Interaction of TWEAK with Fn14 leads to the progression of fibrotic liver disease by directly modulating hepatic stellate cell proliferation

Tumour necrosis factor‐like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor‐inducible 14 (Fn14) have been associated with liver regeneration in vivo. To further investigate the role of this pathway we examined their expression in human fibrotic liver disease and the effect of pathway deficiency in a murine model of liver fibrosis. The expression of Fn14 and TWEAK in normal and diseased human and mouse liver tissue and primary human hepatic stellate cells (HSCs) were investigated by qPCR, western blotting and immunohistochemistry. In addition, the levels of Fn14 in HSCs following pro‐fibrogenic and pro‐inflammatory stimuli were assessed and the effects of exogenous TWEAK on HSCs proliferation and activation were studied in vitro. Carbon tetrachloride (CCl(4)) was used to induce acute and chronic liver injury in TWEAK KO mice. Elevated expression of both Fn14 and TWEAK were detected in acute and chronic human liver injury, and co‐localized with markers of activated HSCs. Fn14 levels were low in quiescent HSCs but were significantly induced in activated HSCs, which could be further enhanced with the profibrogenic cytokine TGFβ in vitro. Stimulation with recombinant TWEAK induced proliferation but not further HSCs activation. Fn14 gene expression was also significantly up‐regulated in CCl(4) models of hepatic injury whereas TWEAK KO mice showed reduced levels of liver fibrosis following chronic CCl(4) injury. In conclusion, TWEAK/Fn14 interaction leads to the progression of fibrotic liver disease via direct modulation of HSCs proliferation, making it a potential therapeutic target for liver fibrosis. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

New immune-therapeutic approaches for treatment of type-1-diabetes mellitus in the non-obese-diabetic-mouse-model with special focus on antigen presenting cells

