Cholestatic liver disease leads to significant adaptative changes in neural circuits regulating social behavior in mice to enhance sociability.

BACKGROUND & AIMS: Cholestatic liver diseases (CLD) are commonly associated with behavioral changes, including social isolation, that negatively affects patient quality of life and remains unaltered by current therapies. It remains unclear whether CLD-associated social dysfunction stems from a direct effect on the brain, or from the psychological impact of CLD. The psychological component of disease is absent in animals, so we investigated the impact of CLD on social behavior and gene expression profiles in key social behavior-regulating brain regions in a mouse model. METHODS: CLD due to bile duct ligation was used with the three-chamber sociability test for behavioral phenotyping. Differentially expressed gene (DEG) signatures were delineated in 3 key brain regions regulating social behavior using RNA-seq. Ingenuity Pathway Analysis (IPA®) was applied to streamline DEG data interpretation and integrate findings with social behavior-regulating pathways to identify important brain molecular networks and regulatory mechanisms disrupted in CLD. RESULTS: CLD mice exhibited enhanced social interactive behavior and significantly altered gene expression in each of the three social behavior-regulating brain regions examined. DEG signatures in BDL mice were associated with key IPA®-identified social behavior-regulating pathways including Oxytocin in Brain Signaling, GABA Receptor Signaling, Dopamine Receptor Signaling, and Glutamate Receptor Signaling. CONCLUSIONS: CLD causes complex alterations in gene expression profiles in key social behavior-regulating brain areas/pathways linked to enhanced social interactive behavior. These findings, if paralleled in CLD patients, suggest that CLD-associated reductions in social interactions predominantly relate to psychological impacts of disease and may inform new approaches to improve management.

Recruitment of α4ß7 monocytes and neutrophils to the brain in experimental colitis is associated with elevated cytokines and anxiety-like behavior.

BACKGROUND: Behavioral comorbidities, such as anxiety and depression, are a prominent feature of IBD. The signals from the inflamed gut that cause changes in the brain leading to these behavioral comorbidities remain to be fully elucidated. We tested the hypothesis that enhanced leukocyte-cerebral endothelial cell interactions occur in the brain in experimental colitis, mediated by α4ß7 integrin, to initiate neuroimmune activation and anxiety-like behavior. METHODS: Female mice treated with dextran sodium sulfate were studied at the peak of acute colitis. Circulating leukocyte populations were determined using flow cytometry. Leukocyte-cerebral endothelial cell interactions were examined using intravital microscopy in mice treated with anti-integrin antibodies. Brain cytokine and chemokines were assessed using a multiplex assay in animals treated with anti-α4ß7 integrin. Anxiety-like behavior was assessed using an elevated plus maze in animals after treatment with an intracerebroventricular injection of interleukin 1 receptor antagonist. RESULTS: The proportion of classical monocytes expressing α4ß7 integrin was increased in peripheral blood of mice with colitis. An increase in the number of rolling and adherent leukocytes on cerebral endothelial cells was observed, the majority of which were neutrophils. Treatment with anti-α4ß7 integrin significantly reduced the number of rolling leukocytes. After anti-Ly6C treatment to deplete monocytes, the number of rolling and adhering neutrophils was significantly reduced in mice with colitis. Interleukin-1ß and CCL2 levels were elevated in the brain and treatment with anti-α4ß7 significantly reduced them. Enhanced anxiety-like behavior in mice with colitis was reversed by treatment with interleukin 1 receptor antagonist. CONCLUSIONS: In experimental colitis, α4ß7 integrin-expressing monocytes direct the recruitment of neutrophils to the cerebral vasculature, leading to elevated cytokine levels. Increased interleukin-1ß mediates anxiety-like behavior.

The Antidepressant Mirtazapine Rapidly Shifts Hepatic B Cell Populations and Functional Cytokine Signatures in the Mouse.

