylation. Moreover, we found that IL-17A stimulated the concentration-and time-dependent phosphorylation of STAT3 in AML-12 liver cells. Blocking STAT3 with a specific inhibitor STATTIC or STAT3 siRNA protected from the IL-17A-induced autophagy suppression in AML-12 cells, indicating that STAT3 mediates IL-17A-suppressed autophagy. Administration of IL-10, which activated STAT3 and inhibited autophagy, reversed the therapeutic effect of IL-17A antagonism in vivo. Our study suggests that the IL-17A/STAT3 signaling pathway plays a crucial role in the pathogenesis of hepatic fibrosis through suppressing hepatocellular autophagy and that blocking this pathway may provide therapeutic benefits for the treatment of hepatic fibrosis.

The aim of this study was to investigate the effect of betaine (BET) on alcoholic liver fibrosis in rats. Fibrosis was experimentally generated with ethanol plus carbon tetrachloride (ETH+CCl4) treatment. Rats were treated with ETH (5% v/v in drinking water) for 14 weeks. CCl4 was administered intraperitoneally (i.p.) 0.2mL/kg twice a week to rats in the last 6 weeks with/without commercial food containing BET (2% w/w). Serum hepatic damage markers, tumor necrosis factor-α, hepatic triglyceride (TG) and hydroxyproline (HYP) levels, and oxidative stress parameters were measured together with histopathologic observations. In addition, α-smooth muscle-actin (α-SMA), transforming growth factor-β1 (TGF-β1) and type I collagen (COL1A1) protein expressions were assayed immunohistochemically to evaluate stellate cell (HSC) activation. mRNA expressions of matrix metalloproteinase-2 (MMP-2) and its inhibitors (TIMP-1 and TIMP-2) were also determined. BET treatment diminished TG and HYP levels; prooxidant status and fibrotic changes; α-SMA, COL1A1 and TGF-β protein expressions; MMP-2, TIMP-1 and TIMP-2 mRNA expressions in the liver of fibrotic rats. In conclusion, these results indicate that the antifibrotic effect of BET may be related to its suppressive effects on oxidant and inflammatory processes together with HSC activation in alcoholic liver fibrosis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Aim: To evaluate the effect of artesunate (AS) supplementation on bacterial translocation (BT) and gut microbiota in a rat model of liver cirrhosis., Methods: Fifty-four male Sprague-Dawley rats were randomly divided into a normal control group (N), a liver cirrhosis group (M) and a liver cirrhosis group intervened with AS (MA). Each group was sampled at 4, 6 and 8 wk. Liver cirrhosis was induced by injection of carbon tetrachloride (CCl4), intragastric administration of 10% ethanol, and feeding a high fat diet. Rats in the MA group were intragastrically administered with AS (25 mg/kg body weight, once daily). Injuries of the liver and intestinal mucosa were assessed by hematoxylin-eosin or Masson's trichrome staining. Liver index was calculated as a ratio of the organ weight (g) to body weight (g). The gut microbiota was examined by automated ribosomal intergenic-spacer analysis of fecal DNA. BT was assessed by standard microbiological techniques in the blood, mesenteric lymph nodes (MLNs), liver, spleen, and kidney., Results: Compared to group N, the body weight was reduced significantly in groups M and MA due to the development of liver cirrhosis over the period of 8 wk. The body weight was higher in group MA than in group M. The liver indices were significantly elevated at 4, 6 and 8 wk in groups M and MA compared to group N. AS supplementation partially decreased the liver indices in group MA. Marked histopathologic changes in the liver and small intestinal mucosa in group M were observed, which were alleviated in group MA. Levels of pro-inflammatory interleukin-6 and tumor necrosis factor-α were significantly elevated at 8 wk in ileal homogenates in group M compared to group N, which were decreased after AS supplementation in group MA. The dysbiosis of gut microbiota indicated by the mean diversity (Shannon index) and mean similarity (Sorenson index) was severe as the liver cirrhosis developed, and AS supplementation had an apparent intervention effect on the dysbiosis of gut microbiota at 4 wk. The occurrence of BT was increased in the liver of group M compared to that of group N. AS supplementation reduced BT in group MA at 8 wk. BT also occurred in the MLNs, spleen, and kidney, which was reduced by AS supplementation. BT was not detected in the blood in any group., Conclusion: Dysbiosis of gut microbiota, injury of intestinal mucosal barrier and BT occurred as liver cirrhosis progressed, which might enhance inflammation and aggravate liver injury. AS may have other non-antimalarial effects that modulate gut microbiota, inhibit BT and alleviate inflammation, resulting in a reduction in CCl4, alcohol and high fat-caused damages to the liver and intestine.

Chronic liver diseases are characterized by a sustained inflammatory response in which chemokines and chemokine-receptors orchestrate inflammatory cell recruitment. In this study we investigated the role of the chemokine receptor CCR6 in acute and chronic liver injury. In the absence of liver injury Ccr6-/- mice presented a higher number of hepatic macrophages and increased expression of pro-inflammatory cytokines and M1 markers Tnf-α, Il6 and Mcp1. Inflammation and cell recruitment were increased after carbon tetrachloride-induced acute liver injury in Ccr6-/- mice. Moreover, chronic liver injury by carbon tetrachloride in Ccr6-/- mice was associated with enhanced inflammation and fibrosis, altered macrophage recruitment, enhanced CD4+ cells and a reduction in Th17 (CD4+IL17+) and mature dendritic (MHCII+CD11c+) cells recruitment. Clodronate depletion of macrophages in Ccr6-/- mice resulted in a reduction of hepatic pro-inflammatory and pro-fibrogenic markers in the absence and after liver injury. Finally, increased CCR6 hepatic expression in patients with alcoholic hepatitis was found to correlate with liver expression of CCL20 and severity of liver disease. In conclusion, CCR6 deficiency affects hepatic inflammatory cell recruitment resulting in the promotion of hepatic inflammation and fibrosis.

Acute schistosomiasis is characterized by pro-inflammatory responses against tissue- or organ-trapped parasite eggs along with granuloma formation. Here, we describe studies in Cx3cr1(-/-) mice and demonstrate the role of Cx3cr1 in the pathoetiology of granuloma formation during acute schistosomiasis. Mice deficient in Cx3cr1 were protected from granuloma formation and hepatic injury induced by Schistosoma japonicum eggs, as manifested by reduced body weight loss and attenuated hepatomegaly along with preserved liver function. Notably, S. japonicum infection induced high levels of hepatic Cx3cr1 expression, which was predominantly expressed by infiltrating macrophages. Loss of Cx3cr1 rendered macrophages preferentially towards M2 polarization, which then led to a characteristic switch of the host immune defense from a conventional Th1 to a typical Th2 response during acute schistosomiasis. This immune switch caused by Cx3cr1 deficiency was probably associated with enhanced STAT6/PPAR-γ signaling and increased expression of indoleamine 2,3-dioxygenase (IDO), an enzyme that promotes M2 polarization of macrophages. Taken together, our data provide evidence suggesting that CX3CR1 could be a viable therapeutic target for treatment of acute schistosomiasis., (© 2015. Published by The Company of Biologists Ltd.)

