Impact of interleukin-27 on replication of hepatitis C virus

Recently, different interleukins have been associated with responses to PEGylated interferon and ribavirin and spontaneous clearance of acute hepatitis C virus (HCV) infection (interleukin (IL)-28B) or with the development of a novel immunotherapeutic strategy for HCV infection (IL-27). IL-27 is a helical cytokine belonging to the IL-6/IL-12 cytokine family with a broad range of anti-inflammatory properties. Some studies demonstrated that IL-27 stimulates hepatoma cells and hepatocytes by inducing a sustained signal transducer and activator of transcription (STAT1 and STAT3) activation. Moreover, IL-27 induces interferon-α-like responses including the induction of antiviral genes (ribonucleic acid-dependent protein kinase), oligoadenylate synthetase, and myxovirus protein. In this review we examine the research on IL-27 and its potential role in therapy for HCV, including the capability to inhibit replication of HCV

The Myeloid Receptor PILRβ Mediates the Balance of Inflammatory Responses through Regulation of IL-27 Production

Paired immunoglobulin-like receptors beta, PILRβ, and alpha, PILRα, are related to the Siglec family of receptors and are expressed primarily on cells of the myeloid lineage. PILRβ is a DAP12 binding partner expressed on both human and mouse myeloid cells. The potential ligand, CD99, is found on many cell types, such as epithelial cells where it plays a role in migration of immune cells to sites of inflammation. Pilrb deficient mice were challenged with the parasite Toxoplasma gondii in two different models of infection induced inflammation; one involving the establishment of chronic encephalitis and a second mimicking inflammatory bowel disease in order to understand the potential role of this receptor in persistent inflammatory responses. It was found that in the absence of activating signals from PILRβ, antigen-presenting cells (APCs) produced increased amounts of IL-27, p28 and promoted IL-10 production in effector T cells. The sustained production of IL-27 led ultimately to enhanced survival after challenge due to dampened immune pathology in the gut. Similar protection was also observed in the CNS during chronic T. gondii infection after i.p. challenge again providing evidence that PILRβ is important for regulating aberrant inflammatory responses

Matrine Inhibits CNS Autoimmunity Through an IFN-β-Dependent Mechanism

Matrine (MAT), a quinolizidine alkaloid component derived from the root of Sophora flavescens, suppresses experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS), by inducing the production of immunomodulatory molecules, e.g., IL-10. In an effort to find the upstream pathway(s) of the mechanism underlying these effects, we have tested certain upregulated immunomodulatory molecules. Among them, we found increased levels of IL-27 and IFN-β, one of the first-line MS therapies. Indeed, while low levels of IFN-β production in sera and type I interferon receptor (IFNAR1) expression in spinal cord of saline-treated control EAE mice were detected, they were significantly increased after MAT treatment. Increased numbers of CD11b+IFN-β+ microglia/infiltrating macrophages were observed in the CNS of MAT-treated mice. The key role of IFN-β induction in the suppressive effect of MAT on EAE was further verified by administration of anti-IFN-β neutralizing antibody, which largely reversed the therapeutic effect of MAT. Further, we found that, while MAT treatment induced production of IL-27 and IL-10 by CNS microglia/macrophages, this effect was significantly reduced by IFN-β neutralizing antibody. Finally, the role of IFN-β in MAT-induced IL-27 and IL-10 production was further confirmed in human monocytes in vitro. Together, our study demonstrates that MAT exerts its therapeutic effect in EAE through an IFN-β/IL-27/IL-10 pathway, and is likely a novel, safe, low-cost, and effective therapy as an alternative to exogenous IFN-β for MS

