Interleukin-l0 (IL-10) helps to limit the duration of potentially harmful inflammatory responses but has also been implicated in the persistence of a number of chronic viral infections. This thesis aimed to investigate the phenotype and function of mv -specific IL-l0-producing cells in chronic HIV-I infection, and the effect of IL-10 blockade on responses to candidate HIV -I vaccines. A cytokine capture assay was used to determine the HIV -specific cellular sources of IL- 10 in PBMC from 55 chronically infected individuals. A rare subset of CD8+ T cells was found to be the major HIV -I Gag-specific IL-10-producing population; these cells were restricted to ART-naive individuals and did not express the regulatory T cell markers CD25 or FoxP3 but could co-express IFN-y. A proportion of the population (median 48% and 9% respectively) expressed the P7 chain of the gut-homing integrin a4p7 and the chemokine receptor CXCR3, which mediates lymphocyte migration to sites of inflammation. Experimental depletion of Gag-specific IL-10+ CD8+ T cells did not affect T cell activation, or the production of cytokines such as IL-2 or IFN-y during short-term culture. However, depletion was associated with a significant increase in CD38 expression on CDI4+ monocytes, a trend towards increased HLA-DR expression on the same cells, and a significant increase in the concentration of the pro-inflammatory cytokine IL-6 in culture supernatants. There was also a significant increase in the number of HIV-infected (p24 antigen+) CD4+ T cells in cultures depleted of Gag- specific IL-10+ CD8+ T cells after 3 days, indicating that this population may contribute to control of viral replication. In order to determine the effect of IL-10 blockade on vaccine immunogenicity, IL-10R blocking antibody was administered to BALB/c mice prior to immunisation with two mV-I candidate vaccines, HIVA and HIVconsv. IL-10R blockade resulted in a trend towards increased IFN-y production by CD8+ T cells in response to the dominant H (Env) and P (Pol) epitopes of HIV A, and a significant increase in IFN-y ELISPOT responses to the subdominant Gl (Gag) epitope of HIV consv in vitro. Collectively, these data suggest that IL-10 producing cell populations may play critical but different roles in chronic infection and vaccination. Further research into how the timing of IL-10 responses affects disease outcome may allow IL-IO blockade to be explored as a therapeutic strategy in humans

Master of Science / Department of Anatomy and Physiology / Lei Wang / Jishu Shi / Interleukin-1 beta (IL-1β) is an important mediator of inflammatory response, and the elevated expression of IL-1β is correlated with tumor growth and metastasis. Epithelial-mesenchymal transition (EMT) is a reversible transition between epithelial phenotype and mesenchymal phenotype. Usually, EMT can be identified by its unique morphology change and expression of EMT markers. In our study, we have found after treated HCT-116, a colon cancer cell line, and human primary colon cancer cells with IL-1β, cells began to display mesenchymal phenotype with highly down-regulated E-cadherin expression and up-regulated ZEB factors expression. For colon cancer cells, sphere formation assay in serum free medium (SFM) with the presence of growth factors is used to identify cancer stem cell population. We have shown that IL-1β can induce colon cancer stem cell proliferation and express stem cell markers (Bmi1, Nanog, and Nestin). In addition, besides the stem cell markers, we also found ZEB factors were highly up-regulated in spheroid cells as well. We silenced Zeb1 expression and investigated the effect of IL-1β on shZeb1 HCT-116 cells. The results indicated Zeb1 knockdown not only inhibited IL-1β-induced EMT but also reduced proliferation of spheroid cells and inhibited Bmi1 expression. Therefore, ZEB factors must play an important role in both EMT process and cancer stem cell development. From our data, we conclude that IL-1β promotes epithelial-mesenchymal transition and a stem cell phenotype in colon cancer via ZEB factors.

