Serotonin protects mouse liver from cholestatic injury by decreasing bile salt pool after bile duct ligation.

UNLABELLED: Obstructive cholestasis induces liver injury, postoperative complications, and mortality after surgery. Adaptive control of cholestasis, including bile salt homeostasis, is necessary for recovery and survival. Peripheral serotonin is a cytoprotective neurotransmitter also associated with liver regeneration. The effect of serotonin on cholestatic liver injury is not known. Therefore, we tested whether serotonin affects the severity of cholestatic liver injury. We induced cholestasis by ligation of the bile duct (BDL) in either wild-type (WT) mice or mice lacking peripheral serotonin (Tph1(-/-) and immune thrombocytopenic [ITP] mice). Liver injury was assessed by the levels of plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT) and tissue necrosis. Bile salt-regulating genes were measured by quantitative polymerase chain reaction and confirmed by western blotting and immunohistochemistry. Tph1(-/-) mice displayed higher levels of plasma AST, ALT, bile salts, and hepatic necrosis after 3 days of BDL than WT mice. Likewise, liver injury was disproportional in ITP mice. Moreover, severe cholestatic complications and mortality after prolonged BDL were increased in Tph1(-/-) mice. Despite the elevation in toxic bile salts, expression of genes involved in bile salt homeostasis and detoxification were not affected in Tph1(-/-) livers. In contrast, the bile salt reabsorption transporters Ostα and Ostß were up-regulated in the kidneys of Tph1(-/-) mice, along with a decrease in urinary bile salt excretion. Serotonin reloading of Tph1(-/-) mice reversed this phenotype, resulting in a reduction of circulating bile salts and liver injury. CONCLUSION: We propose a physiological function of serotonin is to ameliorate liver injury and stabilize the bile salt pool through adaptation of renal transporters in cholestasis.

Partial bile duct ligation in mice: a novel model of acute cholestasis.

BACKGROUND: The standard model for research in cholestasis is the total ligation of the bile duct (tBDL). Because this model causes severe hepatic injury in mice, we developed a novel model of cholestasis using a partial bile duct ligation (pBDL) and evaluate different mechanisms of injury. METHODS: Male C57Bl/6 mice were subjected to sham operation, tBDL, or pBDL. Blood from tail veins was taken repeatedly until day 14 after surgery to assess markers of tissue injury (aspartate aminotransferase [AST]) and cholestasis (bilirubin, alkaline phosphatase [AP]). Also, liver samples were obtained at various time points to determine the histologic injury (hematoxylin and eosin) and tissue repair (Ki67). In addition, the biliary pressure and serum bile acids were evaluated as potential mechanisms of injury. RESULTS: Both models of cholestasis were equal in terms of bilirubin, AST, and AP serum levels during the first week of the experiment. Although these parameters remained constantly elevated thereafter in the tBDL model, all parameters normalized within the second week after pBDL. Moreover, pBDL resulted in significantly less necrosis formation (P = .001) and consequent hepatocyte proliferation (P= .01). Most important, serum bile acid levels (P = .04) and biliary pressures (P = .02) were significantly lower after pBDL than after tBDL and were the best predictors for hepatic necrosis formation. CONCLUSION: We established a model of acute cholestasis, which is ideal for research in resolved acute cholestasis (eg, surgery for Klatskin tumors). Moreover, biliary pressure and toxic bile acid serum levels may be better predictors of cholestatic liver injury than standard laboratory parameters.

Serotonin regulates macrophage-mediated angiogenesis in a mouse model of colon cancer allografts.

Serotonin, a neurotransmitter with numerous functions in the central nervous system (CNS), is emerging as an important signaling molecule in biological processes outside of the CNS. Recent advances have implicated serotonin as a regulator of inflammation, proliferation, regeneration, and repair. The role of serotonin in tumor biology in vivo has not been elucidated. Using a genetic model of serotonin deficiency (Tph1(-/-)) in mice, we show serotonin to be crucial for the growth of s.c. colon cancer allografts in vivo. Serotonin does not enhance tumor cell proliferation but acts as a regulator of angiogenesis by reducing the expression of matrix metalloproteinase 12 (MMP-12) in tumor-infiltrating macrophages, entailing lower levels of angiostatin-an endogenous inhibitor of angiogenesis. Accordingly, serotonin deficiency causes slower growth of s.c. tumors by reducing vascularity, thus increasing hypoxia and spontaneous necrosis. The biological relevance of these effects is underscored by the reconstitution of serotonin synthesis in Tph1(-/-) mice, which restores allograft phenotype in all aspects. In conclusion, we show how serotonin regulates angiogenesis in s.c. colon cancer allografts by influencing MMP-12 expression in tumor-infiltrating macrophages, thereby affecting the production of circulating angiostatin.