Antiinflammatory effect of Triptolide on rat microglial cells

In dieser Arbeit wurde die Wirkung von Triptolid auf Mikrogliazellen in einem in vitro Modell untersucht mit der Fragestellung, ob diese Substanz in der Therapie neurodegenerativer Erkrankungen eingesetzt werden kann. Die steigende Prävalenz neurodegenerativer Erkrankungen wie der M. Alzheimer und der M. Parkinson macht die Erforschung neuer Therapieoptionen notwendig. Neurodegenerativen Erkrankungen ist gemeinsam, dass diesen eine Dysregulation des lokalen Immunsystems zugrunde liegt, die zu einer chronischen Inflammation mit Neuronenverlust und damit Progredienz der Erkrankung führt. Eine Eindämmung der Neuroinflammation, an der hauptsächlich Mikrogliazellen beteiligt sind, ist somit ein vielversprechender Therapieansatz. Triptolid, eine bioaktive Komponente aus der chinesischen Kletterpflanze Wilfords Dreiflügelfrucht (Tripterygium wilfordii) wird traditionell in der chinesischen Medizin zur Behandlung von entzündungsvermittelten Erkrankungen, wie bspw. Arthritis, eingesetzt. In dieser Arbeit wurde Triptolid in einem neuroinflammatorischen in vitro Modell untersucht, ob es zu einer Veränderung der Zytokinsekretion, der Proliferation und Migration sowie der Phagozytoseaktivität kommt, wenn primäre Mikrogliazellen aus den Hirnen neugeborener Ratten mit LPS stimuliert und mit Triptolid koinkubiert wurden. Es wurde eine signifikante eine Reduktion der Expression von iNOS in LPS-stimulierten Mikroglia nach Koinkubation mit Triptolid nachgewiesen. Auch die Untersuchungen des Genexpressionsprofils der proinflammatorischen Zytokine in aktivierten Mikrogliazellen zeigte, dass die Expression der Zytokine IL-6, IL-1β und TNF-α durch Triptolid signifikant gesenkt wurde. Auf Proteinebene wurde ebenfalls eine signifikante Reduktion der Freisetzung von IL-6 in Mikrogliazellen nachgewiesen. Neben proinflammatorischen Zytokinen ist auch das Chemokinsystem mit dem neuroinflammatorischen und neurodegenerativem Geschehen im ZNS assoziiert. Veränderungen im Chemokinmuster kann eine Grundlage der chronischen Inflammationsreaktion darstellen. In dieser Arbeit wurde gezeigt, dass Triptolid modulierend auf das Chemokinsystem wirkt, indem es die Genexpression der Chemokine CCL2, CCL3, CCL5, CCL7 und CXCL10 in aktivierten Mikrogliazellen signifikant reduziert. Einen weniger ausgeprägten Effekt hat Triptotlid auf die Expression der Chemokinrezeptoren CCR5 und CXCR4. Hier reduziert Triptolid lediglich die Genexpression des Rezeptors CXCR4 im Vergleich zu ruhenden Mikrogliazellen. Ein weiterer Faktor, der eine Rolle in der Pathogenese neurodegenerativer Erkrankungen spielt, ist eine veränderte Phagozytoseaktivität von Mikrogliazellen. In dieser Arbeit wurde gezeigt, dass Triptolid keinen Einfluss auf die Genexpression des Phagozytoserezeptors FcγR1 in LPS-aktivierten Mikrogliazellen hat und dass es tendenziell die durch LPS-induzierte verminderte Phagozytoseaktivität von Mikrogliazellen wieder aufheben kann. Zusammenfassend ist die Neuroinflammation ein komplexer Prozess, der eine zentrale Rolle bei verschiedenen ZNS-Erkrankungen spielt. Triptolid stellt aufgrund seiner antiinflammatorischen Wirkungsweise und seinen modulierenden Effekt auf Mikrogliazellen eine vielversprechende Substanz in der Therapie neurodegenerativer Erkrankungen dar