Interleukin-5 (IL-5) is a member of the hematopoietic class I cytokines and is specifically involved in eosinophil activation. IL-5 plays an important role in disease conditions such as allergic asthma and other hypereosinophilias, which are characterized by highly increased levels of eosinophils in peripheral blood and tissues. The IL-5 receptor is a heterodimer consisting of a binding alpha subunit (IL- 5Rα) and a common beta subunit (IL-5Rβ). This IL-5Rβ is shared with the IL-3 and GM-CSF receptors. The IL-5Rα is required for ligand-specific binding, whereas the association of the IL-5Rβ subunit triggers intracellular signal transduction. Previous studies have described the crystallographic structure of human IL-5 (hIL-5), as well as that of the common IL-5Rβ chain (IL-5Rβc) However, no experimental structural data are yet available for the interaction of the high-affinity IL-5 receptor IL-5Rα with its ligand IL-5. Therefore, this thesis had the principle objective to gain new insights into the basis of this important agonist-receptor interaction. In particular, data on the recombinant expression, purification and preparation of the binary complex of hIL-5 bound to the receptor ectodomain of hIL-5Rα are shown, as well as the subsequent crystal structure analysis of the binary ligand-receptor (hIL-5Rα/hIL-5) complex. Both proteins were expressed in an Escherichia coli expression system, purified to homogeneity, and crystallized. However, since the initial analysis of these crystals did not show any X-ray diffraction, each step of the preparation and crystallization procedure had to be stepwise optimized. Several improvements proved to be crucial for obtaining crystals suitable for structure analysis. A free cysteine residue in the N-terminal domain of the hIL-5Rα ectodomain protein was mutated to alanine to remove protein heterogeneity. In addition, hIL-5 affinity chromatography of the receptor protein proved to be absolutely crucial for crystal quality. Additive screening using the initial crystallization condition finally yielded crystals of the binary complex, which diffracted to 2.5Å resolution and were suitable for structure analysis. The preliminary structure data demonstrate a new receptor architecture for the IL-5Rα ligand-binding domain, which has no similarities to other cytokine class I receptor structures known so far. The complex structure demonstrates that the ligand-binding region of human IL-5Rα is dispersed over all three extracellular domains, and adopts a binding topology in which the cytokine recognition motif (CRM) needs the first Fn-III domain of the human IL-5Rα to bind the ligand. In a second project, a prokaryotic expression system for murine IL-5 (mIL-5) was established to allow the production of mIL-5 and mIL-5 antagonist that should facilitate functional studies in mice. Since the expression of mIL-5 in E. coli had never been successful so far, a fusion protein system was generated expressing high yields of mIL-5. Chemical cleavage with cyanogen bromide (CNBr) was used to release mIL-5 monomers, which were subsequently purified and refolded. This technique yielded an active murine IL-5 dimer as confirmed by TF-1 cell proliferation assays. The protein was crystallized and the structure of mIL-5 could be determined at 2.5Å resolution. The molecular structure revealed a symmetrical left-handed four helices bundle dimer similar to human IL-5. Analysis of the structure-/function relationship allowed us to design specific mIL-5 antagonist molecules, which are still under examination. Taken together, these findings provide further insights in the IL-5 and IL-5R interaction which may help to further understand and depict this and other cytokine-receptor interactions of similar architecture, e.g. the IL-13 ligand-receptor system. Ultimately, this may represent another piece of puzzle in the attempts to rationally design and engineer novel IL-5-related pharmacological therapeutics. / Interleukin-5 (IL-5) ist ein Mitglied der Gruppe der hematopoetischen Zytokine der Klasse I und spielt eine Schlüsselrolle bei der Aktivierung von eosinophilen Granulozyten. IL-5 hat damit ein wichtige pathophysiologische Funktion bei der Entstehung von Krankheiten wie allergischem Asthma und anderen Hypereosinophilien, die alle durch eine stark erhöhte Zahl von Eosinophilen in peripherem Blut und Geweben charakerisiert sind. Der IL-5 Rezeptor ist ein Heterodimer, der aus einer alpha-Untereinheit (IL-5Rα) und einer mit den IL-3 und GM-CSF Rezeptoren gemeinsamen beta-Untereinheit (IL-5Rβ) besteht. Der IL-5Rα ist für die spezifische Liganden-Bindung notwendig, während der mit der IL-5Rβ Untereinheit assozierte Komplex die intrazelluläre Signaltransduktion einleitet. In früheren Studien konnte bereits die Kristallstruktur des menschlichen IL-5 (hIL-5) und der gemeinsamen IL-5Rβ-Kette (IL-5Rβc) aufgeklärt werden. Allerdings liegen bisher noch keine experimentellen Strukturdaten für die Interaktionen des hochaffinen IL-5 Rezeptor IL-5Rα mit seinem Ligand IL-5 vor. Deshalb war es die Hauptzielsetzung dieser Arbeit, neue Einblicke in die