Neuroinflammatory Signals during Acute and Chronic Liver Diseases

A spectrum of neurological complications can result from acute and chronic liver diseases and is termed hepatic encephalopathy. The precise pathogenic mechanisms by which hepatic encephalopathy occurs is unclear. However, it is commonly accepted that the development of hepatic encephalopathy shares a long-standing relationship with neuroinflammation. This chapter will outline the evidence for a role of neuroinflammation and proinflammatory cytokines in the pathogenesis of hepatic encephalopathy. Furthermore, we will identify the possible circulating factors, released from the liver after damage, that may contribute to the neurological complications of hepatic encephalopathy, including neuroinflammation. Lastly, we discuss the current and experimental treatment options aimed at reducing neuroinflammation for the management of hepatic encephalopathy

Hypothalamus-Pituitary-Adrenal Dysfunction in Cholestatic Liver Disease

The Hypothalamic-Pituitary-Adrenal (HPA) axis has an important role in maintaining the physiological homeostasis in relation to external and internal stimuli. The HPA axis dysfunctions were extensively studied in neuroendocrine disorders such as depression and chronic fatigue syndrome but less so in hepatic cholestasis, cirrhosis or other liver diseases. The HPA axis controls many functions of the liver through neuroendocrine forward signaling pathways as well as negative feedback mechanisms, in health and disease. This review describes cell and molecular mechanisms of liver and HPA axis physiology and pathology. Evidence is presented from clinical and experimental model studies, demonstrating that dysfunctions of HPA axis are correlated with liver cholestatic disorders. The functional interactions of HPA axis with the liver and immune system in cases of bacterial and viral infections are also discussed. Proinflammatory cytokines stimulate glucocorticoid (GC) release by adrenals but they also inhibit bile acid (BA) efflux from liver. Chronic hepatic inflammation leads to cholestasis and impaired GC metabolism in the liver, so that HPA axis becomes depressed. Recently discovered interactions of GC with self-oscillating transcription factors that generate circadian rhythms of gene expression in brain and liver, in the context of GC replacement therapies, are also outlined

Modulation of the biliary expression of arylalkylamine N-acetyltransferase alters the autocrine proliferative responses of cholangiocytes

Cholangiocytes secrete several neuroendocrine factors regulating biliary functions by autocrine/paracrine mechanisms (Alpini et al., 1994). Melatonin inhibits biliary growth and secretin-stimulated choleresis in cholestatic rats by interaction with melatonin recepror 1 (MT1) (Renzi et al., 2011). We will try to localize the key enzyme involved in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT), in cholangiocytes and, possibly, we will try to determine the effect of modulation of AANAT on the autocrine proliferative/secretory responses of cholangiocytes. In liver sections we found that: (i) AANAT is expressed by cholangiocytes and hepatocytes; (ii) the cholangiocytes expression of AANAT decreased in morpholino (AANAT down regulator)-treated rats; and (iii) the decrease in AANAT expression and subsequent lower melatonin secretion by cholangiocytes is associated with increased biliary proliferation and increased expression of Secretin Receptor (SR) and VEGFA/ C. In vitro, we observed that overexpression of AANAT in large cholangiocyte (LC) decreased proliferation and ablated secretin-stimulated biliary secretion. These results indicate that: in vivo down-regulation of biliary AANAT stimulates cholangiocyte proliferation by an autocrine loop, and in vitro overexpression of AANAT in LC decreases proliferation. Local targeting of AANAT in cholangiocytes may be important for the management of cholangiopathies

Fractalkine suppression during hepatic encephalopathy promotes neuroinflammation in mice