Aim: To investigate the effect of interleukin (IL)-22 on hepatic fibrosis in mice and the possible mechanism involved., Methods: Liver fibrosis was induced in male BALB/c mice by CCl₄. Recombinant IL-22 (rmIL-22) was administered intraperitoneally in CCl₄-treated mice. Fibrosis was assessed by histology and Masson staining. The activation of hepatic stellate cells (HSCs) was investigated by analysis of α-smooth muscle actin expression. The frequencies of T helper (Th) 22 cells, Th17 cells and Th1 cells, the expression of inflammatory cytokines [IL-22, IL-17A, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-6, IL-1β] and transcription factors [aryl hydrocarbon receptor (AHR), RAR-related orphan receptor (RORγt), T-bet] mRNA in the liver were investigated. In addition, the plasma levels of IL-22, IL-17A, IFN-γ, TNF-α, IL-6 and IL-1β were evaluated., Results: Significant elevations in circulating Th22 cells, Th17 cells, Th1 cells, IL-22, IL-17A, and IFN-γ were observed in the hepatic fibrosis group compared with the control group (P < 0.01). Treatment with rmIL-22 in mice with hepatic fibrosis ameliorated the severity of hepatic fibrosis, which was confirmed by lower hepatic fibrosis pathological scores (P < 0.01). RmIL-22 decreased the frequencies of Th22 cells (6.71% ± 0.97% vs 8.09% ± 0.74%, P < 0.01), Th17 cells (4.34% ± 0.37% vs 5.71% ± 0.24%, P < 0.01), Th1 cells (3.09% ± 0.49% vs 4.91% ± 0.73%, P < 0.01), and the levels of IL-22 (56.23 ± 3.08 vs 70.29 ± 3.01, P < 0.01), IL-17A (30.74 ± 2.77 vs 45.68 ± 2.71, P < 0.01), and IFN-γ (74.78 ± 2.61 vs 124.89 ± 2.82, P < 0.01). Down-regulation of IL-22, IL-17A, IFN-γ, TNF-α, IL-6, IL-1β, AHR RORγt, and T-bet gene expression in the liver was observed in the rmIL-22 group (P < 0.01)., Conclusion: The frequencies of Th22, Th17 and Th1 cells are elevated in hepatic fibrosis. RmIL-22 can attenuate HSC activation and down-regulate the levels of inflammatory cytokines, thereby ameliorating liver fibrogenesis.

Background & Aims: Vascular endothelial growth factor (VEGF)-induced angiogenesis is implicated in fibrogenesis and portal hypertension. However, the function of VEGF in fibrosis resolution has not been explored., Methods: We developed a cholecystojejunostomy procedure to reconstruct biliary flow after bile duct ligation in C57BL/6 mice to generate a model of fibrosis resolution. These mice were then given injections of VEGF-neutralizing (mcr84) or control antibodies, and other mice received an adenovirus that expressed mouse VEGF or a control vector. The procedure was also performed on macrophage fas-induced apoptosis mice, in which macrophages can be selectively depleted. Liver and blood samples were collected and analyzed in immunohistochemical, morphometric, vascular permeability, real-time polymerase chain reaction, and flow cytometry assays., Results: VEGF-neutralizing antibodies prevented development of fibrosis but also disrupted hepatic tissue repair and fibrosis resolution. During fibrosis resolution, VEGF inhibition impaired liver sinusoidal permeability, which was associated with reduced monocyte migration, adhesion, and infiltration of fibrotic liver. Scar-associated macrophages contributed to this process by producing the chemokine (C-X-C motif) ligand 9 (CXCL9) and matrix metalloproteinase 13. Resolution of fibrosis was impaired in macrophage fas-induced apoptosis mice but increased after overexpression of CXCL9., Conclusions: In a mouse model of liver fibrosis resolution, VEGF promoted fibrogenesis, but was also required for hepatic tissue repair and fibrosis resolution. We observed that VEGF regulates vascular permeability, monocyte infiltration, and scar-associated macrophages function., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Aim: To investigate long-term effects of Garcinia Cambogia (GC), weight-loss supplement, on adiposity and non-alcoholic fatty liver disease in obese mice., Methods: Obesity-prone C57BL/6J mice were fed a high-fat diet (HFD, 45 kcal% fat) with or without GC (1%, w/w) for 16 wk. The HFD contained 45 kcal% fat, 20 kcal% protein and 35 kcal% carbohydrate. They were given free access to food and distilled water, and food consumption and body weight were measured daily and weekly, respectively. Data were expressed as the mean ± SE. Statistical analyses were performed using the statistical package for the social science software program. Student's t test was used to assess the differences between the groups. Statistical significance was considered at P < 0.05., Results: There were no significant changes in body weight and food intake between the groups. However, the supplementation of GC significantly lowered visceral fat accumulation and adipocyte size via inhibition of fatty acid synthase activity and its mRNA expression in visceral adipose tissue, along with enhanced enzymatic activity and gene expression involved in adipose fatty acid β-oxidation. Moreover, GC supplementation resulted in significant reductions in glucose intolerance and the plasma resistin level in the HFD-fed mice. However, we first demonstrated that it increased hepatic collagen accumulation, lipid peroxidation and mRNA levels of genes related to oxidative stress (superoxide dismutase and glutathione peroxidase) and inflammatory responses (tumor necrosis factor-α and monocyte chemoattractant protein-1) as well as plasma alanine transaminase and aspartate transaminase levels, although HFD-induced hepatic steatosis was not altered., Conclusion: GC protects against HFD-induced obesity by modulating adipose fatty acid synthesis and β-oxidation but induces hepatic fibrosis, inflammation and oxidative stress.

Aim: To investigate the expression of dipeptidyl peptidase (DPP) 8 and DPP9 in lymphocytes and various models of liver fibrosis., Methods: DPP8 and DPP9 expression were measured in mouse splenic CD4⁺ T-cells, CD8⁺ T-cells and B-cells (B220⁺), human lymphoma cell lines and mouse splenocytes stimulated with pokeweed mitogen (PWM) or lipopolysaccharide (LPS), and in dithiothreitol (DTT) and mitomycin-C treated Raji cells. DPP8 and DPP9 expression were measured in epidermal growth factor (EGF) treated Huh7 hepatoma cells, in fibrotic liver samples from mice treated with carbon tetrachloride (CCl₄) and from multidrug resistance gene 2 (Mdr2/Abcb4) gene knockout (gko) mice with biliary fibrosis, and in human end stage primary biliary cirrhosis (PBC)., Results: All three lymphocyte subsets expressed DPP8 and DPP9 mRNA. DPP8 and DPP9 expression were upregulated in both PWM and LPS stimulated mouse splenocytes and in both Jurkat T- and Raji B-cell lines. DPP8 and DPP9 were downregulated in DTT treated and upregulated in mitomycin-C treated Raji cells. DPP9-transfected Raji cells exhibited more annexin V⁺ cells and associated apoptosis. DPP8 and DPP9 mRNA were upregulated in CCl₄ induced fibrotic livers but not in the lymphocytes isolated from such livers, while DPP9 was upregulated in EGF stimulated Huh7 cells. In contrast, intrahepatic DPP8 and DPP9 mRNA expression levels were low in the Mdr2 gko mouse and in human PBC compared to non-diseased livers., Conclusion: These expression patterns point to biological roles for DPP8 and DPP9 in lymphocyte activation and apoptosis and in hepatocytes during liver disease pathogenesis.