Aggravation of viral hepatitis by platelet-derived serotonin.

More than 500 million people worldwide are persistently infected with hepatitis B virus or hepatitis C virus. Although both viruses are poorly cytopathic, persistence of either virus carries a risk of chronic liver inflammation, potentially resulting in liver steatosis, liver cirrhosis, end-stage liver failure or hepatocellular carcinoma. Virus-specific T cells are a major determinant of the outcome of hepatitis, as they contribute to the early control of chronic hepatitis viruses, but they also mediate immunopathology during persistent virus infection. We have analyzed the role of platelet-derived vasoactive serotonin during virus-induced CD8(+) T cell-dependent immunopathological hepatitis in mice infected with the noncytopathic lymphocytic choriomeningitis virus. After virus infection, platelets were recruited to the liver, and their activation correlated with severely reduced sinusoidal microcirculation, delayed virus elimination and increased immunopathological liver cell damage. Lack of platelet-derived serotonin in serotonin-deficient mice normalized hepatic microcirculatory dysfunction, accelerated virus clearance in the liver and reduced CD8(+) T cell-dependent liver cell damage. In keeping with these observations, serotonin treatment of infected mice delayed entry of activated CD8(+) T cells into the liver, delayed virus control and aggravated immunopathological hepatitis. Thus, vasoactive serotonin supports virus persistence in the liver and aggravates virus-induced immunopathology.

Extralymphatic virus sanctuaries as a consequence of potent T-cell activation.

T helper cells can support the functions of CD8(+) T cells against persistently infecting viruses such as murine lymphocytic choriomeningitis virus (LCMV), cytomegalovirus, hepatitis C virus and HIV. These viruses often resist complete elimination and remain detectable at sanctuary sites, such as the kidneys and other extralymphatic organs. The mechanisms underlying this persistence are not well understood. Here we show that mice with potent virus-specific T-cell responses have reduced levels and delayed formation of neutralizing antibodies, and these mice fail to clear LCMV from extralymphatic epithelia. Transfer of virus-specific B cells but not virus-specific T cells augmented virus clearance from persistent sites. Virus elimination from the kidneys was associated with the formation of IgG deposits in the interstitial space, presumably from kidney-infiltrating B cells. CD8(+) T cells in the kidneys of mice that did not clear virus from this site were activated but showed evidence of exhaustion. Thus, we conclude that in this model of infection, site-specific virus persistence develops as a consequence of potent immune activation coupled with reductions in virus-specific neutralizing antibodies. Our results suggest that sanctuary-site formation depends both on organ anatomy and on the induction of different adaptive immune effector mechanisms. Boosting T-cell responses alone may not reduce virus persistence.

Routine stenting reduces urologic complications as compared with stenting "on demand" in adult kidney transplantation.

OBJECTIVES: To examine the impact of the chosen surgical technique and of systematic versus "on-demand" placement of a primary stent on the incidence of urologic complications in adult kidney transplantation. METHODS: Data of 497 consecutive patients undergoing kidney transplantation at a single center were retrospectively analyzed with respect to urologic complications. Three different surgical strategies for the ureteroneocystostomy were compared: (1) transvesical anastomosis with stenting "on demand," (2) extravesical anastomosis with stenting "on demand," and (3) extravesical anastomosis with routine stenting. Nine parameters were evaluated by logistic regression for a possible contribution to the development of urologic complications. RESULTS: Routine placement of a stent significantly reduced the number of urologic complications compared with both transvesical or extravesical anastomoses with stenting "on demand" (20.8% in transvesical "on demand," 17.9% in extravesical "on demand," and 5.8% in extravesical "routine"). Logistic regression analysis revealed that routine stenting versus stenting "on demand" (P = 0.001) and living donor transplantation (P = 0.038) are two independent factors associated with a significantly lower incidence of urologic complications. Although routine stenting was not associated with an increased incidence of urinary tract infections, female gender was the only independent factor associated with this complication (P = 0.001). CONCLUSIONS: Routine stenting of the ureteroneocystostomy is superior to stenting "on demand" in adult kidney transplantation, suggesting that the intraoperative decision of whether to stent is insufficient to avoid urologic complications.

