Background: Candida albicans is an opportunistic fungal pathogen that induces strong proinflammatory responses, such as IL-1β production. Much less is known about the induction of immune modulatory cytokines, such as the IL-1 receptor antagonist (IL-1Ra) that is the main natural antagonist of IL-1, by C. albicans. Methods: Peripheral blood mononuclear cells (PBMC) of healthy individuals were stimulated with C. albicans and different components of the fungal cell wall. The role of pathogen recognition receptors (PRRs) for the induction of IL-1β and IL-1Ra was investigated by using specific blockers or in PBMC from Dectin-1 deficient patients. Results: C. albicans induced a strong IL-1Ra response, and this induction was primarily induced by the cell-wall component β-glucan. Blocking IL-1Ra significantly increased C. albicans β-glucan hyphae induced IL-1β and IL-6 production. Surprisingly, blocking the β-glucan receptor Dectin-1 or the downstream Syk or Raf-1 pathways only marginally reduced C. albicans-induced IL-1Ra production, while blocking of the complement receptor 3 (CR3), TLR2 or TLR4 had no effect. In line with this, blocking MAP kinases had little effect on Candida-induced IL-1Ra production. PBMC isolated from Dectin-1 deficient patients produced normal IL-1Ra amounts in response to C. albicans stimulation. Interestingly, the IL-1Ra synthesis induced by β-glucan was blocked by inhibitors of the Akt/PI3. K pathway. Conclusions: β-glucan of C. albicans induces a strong IL-1Ra response, which is independent of the β-glucan receptors dectin-1 and CR3. These data strongly argue for the existence of an unknown β-glucan receptor that specifically induces an Akt/PI3. K-dependent anti-inflammatory IL-1Ra response upon recognition of C. albicans.

O cavalo tem sido há milhares de anos um dos animais de maior utilidade para o homem, tanto no trabalho quanto no esporte. A integridade de sua estrutura física é determinante para a qualidade de seu desempenho, sendo alvo importante das estratégias terapêuticas e preventivas da atualidade. Diversos estudos têm esclarecido parte da cascata de eventos que ocorre em ambiente intra-articular, revelando os principais mediadores nocivos e ampliando as opções para tratamentos mais eficientes. Experimentos utilizando a citometria de fluxo foram capazes de demonstrar que a adição de plasma às células de líquido sinovial (LS) previamente inflamadas diminui a produção de espécies reativas de oxigênio durante o burst oxidativo, conferindo capacidade antioxidante a este hemoderivado. Pouco se sabe, no entanto, sobre seu potencial pró ou anti-inflamatório, pois não existem relatos na literatura investigando tal atividade. Assim, desenvolvemos este estudo com o objetivo de acompanhar os efeitos exercidos pelo plasma autólogo condicionado (ACP) sobre os tecidos articulares, reportando as alterações clínicas e no LS das articulações metacarpofalangeanas hígidas que receberam este tratamento. Foram administrados 4 ml de ACP em 10 articulações metacarpofalangeanas de equinos saudáveis, enquanto as articulações contralaterais receberam 4 ml de solução fisiológica, como controle. LS foi coletado antes de cada aplicação e nos momentos 3, 6, 24, 48 e 168 horas após. Foi realizada avaliação física diária, e as amostras coletadas foram submetidas imediatamente a análise física (volume, cor, aspecto e viscosidade), teste da qualidade do precipitado de mucina, contagem total e diferencial das células nucleadas. O sobrenadante foi congelado para dosagem posterior de ureia, proteína total, ácido hialurônico (AH), condroitim sulfato (CS), prostaglandina E2 (PGE2) e das citocinas fator de necrose tumoral alfa (TNF-α), interleucina 1 beta (IL-1β) e antagonista do receptor