molekulare Basis der Interaktion von IL-5 Rezeptor und Agonisten zu gewinnen. Im Einzelnen beschreibe ich in dieser Arbeit die rekombinante Expression, Aufreinigung und Herstellung des binären Komplexes von der hIL-5 Bindung an die extrazellulären Domäne des Rezeptors hIL-5Rα sowie die anschließende kristallographische Strukturanalyse dieses binären Ligand-Rezeptor-Komplexes (hIL-Rα/hIL-5). Beide Proteine wurden in einem Escherichia coli-Expressionssystem rekombinant hergestellt, bis zur Homogenität gereinigt und anschließend kristallisiert. Die Analysen dieser ersten Kristalle zeigten nicht die gewünschte Beugung der Röntgenstrahlung, weshalb in allen anschließenden Schritten eine schrittweise Optimierung der Produktions- und Kristallisationsbedingungen durchgeführt wurde. Als Ergebnis dieser Optimierungsstrategie konnten schließlich Kristalle erhalten werden, die für eine Strukturanalyse geeignet waren. Ein ungepaartes Cystein in der N-terminalen Domäne des extrazellulären hIL-5Rα-Protein wurde durch Alanin ersetzt, um so die Protein-Heterogenität durch Cystein-Oxidationsprodukte zu verringern. Die affinitätschromatographische Aufreinigung des Rezeptorproteins war ebenfalls entscheidend, um eine hohe Kristallqualität zu erreichen. Die Verwendung verschiedener Additivsubstanzen zusätzlich zu der initialen Kristallisationsbedingungen führte letztlich zur Bildung für die Strukturanalyse geeigneter Einzelkristallen des binären Komplexes (hIL-5Rα/hIL-5). Ihre Messung ergab Beugungsdaten mit einer maximalen Auflösung von 2.5Å. Eine erste Strukturanalyse zeigt klar, dass die Liganden-bindende Domäne des IL-5Rα Rezeptors eine bisher unbekannte, neuartige Rezeptor-Architektur aufweist, die keinerlei Ähnlichkeit zu bisher bekannten Zytokinrezeptor-strukturen der Klasse I hat. Die Struktur des Komplexes zeigt zudem, dass das Liganden-bindende Epitop von IL-5Rα über alle drei extrazelluläre Domänen verteilt ist und eine Topologie aufweist, in der zusätzlich zu dem Zytokin-Erkennungsmotiv (CRM) die erste Fn-III Domäne von hIL-5Rα benötigt wird, um den Liganden hochaffin binden zu können. In einem zweiten Projekt wurde ein prokaryotisches Expressionssystem für murines Interleukin-5 (mIL-5) entwickelt, welches die Produktion von mIL-5 und eines mIL-5 Antagonisten für funktionelle Studien in einem Mausmodell ermöglichen sollte. Da eine rekombinante Produktion von mIL-5 in E.coli bisher nicht erfolgreich war, wurde ein Fusionsproteinsystem entwickelt, welches die Produktion großer Mengen von mIL-5 Protein erlaubt. Es wurde eine chemische Spaltung mit Cyanbromid (CNBr) durchgeführt, um das Monomer aus dem Fusionsprotein freizusetzen. Das so erhaltene mIL-5 Monomer wurde gereinigt und renaturiert. Nach Rückfaltung zeigt das dimere Protein in TF-1 Zellen eine mit den Literaturwerten vergleichbare biologische Aktivität. Das so erhaltene mIL-5 Protein wurde kristallisiert und mittels Röntgenbeugung analysiert. Auf diese Weisen konnten Beugungsdaten von mIL-5 Kristallen mit einer maximalen Auflösung von 2.5Å erhalten werden. Die Struktur weist ähnlich wie das humane IL-5 ein symmetrisches Vier-Helix Bündel auf. Die Struktur-/Funktionsanalyse ermöglichte daraufhin, definierte mIL-5-Antagonisten zu entwickeln, die sich derzeit noch in der Untersuchung befinden. Zusammengefasst tragen die hier präsentierten Ergebnisse dazu bei, die molekularen Grundlagen der spezifischen IL-5 und IL-5R Bindung sowie Interaktionen ähnlichen Liganden-Rezeptor-Typen zu verstehen. Letztendlich besteht die Hoffnung, dass diese und ähnliche Arbeiten ein weiteres wichtiges Puzzlestück darstellen bei dem Versuch, neue und innovative pharmakologische Therapieansätze zu entwickeln, die bei dem für die Pathophysiologie von Asthma wichtigen Schlüsselmolekül IL-5 angreifen.

Macrophages are important effector cells of the innate and adaptive immune response and exert a wide variety of immunological functions which necessitates a high level of plasticity on the chromatin level. In response to pathogen-associated molecular patterns (PAMPs) or inflammatory signals macrophages undergo a process of cellular activation which is associated with morphologic, functional and biochemical changes. Toll-like receptors (TLR) are able to sense many different PAMPs. TLR4 is an important sensor for lipopolysaccharide (LPS) which elicits a major portion of the host’s inflammatory response through the activation of many different signaling pathways such as the NF-κB and the MAPK protein kinase pathways RASRAF- MEK-ERK, p38 and JNK. Polycomb group (PcG) proteins are well known chromatin modifiers which function in large complexes and are required to maintain chromatin structure in a transcriptionally repressed state. It has previously been shown that the PcG protein Bmi1 is phosphorylated by 3pK, a downstream effector kinase of the MAPK protein kinase pathways RAS-RAF-MEK-ERK, p38 and JNK. In this work I analyzed the role of Bmi1 as a downstream effector of MAPK signaling during macrophage activation. Unexpectedly a rapid up-regulation on the Bmi1 protein level was observed in bone marrow derived macrophages (BMDMs) after LPS treatment. The Bmi1 induction was associated with transient protein phosphorylation that occured downstream of MAPK signaling. LPS treatment of BMDMs in the absence of Bmi1 resulted in a pronounced increase of IL-10 secretion. This secretion of the anti-inflammatory cytokine IL-10 was associated with increased IL-10 mRNA levels. Furthermore, siRNA mediated knock down of Bmi1 in J774A.1 macrophages also resulted in elevated IL-10 mRNA levels in response to LPS. ChIP analysis revealed that Bmi1 binds to throughout the il-10 locus. Alternative activation of wild type BMDMs via concomitant TLR4 and FcγR activation which triggers high IL-10 expression is paralleled by an attenuated Bmi1 protein expression. These results identify Bmi1 as a repressor of IL-10 expression during activation of macrophages. / Makrophagen sind wichtige Effektorzellen der angeborenen und adaptiven Immunantwort und üben eine große Fülle von immunologischen Funktionen aus. Deshalb benötigen sie eine hohe Plastizität auf Chromatinebene. Als Antwort auf pathogen-assoziierte molekulare Muster (PAMPs) oder andere inflammatorische Signale machen Makrophagen einen zellulären Aktivierungsprozess durch, der mit morphologischen, funktionellen und biochemischen Veränderungen assoziiert ist. Toll-like Rezeptoren (TLR) sind fähig, solche PAMPs zu erkennen. Der TLR4 ist ein wichtiger Sensor für Lipopolysaccharid (LPS). LPS löst eine inflammatorische Antwort des Wirtes durch die Aktivierung vieler verschiedener Signalwege wie zum Beispiel des NF-κB Signalwegs und der MAPK Proteinkinasensignalwege RAS-RAFMEK- ERK, p38 und JNK aus. Polycomb Gruppen (PcG) Proteine arbeiten in großen Proteinkomplexen zusammen und werden benötigt, um das Chromatin in einem transkriptionell reprimierten Zustand beizubehalten. Es konnte gezeigt werden, dass das PcG Protein Bmi1 von 3pK, einer Effektorkinase der MAPK Proteinkinasensignalwege RAS-RAF-MEK-ERK, p38 und JNK, phosphoryliert wird. In meiner Doktorabeit analysierte ich die Rolle von Bmi1 als downstream-Effektor der MAPKSignaltransduktion während der Makrophagenaktivierung. Es wurde unerwarteterweise eine schnelle Induktion von Bmi1 auf Proteinebene in Knochenmarks-Makrophagen (BMDMs) beobachtet. Diese Induktion war MAPK-abhängig und mit einer transienten Proteinphosphorylierung assoziiert. Die LPS-Behandlung der BMDMs in Abwesenheit von Bmi1 hatte erhöhte IL-10 Sekretion zur Folge. Diese erhöhte Sekretion des antiinflammatorischen Zytokines IL-10 war mit erhöhten IL-10 mRNA Expression verbunden. Des Weiteren hatte ein Bmi1 siRNA-vermittelter Gen-Knockdown in J774A.1 Makrophagen eine erhöhte IL-10 mRNA-Expression zur Folge. ChIP Analysen haben gezeigt, dass Bmi1 am ganzen il-10-Locus verteilt binden kann. Eine TLR4 und FcγR vermittelte alternative Aktivierung von BMDMs, die mit hoher IL-10 Expression verbunden ist, führte zu einer attenuierten Bmi1 Proteinexpression. Diese Ergebnisse zusammengenommen weisen Bmi1 als Repressor von IL-10 während der Makrophagenaktivierung aus.

Orientador: Luiz Henrique de Araújo Machado / Resumo: Dermatite atópica (DA) canina é uma enfermidade alergoinflamatória de caráter crônico, da pele e orelha externa, geralmente de cães adultos jovens, induzida por distintos alérgenos. A afecção tem etiopatologia complexa e ainda não totalmente elucidada, dificilmente atingindo cura. Porém, há hoje, várias opções medicamentosas para o seu controle. O oclacitinib faz parte da recente remessa de medicamentos para tratamento e controle do prurido dos cães atópicos, com poucos efeitos colaterais. É um fármaco inibidor de janus quinase, com rápida ação e que age na ligação das citocinas aos receptores JAKs. Este trabalho teve como intuito avaliar a resposta inflamatória da pele de animais atópicos, antes e 30 dias após o tratamento com Oclacitinib comercial (Apoquel®), através da mensuração das interlucinas IL-1, IL-4, IL-6, IL-10, IL-17, IL-31, TNF-, IFN- e filagrina, pela técnica de imuno-histoquímica, além da resposta clinica ao tratamento pela avaliação do CADESI-4 e escore do prurido. Os 10 cães utilizados no estudo são provenientes do atendimento dermatológico do HV da UNESP-Botucatu e clínica particular de Sorocaba-SP. Apenas os escores do CADESI-4, do prurido e da IL-1, citocina inflamatória chave da imunidade inata e na patogênese da DA, apresentaram redução na análise estatística. Concluise que a IL-1 pode ter um papel importante na farmacocinética do oclacitinib e maiores estudos são necessários para confirmar a ação do oclacitinib sobre a produção e ligação da IL-1 aos ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Canine atopic dermatitis (DA) is a chronic inflammatory alergo disease of the skin and external ear, usually of young adult dogs, induced by distinct allergens. The disease has complex and still not fully elucidated etiopatology, hardly reaching cure. However, there are today several drug options for your control. Oclacitinib is part of the recent consignment of medications for treatment and control of the itching of atopic dogs, with few side effects. It is an inhibitor drug of