Novel mechanisms regulating inflammatory gene expression in adipocytes

Obesity and diabetes are major public health concerns worldwide that contribute to cardiovascular disease, hypertension, and stroke. Research over the last two decades has revealed an important role for adipose tissue (AT) in the regulation and control of whole-body metabolic homeostasis and that chronic AT inflammation is an important pathogenic mechanism that links obesity and diabetes. While it is now recognized that inflammation is increased in AT with obesity, studies are underway to identify key inflammatory mediators and their downstream pathways that contribute to adipocyte inflammation. Therefore, data presented in this dissertation demonstrate that three novel mechanisms that may mediate obesity-induced inflammation in adipocytes. Interleukin (IL)-12 family cytokines are heterodimeric proteins mostly expressed in classic immune cells and play critical roles in innate and adaptive immunity. However, recent evidence has shown that plasma levels of IL-12 family cytokines are elevated in obesity and diabetes, yet little has addressed a role for IL-12 family cytokines in adipocytes under any condition. Data presented in this dissertation demonstrate that various IL-12 family members are highly induced in AT under conditions of genetic and diet-induced obesity that is associated with increased inflammation and IR. Subsequently, we show that both preadipocytes (PAs) and adipocytes (ADs) secrete IL-27 in response to inflammatory stress and demonstrate a novel function of IL-27 in adipocytes that is dependent on environmental cues. While PAs and ADs both secrete IL-27 protein, divergent mRNA regulation of the IL-27 subunits (i.e., p28 and EBI3) was observed in response to tumor necrosis factor a (TNFa) in adipocytes. As our data indicated a role for epigenetic modifications in differential IL-27 mRNA expression, we further determined that histone deacetylases (HDACs) play a key role in TNFa-induced p28 gene expression. Thus, these data demonstrate that epigenetic modification of histones potentially regulates the divergent outcomes observed with p28 and EBI3 gene expression during inflammatory stress. While extensive studies have identified TNFa as a master cytokine involved in AT inflammation, questions regarding its inflammatory actions on gene expression remain unclear. Emerging evidence highlights an important role for the Golgi apparatus (GA) in TNFa-induced inflammation, in part, through TNF receptor (TNFR) storage and secretion to the cell-surface, thus allowing for TNFa signaling. Our findings demonstrate a critical role for the GA in TNFR cell-surface expression and TNFa signaling as well as suggest a novel mechanism for the GA in TNFa-induced inflammation that potentially involves Golgi-localized co-factor(s) necessary for transcriptional gene expression. Collectively, data presented in this dissertation provide seminal evidence for novel mechanisms regulating adipocyte inflammation, potentially linking obesity with metabolic diseases

Role Of Il-27 And Tcr Stimulation In Inhibitory Receptor Expression

The adaptive immune response is necessary for control of pathogen burden in a wide range of infections. However, in the absence of active regulatory mechanisms, this protective response can lead to immune pathology. The cytokine interleukin (IL)-27 is required for control of exaggerated immune responses during toxoplasmosis and other infections and autoimmune settings. Multiple regulatory pathways have been found to be controlled by IL-27. However, there are gaps in our knowledge of the mechanisms by which IL-27 limits T cell responses. The present work focuses on the ability of IL-27 to promote expression of inhibitory receptors on T cells. The studies presented here establish that TCR and cytokines have distinct and complementary roles in promoting inhibitory receptor expression. In vitro, IL-27, type I IFN, and IFN-g induced expression of PD-L1 and Sca-1 on naïve murine CD4+ and CD8+ T cells in the absence of TCR stimulation. TCR stimulation induced expression of multiple inhibitory receptors and IL- 27 combined synergistically with TCR stimulation to further upregulate Ly6C, LAG-3, CTLA-4, TIGIT and TIM-3. This IL-27-mediated inhibitory expression was STAT1- dependent. The response to TCR stimulation was graded and thus a stronger TCR stimulus resulted in greater inhibitory receptor expression. In vivo, during infection with Toxoplasma gondii, IL-27 was required for full expression of Ly6C, PD-L1, LAG-3, CTLA-4, and TIGIT by parasite-specific T cells in the lung, a local site of infection, but not in the spleen. STAT1 was also required for full expression of LAG-3, CTLA-4, and TIGIT at local sites of infection. Taken together, these studies suggest a model in which inhibitory receptor expression on T cells is a graded regulatory pathway that is upregulated by exposure to increasing levels of TCR stimulation and cytokines present at sites of inflammation

Immunmodulatorische Wirkungen von repetitiver CpG-ODN-Gabe auf das murine Gehirn und die Leber