Introduction: B cells are important regulators of both adaptive and innate immunity. The normal liver contains significant numbers of B cells, and their numbers increase dramatically in immune-mediated liver diseases. Our previous observations suggest a hepatoprotective effect of the antidepressant mirtazapine in human and experimental immune-mediated liver disease. Therefore, we performed a series of experiments to determine the impact of mirtazapine treatment on hepatic B cell homeostasis, as reflected by B cell number, trafficking and phenotype using flow cytometry (FCM) and intravital microscopy (IVM) analysis. Mirtazapine treatment rapidly induced a significant reduction in total hepatic B cell numbers, paralleled by a compositional shift in the predominant hepatic B cell subtype from B2 to B1. This shift in hepatic B cells induced by mirtazapine treatment was associated with a striking increase in total hepatic levels of the chemokine CXCL10, and increased production of CXCL10 by hepatic macrophages and dendritic cells. Furthermore, mirtazapine treatment led to an upregulation of CXCR3, the cognate chemokine receptor for CXCL10, on hepatic B cells that remained in the liver post-mirtazapine. A significant role for CXCR3 in the hepatic retention of B cells post-mirtazapine was confirmed using CXCR3 receptor blockade. In addition, B cells remaining in the liver post-mirtazapine produced lower amounts of the proinflammatory Th1-like cytokines IFNγ, TNFα, and IL-6, and increased amounts of the Th2-like cytokine IL-4, after stimulation in vitro. Conclusion: Mirtazapine treatment rapidly alters hepatic B cell populations, enhancing hepatic retention of CXCR3-expressing innate-like B cells that generate a more anti-inflammatory cytokine profile. Mirtazapine-induced hepatic B cell shifts could potentially represent a novel therapeutic approach to immune-mediated liver diseases characterized by B cell driven pathology.

ADAM Metalloproteinase Domain 17 Regulates Cholestasis-Associated Liver Injury and Sickness Behavior Development in Mice.

Disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) is a ubiquitously expressed membrane-bound enzyme that mediates shedding of a wide variety of important regulators in inflammation including cytokines and adhesion molecules. Hepatic expression of numerous cytokines and adhesion molecules are increased in cholestatic liver diseases including primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), however, the pathophysiological role of ADAM17 in regulating these conditions remains unknown. Therefore, we evaluated the role of ADAM17 in a mouse model of cholestatic liver injury due to bile duct ligation (BDL). We found that BDL enhanced hepatic ADAM17 protein expression, paralleled by increased ADAM17 bioactivity. Moreover, inhibition of ADAM17 bioactivity with the specific inhibitor DPC 333 significantly improved both biochemical and histological evidence of liver damage in BDL mice. Patients with cholestatic liver disease commonly experience adverse behavioral symptoms, termed sickness behaviors. Similarly, BDL in mice induces reproducible sickness behavior development, driven by the upregulated expression of cytokines and adhesion molecules that are in turn regulated by ADAM17 activity. Indeed, inhibition of ADAM17 activity significantly ameliorated BDL-associated sickness behavior development. In translational studies, we evaluated changes in ADAM17 protein expression in liver biopsies obtained from patients with PBC and PSC, compared to normal control livers. PSC and PBC patients demonstrated increased hepatic ADAM17 expression in hepatocytes, cholangiocytes and in association with liver-infiltrating immune cells compared to normal controls. In summary, cholestatic liver injury in mice and humans is associated with increased hepatic ADAM17 expression. Furthermore, inhibition of ADAM17 activity improves both cholestatic liver injury and associated sickness behavior development, suggesting that ADAM17 inhibition may represent a novel therapeutic approach for treating patients with PBC/PSC.

The Antidepressant Mirtazapine Activates Hepatic Macrophages, Facilitating Pathogen Clearance While Limiting Tissue Damage in Mice.