BACKGROUND: Acute liver failure is associated with numerous systemic consequences including neurological dysfunction, termed hepatic encephalopathy, which contributes to mortality and is a challenge to manage in the clinic. During hepatic encephalopathy, microglia activation and neuroinflammation occur due to dysregulated cell signaling and an increase of toxic metabolites in the brain. Fractalkine is a chemokine that is expressed primarily in neurons and through signaling with its receptor CX3CR1 on microglia, leads to microglia remaining in a quiescent state. Fractalkine is often suppressed during neuropathies that are characterized by neuroinflammation. However, the expression and subsequent role of fractalkine on microglia activation and the pathogenesis of hepatic encephalopathy due to acute liver failure is unknown. METHODS: Hepatic encephalopathy was induced in mice via injection of azoxymethane (AOM) or saline for controls. Subsets of these mice were implanted with osmotic minipumps that infused soluble fractalkine or saline into the lateral ventricle of the brain. Neurological decline and the latency to coma were recorded in these mice, and brain, serum, and liver samples were collected. Neurons or microglia were isolated from whole brain samples using immunoprecipitation. Liver damage was assessed using hematoxylin and eosin staining and by measuring serum liver enzyme concentrations. Fractalkine and CX3CR1 expression were assessed by real-time PCR, and proinflammatory cytokine expression was assessed using ELISA assays. RESULTS: Following AOM administration, fractalkine expression is suppressed in the cortex and in isolated neurons compared to vehicle-treated mice. CX3CR1 is suppressed in isolated microglia from AOM-treated mice. Soluble fractalkine infusion into the brain significantly reduced neurological decline in AOM-treated mice compared to saline-infused AOM-treated mice. Infusion of soluble fractalkine into AOM-treated mice reduced liver damage, lessened microglia activation, and suppressed expression of chemokine ligand 2, interleukin-6, and tumor necrosis factor alpha compared to saline-infused mice. CONCLUSIONS: These findings suggest that fractalkine-mediated signaling is suppressed in the brain following the development of hepatic encephalopathy. Supplementation of AOM-treated mice with soluble fractalkine led to improved outcomes, which identifies this pathway as a possible therapeutic target for the management of hepatic encephalopathy following acute liver injury

Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms

Although large cholangiocytes exert their functions by activation of cyclic adenosine 3',5'-monophosphate (cAMP), Ca(2+)-dependent signaling regulates the function of small cholangiocytes. Histamine interacts with four receptors, H1-H4HRs. H1HR acts by Gαq activating IP(3)/Ca(2+), whereas H2HR activates Gα(s) stimulating cAMP. We hypothesize that histamine increases biliary growth by activating H1HR on small and H2HR on large cholangiocytes. The expression of H1-H4HRs was evaluated in liver sections, isolated and cultured (normal rat intrahepatic cholangiocyte culture (NRIC)) cholangiocytes. In vivo, normal rats were treated with histamine or H1-H4HR agonists for 1 week. We evaluated: (1) intrahepatic bile duct mass (IBDM); (2) the effects of histamine, H1HR or H2HR agonists on NRIC proliferation, IP(3) and cAMP levels and PKCα and protein kinase A (PKA) phosphorylation; and (3) PKCα silencing on H1HR-stimulated NRIC proliferation. Small and large cholangiocytes express H1-H4HRs. Histamine and the H1HR agonist increased small IBDM, whereas histamine and the H2HR agonist increased large IBDM. H1HR agonists stimulated IP(3) levels, as well as PKCα phosphorylation and NRIC proliferation, whereas H2HR agonists increased cAMP levels, as well as PKA phosphorylation and NRIC proliferation. The H1HR agonist did not increase proliferation in PKCα siRNA-transfected NRICs. The activation of differential signaling mechanisms targeting small and large cholangiocytes is important for repopulation of the biliary epithelium during pathologies affecting different-sized bile ducts

Bile Acid-Mediated Sphingosine-1-Phosphate Receptor 2 Signaling Promotes Neuroinflammation during Hepatic Encephalopathy in Mice

Hepatic encephalopathy (HE) is a neuropsychiatric complication that occurs due to deteriorating hepatic function and this syndrome influences patient quality of life, clinical management strategies and survival. During acute liver failure, circulating bile acids increase due to a disruption of the enterohepatic circulation. We previously identified that bile acid-mediated signaling occurs in the brain during HE and contributes to cognitive impairment. However, the influences of bile acids and their downstream signaling pathways on HE-induced neuroinflammation have not been assessed. Conjugated bile acids, such as taurocholic acid (TCA), can activate sphingosine-1-phosphate receptor 2 (S1PR2), which has been shown to promote immune cell infiltration and inflammation in other models. The current study aimed to assess the role of bile-acid mediated S1PR2 signaling in neuroinflammation and disease progression during azoxymethane (AOM)-induced HE in mice. Our findings demonstrate a temporal increase of bile acids in the cortex during AOM-induced HE and identified that cortical bile acids were elevated as an early event in this model. In order to classify the specific bile acids that were elevated during HE, a metabolic screen was performed and this assay identified that TCA was increased in the serum and cortex during AOM-induced HE. To reduce bile acid concentrations in the brain, mice were fed a diet supplemented with cholestyramine, which alleviated neuroinflammation by reducing proinflammatory cytokine expression in the cortex compared to the control diet-fed AOM-treated mice. S1PR2 was expressed primarily in neurons and TCA treatment increased chemokine ligand 2 mRNA expression in these cells. The infusion of JTE-013, a S1PR2 antagonist, into the lateral ventricle prior to AOM injection protected against neurological decline and reduced neuroinflammation compared to DMSO-infused AOM-treated mice. Together, this identifies that reducing bile acid levels or S1PR2 signaling are potential therapeutic strategies for the management of HE