Hintergrund/Ziele Antigen präsentierende Zellen nehmen bei Autoimmunerkrankungen wie dem Diabetes mellitus Typ 1 eine Schlüsselrolle ein. In den hier abgehandelten Studien wurde daher an einem etablierten Mausmodell des Autoimmundiabetes, der „non-obese-diabetic“ (NOD) Maus, zunächst untersucht, ob Antigen präsentierende Zellen myeloiden Ursprungs mit immunsuppressivem Potential einen Einfluss auf die Immunpathogenese des Autoimmundiabetes haben. Verwendet wurden hierfür Vorläufer dendritischer Zellen, sog. Myeloide Suppressorzellen (engl. myeloid derived suppressor cells, MDSC), die bereits in verschiedenen Tumormodellen ein besonderes immunsuppressives Potential gezeigt haben. Des Weiteren wurde in der NOD-Maus untersucht, ob eine pharmakologische Beeinflussung Antigen präsentierender Zellen durch das makrozyklische Kohlenstoff-Suboxid 18 (MCS-18) einen positiven Einfluss auf den Autoimmundiabetes hat, zumal diese Substanz bereits im Tiermodell der multiplen Sklerose eine günstige Beeinflussung des Krankheitsverlaufs gezeigt hat. Methoden Weibliche NOD-Mäuse, die unter speziell keimarmen Verhältnissen in mindestens 80% der Fälle bis zur 30. Lebenswoche einen manifesten Diabetes mellitus Typ 1 entwickeln, erhielten MDSC intravenös (i.v.) zu verschiedenen Zeitpunkten der Insulitis (frühe Phase der Insulitis: 8. Lebenswoche; späte Phase der Insulitis: 12. Lebenswoche) in verschiedenen Konzentrationen oder MCS-18 intraperitoneal (i.p.) in verschiedenen Dosen in der frühen Phase der Insulitis. Es erfolgte ein wöchentliches Screening bzgl. einer Glukosurie als Ausdruck eines manifesten Diabetes mellitus. Bei Erreichen der 30. Lebenswoche oder bei Diabetesmanifestation wurde das Ausmaß der Insulitis anhand einer histologischen Untersuchung des Pankreas sowie der Einfluss einer Behandlung auf die Stimulierbarkeit von Lymphozyten der Milz mit Inselzellantigenen untersucht. Zusätzlich wurde das Migrationsverhalten von MDSC nach i.v.-Applikation mittels fluoreszenz-aktivierter Zellsortierung (FACS) der Milz-Monozyten und immunhistochemischer Untersuchungen des Pankreas analysiert, um Rückschlüsse auf einen primären Wirkort ziehen zu können. Beobachtungen und Ergebnisse Eine Behandlung mit MDSC konnte sowohl in der frühen als auch in der fortgeschrittenen Insulitis die Diabetesinzidenz signifikant senken. Morphologisch fand dies Niederschlag in einer Verminderung der Intrainsulitis bei gleichzeitiger Expansion der Fraktion peripher infiltrierter Inseln, was als protektive Insulitis gewertet wurde. In Korrelation hierzu zeigten Milz-Lymphozyten eine Th2-polarisierte Zytokinsekretion nach Stimulation mit definierten Inselzellantigenen. Zwar konnten MDSC unmittelbar nach Transfer in der Milz und in späteren Phasen auch im Randsaum der Langerhansinseln nachgewiesen werden, ein eindeutiger Wirkort konnte allerdings nicht identifiziert werden. Die Behandlung mit MCS-18 in der frühen Phase der Insulitis konnte ebenfalls eine signifikante Erniedrigung der Diabetesinzidenz bewirken. Hierbei ließ sich eine Korrelation zu einer Verminderung autoreaktiver Milz-Lymphozyten beobachten. Zugleich zeigte sich eine signifikante Reduktion der Intrainsulitis bei gleichzeitiger Expansion der Fraktion nicht infiltrierter Inseln. Praktische Schlussfolgerung/Diskussion Selbst die fortgeschrittene Autoimmuninsulitis ließ sich durch den Einsatz von MDSC günstig beeinflussen, weshalb die Anwendung dieser Zellen eine vielversprechende Therapiestrategie darstellt. Für einen sicheren Einsatz dieser Zellen beim Menschen bedarf es allerdings weiterer Kenntnisse über die Dynamik dieser Zellen, insbesondere bzgl. ihrer Fähigkeit, zu dendritischen Zellen ausreifen zu können. Demgegenüber hat MCS-18 in klinischen Studien bereits ein günstiges Risiko-/Nutzenprofil gezeigt, weshalb ein klinischer Einsatz zur Behandlung des Diabetes mellitus Typ 1 aufgrund der hier dargelegten Ergebnisse erwogen werden sollte.Background and aims Antigen-presenting cells exert a key role in type-1-diabetes as well as in other autoimmune diseases. Hence, successful therapy might depend on these cells to a high extend. In this context, myeloid-derived suppressor cells (MDSC) display progenitors of dendritic cells (DC) with extraordinary immunosuppressive potential as observed earlier in various tumour models. Therefore, it was one aim of this study to evaluate the influence of MDSC on type-1-diabetes in the non-obese-diabetic-(NOD) mouse-model, an established model of autoimmune diabetes. Another therapeutic approach might be based on the pharmacological manipulation of antigen-presenting cells. In this respect, the macrocyclic carbon suboxide MCS-18 has shown to ameliorate encephalitis disseminata in the EAE-mouse-model by impairing dendritic cell functions. Therefore, the influence of MCS-18 treatment on type-1-diabetes in the NOD-mouse-model was investigated in the second part of this study. Methods Female NOD mice are known to develop type-1-diabetes until the 30th week of life with a frequency of 80% if kept under special pathogen-free conditions. Treatment with MDSC was performed in different concentrations and at different stages of Insulitis (8th week: early stage insulitis; 12th week: late stage insulitis). Treatment with MCS-18 was performed at different concentrations at early stage insulitis. Screening for diabetes-manifestation was conducted weekly using a glucose measuring urin-stix. Either at the age of 30 weeks or at onset of diabetes mellitus insulitis was histological scored by analysing the degree of lymphocyte infiltration of the islets of Langerhans. Autoreactivity was measured by analysing the secretion of distinct cytokines upon stimulation with specific antigens of the islets of Langerhans. Moreover, the site of action of MDSC was investigated using fluorescence-activated cell sorting (FACS) and immunohistochemistry. Results At early as well as at late stage insulitis treatment with MDSC resulted in significant reduction of diabetes incidence. Further, intrainsulitits was reduced and periinsulitits was pronounced after treatment with MDSC indicating a protective type of insulitis. Moreover, splenocytic lymphocytes showed an autoprotective Th2-phenotype upon stimulation with islet-antigens when MDSC-treatment had been conducted. Although MDSC were detected in the spleen at day 7 after application and in the islet of Langerhans later, the site of action is still elusive. Treatment with MCS-18 led to a significant dose-depended reduction of diabetes-onset. Furthermore, a reduction of autoreactive splenocytic lymphocytes and a decrease in intrainsulitis was found after treatment with MCS-18. Discussion Treatment with MDSC led to significant reduction of diabetes incidence even when performed at late stage insulitis. However, the capacity of MDSC to mature to dendritic cells imposes an obstacle for the clinical use of these cells to date. Moreover, due to good experience in clinical practice MCS-18 should be applied for treatment of type-1-diabetes as it has proved efficacy in this study