Background & Aims: Interleukin (IL)-17 signaling has been implicated in lung and skin fibrosis. We examined the role of IL-17 signaling in the pathogenesis of liver fibrosis in mice., Methods: Using cholestatic and hepatotoxic models of liver injury, we compared the development of liver fibrosis in wild-type mice with that of IL-17RA(-/-) mice and of bone marrow chimeric mice devoid of IL-17 signaling in immune and Kupffer cells (IL-17RA(-/-) to wild-type and IL-17A(-/-) to wild-type mice) or liver resident cells (wild-type to IL-17RA(-/-) mice)., Results: In response to liver injury, levels of Il-17A and its receptor increased. IL-17A increased appeared to promote fibrosis by activating inflammatory and liver resident cells. IL-17 signaling facilitated production of IL-6, IL-1, and tumor necrosis factor-α by inflammatory cells and increased the expression of transforming growth factor-1, a fibrogenic cytokine. IL-17 directly induced production of collagen type I in hepatic stellate cells by activating the signal transducer and activator of transcription 3 (Stat3) signaling pathway. Mice devoid of Stat3 signaling in hepatic stellate cells (GFAPStat3(-/-) mice) were less susceptible to fibrosis. Furthermore, deletion of IL-23 from immune cells attenuated liver fibrosis, whereas deletion of IL-22 exacerbated fibrosis. Administration of IL-22 and IL-17E (IL-25, a negative regulator of IL-23) protected mice from bile duct ligation-induced liver fibrosis., Conclusions: IL-17 induces liver fibrosis through multiple mechanisms in mice. Reagents that block these pathways might be developed as therapeutics for patients with cirrhosis., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Although bacterial endotoxin, such as lipopolysaccharide (LPS), plays a key role in the pathogenesis of nonalcoholic steatohepatitis (NASH), detailed mechanisms of this pathogenesis remain unclear. Here, we demonstrate that upregulation of CD14 by leptin-mediated signaling is critical to hyperreactivity against endotoxin during NASH progression. Upregulation of CD14 in Kupffer cells and hyperreactivity against low-dose LPS were observed in high-fat diet (HFD)-induced steatosis mice, but not chow-fed-control mice. Hyperresponsivity against low-dose LPS led to accelerated NASH progression, including liver inflammation and fibrosis. Administering leptin in chow-fed mice caused increased hepatic expression of CD14 via STAT3 signaling, resulting in hyperreactivity against low-dose LPS without steatosis. In contrast, a marked decrease in hepatic CD14 expression was observed in leptin-deficient ob/ob mice, despite severe steatosis. Our results indicate that obesity-induced leptin plays a crucial role in NASH progression via enhanced responsivity to endotoxin, and we propose a mechanism of bacteria-mediated progression of NASH., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cannabinoid 2 (CB2) receptors expressed on immune cells are considered to be antifibrogenic. Hepatic stellate cells (HSCs) directly interact with phagocytosis lymphocytes, but the nature of this interaction is obscure. We aimed to study the effects of CB2 receptors on hepatic fibrosis via their role in mediating immunity. Hepatic fibrosis was induced by carbon-tetrachloride (CCl(4)) administration in C57BL/6 wild-type (WT) and CB2 knockout (CB2(-/-)) mice. Irradiated animals were reconstituted with WT or CB2(-/-) lymphocytes. Lymphocytes from naïve/fibrotic WT animals and healthy/cirrhotic hepatitis C virus were preincubated in vitro with or without CB2 antagonist, evaluated for proliferation and apoptosis, and then cocultured with primary mouse HSCs or a human HSC line (LX2), respectively. Lymphocyte phagocytosis was then evaluated. Following CCl(4)-administration, CB2(-/-) mice developed significant hepatic fibrosis but less necroinflammation. WT mice harbored decreased liver CD4(+) and NK(+) cells but increased CD8(+) subsets. Naïve CB2(-/-) mice had significantly decreased T cell subsets. Adoptive transfer of CB2(-/-) lymphocytes led to decreased fibrosis in the irradiated WT recipient compared with animals receiving WT lymphocytes. Moreover, necroinflammation also tended to decrease. In vitro, a CB2-antagonist directly increased human HSC activation and increased apoptosis and decreased proliferation of mice/human T cells (healthy/fibrotic) and their phagocytosis. We concluded that CB2(-/-) lymphocytes exert an antifibrotic activity, whereas lack of CB2 receptor in HSCs promotes fibrosis. These findings broaden our understanding of cannabinoid signaling in hepatic fibrosis beyond their activity solely in HSCs.

Females with sepsis have a better prognosis than males, while those of both genders with cirrhosis have a high mortality. Impaired immunity accompanies liver cirrhosis. The potential association between sex and immunologic response of cirrhotic rats in sepsis following immunonutrition was investigated. One hundred and forty-three rats were randomly divided into groups. Liver cirrhosis was produced by weekly feeding of CCl(4) for 8 weeks. Among them, 24 male and 19 female underwent castration one month before studying. The rats were fed with either immune enhancing diet or control diet for five days, then sepsis was induced with cecal ligation and two holes puncture. Main outcomes included mortality and serum cytokines (IL-1β, 6, and 10). Comparisons were made both within and between genders. Cirrhotic non-castrated male rats showed a significant decrease in mortality (64.1% vs. 32.1%, p = 0.032) with better survival than control diet following immune enhancing diet. Lower mortality of cirrhotic non-castrated female rats was found after immune enhancing diet (69.6% vs. 52.1%, p = 0.365). Cirrhotic castrated male rats showed a lower mortality (44.4%) following immune enhancing diet, and cirrhotic castrated female rats also showed significantly lower mortality and better survival than control diet after immune enhancing diet (87.5% vs. 33.3%, p = 0.004). Plasma concentrations of IL-1β were higher in non-oophorectomized female rats fed with control diet compared to immune enhancing diet. Non-orchidectomized males and non-oophorectomized females exhibited similar increases in IL-10 after immune enhancing diet. Our results demonstrated that immunonutrition was more beneficial for male than female cirrhotic rats following sepsis. Though orchidectomy was not found to be more advantageous for the normal male rats in sepsis, immunonutrition seemed to be as important as sex hormone for female rats in sepsis.