Background and Aims: Mirtazapine is an atypical antidepressant with antagonist activity for serotonin and histamine receptors. Clinical and experimental evidence suggests that, in addition to treating depression, mirtazapine also alters liver innate immunity and suppresses immune-driven hepatic macrophage activation. Liver macrophages, Kupffer cells, represent the largest collection of fixed macrophages in the body and are critical in regulating hepatic immunity. In addition to their capacity to regulate inflammation, Kupffer cells are key sentinels for clearing blood-borne pathogens, preventing their dissemination within the body. This process involves pathogen capture, phagocytosis, and activation-induced killing via reactive oxygen species (ROS) production. Therefore, we speculated that mirtazapine might adversely alter Kupffer cell pathogen-associated activation and killing. Methods: Mice were treated with mirtazapine and time-dependent changes in Kupffer cells were characterized using intravital microscopy. Macrophage and neutrophil responses, bacterial dissemination, and liver damage were assessed following i.v. infection with a pathogenic strain of S. aureus. Results: Mirtazapine rapidly (within 1.5 h) activates Kupffer cells, indicated by a loss of elongated shape with cellular rounding. However, this shape change did not result in impaired pathogen capture function, and, in fact, generated enhanced ROS production in response to S. aureus-induced sepsis. Neutrophil dynamics were altered with reduced cellular recruitment to the liver following infection. Bacterial dissemination post-intravenous administration was not altered by mirtazapine treatment; however, hepatic abscess formation was significantly reduced. Conclusions: Mirtazapine rapidly activates Kupffer cells, associated with preserved bacterial capture functions and enhanced ROS generation capacity. Moreover, these changes in Kupffer cells were linked to a beneficial reduction in hepatic abscess size. In contrast to our initial speculation, mirtazapine may have beneficial effects in sepsis and warrants further exploration.

Brain TNF drives post-inflammation depression-like behavior and persistent pain in experimental arthritis.

Patients with rheumatoid arthritis experience chronic pain, depression and fatigue, even when inflammation of the joints is well controlled. To study the relationship between arthritis, depression, and sustained pain when articular inflammation is no longer observed, we tested the hypothesis that brain TNF drives post-inflammation depression-like behavior and persistent pain in experimental arthritis. The murine model of antigen-induced arthritis (AIA) was used to evaluate the effects of knee inflammation on sustained pain and depression-like behavior. We measured joint pain using an automated dynamic plantar algesiometer and depression-like behavior with the tail suspension test. Cytokines were measured by Luminex assay and ELISA. TNF in the brain was blocked by intracerebroventricular injection of anti-TNF antibodies. Histological damage and elevated levels of cytokines were observed in the knee 24 h after antigen treatment, but not at 13 days. Reduced pain thresholds were seen 24 h and 13 days after treatment. Depression-like behavior was observed on day 13. Treatment with the antidepressant imipramine reduced both depression-like behavior and persistent pain. However, blocking joint pain with the analgesic dipyrone did not alter depression-like behavior. Elevated levels of TNF, CCL2, and CXCL-1 were observed in the hippocampus 24 h after treatment, with TNF remaining elevated at day 13. Intracerebroventricular infusion of an anti-TNF antibody blocked depression-like behavior and reduced persistent pain. We have demonstrated that depression-like behavior and pain is sustained in AIA mice after the resolution of inflammation. These changes are associated with elevated levels of TNF in the hippocampus and are dependent upon brain TNF. The findings reveal an important mechanistic link between the expression of chronic pain and depression in experimental arthritis. Furthermore, they suggest treating depression in rheumatoid arthritis may positively impact other debilitating features of this condition.

Corynebacterium tuberculostearicum, a human skin colonizer, induces the canonical nuclear factor-κB inflammatory signaling pathway in human skin cells.