Hydrogen-Peroxide Synthesis and LDL-Uptake Controls Immunosuppressive Properties in Monocyte-Derived Dendritic Cells

Background and Aims: Induction of myeloid-derived suppressor cells (MDSC) is a critical step in immune cell evasion by different cancer types, including liver cancer. In the liver, hepatic stromal cells orchestrate induction of MDSCs, employing a mechanism dependent on hydrogen peroxide (H2O2) depletion. However, the effects on monocyte-derived dendritic cells (moDCs) are unknown. Methods: Monocytes from healthy donors were differentiated to moDCs in the presence of extracellular enzymatic H2O2-depletion (hereinafter CAT-DCs), and studied phenotypically and functionally. To elucidate the underlying molecular mechanisms, we analyzed H2O2- and LDL-metabolism as they are interconnected in monocyte-driven phagocytosis. Results: CAT-DCs were of an immature DC phenotype, particularly characterized by impaired expression of the costimulatory molecules CD80/86. Moreover, CAT-DCs were able to suppress T-cells using indoleamine 2,3-dioxygenase (IDO), and induced IL10/IL17-secreting T-cells—a subtype reported to exert immunosuppression in acute myeloid leukemia. CAT-DCs also displayed significantly increased NADPH-oxidase-driven H2O2-production, enhancing low-density lipoprotein (LDL)-uptake. Blocking LDL-uptake restored maturation, and attenuated the immunosuppressive properties of CAT-DCs. Discussion: Here, we report a novel axis between H2O2- and LDL-metabolism controlling tolerogenic properties in moDCs. Given that moDCs are pivotal in tumor-rejection, and lipid-accumulation is associated with tumor-immune-escape, LDL-metabolism appears to play an important role in tumor-immunology

Molecular Rotors for Universal Quantitation of Nanoscale Hydrophobic Interfaces in Microplate Format

Hydrophobic self-assembly pairs diverse chemical precursors and simple formulation processes to access a vast array of functional colloids. Exploration of this design space, however, is stymied by lack of broadly general, high-throughput colloid characterization tools. Here, we show that a narrow structural subset of fluorescent, zwitterionic molecular rotors, dialkylaminostilbazolium sulfonates [DASS] with intermediate-length alkyl tails, fills this major analytical void by quantitatively sensing hydrophobic interfaces in microplate format. DASS dyes supersede existing interfacial probes by avoiding off-target fluorogenic interactions and dye aggregation while preserving hydrophobic partitioning strength. To illustrate the generality of this approach, we demonstrate (i) a microplate-based technique for measuring mass concentration of small (20–200 nm), dilute (submicrogram sensitivity) drug delivery nanoparticles; (ii) elimination of particle size, surfactant chemistry, and throughput constraints on quantifying the complex surfactant/metal oxide adsorption isotherms critical for environmental remediation and enhanced oil recovery; and (iii) more reliable self-assembly onset quantitation for chemically and structurally distinct amphiphiles. These methods could streamline the development of nanotechnologies for a broad range of applications

Midkine Promotes Metastasis and Therapeutic Resistance via mTOR/RPS6 in Uveal Melanoma