de IL-1 (IL-1ra). A avaliação física dos animais detectou presença de claudicação nas articulações metacarpofalangeanas tratadas com ACP nos momentos 24 e 48 horas após sua administração. A análise física do LS das mesmas articulações revelou maior contaminação das amostras com sangue às 6 horas e maior contagem de células nucleadas às 3, 6, 24 e 48 horas, com predomínio de leucócitos polimorfonucleares. Ainda, o ACP induziu maiores concentrações de proteína total e PGE2 às 3 e 6 horas, e maior concentração de CS às 24 horas. Houve também tendência ao aumento de TNF-α às 24 horas. Às 168 horas, todavia, nenhuma alteração foi observada em quaisquer dos itens avaliados. Não ocorreram alterações nos demais aspectos físicos do LS nem na qualidade do precipitado de mucina, tampouco nas concentrações de ureia e AH. Estes resultados indicam que em articulações hígidas o ACP possui ação pró-inflamatória transitória, com maiores concentrações de PGE2 induzindo o catabolismo dos PGs da matriz, aumentando particularmente o CS. / History has connected human and equine species, for work and pleasure purposes and in both scenarios, horses\' physical integrity is of paramount importance for adequate performance. Soundness has been the target of many studies in orthopedic therapeutics and preventive equine medicine. Several of these studies have thrown light on the sequence of deleterious intra-articular events that take place after an insult, revealing key mediators of joint destruction and widening treatment options. Experiments evaluating the effects of plasma on reactive oxygen species production by chemically stimulated synovial fluid (SF) cells revealed a potent antioxidant effect. Little is known, however about plasma´s anti or pro inflammatory activities, still an unexplored property of plasma. This study was to designed to observe the effects of autologous conditioned plasma (ACP) on articular components, reporting findings of serial SF analysis and clinical evaluations, before and after its administration in healthy metacarpophalangeal joints. Four mililiters of ACP were injected in 10 healthy metacarpophalangeal joints, and the contralateral joints were injected with 4 ml of saline, serving as controls. SF was obtained for analysis before, and then 3, 6, 24, 48 and 168 hours after treatment injection. Horses were subjected to daily clinical evaluations and synovial fluid samples were immediately analyzed for color, viscosity, volume, aspect, quality of mucin clot and total and differential nucleated cell counts. The supernatant was frozen and stored for posterior dosages of urea, total protein, hyaluronic acid (HA), chondroitin sulphate (CS), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL- 1β), and interleukin 1 receptor antagonist (IL-1ra). Physical evaluation of ACP treated subjects detected mild lameness at 24 and 48 hours observation points. SF analysis of ACP treated joints revealed blood contamination and higher total nucleated cell counts at 3, 6, 24 and 48 hours, with predominance of polymorphonuclear cells. ACP treatment has also induced higher protein concentrations and PGE2 levels at 3 and 6 hours and higher CS levels at 24 hours in synovial fluid analysis. At 24 hours, TNFα concentrations were higher, although not significantly. At 168 hours post ACP treatment, however, no change was observed in any parameter of synovial fluid analysis. No alterations were detected in the remaining items evaluated, nor in the quality of the mucin clot, urea concentration or HA. These results indicate that, when injected into healthy joints, ACP elicits a transient inflammatory response, characterized by higher PGE2 concentrations, matrix catabolism, with particular increase in CS.