Unmethylierte bakterielle DNA, die reich an CG Sequenzen ist stimuliert das angeborene Immunsystem. Die Vermittlung dieser Wirkung erfolgt über den Toll-like Rezeptor 9. Reine CG Sequenzen sind im Organismus nicht stabil, da sie sehr rasch von den körpereigenen Nukleasen abgebaut werden. Mit synthetisch hergestellten Oligodeoxyribonukleotiden, sogenannten CpG-ODNs, kann eine Nukleaseresistenz erzielt werden und der immunstimulatorische Effekt nachgeahmt werden. Weiterhin wirken sich CpG-ODNs positiv auf den Verlauf von bakteriellen Infektionen, Tumoren und Prioninfektionen aus. Man weiß, dass eine Aktivierung des Immunsystems durch einmalige Gabe von CpG-ODNs nur über einen kurzen Zeitraum stattfindet. Die CpG-ODNs führen im Organismus innerhalb von Minuten zu einem mRNA Anstieg und innerhalb von Stunden zu einer kurzfristigen Zytokinsekretion und IgM Produktion, werden dann aber rasch abgebaut, so dass der immunstimulatorische Effekt in der Regel nur kurz anhält. Um CpG-ODN als Therapeutikum besser nutzen zu können liegt der Wunsch nahe, die Wirkung von CpG-ODN zu verlängern. Eine mögliche Strategie ist hierbei eine repetitive Applikation. Ziel dieser Arbeit war, den immunstimulatorischen Effekt von repetitiver CpG-ODN-Gabe besser zu verstehen und mit der Repetition die Wirkung zu verlängern. Damit könnte CpG-ODN als Therapeutikum bei bakteriellen Infektionen, Tumoren und bei Prionerkrankungen wirkungsvoll eingesetzt werden. In dieser Arbeit wurden jeweils 12 Gruppen zu je 5 Mäusen gebildet, wobei die jeweiligen Gruppen an 5, 7 oder 21 aufeinanderfolgenden Tagen jeweils eine CpG-ODN, NaCl oder Neg-ODN Applikation i.p. erhielten. Die Mäuse wurden anschließend an Tag 7 oder 28 während bzw. nach der Behandlung getötet. So erhielt man folgende Gruppen: 5x-CpG-ODN, NaCl oder Neg-ODN behandelte, an Tag 7 getötete Mäuse 5x-CpG-ODN, NaCl oder Neg-ODN behandelte, an Tag 28 getötete Mäuse 7x-CpG-ODN, NaCl oder Neg-ODN behandelte, an Tag 7 getötete Mäuse 21x-CpG-ODN, NaCl oder Neg-ODN behandelte, an Tag 28 getötete Mäuse Im Verlauf der Arbeit, die die immunmodulatorische Wirkung auf das Gehirn und die Leber der Maus untersucht, konnten zusammenfassend folgende Ergebnisse herausgearbeitet werden, die mittels Real time PCR und Immunhistologie gewonnen wurden. Im Gehirn führt CpG-ODN zu einer mindestens einwöchigen Hochregulierung der mRNA von TNFα. Des Weiteren kommt es zu einer mindestens zweitägigen C1q und IFNg Hochregulierung. Eine IL-12p40 Hochregulierung findet nur ca. 18 Stunden statt, während eine STAT3 Hochregulierung nicht nachweisbar ist. In der histologischen Betrachtung finden sich in der HE Färbung keine pathologischen Veränderungen der Hirnarchitektur und in einer immunhistologischen Färbung mit CD 11b und GFAP bindenden Antikörpern keine Unterschiede zwischen den CpG-ODN, NaCl oder Neg-ODN behandelten Tieren. In der Leber findet sich eine signifikante Hochregulierung von IL-12p40 über mindestens drei Wochen und eine C1q, TNFα und IFNg Hochregulierung von mindestens einer Woche. Histologisch finden sich in der HE Färbung massive Leukozyteninfiltrate über mindestens zwei Tage. In der Immunhistologie sieht man an den Infiltraten beteiligte aktivierte Makrophagen, T und B-Lymphozyten, jeweils dargestellt mit CD 11b, CD 8 und B220 bindenden Antikörpern. Es zeigte sich, dass eine repetitive CpG-ODN-Gabe eine verlängerte stimulatorische Wirkung auf das