Introduction: Immunomodulatory drugs have been reported to have anti-inflammatory and anti-fibrotic properties. Thymic humoral factor (THF), a peptide produced in the thymus, causes a potent immunomodulatory effect on different components of the immune system., Objective: To evaluate the effect of THF on different stages of liver damage and fibrosis induced in rats through the administration of porcine serum (PS)., Material and Methods: PS-induced liver fibrosis models serve as a primarily immunological mechanism in the development of liver damage and fibrosis., Results: The intraperitoneal administration of THF in rats with PS-induced liver damage produced a reduction of ALT and AST after 60 days. Histopathological changes in liver sections showed an improved histological appearance and lower % of fibrosis after 60 days in liver damaged rats that received THF treatment. Serum IL-6 levels were visibly reduced by THF administration after 60 days and in comparison with rats that did not receive the treatment. This was due to an increment in serum IL-10 levels caused by the administration of THF, which appears to reduce the inflammatory process by decreasing immune response., Conclusion: THF had beneficial effects in combating liver damage and fibrosis processes in an autoimmune model of PS-induced liver fibrosis in rats.

Aim: To study the effect of salvianolate on expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 mRNA in small intestine of cirrhotic rats., Methods: Cirrhosis in rats was induced using CCl4 (0.3 mL/kg). Rats were randomly divided into non-treatment group, low-dose salvianolate (12 mg/kg) treatment group, medium-dose salvianolate (24 mg/kg) treatment group, and high-dose salvianolate (48 mg/kg) treatment group, and treated for 2 wk. Another 10 healthy rats served as a normal control group. Mortality of cirrhotic rats in each group was evaluated after treatment with salvianolate. Serum samples were taken from portal vein for the detection of endotoxin. Morphological changes in tissue samples from the ileocecum were observed under a light microscope. Expression of TNF-α and IL-6 mRNA in the small intestine of rats was analyzed by real-time reverse-transcriptase polymerase chain reaction., Results: The mortality of cirrhotic rats in the non-treatment group was 37.5%. No cirrhotic rat died in the high-dose salvianolate treatment group. The serum endotoxin level was significantly higher in the non-treatment group than in the salvianolate treatment and normal control groups. The intestinal mucosal and villous atrophy, necrosis and shedding of the intestinal mucosal epithelium, observed in the non-treatment group, were reversed in different salvianolate treatment groups. The TNF-α and IL-6 mRNA expression levels in small intestine were significantly lower in different salvianolate treatment groups than in the non-treatment group., Conclusion: Salvianolate can reduce the endotoxin level, ameliorate the injury of intestinal mucosa, and inhibit the expression of TNF-α and IL-6 mRNA in small intestine of cirrhotic rats.

Toll-like receptor 4 (TLR4) and its coreceptor, myeloid differentiation factor-2 (MD-2), are key in recognition of lipopolysaccharide (LPS) and activation of proinflammatory pathways. Here we tested the hypothesis that TLR4 and its coreceptor MD-2 play a central role in nonalcoholic steatohepatitis (NASH) and liver fibrosis in nonalcoholic fatty liver disease. Mice of control genotypes and those deficient in MD-2 or TLR4 [knockout (KO)] received methionine choline-deficient (MCD) or methionine choline-supplemented (MCS) diet. In mice of control genotypes, MCD diet resulted in NASH, liver triglycerides accumulation, and increased thiobarbituric acid reactive substances, a marker of lipid peroxidation, compared with MCS diet. These features of NASH were significantly attenuated in MD-2 KO and TLR4 KO mice. Serum alanine aminotransferase, an indicator of liver injury, was increased in MCD diet-fed genotype controls but was attenuated in MD-2 KO and TLR4 KO mice. Inflammatory activation, indicated by serum TNF-α and nictoinamide adenine dinucleotide phosphate oxidase complex mRNA expression and activation, was significantly lower in MCD diet-fed MD-2 KO and TLR4 KO compared with corresponding genotype control mice. Markers of liver fibrosis [collagen by Sirius red and α-smooth muscle actin (SMA) staining, procollagen-I, transforming growth factor-β1, α-SMA, matrix metalloproteinase-2, and tissue inhibitor of matrix metalloproteinase-1 mRNA] were attenuated in MD-2 and TLR4 KO compared with their control genotype counterparts. In conclusion, our results demonstrate a novel, critical role for LPS recognition complex, including MD-2 and TLR4, through NADPH activation in liver steatosis, and fibrosis in a NASH model in mice.

Objective: To observe the occurrence and progression of liver fibrosis induced by pig serum exposure and bile duct ligation, and analyze the relationship between hepatic inflammation and liver fibrosis., Methods: Chronically immune-mediated liver fibrosis was induced in rats by weekly injection of pig serum (IPS) into the peritoneal cavity at 3 ml/kg for 12 weeks. Cholestatic fibrosis was induced by common bile duct ligation (BDL). The Knodell score was used to evaluate the histological changes in the liver, and immunohistochemistry was performed using anti-SMA, anti-ED1, anti-CK7, and anti-CD45 antibodies. Quantitative real time PCR (qPCR) analysis was employed to quantify the mRNA expression of the genes related to inflammation, including interleukin-1beta (IL-1beta), IL-6, monocyte chemotactic protein-1, tumor necrosis factor-alpha, regulated upon activation normal T cell expressed and secreted (RANTES), transforming growth factor-beta, platelet-derived growth factor A, as well as the genes associated with fibrogenesis, namely collagen 1, alphaSMA, MMP-9 and TIMP-1., Results: Knodell scores for periportal necrosis, intralobular degeneration and focal necrosis, and portal inflammation were all significantly higher in the BDL group than in the IPS group (P<0.01), whereas the scores for fibrosis was higher in the IPS group (P<0.05). Immunohistochemistry showed obvious inflammation with numerous alphaSMA-positive cells in the liver of the rats in BDL group; the liver of the rats in IPS group showed numerous alphaSMA-positive myofibroblasts with limited inflammatory cell infiltration. qPCR demonstrated a significant up-regulation of the genes related to extracellular matrix remodeling such as collagen 1 (P<0.01), alphaSMA (P<0.01), MMP-9 (P<0.01) and TIMP-1 (P<0.01) in the rat liver in IPS group compared with those in the normal control group, and the mRNA expressions of the inflammation-related cytokines, except for RANTES, were comparable with those in the control. In contrast, the BDL group showed a significant up-regulation of all the pro-inflammatory genes examined with also increased expression of the fibrogenesis-related genes (P<0.05)., Conclusion: Liver fibrosis induced by IPS is characterized by active ECM remodeling in the absence of obvious inflammation, indicating that chronic development of liver fibrosis can be independent of active hepatic inflammation. BDL-induced liver fibrosis highlights obvious inflammation and fibrous proliferation in the liver.

Objective: To observe the changes in the hemodynamics of rats with immunological liver fibrosis and explore the pathogenesis of "blood stasis" in liver fibrosis., Methods: Rat models of liver fibrosis were established by multiple intraperitoneal injections of pig serum. The hematocrit, blood viscosity at the shear rate of 150/s, 30/s, 5/s, and 1/s, serum markers for liver fibrosis, and serum transaminase levels were measured in the control and model rats., Results: The hematocrit, blood viscosity at different shear rates, hyaluronic acid (HA), laminin (LN), procollagen type III (PCIII), type IV collagen (CIV), glutamic-pyruvic transaminase (ALT) and glutamic-oxaloacetic transaminase (AST) increased significantly in the rats with experimental liver fibrosis appeared as compared with those in the control rats. Positive correlations were noted between blood viscosity at different shear rates and serum concentrations of the fibrosis markers (HA, LN, PCIII, and CIV) in the model rats., Conclusion: The changes in the hemodynamics in rats with immunological liver fibrosis suggests the role of "blood stasis" in the pathogenesis of liver fibrosis and provide experimental evidence for therapies to "activate the blood circulation and dissipate blood stasis" for treatment of liver fibrosis.