O cavalo tem sido há milhares de anos um dos animais de maior utilidade para o homem, tanto no trabalho quanto no esporte. A integridade de sua estrutura física é determinante para a qualidade de seu desempenho, sendo alvo importante das estratégias terapêuticas e preventivas da atualidade. Diversos estudos têm esclarecido parte da cascata de eventos que ocorre em ambiente intra-articular, revelando os principais mediadores nocivos e ampliando as opções para tratamentos mais eficientes. Experimentos utilizando a citometria de fluxo foram capazes de demonstrar que a adição de plasma às células de líquido sinovial (LS) previamente inflamadas diminui a produção de espécies reativas de oxigênio durante o burst oxidativo, conferindo capacidade antioxidante a este hemoderivado. Pouco se sabe, no entanto, sobre seu potencial pró ou anti-inflamatório, pois não existem relatos na literatura investigando tal atividade. Assim, desenvolvemos este estudo com o objetivo de acompanhar os efeitos exercidos pelo plasma autólogo condicionado (ACP) sobre os tecidos articulares, reportando as alterações clínicas e no LS das articulações metacarpofalangeanas hígidas que receberam este tratamento. Foram administrados 4 ml de ACP em 10 articulações metacarpofalangeanas de equinos saudáveis, enquanto as articulações contralaterais receberam 4 ml de solução fisiológica, como controle. LS foi coletado antes de cada aplicação e nos momentos 3, 6, 24, 48 e 168 horas após. Foi realizada avaliação física diária, e as amostras coletadas foram submetidas imediatamente a análise física (volume, cor, aspecto e viscosidade), teste da qualidade do precipitado de mucina, contagem total e diferencial das células nucleadas. O sobrenadante foi congelado para dosagem posterior de ureia, proteína total, ácido hialurônico (AH), condroitim sulfato (CS), prostaglandina E2 (PGE2) e das citocinas fator de necrose tumoral alfa (TNF-α), interleucina 1 beta (IL-1β) e antagonista do receptor de IL-1 (IL-1ra). A avaliação física dos animais detectou presença de claudicação nas articulações metacarpofalangeanas tratadas com ACP nos momentos 24 e 48 horas após sua administração. A análise física do LS das mesmas articulações revelou maior contaminação das amostras com sangue às 6 horas e maior contagem de células nucleadas às 3, 6, 24 e 48 horas, com predomínio de leucócitos polimorfonucleares. Ainda, o ACP induziu maiores concentrações de proteína total e PGE2 às 3 e 6 horas, e maior concentração de CS às 24 horas. Houve também tendência ao aumento de TNF-α às 24 horas. Às 168 horas, todavia, nenhuma alteração foi observada em quaisquer dos itens avaliados. Não ocorreram alterações nos demais aspectos físicos do LS nem na qualidade do precipitado de mucina, tampouco nas concentrações de ureia e AH. Estes resultados indicam que em articulações hígidas o ACP possui ação pró-inflamatória transitória, com maiores concentrações de PGE2 induzindo o catabolismo dos PGs da matriz, aumentando particularmente o CS. / History has connected human and equine species, for work and pleasure purposes and in both scenarios, horses\' physical integrity is of paramount importance for adequate performance. Soundness has been the target of many studies in orthopedic therapeutics and preventive equine medicine. Several of these studies have thrown light on the sequence of deleterious intra-articular events that take place after an insult, revealing key mediators of joint destruction and widening treatment options. Experiments evaluating the effects of plasma on reactive oxygen species production by chemically stimulated synovial fluid (SF) cells revealed a potent antioxidant effect. Little is known, however about plasma´s anti or pro inflammatory activities, still an unexplored property of plasma. This study was to designed to observe the effects of autologous conditioned plasma (ACP) on articular components, reporting findings of serial SF analysis and clinical evaluations, before and after its administration in healthy metacarpophalangeal joints. Four mililiters of ACP were injected in 10 healthy metacarpophalangeal joints, and the contralateral joints were injected with 4 ml of saline, serving as controls. SF was obtained for analysis before, and then 3, 6, 24, 48 and 168 hours after treatment injection. Horses were subjected to daily clinical evaluations and synovial fluid samples were immediately analyzed for color, viscosity, volume, aspect, quality of mucin clot and total and differential nucleated cell counts. The supernatant was frozen and stored for posterior dosages of urea, total protein, hyaluronic acid (HA), chondroitin sulphate (CS), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL- 1β), and interleukin 1 receptor antagonist (IL-1ra). Physical evaluation of ACP treated subjects detected mild lameness at 24 and 48 hours observation points. SF analysis