Serotonin mediates oxidative stress and mitochondrial toxicity in a murine model of nonalcoholic steatohepatitis.

BACKGROUND AND AIMS: Nonalcoholic steatohepatitis (NASH) is one of the most common causes of liver enzyme elevation in the West. Its prevalence is likely to increase further, paralleling the epidemic increase of the metabolic syndrome. Serotonin degradation by monoamine oxidase A (MAO-A) was recently implicated as an important source of reactive oxygen species. We therefore tested the pathogenetic role of serotonin in a murine model of diet-induced steatohepatitis. METHODS: Wild-type and serotonin-deficient mice, tryptophan hydroxylase 1 (Tph1(-/-)) were fed a choline-methionine-deficient diet for 2 and 6 weeks. MAO-A was inhibited with clorgyline. Steatosis, hepatocyte injury, and hepatic inflammation were assessed by histology, immunohistochemistry, and biochemical analysis. Expression levels of MAO-A and serotonin transporter were analyzed by reverse-transcription polymerase chain reaction and Western blot. Oxidative stress was detected by measuring lipid peroxidation. Mitochondrial damage was determined by electron microscopy and quantification of cytochrome c release. RESULTS: After choline-methionine-deficient diet, Tph1(-/-) mice displayed an equal degree of steatosis, yet reduced hepatocellular injury and less severe inflammation. The difference in these NASH-defining features could be attributed to an increased uptake and catabolism of serotonin, yielding enhanced levels of reactive oxygen species and lipid peroxides, which mediated hepatocellular injury by mitochondrial damage and inflammation. Inhibition of MAO-A reduced hepatocellular damage in wild-type mice. Correspondingly, MAO-A expression was up-regulated significantly in human NASH. CONCLUSIONS: This study provides evidence that serotonin plays a role in the pathogenesis of steatohepatitis, and therefore might represent a novel target for the prevention and treatment of NASH.

Platelets and platelet-derived serotonin promote tissue repair after normothermic hepatic ischemia in mice.

UNLABELLED: Hepatic ischemia and reperfusion (I/R) leads to the formation of leukocyte-platelet aggregates. Upon activation, platelets generate reactive oxygen species and release proapoptotic and proinflammatory mediators as well as growth factors. In cold hepatic ischemia, adhesion of platelets to endothelial cells mediates sinusoidal endothelial cell apoptosis. Furthermore, platelet-derived serotonin mediates liver regeneration. We hypothesized that platelets may contribute to reperfusion injury and repair after normothermic hepatic ischemia. The aim of this study was to assess the impact of platelets in normothermic hepatic I/R injury using models of impaired platelet function and immune thrombocytopenia. Inhibition of platelet function in mice was achieved via clopidogrel feeding. Immune thrombocytopenia was induced via intraperitoneal injection of anti-CD41 antibody. Platelet-derived serotonin was investigated using mice lacking tryptophan hydroxylase 1. Mice were subjected to 60 minutes of partial hepatic ischemia and various time points of reperfusion. Hepatic injury was determined via AST and histological analysis of the necrotic area as well as leukocyte infiltration. Liver regeneration was determined via proliferating cell nuclear antigen and Ki67 immunohistochemistry. Neither inhibition of platelet function nor platelet depletion led to a reduction of I/R injury. Liver regeneration and repair were significantly impaired in platelet-depleted animals. Mice lacking peripheral serotonin were deficient in hepatocyte proliferation, but otherwise displayed normal tissue remodeling. CONCLUSION: Platelets have no direct impact on the pathogenesis of normothermic I/R injury. However, they mediate tissue repair and liver regeneration. Furthermore, platelet-derived serotonin is a mediator of hepatocyte proliferation in the postischemic liver, but has no impact on tissue remodeling.