Neuronal CCL2 is upregulated during hepatic encephalopathy and contributes to microglia activation and neurological decline

BACKGROUND: Acute liver failure leads to systemic complications with one of the most dangerous being a decline in neurological function, termed hepatic encephalopathy. Neurological dysfunction is exacerbated by an increase of toxic metabolites in the brain that lead to neuroinflammation. Following various liver diseases, hepatic and circulating chemokines, such as chemokine ligand 2 (CCL2), are elevated, though their effects on the brain following acute liver injury and subsequent hepatic encephalopathy are unknown. CCL2 is known to activate microglia in other neuropathies, leading to a proinflammatory response. However, the effects of CCL2 on microglia activation and the pathogenesis of hepatic encephalopathy following acute liver injury remain to be determined. METHODS: Hepatic encephalopathy was induced in mice via injection of azoxymethane (AOM) in the presence or absence of INCB 3284 dimesylate (INCB), a chemokine receptor 2 inhibitor, or C 021 dihydrochloride (C021), a chemokine receptor 4 inhibitor. Mice were monitored for neurological decline and time to coma (loss of all reflexes) was recorded. Tissue was collected at coma and used for real-time PCR, immunoblots, ELISA, or immunostaining analyses to assess the activation of microglia and consequences on pro-inflammatory cytokine expression. RESULTS: Following AOM administration, microglia activation was significantly increased in AOM-treated mice compared to controls. Concentrations of CCL2 in the liver, serum, and cortex were significantly elevated in AOM-treated mice compared to controls. Systemic administration of INCB or C021 reduced liver damage as assessed by serum liver enzyme biochemistry. Administration of INCB or C021 significantly improved the neurological outcomes of AOM-treated mice, reduced microglia activation, reduced phosphorylation of ERK1/2, and alleviated AOM-induced cytokine upregulation. CONCLUSIONS: These findings suggest that CCL2 is elevated systemically following acute liver injury and that CCL2 is involved in both the microglia activation and neurological decline associated with hepatic encephalopathy. Methods used to modulate CCL2 levels and/or reduce CCR2/CCR4 activity may be potential therapeutic targets for the management of hepatic encephalopathy due to acute liver injury

Melatonin inhibits cholangiocyte hyperplasia in cholestatic rats by downregulation of CLOCK genes

Background and aims. In the experimental model of bile duct ligated (BDL) rats, the growth of large cholangiocytes is regulated by activation of cAMP-dependent signalling pathway1-2. Melatonin, which is secreted from pineal gland as well as extrapineal tissues, regulates cell proliferation by interacting with its receptors (MT1 and MT2) via the modulation of cAMP levels and the expression of the CLOCK genes3. We studied the mechanism by which melatonin regulates the growth of the biliary epithelium. Methods. Normal and BDL rats were treated in vivo with melatonin for 7 days before evaluating: (i) intrahepatic bile duct mass (IBDM) in liver sections by immunohistochemistry for citokeratin-19 (CK-19), a specific marker of cholangiocytes; and (ii) expression of MT1, MT2 and CLOCK genes by immunohistochemistry and real time PCR. In vitro, large cholangiocytes were stimulated with melatonin in the absence/presence of luzindole (a MT1/MT2 antagonist) and 4-P-PDOT (a specific MT2 antagonist) before assessing cellular growth by MTS proliferation assay, cAMP levels by a RIA kit and ERK1/2 phosphorylation by western blots. Results. Cholangiocytes express MT1, MT2 and CLOCK genes that were all upregulated following BDL. Administration of melatonin to BDL rats decreased IBDM and the expression of all CLOCK genes. In cells, melatonin decreased the proliferation, cAMP levels and the activation of ERK2, decreases that were blocked by luzindole. Conclusion. Supporting other studies we found that melatonin-inhibition of biliary hyperplasia is associated with downregulation of CLOCK gene expression. These findings have important clinical implications since melatonin may be an important therapeutic tool for managing cholangiocyte hyperproliferation in biliary disorders

Hepatic alterations are accompanied by changes to bile acid transporter-expressing neurons in the hypothalamus after traumatic brain injury

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