Janus Kinase, with rapid action and acting in the binding of cytokines to jaks receptors. The objective of this work was to evaluate the inflammatory response of the skin of atopic animals before and 30 days after treatment with commercial oclacitinib (Apoquel®), through the measurement of interlukines il-1, IL-4, IL-6, IL-10, IL-17, IL-31, TNF-α, IFN-γ and Filagrin, by immunohistochemistry technique, in addition to clinical response to treatment by evaluation of Cadesi-4 and pruritus score. The 10 dogs used in the study are from the dermatological treatment of HV of UNESP-Botucatu and private Clinic of Sorocaba-SP. Only the scores of Cadesi4, pruritus and IL-1, the key inflammatory cytokine of innate immunity and the pathogenesis of DA, showed a reduction in the statistical analysis. It is concluded that IL-1 may play an important role in the pharmacokinetics of oclacitinib and further studies are needed to confirm the action of oclacitinib on the production and binding of IL-1 to cutaneous receptors... (Complete abstract click electronic access below) / Mestre

O Diabetes Mellitus tipo 1 (DM1A) é uma doença autoimune caracterizada pela infiltração pancreática de linfócitos T e B, macrófagos e células dendríticas, levando à perda progressiva da capacidade de secreção de insulina pelas células beta pancreáticas. A homeostase das células T, ou seja, o desenvolvimento e manutenção apropriados dos números e funções das células T são essenciais para a integridade do sistema imune. Classicamente acreditava-se que as células T CD4+ poderiam se subdividir em duas populações efetoras distintas, T helper 1 e T helper 2. Recentemente, foram descritas duas novas vias de ativação de linfócitos T CD4+: a via Th17, que tem papel fundamental na autoimunidade; a via T regulatória, onde células T CD4+CD25+ high são essenciais na tolerância periférica e proteção contra autoimunidade. As Interleucinas (IL) de cadeia gama comum agem em várias etapas desta diferenciação linfocítica. A IL-21 é o membro mais recente desta família de citocinas, que inclui também: IL-2, IL-4, IL-7 , IL-9 e IL-15. A IL-21 atua através da interação com seu receptor, o IL-21R, apresentando ações pleiotrópicas e, como regra, pró-inflamatórias. Em estudos com modelos animais de diabetes autoimune verificou-se que a IL-21 e seu receptor são essenciais para o desenvolvimento da doença, porém ainda não há estudos sobre a ação desta interleucina no DM1 em humanos. O objetivo de nosso estudo foi avaliar o papel dos receptores das interleucinas de cadeia gama comum na patogênese do DM1A através da determinação da expressão da proteína de superfície e do RNA mensageiro destes receptores em pacientes com DM1A de início recente, em comparação com indivíduos controles normais, e da correlação destes valores com títulos de autoanticorpos pancreáticos. Estudamos a expressão da proteína de superfície do IL-21R, IL-2R (CD25), IL-2R (CD122), IL-4R (CD124) e IL-7R (CD127) em linfócitos T periféricos de 35 pacientes com DM1 e 25 controles sadios utilizando citometria de fluxo. O tempo médio de diagnóstico do DM1 foi de 3 meses, e todos os pacientes estavam em uso de insulina no momento da coleta de sangue. Auto-anticorpos pancreáticos (anti-GAD65 e anti-IA2) foram dosados através de radioimunoensaio. A expressão do RNAm de IL-21R, IL-2R e IL-2R foi quantificada por PCR em tempo real em 23 dos pacientes portadores de DM1A. Detectamos, pela primeira vez, diminuição significativa na expressão proporcional de IL-21R, CD25 e CD122 em linfócitos TCD3+ e TCD4+, além de diminuição na expressão de CD124 em linfócitos T CD4+ e CD127 em linfócitos T CD3+. Verificamos também redução significativa na quantidade de células TCD4+CD25+high (T regulatórias) nos pacientes DM1A. Não houve correlação entre expressão dos receptores de superfície das interleucinas de cadeia gama comum e títulos de autoanticorpos pancreáticos. Realizamos o PCR em tempo real para quantificar a expressão do RNA mensageiro (RNAm) dos receptores de interleucinas de cadeia gama comum, e avaliar se esta correspondia à expressão das proteínas de superfície obtida através de citometria de fluxo. Comparamos a expressão do RNAm de IL-21R, IL-2R e IL-2R nos pacientes DM1A dividindo-os em tercis de acordo com os valores de expressão de proteína de superfície obtidos por citometria de fluxo em linfócitos T CD3+, e verificamos que não houve diferença entre os 3 grupos na expressão relativa dos genes estudados. Portanto, em nossa casuística a redução da expressão da proteína de superfície dos receptores de interleucinas de cadeia gama comum possivelmente decorreu de alterações posteriores à transcrição do RNA mensageiro / Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by pancreatic infiltration of T and B lymphocytes, macrophages and dendritic cells, leading to a progressive destruction of the insulin-producing -cells. Homeostasis of T cells can be defined as the ability of the immune system to maintain normal T-cell counts and to restore T-cell numbers following T-cell depletion or expansion. It was classically believed that the CD4+ T cells