Increased susceptibility to bacterial superinfection as a consequence of innate antiviral responses.

The reason why severe localized or systemic virus infections enhance and aggravate bacterial superinfection is poorly understood. Here we show that virus-induced IFN type I caused apoptosis in bone marrow granulocytes, drastically reduced granulocyte infiltrates at the site of bacterial superinfection, caused up to 1,000-fold higher bacterial titers in solid organs, and increased disease susceptibility. The finding that the innate antiviral immune response reduces the antibacterial granulocyte defense offers an explanation for enhanced susceptibility to bacterial superinfection during viral disease.

Immunoprivileged status of the liver is controlled by Toll-like receptor 3 signaling.

The liver is known to be a classical immunoprivileged site with a relatively high resistance against immune responses. Here we demonstrate that highly activated liver-specific effector CD8+ T cells alone were not sufficient to trigger immune destruction of the liver in mice. Only additional innate immune signals orchestrated by TLR3 provoked liver damage. While TLR3 activation did not directly alter liver-specific CD8+ T cell function, it induced IFN-alpha and TNF-alpha release. These cytokines generated expression of the chemokine CXCL9 in the liver, thereby enhancing CD8+ T cell infiltration and liver disease in mice. Thus, nonspecific activation of innate immunity can drastically enhance susceptibility to immune destruction of a solid organ.

Kupffer cell-dependent TNF-alpha signaling mediates injury in the arterialized small-for-size liver transplantation in the mouse.

Implantation of small liver grafts causes liver injury and defective regeneration leading to graft failure. We investigated whether Kupffer cell-dependent TNF-alpha signaling contributes to this poor outcome. Partial 30% liver transplantation was performed in C57BL/6 wild-type mice (control group), and in three groups with down-regulation of the TNF-alpha pathway: (i) TNF receptor 1 knockout [TNFR-1(-/-)] mice, and mice pretreated with (ii) gadolinium chloride or (iii) pentoxifylline (PTX). Fifty-percent partial liver transplantation, a model associated with full recovery, and transplantation in IL-6 knockout [IL-6(-/-)] mice were performed in some experiments. Graft injury, regeneration, portal flow, liver microcirculation, leukocyte adhesion, and animal survival were assessed. Animal survival rates were 14% in the control group vs. 43% in the gadolinium chloride group, 57% for the TNFR-1(-/-) group, and 86% in the PTX group (P < 0.001). Markers of liver injury were reduced in all treated groups when compared with controls. Each treated group disclosed better portal flow and sinusoid perfusion, decreased leukocyte adherence, particularly in the PTX group. Liver regeneration occurred only in the treated groups. IL-6 and IL-10 levels were dramatically up-regulated (50x) in the PTX group, and at lower levels in other experimental groups. The protective effect of PTX was lost in IL-6(-/-) mice and protection was restored by a single dose of r-IL-6. In conclusion, interruption of TNF-alpha signaling or depletion of Kupffer cells improves survival after 30% liver transplantation, reduces liver injury, and enhances regeneration. The superior effects of PTX are mediated by IL-6.

Small-for-size syndrome after partial liver transplantation: definition, mechanisms of disease and clinical implications.

Widespread application of cadaveric split or living donor liver transplantation bears considerable potential to increase the pool of available organs and thus alleviate the problem of organ shortage. Although splitting of a cadaveric liver into two grafts for adult recipients can be performed successfully, sufficient function of undersized grafts is a major concern. To minimize the risk for living donors, transplant surgeons aim at procuring the least necessary liver volume, also leading to potentially small grafts. When small partial grafts are unable to meet the functional demands, the recipients can develop a so-called small-for-size syndrome (SFSS). There is currently limited data on the pathogenesis of SFSS, with clinical studies mainly focusing on portal hyperperfusion. Additional aspects include graft-related factors such as functional and regenerative capacity, as well as recipient-related factors, such as overall health status and severity of cirrhosis. However, there is currently no consensus on the definition of SFSS. We propose a novel definition, based on simple clinical criteria, which divides the syndrome into either nonfunction or dysfunction of a small graft after the exclusion of other causes. This definition should ease comparability of future clinical trials, and thus improve understanding of the pathogenesis of SFSS.