of ACP treated joints revealed blood contamination and higher total nucleated cell counts at 3, 6, 24 and 48 hours, with predominance of polymorphonuclear cells. ACP treatment has also induced higher protein concentrations and PGE2 levels at 3 and 6 hours and higher CS levels at 24 hours in synovial fluid analysis. At 24 hours, TNFα concentrations were higher, although not significantly. At 168 hours post ACP treatment, however, no change was observed in any parameter of synovial fluid analysis. No alterations were detected in the remaining items evaluated, nor in the quality of the mucin clot, urea concentration or HA. These results indicate that, when injected into healthy joints, ACP elicits a transient inflammatory response, characterized by higher PGE2 concentrations, matrix catabolism, with particular increase in CS.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Immunological stressors, in the form of clinical and sub-clinical disease are currently controlled using both prophylactic antibiotics in-feed, and therapeutic antibiotic treatment. Respiratory disease, primarily Actinobacillus pleuropneumoniae (App) infection, is recognised as a major factor causing reduced productivity in pigs. This thesis reports investigations into the use of novel immunomodulators in particular Interleukin 4 (IL-4) and Interleukin 1 receptor antagonist (IL-1ra) as alternatives to antibiotics to treat App infection. Immunological and molecular biological assays were used to investigate and accumulate data. An in vitro study undertaken to find potential anti-inflammatory substances, revealed that Interleukin 8 (IL-8) mRNA production stimulated by PMA or LPS in whole pigs' blood was suppressed by IL-4. IL-1ra also suppressed stimulated IL-8 mRNA production by heat killed App bacteria (KB) in vitro. An acute LPS challenge in pigs in vivo however, showed no variation in illness or weight loss between pigs treated prophylactically with anti-inflammatory substance (IL-4 and IL-1ra) and saline treated pigs. The use of plasmids as a delivery system for anti-inflammatory substance did not show promise since it did not enhance growth or prolong the expression of the substances in the pigs. However, in the chronic App challenge model IL-4 and IL-1ra administered prophylactically in vivo showed an ability to improve growth. The therapeutic administration of IL-4 and IL-1ra to App challenged pigs showed no difference in pigs' growth, regardless of the treatment or control administered. To conclude, IL-4 and IL-1ra showed promise when administered prophylactically and improved growth and abrogated disease under conditions of App challenge. However when IL-4 and IL-1ra where administered therapeutically they did not perform as well. Moreover these compounds have potential as a commercial application to reduce the growth reduction caused by disease such as App.

Immunological stressors, in the form of clinical and sub-clinical disease are currently controlled using both prophylactic antibiotics in-feed, and therapeutic antibiotic treatment. Respiratory disease, primarily Actinobacillus pleuropneumoniae (App) infection, is recognised as a major factor causing reduced productivity in pigs. This thesis reports investigations into the use of novel immunomodulators in particular Interleukin 4 (IL-4) and Interleukin 1 receptor antagonist (IL-1ra) as alternatives to antibiotics to treat App infection. Immunological and molecular biological assays were used to investigate and accumulate data. An in vitro study undertaken to find potential anti-inflammatory substances, revealed that Interleukin 8 (IL-8) mRNA production stimulated by PMA or LPS in whole pigs' blood was suppressed by IL-4. IL-1ra also suppressed stimulated IL-8 mRNA production by heat killed App bacteria (KB) in vitro. An acute LPS challenge in pigs in vivo however, showed no variation in illness or weight loss between pigs treated prophylactically with anti-inflammatory substance (IL-4 and IL-1ra) and saline treated pigs. The use of plasmids as a delivery system for anti-inflammatory substance did not show promise since it did not enhance growth or prolong the expression of the substances in the pigs. However, in the chronic App challenge model IL-4 and IL-1ra administered prophylactically in vivo showed an ability to improve growth. The therapeutic administration of IL-4 and IL-1ra to App challenged pigs showed no difference in pigs' growth, regardless of the treatment or control administered. To conclude, IL-4 and IL-1ra showed promise when administered prophylactically and improved growth and abrogated disease under conditions of App challenge. However when IL-4 and IL-1ra where administered therapeutically they did not perform as well. Moreover these compounds have potential as a commercial application to reduce the growth reduction caused by disease such as App.