Aim: To investigate the correlation between the antifibrotic effect of baicalin and serum cytokine production in rat hepatic fibrosis., Methods: Forty male Sprague-Dawley rats were divided randomly into four groups: normal control group, model group, baicalin-treated group, and colchicine-treated group. Except for the normal control group, all rats in the other groups were administered with carbon tetrachloride to induce hepatic fibrosis. At the same time, the last two groups were also treated with baicalin or colchicine. At the end of the 8 wk, all animals were sacrificed. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), transforming growth factor (TGF)-beta1, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-10 were measured. Liver index, hepatic hydroxyproline content and the degree of liver fibrosis were also evaluated., Results: The levels of ALT, AST and liver index in the baicalin-treated group were markedly lower than those in the model group (ALT: 143.88 +/- 14.55 U/L vs 193.58 +/- 24.35 U/L; AST: 263.66 +/- 44.23 U/L vs 404.37 +/- 68.29 U/L; liver index: 0.033 +/- 0.005 vs 0.049 +/- 0.009, P < 0.01). Baicalin therapy also significantly attenuated the degree of hepatic fibrosis, collagen area and collagen area percentage in liver tissue (P < 0.01). Furthermore, the levels of serum TGFbeta1, TNF-alpha and IL-6 were strikingly reduced in the baicalin-treated group compared with the model group, while the production of IL-10 was up-regulated: (TGF-beta1: 260.21 +/- 31.01 pg/mL vs 375.49 +/- 57.47 pg/mL; TNF-alpha: 193.40 +/- 15.18 pg/mL vs 260.04 +/- 37.70 pg/mL; IL-6: 339.87 +/- 72.95 pg/mL vs 606.47 +/- 130.73 pg/mL; IL-10: 506.22 +/- 112.07 pg/mL vs 316.95 +/- 62.74 pg/mL, P < 0.01)., Conclusion: Baicalin shows certain therapeutic effects on hepatic fibrosis, probably by immunoregulating the imbalance between profibrotic and antifibrotic cytokines.

Background & Aims: c-Jun N-terminal kinase (JNK) plays a pivotal role in the development of the metabolic syndrome including nonalcoholic fatty liver disease. However, the mechanism underlying the contribution of JNK to the progression from simple steatosis to steatohepatitis and liver fibrosis is unresolved., Methods: Hepatic steatosis, inflammation, and fibrosis were examined in wild-type, jnk1(-/-), or jnk2(-/-) mice fed a choline-deficient L-amino acid-defined (CDAA) diet for 20 weeks. The functional contribution of JNK isoforms in Kupffer cells was assessed in vitro and in vivo using chimeric mice in which the hematopoietic compartment including Kupffer cells was replaced by wild-type, jnk1(-/-), or jnk2(-/-) cells., Results: CDAA diet induced significantly less hepatic inflammation and less liver fibrosis despite a similar level of hepatic steatosis in jnk1(-/-) mice as compared with wild-type or jnk2(-/-) mice. CDAA diet-induced hepatic inflammation was chronic and mediated by Kupffer cells. Pharmacologic inhibition of JNK or gene deletion of jnk1 but not jnk2 repressed the expression of inflammatory and fibrogenic mediators in primary Kupffer cells. In vivo, CDAA diet induced less hepatic inflammation and liver fibrosis despite an equivalent level of hepatic steatosis in chimeric mice with jnk1(-/-) hematopoietic cells as compared with chimeric mice with wild-type or jnk2(-/-) hematopoietic cells., Conclusions: jnk1(-/-) mice are resistant to diet-induced steatohepatitis and liver fibrosis. JNK1 in hematopoietic cells, especially in Kupffer cells, contributes to the development of liver fibrosis by inducing chronic inflammation.

Cellular senescence acts as a potent mechanism of tumor suppression; however, its functional contribution to noncancer pathologies has not been examined. Here we show that senescent cells accumulate in murine livers treated to produce fibrosis, a precursor pathology to cirrhosis. The senescent cells are derived primarily from activated hepatic stellate cells, which initially proliferate in response to liver damage and produce the extracellular matrix deposited in the fibrotic scar. In mice lacking key senescence regulators, stellate cells continue to proliferate, leading to excessive liver fibrosis. Furthermore, senescent activated stellate cells exhibit gene expression profile consistent with cell-cycle exit, reduced secretion of extracellular matrix components, enhanced secretion of extracellular matrix-degrading enzymes, and enhanced immune surveillance. Accordingly natural killer cells preferentially kill senescent activated stellate cells in vitro and in vivo, thereby facilitating the resolution of fibrosis. Therefore, the senescence program limits the fibrogenic response to acute tissue damage.

Tissue-type plasminogen activators (tPA) and urokinase-type plasminogen activators (uPA) are involved in liver repair. We examined the potential immunomodulatory actions of uPA, tPA and uPA-receptor (uPAR) in carbon-tetrachloride-induced hepatic fibrosis in wild-type (WT), tPA-/-, uPA-/- and uPAR-/- mice. Carbon-tetrachloride treatment increased fibrosis in four groups but significantly less in three knock-out models. Serum cytokines and intrahepatic T cells elevated significantly following fibrosis process in WT animals but not in the knock-out groups. In culture, uPA increased lymphocyte proliferation significantly in WT and uPA-/- but not uPAR-/- animals. Following uPA exposure in vivo, there was CD8 predominance. To isolate uPA's effect on lymphocytes, WT mice were irradiated sublethally and then reconstituted with WT or uPA-/- lymphocytes. In these animals fibrosis was decreased and T cells were reduced in the uPA-/- recipients. Based on these data we postulate that plasminogen activators affect fibrosis in part by liver-specific activation of CD8 subsets that govern the fibrogenic activity of hepatic stellate cells.

Objective: To investigate the phenotypes of T lymphocytes in the intestinal epithelium in cirrhosis., Methods: Forty Wistar rats were randomly divided into 2 groups: cirrhosis group (n = 25) undergoing subcutaneous injection of 40% carbon tetrachloride twice a week for 10-12 weeks to establish cirrhosis models, and control group (n = 15). Twelve weeks later the rats were killed with their levers taken out. The T lymphocytes in the intestinal epithelium were isolated and labeled with mouse anti-rat CD3, CD4 and CD8 monoclonal antibody, Flow cytometry was performed., Results: The number of intestinal intraepithelial lymphocytes of the cirrhosis group was (3.4 +/- 1.1) x 10(5)/cm, significantly higher than that of the control group [(2.3 +/- 0.5) x 10(5)/cm, P < 0.05]. The proportion of CD3(+) cells of the cirrhosis group were (76 +/- 8)%, not significantly different from that of the control group [(80 +/- 6)%, P > 0.05]. There were no significant different between two group (P > 0.05). The proportion of CD4(+)CD8(+) subpopulation of the cirrhosis group was (6.9 +/- 3.3)%, significantly higher than that of the control group [(3.7 +/- 1.8)%, P < 0.05]. The proportion of CD4(+)CD8(-) subpopulation of the cirrhosis group was (6.9 +/- 3.0)%, significantly higher than that of the control group [(4.4 +/- 1.4)%, P < 0.05]., Conclusion: There were alterations of the immune barrier function in cirrhosis. The increase of the proportion of CD4(+)CD8(+) subpopulation may be associated with the increases of the number of intestinal bacteria.