Immunsystem ausübt, was Voraussetzung für den Einsatz als Therapeutikum ist. Besonders bemerkenswert ist die Tatsache, dass man durch periphere Gabe von CpG-ODN im Gehirn eine Immunstimulierung erreichen kann. Diese könnte im Rahmen einer Therapie von Prionerkrankungen, anderen Gehirninfektionen, Morbus Alzheimer oder Gehirntumoren Anwendung finden. Allerdings sind noch weitere Studien nötig, um Risiken wie Hepatotoxizität oder Autoimmunität besser abschätzen zu können und den Mechanismus zu erforschen, wie durch periphere Gabe von CpG-ODN eine immunologische Gehirnaktivierung erreichen wird. Eine wichtige Frage für die Zukunft ist hierbei, ob die Wirkung von CpG-ODN direkt auf die Gehirnzellen wirkt oder ob es einen second messenger gibt, der die Blut-Hirn-Schranke überwindet.It is well known, that unmethylated bacterial DNA, which contains CG motifs has a stimulatory effect to the immune system. The recognition of this effect is mediated via the toll-like receptor 9. This immunstimulatory effect was mimicked by synthetic oligodeoxyribonucleotides (CpG-ODNs), which are much more stable in the living organism. It has been shown that CpG-ODN administration has a positive effect on infection diseases, tumors and prion infections. This study wanted to investigate whether the strategy of consecutive CpG-ODN administration could be used to induce brain relevant immune activation as prion infection predominantely effects the brain. Furthermore it was investigated whether the consecutive administration of CpG-ODN led to detrimental sideeffects in brain and liver tissue. We choose four application patterns for CpG-ODN including a saline or Neg-ODN treated control respectively. So we got 12 groups following the proximate pattern: 5x-CpG-ODN, saline or Neg-ODN treated mice, sacrificed at day 7 5x-CpG-ODN, saline or Neg-ODN treated mice, sacrificed at day 28 7x-CpG-ODN, saline or Neg-ODN treated mice, sacrificed at day 7 21x-CpG-ODN, saline or Neg-ODN-treated mice, sacrificed at day 28 We were able to detect the following results by Real-time PCR and immunohistology on the immunmodulatory effect and chronological sequence on the murine brain and the liver. In the brain mRNA TNFα levels are upregulated up to 1 week, mRNA levels of C1q und IFNg are upregulated up to at least two days. Upregulation of IL-12p40 is detectable for a minimum of 18 h. An upregulation of STAT3 was not detectable in the brain. In immunohistology we didn’t observe any pathological changes in brain morphology and no enhanced staining of microglia and astrocytes using antibodies for GFAP and CD 11b. In the liver a significant upregulation for IL-12p40 mRNA for a minimum of 3 weeks was detected. Furthermore we observed upregulated mRNA levels for C1q, TNFα and IFNg for minimum 1 week. STAT3 mRNA levels were upregulated up to two days. By HE staining we observed massive infiltrates of leukocytes for at least 2 days. In immunohistology we detected active macrophages, T-and B-Lymphozytes by the antibodies CD 11b, CD 8 and B220. Our study showed that it was possible to induce brain specific immune activation without observable side effects this may indicate the possibility to use such a strategy to combat brain diseases like prion infection, Alzheimer`s disease etc. Regarding the liver no longterm side effects were observed however further studies are needed to completely rule out relevant liver toxicity