Uveal melanoma is a rare form of melanoma that originates in the eye, exerts widespread therapeutic resistance, and displays an inherent propensity for hepatic metastases. Because metastatic disease is characterized by poor survival, there is an unmet clinical need to identify new therapeutic targets in uveal melanoma. Here, we show that the pleiotropic cytokine midkine is expressed in uveal melanoma. Midkine expression in primary uveal melanoma significantly correlates with poor survival and is elevated in patients that develop metastatic disease. Monosomy 3 and histopathologic staging parameters are associated with midkine expression. In addition, we demonstrate that midkine promotes survival, migration across a barrier of hepatic sinusoid endothelial cells and resistance to AKT/mTOR inhibition. Fur-thermore, midkine is secreted and mediates mTOR activation by maintaining phosphorylation of the mTOR target RPS6 in uveal melanoma cells. Therefore, midkine is identified as a uveal melanoma cell survival factor that drives metastasis and therapeutic resistance, and could be exploited as a biomarker as well as a new therapeutic target. Implications: Midkine is identified as a survival factor that drives liver metastasis and therapeutic resistance in melanoma of the eye

Contact-Dependent Depletion of Hydrogen Peroxide by Catalase Is a Novel Mechanism of Myeloid-Derived Suppressor Cell Induction Operating in Human Hepatic Stellate Cells

Abstract Myeloid-derived suppressor cells (MDSC) represent a unique cell population with distinct immunosuppressive properties that have been demonstrated to shape the outcome of malignant diseases. Recently, human hepatic stellate cells (HSC) have been reported to induce monocytic-MDSC from mature CD14+ monocytes in a contact-dependent manner. We now report a novel and unexpected mechanism by which CD14+HLADRlow/− suppressive cells are induced by catalase-mediated depletion of hydrogen peroxide (H2O2). Incubation of CD14+ monocytes with catalase led to a significant induction of functional MDSC compared with media alone, and H2O2 levels inversely correlated with MDSC frequency (r = −0.6555, p &amp;lt; 0.05). Catalase was detected in primary HSC and a stromal cell line, and addition of the competitive catalase inhibitor hydroxylamine resulted in a dose-dependent impairment of MDSC induction and concomitant increase of H2O2 levels. The NADPH-oxidase subunit gp91 was significantly increased in catalase-induced MDSC as determined by quantitative PCR outlining the importance of oxidative burst for the induction of MDSC. These findings represent a so far unrecognized link between immunosuppression by MDSC and metabolism. Moreover, this mechanism potentially explains how stromal cells can induce a favorable immunological microenvironment in the context of tissue oxidative stress such as occurs during cancer therapy.</jats:p

Impaired Transmigration of Myeloid-Derived Suppressor Cells across Human Sinusoidal Endothelium Is Associated with Decreased Expression of CD13

Abstract Human monocytic myeloid-derived suppressor cells (MO-MDSCs) within the hepatic compartment suppress inflammation and impair immune surveillance in liver cancer. It is currently not known whether recruitment of MO-MDSCs from blood via hepatic sinusoidal endothelium (HSEC) contributes to their enrichment within the hepatic compartment. We compared the transmigratory potential of MO-MDSCs and monocytes after adhesion to hepatic endothelial monolayers in flow-based assays that mimic in vivo shear stress in the sinusoids. Despite comparable binding to HSEC monolayers, proportionally fewer MO-MDSCs underwent transendothelial migration, indicating that the final steps of extravasation, where actin polymerization plays an important role, are impaired in MO-MDSCs. In this article, we found reduced levels of CD13 on MO-MDSCs, which has recently been reported to control cell motility in monocytes, alongside reduced VLA-4 expression, an integrin predominantly involved in adherence to the apical side of the endothelium. CD13 and VLA-4 blocking and activating Abs were used in flow-based adhesion assays, live-cell imaging of motility, and actin polymerization studies to confirm a role for CD13 in impaired MO-MDSC transmigration. These findings indicate that CD13 significantly contributes to tissue infiltration by MO-MDSCs and monocytes, thereby contributing to the pathogenesis of hepatic inflammation.</jats:p