Type I interferons (IFNs), IFN-alpha and IFN-beta, are widely used for treating chronic hepatitis C. Although retrospective studies have suggested that type I IFNs have direct antifibrotic effects, little is known about these mechanisms. The present study was designed to clarify the preventive mechanisms of type I IFNs in the progression of fibrosis for the establishment of a more effective therapy. A murine fibrosis model comprising immunological reactions was induced by the administration of concanavalin A (0.3 mg/body) into mice once a week for 4 weeks. Liver injury and the degree of fibrosis were determined by measuring the serum alanine aminotransferase activities and liver hydroxyproline contents with or without IFN-beta pretreatment. IFN-beta suppressed the hepatocellular injury and increased the hydroxyproline content induced by repeated concanavalin A injections, but had no effect on established fibrosis. Furthermore, IFN-beta reduced the expressions of transforming growth factor-beta, basic fibroblast growth factor, collagen type I A2 and tissue inhibitor of metalloproteinase 1 messenger RNAs, which are related to the progression of liver fibrosis. The IFN-beta reduced the liver injury and fibrosis induced by immunological reactions. These data suggest that type I IFNs suppress the progression of cirrhosis through inhibition of repeated hepatocellular injury and/or factors that promote the liver fibrosis induced by hepatitis virus infection.

Hepatic injury is associated with a defective intestinal barrier and increased hepatic exposure to bacterial products. Here we report that the intestinal bacterial microflora and a functional Toll-like receptor 4 (TLR4), but not TLR2, are required for hepatic fibrogenesis. Using Tlr4-chimeric mice and in vivo lipopolysaccharide (LPS) challenge, we demonstrate that quiescent hepatic stellate cells (HSCs), the main precursors for myofibroblasts in the liver, are the predominant target through which TLR4 ligands promote fibrogenesis. In quiescent HSCs, TLR4 activation not only upregulates chemokine secretion and induces chemotaxis of Kupffer cells, but also downregulates the transforming growth factor (TGF)-beta pseudoreceptor Bambi to sensitize HSCs to TGF-beta-induced signals and allow for unrestricted activation by Kupffer cells. LPS-induced Bambi downregulation and sensitization to TGF-beta is mediated by a MyD88-NF-kappaB-dependent pathway. Accordingly, Myd88-deficient mice have decreased hepatic fibrosis. Thus, modulation of TGF-beta signaling by a TLR4-MyD88-NF-kappaB axis provides a novel link between proinflammatory and profibrogenic signals.

Background: While CD8 lymphocytes possess pro-fibrogenic properties and NK (non-T) cells are anti-fibrogenic, the role of NKT lymphocytes in liver fibrosis is still unclear. Beta-glucosylceramide (GC), a naturally occurring glycolipid, exerts modulatory effects on these cells., Aim: To explore the role of NKT cells in hepatic fibrosis via GC., Methods: Hepatic fibrosis was induced by biweekly intra-peritoneal (IP) carbon tetrachloride (CCl(4)) administrations for 7 weeks in 5 groups (A-E) of male C57Bl/6 mice. Mice were treated with daily IP GC injections in groups A and C, or daily oral doses in groups B and D. GC was administered either for the duration of the study period (in groups A and B), or for the last 3 weeks of CCl(4) induction (groups C and D). GC-treated mice were compared with non-treated fibrotic controls (group E) and naive rodents (group F). Liver fibrosis, injury parameters and FACS analysis of lymphocytes were assessed., Results: Marked amelioration (P < 0.0001) of hepatic fibrosis observed in all GC-treated mice without altering reactive oxygen species production. As determined by Sirius red-stained liver tissue sections and measured by Bioquant morphometry; all CCl(4)-administered groups significantly (P < 0.0001) increased the relative fibrosis area compared with naive animals. The increases were 14.4 +/- 1.03-fold in group A, 7.9 +/- 0.37-fold in group B, 5.2 +/- 0.2-fold in group C, 10.3 +/- 0.4-fold in group D and 23.8 +/- 1.9-fold in group E. Western blot analysis for alpha smooth muscle actin from liver extracts followed a similar pattern, increasing in groups A-E. A significant decrease in liver damage was observed in all GC-treated groups, as noted by a decrease in transaminase serum levels (P < 0.005). The beneficial effect of GC was associated with a significant decrease in the intra-hepatic NKT and CD8 lymphocytes as well as their attenuation of both T(h)1 and T(h)2 cytokines., Conclusions: Administration of GC had a significant anti-fibrotic effect following CCl(4) administration. This effect was associated with an altered NKT and CD8 lymphocyte distribution and a cytokine shift. Immune modulation using GC may have a role in the treatment of fibrosis and other immune-mediated disorders.

Recent functional research studies suggest an anti-fibrotic role for natural killer (NK) cells coupled with a profibrotic role for CD8 cells. However, the morphological cellular interplay between the different cell types is less clear. To investigate lymphocyte/hepatic stellate cell (HSC) interactions, hepatic fibrosis was induced by administering carbon tetrachloride (CCl4) intraperitoneally (i.p.) for 4 weeks in C57Bl/6 mice. Animals were killed at 0, 1, 2, 3 and 4 weeks. Liver sections were stained for Sirius red. Confocal microscopy was used to evaluate alpha smooth-muscle actin (alphaSMA) and lymphocyte subsets in liver sections. At weeks 0 and 4, liver protein extracts were assessed for alphaSMA by Western blotting and isolated liver lymphocytes as well as HSC were analysed by fluorescence activated cell sorter (FACS). Similar to the results obtained from classical Sirius red staining and alphaSMA blotting, analysis of liver sections by confocal microscopy revealed a marked and continuous accumulation of alphaSMA staining along sequential experimental check-points after administering CCl4. Although the number of all liver lymphocyte subsets increased following fibrosis induction, FACS analysis revealed an increase in the distribution of liver CD8 subsets and a decrease of CD4 T cells. Confocal microscopy showed a significant early appearance of CD8 and NK cells, and to a lesser extent CD4 T cells, appearing only from week 2. Lymphocytes were seen in proximity only to HSC, mainly in the periportal area and along fibrotic septa, suggesting a direct interaction. Notably, lymphocyte subsets were undetectable in naive liver sections. Freshly isolated HCS show high expression of major histocompatibility complex (MHC) class II and CD11c. In the animal model of hepatic fibrosis, lymphocytes infiltrate into the liver parenchyma and it is thought that they attach directly to activated HSC. Because HSCs express CD11c/class II molecules, interactions involving them might reflect that HSCs have an antigen-presenting capacity.