could be activated into two distinct effector populations, T helper1 and T helper2. It was recently described two new pathways of CD4+ T lymphocytes activation: the Th17 pathway, that plays a fundamental role in autoimmunity and the regulatory pathway (Treg), where CD4+CD25+high T cells are essential to maintain peripheral tolerance and therefore protect against autoimmunity. The common gamma chain cytokines interfere with several steps of the CD4+ T lymphocytes differentiation. Interleukin-21 (IL-21) is the most recent member of this family, that also includes IL-2, IL-4, IL-7, IL-9 and IL-15, and has pleiotropic effects on the immune system. Interleukin-21 acts through interaction with its receptor, the IL-21R, which is expressed in a great variety of immune cells. Various studies with animal models of autoimmune diabetes demonstrated that IL-21 and its receptor are essential for the development of the disease, but there are no studies evaluating the role of this interleukin and its receptor in T1DM in humans. The aim of our study was to assess the role of common gamma chain-dependent cytokine receptors in the pathogenesis of T1D, by determining the expression of the surface protein and mRNA of these receptors in recent-onset T1D patients and correlating these values with titles of pancreatic autoantibodies. We studied the surface protein expression of IL-21R, IL-2R (CD25), IL-2R (CD122), IL-4R (CD124) and IL-7R (CD127) in peripheral T lymphocytes of 35 patients with T1D and 25 healthy controls using flow cytometry. Mean T1D duration was 3 months and all patients were using insulin at the time of blood withdraw. Pancreatic autoantibodies (anti-GAD65 and anti-IA2) were assessed by radioimmunoassay. The mRNA expression of IL-21R, IL-2R and IL-2R was quantified by real time PCR in 23 of the T1D patients. We detected for the first time a statistically significant decrease in the proportional expression of IL-21R, CD25 and CD122 on CD3+ and CD4+ T lymphocytes, a decrease in the expression of CD124 on CD4+ T cells and CD127 on CD3+ T lymphocytes. We also observed a significant reduction in the amount of CD4+ CD25+high (T regulatory cells) in T1D patients. There was no correlation between the expression of the surface receptors of common gamma chain cytokines and titles of pancreatic autoantibodies. We performed real-time PCR to quantify RNA expression of common gamma-chain interleukin receptors, and evaluate if these values corresponded to those of surface proteins obtained using flow cytometry. We compared the mRNA expression of IL-21R, IL-2R and IL-2R in T1D patients by dividing them into tertiles according to the expression values of surface protein obtained by flow cytometry in CD3+T lymphocytes. We observed that there was no difference in the relative expression of mRNA among the 3 groups of patients. Therefore, in our study, the reduction of surface protein expression of common gamma chain cytokines receptors was possibly due to alterations that occurred after the transcription of mRNA

Elevated levels of the inflammatory cytokine interleukin-6, IL-6, have been linked with poor prognosis in ovarian cancer patients by influencing tumour growth, invasion, angiogenesis and chemo-resistance. A clinical trial conducted in parallel with pre-clinical studies showed an anti-IL-6 antibody to have some activity in ovarian cancer patients and in xenograft models, via reduction in pro-inflammatory and angiogenic factors such as TNF-α, IL-8 and VEGF. Anti-IL-6 treatment also showed significant reductions in vascular area with decreased expression of an angiogenic factor Jagged1. The aim of my study was to investigate the effects of IL-6 on normal and tumour angiogenesis. I found that recombinant IL-6 stimulates angiogenesis in mouse and rat aortic ring assays and that it can also stimulate growth and migration of endothelial cells in vitro. IL-6 has similar potency as VEGF in inducing vessel sprouting. IL-6 itself does not induce VEGF in the endothelial cells I tested. Investigation of the effects of IL-6 on vessel maturation revealed a significant reduction in pericyte coverage of vessels treated with IL-6 compared with VEGF. Collectively, these data led to my hypothesis that ‘IL-6 drives aberrant angiogenesis, independent of VEGF signalling’. Investigating the mechanism by which IL-6 drives angiogenesis and leads to defective pericyte formation, I showed a link between IL-6 and the Notch ligands, Jagged1 and DLL4. My data suggested that IL-6 could stimulate Jagged1 in endothelial cells, whereas VEGF induces DLL4, the Notch ligand known to be involved in inducing stalk phenotype. Exploring previous findings to get a better understanding of the interaction of Notch ligands and pericyte recruitment also suggested a role of Angiopoeitin-2 in relation to IL-6 signalling. These observations were extended in IGROV-1 ovarian cancer xenografts treated with an anti-IL-6 antibody and by analysis of gene expression datasets from ovarian cancer biopsies. My results suggest therapeutic potential of combining inhibitors of IL-6 and VEGF in ovarian cancer.