INTRODUCTION: Corynebacterium tuberculostearicum (C. t.) is a ubiquitous bacterium that colonizes human skin. In contrast to other members of the genus Corynebacterium, such as toxigenic Corynebacterium diphtheriae or the opportunistic pathogen Corynebacterium jeikeium, several studies suggest that C. t. may play a role in skin health and disease. However, the mechanisms underlying these effects remain poorly understood. METHODS: To investigate whether C. t. induces inflammatory pathways in primary human epidermal keratinocytes (HEKs) and human cutaneous squamous carcinoma cells (SCCs), cell culture, reverse transcription-polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, immunofluorescence microscopy, Western blot, chromatin immunoprecipitation-PCR, small interfering RNA knockdown and luciferase reporter expression system were used. RESULTS: Herein, we demonstrate that C. t. upregulates the messenger RNA (mRNA) and protein levels of inflammatory mediators in two human skin cell lines, HEKs and SCCs. We further show activation of the canonical nuclear factor-κB (NF-κB) pathway in response to C. t. infection, including phosphorylation of the inhibitor of κB (IκB), the nuclear translocation of NF-κB subunit (NF-κB-P65 ) and the recruitment of NF-κB-P65 and RNA polymerase to the NF-κB response elements at the promoter region of the inflammatory genes. Lastly, the data confirm that C. t.-induced tumor necrosis factor mRNA expression in HEKs is toll-like receptor 2 (TLR2 ) dependent. CONCLUSION: Our results offer a mechanistic model for C. t.-induced inflammation in human keratinocytes via TLR2 and activation of IκB kinase and downstream signaling through the canonical NF-κB pathway. Relevance to chronic inflammatory diseases of the skin and cutaneous oncology is discussed.

The Antidepressant Mirtazapine Inhibits Hepatic Innate Immune Networks to Attenuate Immune-Mediated Liver Injury in Mice.

Activation of the innate immune system, including tissue macrophages and associated neutrophil infiltration, is an important driver of subsequent adaptive immune responses in many autoimmune diseases, including autoimmune hepatitis (AIH). The antidepressant mirtazapine has a unique complex pharmacology, altering signaling through a number of serotonin and histamine receptors that can impact macrophage function; an effect potentially influencing AIH outcome. In the mouse model of concanavalin A (Con A) induced liver injury (mimics many aspects of human AIH), in which early innate immune activation (i.e., stimulated hepatic macrophages/monocytes recruit neutrophils and additional monocytes to the liver) critically drives immune-mediated hepatitis induction, mirtazapine strikingly and dose-dependently inhibited Con A-induced liver injury. This inflammation-suppressing effect of mirtazapine was linked to an attenuation of Con A-stimulated early innate immune responses within the liver, including inhibition of hepatic macrophage/monocyte activation, decreased hepatic macrophage/monocyte-derived pro-inflammatory cytokine (e.g., TNFα) and chemokine (e.g., CXCL1 and CXCL2) production, suppression of Con A-induced increases in the hepatic expression of the neutrophil relevant endothelial cell adhesion molecule ICAM-1, with the resultant significant reduction in neutrophil recruitment into the liver. Consistent with our findings in the Con A model, mirtazapine also significantly reduced activation-induced release of cytokine/chemokine mediators from human CD14+ monocytes in vitro. Conclusion: Our data suggest that mirtazapine can attenuate hepatic innate immune responses that critically regulate the subsequent development of autoimmune liver injury. Therefore, given that it is a safe and widely used medication, mirtazapine may represent a novel therapeutic approach to autoimmune liver disease.

The impact of depression and antidepressant usage on primary biliary cholangitis clinical outcomes.

BACKGROUND: Depression is prevalent in primary biliary cholangitis (PBC) patients. Our aims were to examine the effects of depression and antidepressants on hepatic outcomes of PBC patients. METHODS: We used the UK Health Improvement Network database to identify PBC patients between 1974 and 2007. Our primary outcome was one of three clinical events: decompensated cirrhosis, liver transplantation and death. We assessed depression and each class of antidepressant medication in adjusted multivariate Cox proportional hazards models to identify independent predictors of outcomes. In a sensitivity analysis, the study population was restricted to PBC patients using ursodeoxycholic acid (UDCA). RESULTS: We identified 1,177 PBC patients during our study period. In our cohort, 86 patients (7.3%) had a depression diagnosis prior to PBC diagnosis, while 79 patients (6.7%) had a depression diagnosis after PBC diagnosis. Ten-year incidence of mortality, decompensated cirrhosis, and liver transplantation were 13.4%, 6.6%, and 2.0%, respectively. In our adjusted models, depression status was not a predictor of poor outcomes. After studying all classes of antidepressants, using the atypical antidepressant mirtazapine after PBC diagnosis was significantly protective (Adjusted HR 0.23: 95% CI 0.07-0.72) against poor liver outcomes (decompensation, liver transplant, mortality), which remained statistically significant in patients using UCDA (HR 0.21: 95% CI 0.05-0.83). CONCLUSIONS: In our study, depression was not associated with poor clinical outcomes. However, using the antidepressant mirtazapine was associated with decreased mortality, decompensated cirrhosis and liver transplantation in PBC patients. These findings support further assessment of mirtazapine as a potential treatment for PBC patients.