Models of liver fibrosis, which include cell culture models, explanted and biopsied human material, and experimental animal models, have demonstrated that liver fibrosis is a highly dynamic example of solid organ wound healing. Recent work in human and animal models has shown that liver fibrosis is potentially reversible and, in specific circumstances, demonstrates resolution with a restoration of near normal architecture. This Review highlights the manner in which studies of models of liver fibrosis have contributed to the paradigm of dynamic wound healing in this solid organ.

Primary biliary cirrhosis (PBC) is an autoimmune disease with a strong genetic component characterized by biliary ductular inflammation with eventual liver cirrhosis. The serologic hallmark of PBC is antimitochondrial antibodies that react with the pyruvate dehydrogenase complex, targeting the inner lipoyl domain of the E2 subunit (anti-PDC-E2). Herein we demonstrate that NOD.c3c4 mice congenically derived from the nonobese diabetic strain develop an autoimmune biliary disease (ABD) that models human PBC. NOD.c3c4 (at 9-10 wk, before significant biliary pathology) develop antibodies to PDC-E2 that are specific for the inner lipoyl domain. Affected areas of biliary epithelium are infiltrated with CD3+, CD4+, and CD8+ T cells, and treatment of NOD.c3c4 mice with monoclonal antibody to CD3 protects from ABD. Furthermore, NOD.c3c4-scid mice develop disease after adoptive transfer of splenocytes or CD4+ T cells, demonstrating a central role for T cells in pathogenesis. Histological analysis reveals destructive cholangitis, granuloma formation, and eosinophilic infiltration as seen in PBC, although, unlike PBC, the extrahepatic biliary ducts are also affected. Using a congenic mapping approach, we define the first ABD (Abd) locus, Abd1. These results identify the NOD.c3c4 mouse as the first spontaneous mouse model of PBC.

Background & Aims: Viral hepatitis infection, which is a major cause of liver fibrosis, is associated with activation of innate immunity. However, the role of innate immunity in liver fibrosis remains obscure., Methods: Liver fibrosis was induced either by feeding mice with the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet or by injecting them with carbon tetrachloride. The Toll-like receptor 3 ligand, polyinosinic-polycytidylic acid, was used to activate innate immunity cells and mediators, including natural killer cells and interferon gamma., Results: In the mouse model of DDC-induced liver fibrosis, natural killer cell activation by polyinosinic-polycytidylic acid induced cell death to activated hepatic stellate cells and attenuated the severity of liver fibrosis. Polyinosinic-polycytidylic acid treatment also ameliorated liver fibrosis induced by carbon tetrachloride. The observed protective effect of polyinosinic-polycytidylic acid on liver fibrosis was diminished through either depletion of natural killer cells or by disruption of the interferon gamma gene. Expression of retinoic acid early inducible 1, the NKG2D ligand, was undetectable on quiescent hepatic stellate cells, whereas high levels were found on activated hepatic stellate cells, which correlated with the resistance and susceptibility of quiescent hepatic stellate cells and activated hepatic stellate cells to natural killer cell lysis, respectively. Moreover, treatment with polyinosinic-polycytidylic acid or interferon gamma enhanced the cytotoxicity of natural killer cells against activated hepatic stellate cells and increased the expression of NKG2D and tumor necrosis factor-related apoptosis-inducing ligand on liver natural killer cells. Blocking NKG2D or tumor necrosis factor-related apoptosis-inducing ligand with neutralizing antibodies markedly diminished the cytotoxicity of polyinosinic-polycytidylic acid-activated natural killer cells against activated hepatic stellate cells., Conclusions: Our findings suggest that natural killer cells kill activated hepatic stellate cells via retinoic acid early inducible 1/NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent mechanisms, thereby ameliorating liver fibrosis.

Rats infected with the helminth Capillaria hepatica regularly develop septal fibrosis of the liver similar to that induced by repeated ip injections of pig serum. Fibrosis starts when the focal parasitic lesions begin to show signs of resorption, thus suggesting an immunologically mediated pathogenesis of this fibrosis. To explore this possibility, the development of C. hepatica-related hepatic fibrosis was observed in rats exposed to worm antigens from the first neonatal day onward. Wistar rats (150 g) were either injected ip with an extract of C. hepatica eggs (protein concentration: 1 mg/ml) or received immature eggs by gavage from the first neonatal day until adult life and were then infected with 500 embryonated eggs. Changes were monitored on the basis of serum levels of anti-worm antibodies and hepatic histopathology. Rats submitted to immunological oral tolerance markedly suppressed C. hepatica-related serum antibodies and septal fibrosis of the liver when infected with the helminth later on. Tolerance trials with ip injections of worm antigens gave essentially negative results. The partial suppression of septal fibrosis of the liver after the induction of immunological tolerance to C. hepatica antigens in rats indicates an immunological basis for the fibrosis and emphasizes the importance of immunological factors in the pathogenesis of hepatic fibrosis.

Several lines of evidence suggest that interferon (IFN)-alpha is effective in suppression of liver cirrhosis (LC) as well as hepatitis C virus (HCV) infection, which is a major cause of LC in Japan. However, IFN-alpha often causes systemic toxicity such as flu-like symptoms, which precludes the IFN-alpha dose escalation required for clinical efficacy. Since IFN-alpha is rapidly degraded in the blood circulation, only a small amount of subcutaneously injected IFN-alpha protein can reach the target organ, the liver. It is expected that on-site IFN-alpha production in the liver overcomes the limitation of the conventional parenteral IFN-alpha administration. An adenovirus vector expressing the rat IFN-alpha gene (AxCA-rIFN) was injected intravenously into rats with dimethylnitrosamine-induced LC. While the subcutaneous IFN-alpha protein injection led to a transient elevation of the cytokine both in the liver and serum, the vector-mediated IFN-alpha gene transduction induced a significant amount of IFN-alpha detected in the liver but not in the serum. The injection of AxCA-rIFN prevented the progression of the rat LC, and improved the survival rate of the treated rats. Although no significant toxicity was noted in the animals, we showed that IFN-alpha gene expression in the liver can be efficiently downregulated by the Cre/loxP-mediated shut-off system, in case the IFN-alpha overdose becomes a problem. The study suggested for the first time the advantage and feasibility of IFN-alpha gene therapy for LC.

Background & Aims: Hepatocyte transplantation has been proposed as an alternative to liver transplantation for the treatment of hepatic failure. A major limitation to this form of therapy is the availability of human livers as a source of hepatocytes. The use of porcine hepatocytes might address this problem; however, xenogeneic hepatocytes are thought to be functionally incompatible across species and susceptible to irreversible rejection., Methods: Liver cirrhosis was induced with phenobarbital and carbon tetrachloride. Only rats with decompensated liver failure that did not correct 4 weeks after the discontinuation of carbon tetrachloride were subjected to intrasplenic rat or porcine hepatocyte transplantation. The immunologic integrity of cirrhotic rats was assessed by allogeneic skin grafting, and the immune response to transplanted porcine hepatocytes was assessed by enzyme-linked immunosorbent assay., Results: Porcine hepatocytes restored metabolic function and prolonged the survival of cirrhotic rats, as well as rat hepatocytes. Cirrhotic rats retained the ability to reject allogeneic skin grafts and showed an immune response to the engrafted hepatocytes. Despite this, survival of transplanted porcine hepatocytes was accepted in cirrhotic rats for a period of weeks without immunosuppression. Conventional immunosuppression with FK506 allowed successful retransplantation with hepatocytes from a second porcine donor., Conclusions: Hepatocytes transplanted between widely divergent species can function to correct liver failure in cirrhotic rats and prolong their survival. Conventional immunosuppression allows long-term functioning of xenogeneic hepatocyte retransplants and suggests that hepatocyte xenotransplantation might be useful as a bridge to liver transplantation and could potentially provide long-term hepatic support.