Neuroinflammation is a major driver of secondary brain cell death after ischaemic stroke, seizure activity and traumatic brain injury. In a model of excitotoxic neuroinflammation, striatal injection of a toxic dose of AMPA causes cell death in the striatum after 24 hours. Co-injection of AMPA with the pro-inflammatory cytokine interleukin-1β (IL-1β) leads to additional cortical cell death. Injected alone, IL-1β leads to little or no cell death. It is hypothesised that IL-1β may exacerbate cell death by interfering with blood flow coupling. In the first study, two-dimensional optical imaging spectroscopy was used to measure early changes in the haemodynamic response in the anaesthetised rat barrel cortex before and for 6 hours after injection of vehicle, AMPA, IL-1β, or AMPA+IL-1β. After injection of IL-1β, with or without AMPA, the oxygenated blood flow response to mechanical whisker stimulation approximately halved over the course of 6h. In the second study, to determine whether the IL-1β-dependent changes in blood flow response are reflected by altered cellular activity, local field potentials, multi-unit activity and local tissue oxygenation responses to whisker stimulation were recorded simultaneously from the active barrel before and up to 6h after injection. A similar reduction in the size of the oxygenation response was seen again in the IL-1β- and AMPA+IL-1β-treated groups. Importantly, the level of gamma frequency oscillations at stimulus onset decreased within the first hours after injection of AMPA+IL-1β or IL-1β, suggesting a disruption of the fast-spiking interneuron network in the barrel cortex. These findings, along with histological observations of IL-1β-dependent markers of neuroinflammation, suggest that IL-1β may exacerbate AMPA-induced excitotoxicity by potentiating seizure activity and decoupling the neurovascular response in the cortex.

A ozonioterapia consiste na aplicação de ozônio medicinal, uma mistura de ozônio e oxigênio, que através das espécies reativas de oxigênio e produtos de lipoperoxidação exercem diversos efeitos no organismo como, melhora da oxigenação e metabolismo dos tecidos, angiogênese, aumento dos mecanismos antioxidantes, melhora do sistema imune, efeito anti-inflamatório, entre outros. Esta modalidade terapêutica já é amplamente estudada na medicina humana e vem sendo aplicada na medicina esportiva equina no tratamento de osteoartrite, porém sem estudos expressivos que comprovem sua segurança e eficácia. O objetivo do presente estudo é analisar os efeitos inflamatório e oxidante do ozônio medicinal em articulações sinoviais de equinos hígidos. Para tanto foram utilizadas 24 articulações tibiotársicas distribuídas aleatoriamente em três grupos. Nos grupos tratados foram realizadas três aplicações semanais de 15 ml de ozônio medicinal na concentração de 20 (GA) e 40 µg/ml (GB), no total de 10 aplicações. Já no grupo controle, as articulações receberam três aplicações semanais de 15 ml de O2 (GC), também no total de dez aplicações. Foram realizados exames físico, de claudicação e ultrassonográfico, bem como análise do líquido sinovial, incluindo contagem total de células nucleadas e quantificação de proteína total, prostaglandina E2, Substância P, interleucina-1, interleucina-6, interleucina 10, fator de necrose tumoral-α, ácido hialurônico (concentração e peso molecular) e condroitim sulfato. Para avaliação antioxidante no líquido sinovial foi determinada a atividade da superóxido desmutase e o burst oxidativo. Houve aumento da temperatura em GA e GB, os animais de GB apresentaram maior claudicação comparado aos demais grupos e observou-se aumento em todos os grupos dos escores ultrassonográficos. Na análise do líquido sinovial observou-se aumento nas contagens celulares de GA e GB, acompanhado de polimorfonucleares em GB, aumento da concentração de proteína no GA e GB, da atividade da superóxido desmutase e do índice de ativação em GA e diminuição da concentração de ácido hialurônico em todos os grupos e condroitim sulfato em GB e GC. Não houve diferença nas concentrações de PGE2, substância P, IL-1, IL-6, IL-10 e TNF-α. A aplicação consecutiva do ozônio medicinal intra-articular provocou alterações ultrassonográficas e no exame de claudicação, mais perceptível na dose de 40 ug/mL. Estas alterações estão mais relacionadas à distensão articular causada pela infusão de gases do que aos efeitos inflamatórios provindos do O3, uma vez que as análises de líquido sinovial não mostraram relevante inflamação. Conclui-se que a aplicação intra-articular de ozônio medicinal em equinos é segura em ambas as doses, e que experimentos devem ser realizados utilizando-se animais com diferentes doenças articulares, para que os benefícios da ozonioterapia sejam evidenciados e compreendidos. / Ozone therapy consists of the application of medicinal ozone, a mixture of ozone and oxygen, which through reactive oxygen species and products of lipoperoxidation exert various effects on the body, such as improvement of tissue oxygenation and