Interactions Between Platelets and Inflammatory Monocytes Affect Sickness Behavior in Mice With Liver Inflammation.

BACKGROUND & AIMS: Patients with inflammatory liver disease commonly develop debilitating symptoms, called sickness behaviors, which arise via changes in brain function. Monocytes that produce tumor necrosis factor interact with cerebral endothelial cells to activate microglial cells and promote sickness behavior. Platelets regulate inflammation, and aggregates of monocytes and platelets are increased in the circulation of patients with liver disease. We investigated the role of platelets in inducing inflammatory features of circulating monocytes and promoting sickness behaviors in mice with cholestatic liver injury. METHODS: We performed bile-duct ligations or sham surgeries on C57BL/6 or toll-like receptor 4 (TLR4)-knockout mice to induce liver inflammation. Liver inflammation was also induced in a separate group of mice by administration of concanavalin A. Circulating platelets, aggregates of monocytes and platelets, and activation of microglial cells were measured by flow cytometry. To deplete platelets, mice were given anti-thrombocyte serum or normal rabbit serum (control) 4 days after surgery. Interactions between monocytes and cerebral endothelial cells were analyzed by intravital microscopy. Sickness behaviors were quantified based on time spent by adult mice engaging in social behaviors toward a juvenile mouse, compared with time spent in nonsocial behavior or remaining immobile. RESULTS: Aggregates of monocytes and platelets in circulation of mice increased significantly following bile-duct ligation. Platelet-monocyte interactions were required for activation of inflammatory monocytes and production of tumor necrosis factor. Platelet depletion greatly reduced adhesive interactions between inflammatory monocytes and adhesive interactions with cerebral endothelial cells and activation of the microglia, as well as development of sickness behavior. Furthermore, TLR4 signaling was important for aggregation of monocytes and platelets, and development of sickness behavior following bile-duct ligation. These findings were confirmed in mice with concanavalin A-induced liver injury. CONCLUSIONS: In mice with liver inflammation, we found TLR4 and aggregates of monocytes and platelets to regulate microglial activation and development of sickness behavior. These findings might lead to new therapeutic strategies for liver disease-associated symptoms.

TNFα Augments Cytokine-Induced NK Cell IFNγ Production through TNFR2.

NK cells play a central role in innate immunity, acting directly through cell-mediated cytotoxicity and by secreting cytokines. TNFα activation of TNFR2 enhances NK cell cytotoxicity, but its effects on the other essential function of NK cells - cytokine production, for which IFNγ is paramount - are poorly defined. We identify the expression of both TNFα receptors on human peripheral blood NK cells (TNFR2 > TNFR1) and show that TNFα significantly augments IFNγ production from IL-2-/IL-12-treated NK cells in vitro, an effect mimicked by a TNFR2 agonistic antibody. TNFα also enhanced murine NK cell IFNγ production via TNFR2 in vitro. In a mouse model characterized by the hepatic recruitment and activation of NK cells, TNFR2 also regulated NK cell IFNγ production in vivo. Specifically, in this model, after activation of an innate immune response, hepatic numbers of TNFR2-expressing and IFNγ-producing NK cells were both significantly increased; however, the frequency of IFNγ-producing hepatic NK cells was significantly reduced in TNFR2-deficient mice. We delineate an important role for TNFα, acting through TNFR2, in augmenting cytokine-induced NK cell IFNγ production in vivo and in vitro, an effect with significant potential implications for the regulation of innate and adaptive immune responses.