Transplanted hepatocytes are ideal carriers for exogenous genes in liver gene therapy. The present study investigated the anti-fibrogenic effects of intrasplenically transplanted hepatocytes modified with interferon gamma (IFN-gamma) gene on cirrhotic rats. Hepatocytes isolated from normal Sprague-Dawley (SD) rats were transfected with an adenoviral vector encoding human IFN-gamma gene (AdCMVhIFN-gamma) and transplanted into the spleens of syngenic recipients with ongoing liver fibrosis induced by carbon tetrachloride (CCl(4)). Histology was assessed, and liver hydroxyproline was detected. Additionally, serum procollagen type III (PIIINP) levels and hepatic collagenase activity were measured to determine hepatic collagen synthesis and degradation. Transplantation with AdCMVhIFN-gamma transfected hepatocytes ameliorated the histological outcome of liver fibrosis by reducing liver collagen content and decreasing hepatic hydroxyproline. Additionally, IFN-gamma transfected hepatocytes reduced serum PIIINP levels and increased hepatic collagenase activity. Our data suggest that transplantation of IFN-gamma transfected hepatocytes may reduce the pace of liver fibrosis by inhibiting the synthesis and enhancing the degradation of hepatic collagen.

Mice sensitized with SCHISTOSOMA: mansoni eggs and IL-12 develop liver granulomas, on subsequent infection, which are smaller and less fibrotic than those in nonsensitized mice. The protective response is accompanied by a shift in the type-2 cytokine profile to one dominated by type-1 cytokines. The deviated response is associated with marked increases in inducible nitric oxide synthase (NOS-2) activity. Here, we demonstrate, by using NOS-2-deficient mice, that the anti-inflammatory and anti-fibrotic effects of the type-1 response are completely NOS-2-dependent. Strikingly, despite developing a polarized type-1 cytokine response that was similar in magnitude, the egg/IL-12-sensitized NOS-deficient mice developed granulomas 8 times larger than WT mice did. There was also no decrease in hepatic fibrosis in the sensitized mutant animals. Interferon-gamma-deficient mice failed to exhibit the exacerbated inflammatory response, despite displaying a marked deficiency in nitric oxide production. However, immune deviation was unsuccessful in the latter animals, which suggested that the increase in inflammation in NOS-deficient mice resulted from a polarized but nitric oxide-deficient type-1 response. These results reveal a beneficial role for NOS-2 in the regulation of inflammation and suggest that the ultimate success of Th2-to-Th1 immune deviation strategies will rely on the efficient activation of NOS-2 expression in downstream effector cells.

Chronic intermittent injection of carbon tetrachloride (CCl4) for more than 10 weeks induced liver fibrosis in mice, as evidenced by positive Azan staining and increased intrahepatic collagen content. Preceding the onset of liver fibrosis, interleukin-6 (IL-6) gene expression was enhanced in liver and immunoreactive IL-6 was detected in infiltrating inflammatory cells. To delineate the role of IL-6 in this process, we treated IL-6-deficient mice with CCl4 in a similar manner for 12 weeks, after which fibrotic changes were less evident and serum albumin levels were lower in IL-6-deficient than wild-type mice. Moreover, CCl4-induced expression of transforming growth factor beta1 and hepatocyte growth factor genes in liver was significantly reduced in IL-6-deficient mice. Thus, IL-6 may be vitally involved in fibrotic changes and maintenance of serum albumin levels, partly by modulating intrahepatic expression of these cytokines.

Liver fibrosis is commonly observed in chronic liver disease. However, the immunological mechanisms underlying hepatic fibrosis due to chronic inflammation are not well defined, mainly because suitable experimental models have not been established. We have found that weekly i.v. administration of concanavalin A (Con A) in BALB/c mice brought about a striking alanine aminotransferase increase, resulting in piecemeal necrosis with bridging fibrosis in the parenchyma. Using this fibrosis model, we demonstrated the kinetics of cytokine mRNA expression in liver. Transforming growth factor (TGF)-beta1, TGF-alpha, basic fibroblast growth factor (bFGF) and hepatocyte growth factor mRNAs were up-regulated after each Con A administration. Furthermore, either anti-IFN-gamma, anti-tumor necrosis factor (TNF)-alpha or anti-TGF-beta mAb given together with Con A markedly inhibited the development of hepatic fibrosis. Treatment with either anti-IFN-gamma or anti-TNF-alpha mAb also completely prevented hepatic injury; in contrast, treatment with anti-TGF-beta mAb did not. The treatment with anti-TGF-beta mAb did not affect the levels of hepatic mRNAs for either IFN-gamma or TNF-alpha after Con A injection. Treatment with either anti-IFN-gamma or anti-TNF-alpha did not affect the expression levels of TGF-beta in the liver. In conclusion, the continuous presence of both severe liver damage and up-regulation of TGF-beta synthesis is necessary to induce hepatic fibrosis in this model.

We investigated the role of pneumolysin's complement-activating activity during Streptococcus pneumoniae bacteremia in a hypocomplementemic, cirrhotic host. Isogenic mutant pneumococcal strains, in which pneumolysin was expressed from a plasmid, were used. These strains included H+C+, expressing wild-type pneumolysin with both cytolytic and complement-activating activity; PLY-, carrying the plasmid without the pneumolysin gene; and, H+C-, expressing pneumolysin with cytolytic activity only. In control rats, intravenous infection with 2.0 x 10(7) CFU of H+C+ per ml of blood resulted in a decrease in bacteremia of 3.5 log units by 18 h postinfection and 55% mortality. By contrast, cirrhotic rats infected similarly with the H+C+ strain demonstrated a 0.2-log-unit increase in bacteremia by 18 h postinfection and 100% mortality. Both control and cirrhotic rats cleared the PLY- strain more effectively from their bloodstreams by 18 h postinfection (6.2 and 5. 6 log unit decreases, respectively). Infection with the PLY- strain also resulted in low mortality (0 and 14%, respectively) for control and cirrhotic rats. When infected with the H+C- strain (without complement-activating activity), both groups cleared the organism from their bloodstreams nearly as well as they did the PLY- strain. Furthermore, the mortality rate for control and cirrhotic rats was identical after infection with the H+C- strain. These studies suggest that pneumolysin production contributes to decreased pneumococcal clearance from the bloodstream and higher mortality in both control and cirrhotic rats. However, pneumolysin's complement-activating activity may uniquely enhance pneumococcal virulence in the hypocomplementemic, cirrhotic host.