metabolism, angiogenesis, increase of antioxidant mechanisms, improvement of the immune system, anti-inflammatory effect, among others. This therapeutic modality is already widely studied in human medicine and has been applied in equine sports medicine in the treatment of osteoarthritis, but without expressive studies that prove its safety and efficacy. The objective of the present study is to analyze the inflammatory and oxidizing effects of medicinal ozone on synovial joints of healthy horses. Twenty-four tibiotarsic joints were randomly distributed in three groups. In the treated groups three weekly applications of 15 ml of medicinal ozone in the concentration of 20 (GA) and 40µg / ml (GB) were carried out for a total of 10 applications. Already in the control group, the joints received three weekly applications of 15 ml of O2 (GC), also in the total of 10 applications. Physical, lameness and ultrasound examinations were performed, as well as synovial fluid analysis, including total nucleated cell count and quantification of total protein, prostaglandin E2, Substance P, interleukin-1, interleukin-6, interleukin-10, tumor necrosis factor-α, hyaluronic acid (concentration and molecular weight) and chondroitin sulfate. For the antioxidant evaluation in the synovial fluid, the activity of the superoxide dismutase and the oxidative burst was determined. There was an increase in temperature in GA and GB, GB animals presented greater lameness compared to the other groups and an increase was observed in all groups of ultrasound scores. In the synovial fluid analysis, GA and GB cell counts were observed, followed by polymorphonuclear cells in GB, increased protein concentration in GA and GB, superoxide desmutase activity and activation index in GA, and decrease in concentration of Hyaluronic acid in all groups and chondrocyte sulfate in GB and CG. There was no difference in the concentrations of PGE2, substance P, IL-1, IL-6, IL-10 and TNF-α. The consecutive application of intra-articular medicinal ozone caused ultrasonography changes and lameness, more noticeable at 40 ug / mL. These changes related more to joint distension caused by gas infusion than to inflammatory effects from O3, since synovial fluid analyzes did not show relevant inflammation. It is concluded that the intra-articular application of medical ozone in horses is safe in both doses, and that experiments must be performed using animals with different joint diseases, so that the benefits of ozonotherapy are evidenced and understood.

Interleukin-1-beta (IL-1β) is a potent proinflammatory cytokine. A primary outcome of IL-1β signaling is the activation of NFκB, a transcription factor that induces a large number of immune molecules, apoptotic factors, anti-apoptotic factors, and other transcription factors. Recent work has demonstrated that the activation of NFκB involves a multistep redox-signaling cascade that requires endocytosis of the interleukin receptor (IL-1R1)/ligand pair and superoxide production by NADPH oxidase 2 (Nox2) within the resulting newly formed early endosome. Hydrogen peroxide produced by the rapid dismutation of superoxide is necessary for the subsequent downstream recruitment of IL-1R1 effectors (TRAF6, IKK kinases) and ultimately the activation of NFκB. In this thesis, I have further dissected the spatial and temporal events that coordinate signaling processes of the IL-1β pathway. Using a combination of biophotonic imaging, immunofluorescence imaging, and lipid raft density gradient isolation, I demonstrate that both Nox2 and IL-1R1 are constitutively present in lipid raft microdomains on the plasma membrane. Stimulation by IL-1β induces endocytosis of Nox2 and IL-1R1 from the plasma membrane into caveolin-1, lipid raft positive early endosomes. Further, inhibition of lipid raft mediated endocytosis or deletion of caveolin-1 inhibits activation of NFκB, by IL-1β. We have also identified Vav1 as the Rac1 guanine exchange factor that is recruited to caveolin-1 positive lipid rafts following IL-1β stimulation, and demonstrated that dominant negative Vav1 inhibits NFκB activation by IL-1β. Following this work, I utilized assays for redox sensitivity and mass spectrometry to demonstrate that C70, C73, and C105 are hydrogen peroxide sensitive cysteines within the RING domain of TRAF6. I further demonstrate that hydrogen peroxide does not alter the E3 ubiquitin ligase activity associated with the TRAF6 RING domain. My findings suggest that the redox sensitivity of the RING domain mediates TRAF6 recruitment to the receptor complex. This is supported by the observation that hydrogen peroxide treatment of TRAF6, but not early signaling effectors (IL-1R1, IRAK1, IRAK4, MyD88) mediates TRAF6 recruitment to the IL-1 receptor complex. Further, mutation of the identified redox sensitive cysteines inhibits IL-1β signaling and NFκB activation. This research has helped to refine the understanding of the IL-1β signaling pathway, and may ultimately lead to new therapeutic targets for controlling inflammation.