Rapid activation and hepatic recruitment of innate-like regulatory B cells after invariant NKT cell stimulation in mice.

BACKGROUND & AIMS: Invariant natural killer T (iNKT) cells are present within the liver and have been implicated in the development of many liver diseases. Upon activation by glycolipid ligands (including α-galactosylceramide; αGalCer), hepatic iNKT cells produce numerous cytokines and recruit both pro-inflammatory and regulatory immune cells. However, the involvement of B cells in this process is poorly defined. METHODS: Wild-type (male, C57BL/6), B cell deficient, or B cell depleted mice were injected with αGalCer or vehicle, hepatic B cell phenotype and liver injury was subsequently determined. RESULTS: iNKT cell activation resulted in liver injury and the rapid activation and hepatic recruitment of B cells (mainly innate-like B1 and MZ-like B cells) from the spleen and peritoneal cavity. B cells recruited to the liver produce IL-10 and TGFß, and express cell surface CD73 (ectoenzyme which generates adenosine). B cell deficient mice developed augmented αGalCer-induced hepatitis, enhanced neutrophil recruitment and striking alterations in the hepatic cytokine milieu. αGalCer-induced hepatitis was unaltered in IL-10(-/-) mice, or after TGFß neutralization, but was significantly worsened in mice treated with a CD73 inhibitor. CONCLUSIONS: iNKT cell stimulation recruits innate-like regulatory B cells to the liver which suppress hepatic inflammation through IL-10 and TGFß1 independent mechanisms, but involve CD73 activity. These findings highlight an important inflammation suppressing role for B cells at early time points during the development of an innate immune response within the liver, and represent a potential therapeutic target for the treatment of liver disease.

Effects of prostaglandin D2, 15-deoxy-Delta12,14-prostaglandin J2, and selective DP1 and DP2 receptor agonists on pulmonary infiltration of eosinophils in Brown Norway rats.

Prostaglandin (PG) D2 is an arachidonic acid metabolite that is released by allergen-stimulated mast cells. It is a potent in vitro chemoattractant for human eosinophils, acting through the DP2 receptor/chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). Furthermore, there is in vivo evidence that PGD2 contributes to allergen-induced pulmonary eosinophilia via its classic DP1 receptor. The PGD2-derived product 15-deoxy-Delta12,14-PGJ2 is widely used as a peroxisome proliferator-activated receptor gamma agonist and has been shown to have anti-inflammatory properties. However, this substance can also activate eosinophils in vitro through the DP2 receptor. The objectives of the present study were to determine whether PGD2 and 15-deoxy-Delta12,14-PGJ2 can induce pulmonary eosinophilia, and, if so, to examine the abilities of selective DP1 and DP2 receptor agonists to induce this response. Brown Norway rats were treated by intratracheal instillation of PGs. Vehicle and 5-oxo-6,8,11,14-eicosatetraenoic acid were used as negative and positive controls, respectively. Lung eosinophils were identified by immunostaining of lung sections with an antibody to major basic protein. Both PGD2 and 15-deoxy-Delta12,14-PGJ2 induced robust eosinophilic responses that were apparent by 12 h and persisted for at least 48 h. Two selective DP2 receptor agonists, 15R-methyl-PGD2 and 13-14-dihydro-15-keto-PGD2, induced similar responses, the former being more potent than PGD2, whereas the latter was less potent. The selective DP1 receptor agonist BW245C [(4S)-(3-[(3R,S)-3-cyclohexyl-3-hydroxypropyl]-2,5-dioxo)-4-imidazolidineheptanoic acid] was completely inactive. We conclude that PGD2 and 15-deoxy-Delta12,14-PGJ2 induce eosinophil infiltration into the lungs through the DP2 receptor. The potent in vitro DP2 receptor agonist 15R-methyl-PGD2 is also very active in vivo and should be a useful tool in examining the role of this receptor.