The role of IL-27 and IL-35 in inflammatory diseases

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by chronic inflammation within the synovial tissues in multiple joints leading to progressive, erosive destruction of cartilage and underlying joints. The severity of RA is associated with the overexpression of proinflammatory cytokines within the synovial tissue, such as TNF-, IL-1 and IL-6. Recently, IL-17 is thought to play a critical role in maintaining the inflammatory processes within the arthritic joints. Although the etiology and pathogenesis of RA has not been completely elucidated, neutralizing Antibodies against the inflammatory components have been shown to successfully suppress joint inflammation, reduce the relapse rate and delay disease onset in RA patients. Therefore, the expression and regulation of cytokines produced during the disease progression has been the centre of interest in therapeutic studies. Cytokines are important mediators of immune functions in humans and animals. In this thesis, a murine model of RA has been used to investigate the roles of new cytokines Interleukin (IL)-27 and Interleukin (IL)-35. Interleukin (IL)-27, is a heterodimeric cytokine comprised of an IL-12p40 related protein, Epstein-Barr virus-induced gene 3 (EBI3) and a unique IL-12p35 like protein p28. IL-27 is a member of IL-12 family, mainly generated by activated macrophages and dendritic cells. IL-27 binds a receptor composed of WSX-1/TCCR, a ligand-specific chain, and gp130, a signal-transducing molecule shared with other cytokines such as IL-6. IL-27 can promote both pro- and anti-inflammatory immune responses. A novel role of IL-27 regulating autoimmunity has been suggested by experiments on experimental autoimmune encephalomyelitis (EAE) and central nervous system (CNS) inflammation when infected with Toxoplasma gondii. IL-27 suppresses these chronic diseases through inhibiting Th17 activity. Thus, IL-27 may have an important therapeutic potential for treatment of RA in humans. A major aim of this project has been to clone and express a recombinant murine IL-27 in sufficient quantities to study the role of IL-27 in a murine model of RA closely related to the human disease, collagen-induced arthritis (CIA). A short term administration of IL-27 to mice at the onset of the disease had a significantly suppressive effect on disease severity and incidence compared with untreated controls. Mice treated with the recombinant IL-27 also showed reduced serum IL-6, IL-17 and collagen-specific IgG2a. Spleen and lymph node cells from the IL-27-treated mice produced significantly less IFN- and IL-17 compared with cells from the control mice when cultured with collagen in vitro. In contrast, administration of IL-27 to mice during the late phase of CIA significantly exacerbated disease severity. IL-27-treated mice also showed elevated IFN-γ and IL-6 production by the lymphoid cells when compared to untreated mice. However, IL-17 synthesis was not affected between IL-27-treated mice and untreated mice. Consistent with this finding, in vitro IL-27 markedly inhibited the development of Th17 from naïve CD4+, CD4+CD25- and CD4+CD25+ T cells, but had little or no effect on differentiated Th17 cells. Together, these results demonstrated that IL-27 had both pro-inflammatory and anti-inflammatory effects on chronic articular inflammation, mainly associated with Th17 functions. Interleukin (IL)-35, another novel heterodimeric cytokine belonging to IL-12 family, is composed of EBI3 and the IL-12p35 subunit. Little is known about the biological function of IL-35. To study the role of IL-35 in immune responses, murine recombinant IL-35 was cloned and expressed in a mammalian GS system to produce sufficient quantities. IL-35 induced proliferation of murine CD4+CD25+ and CD4+CD25- T cells when stimulated with immobilized anti-CD3 and anti-CD28 antibodies in vitro. The IL-35-expanded CD4+CD25+ T cell population expressed Foxp3 and produced elevated levels of IL-10, whereas the IL-35-induced CD4+CD25- T cells produced IFN-γ but not IL-4. The IL-35-expanded CD4+CD25+ T cells maintained their suppressive functions against CD4+CD25- effector cells. Furthermore, when cultured with soluble anti-CD3 and antigen-presenting cells, IL-35 directly suppressed the proliferation of CD4+CD25- effector cells. Moreover, IL-35 inhibited the differentiation of Th17 cells in vitro. In vivo, IL-35 effectively suppressed established collagen-induced arthritis in mice with the suppression of IL-17 production but enhanced IFN-γ synthesis. Therefore, IL-35 is a novel cytokine suppressing the immune response through the expansion of regulatory T cells and suppression of Th17 cell development. For the future study of human IL-35, human EBI3 and p35 were cloned and linked together with an Fc fusion part. Human IL-35 was expressed in GS system and the function of the recombinant protein needs further study. These data in this thesis provide direct evidence that IL-27 and IL-35 are important mediators in murine collagen-induced arthritis disease. This implicated that IL-27 and IL-35 could represent potential new targets for novel therapeutic agents in human RA. However, the findings on the dual role of IL-27 at the different disease process suggested that the involvement of IL-27 in the pathogenesis of human RA should be carefully investigated before clinical therapy application

Sistema cannabinoide y regulación neuroinmune en un modelo viral de esclerosis múltiple: estudio de la familia de citoquinas heterodiméricas IL-12/IL-23

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina. Departamento de Anatomía, Histología y Neurociencia. Fecha de lectura: 11 de Junio 200