Rodents are a natural host for the dimorphic pathogenic fungi Coccidioides immitis and Coccidioides posadasii, and mice are a good model for human infection. Humans and rodents both express Dectin-1 and Toll-like receptor 2 (TLR2) on myeloid cells, and those receptors collaborate to maximize the cytokine/chemokine responses to spherules (the tissue form of the fungi) and to formalin-killed spherules (FKS). We showed that Dectin-1 is necessary for resistance to pulmonary coccidioidomycosis, but the importance of TLR2 in vivo is uncertain. Myeloid differentiation factor 88 (MyD88) is the adapter protein for TLR2 and -4, interleukin-1R1 (IL-1R1), and IL-18R1. MyD88/TRIF -/- and MyD88 -/- mice were equally susceptible to C. immitis infection, in contrast to C57BL/6 (B6) controls. Of the four surface receptors, only IL-1R1 was required for resistance to C. immitis, partially explaining the susceptibility of MyD88 -/- mice. We also found that FKS stimulated production of IL-1Ra by bone marrow-derived dendritic cells (BMDCs), independent of MyD88 and Dectin-1. There also was a very high concentration of IL-1Ra in the lungs of infected B6 mice, supporting the potential importance of this regulatory IL-1 family protein in the largely ineffective response of B6 mice to coccidioidomycosis. These results suggest that IL-1R1 signaling is important for defense against C. immitis infection.

Einleitung Zytokine bewirken maßgeblich die Kommunikation und Koordination der zellulären und humoralen Effektorsysteme der angeborenen und erworbenen Immunität. Die Immunzellen stellen selbst die Hauptproduzenten der Zytokine dar. Pro- und anti-inflammatorische Zytokine nehmen nicht nur innerhalb der Zellkommunikation ablaufender Immun- und Entzündungsreaktionen eine Schlüsselrolle ein, sondern sind somit ebenso am Ablauf von Pathogenesen zahlreicher Erkrankungen beim Pferd beteiligt. Trotz verschiedener Studien anhand unterschiedlicher Modelle existiert keine einheitliche Datenlage zu validierten, vergleichbaren in vivo-nahen Zellkultursystemen, die es erlauben die Kinetiken equiner Zytokine als Grundlage zur weiterführenden Erforschung von Zytokinwechselwirkungen sowie zur Testung potentieller Arzneimittel abzubilden. Aktuell werden insbesondere Glukokortikoide weiterhin aufgrund ihrer anti-inflammatorischen und immunmodulatorischen Eigenschaften häufig, aber in Bezug auf Zytokine unspezifisch zur Therapie equiner Erkrankungen eingesetzt. Ziele der Untersuchung Das Ziel der vorliegenden Studie bestand darin, eine einfache, schnelle, günstige und reproduzierbare Methodik zur ex vivo-Messung von Zytokinen (Tumornekrosefaktor-alpha [TNF-α], Interleukin-1 Rezeptor-Antagonist [IL-1Ra] und Interferon gamma [IFN-γ]) und deren zeit- und konzentrationsabhängige Freisetzung in der equinen Vollblutzellkultur zu entwickeln. Anhand dessen sollte weiterführend der Effekt der Glukokortikoide Dexamethason (DEX) und Hydrocortison (HC) auf die Produktion von TNF-α, IL-1Ra und IFN-γ im equinen Vollblut untersucht werden. Zurzeit sind Zytokine, ihre Freisetzung sowie das Eintreten ihrer vermittelten Effekte Gegenstand der gegenwärtigen Forschung, mit dem Ziel spezifische Wechselwirkungen aufzuzeigen und somit zielgerichtete Therapeutika etablieren zu können. Insbesondere Pferde, die eine Anfälligkeit gegenüber mit Sepsis einhergehenden Erkrankungen oder für equines Asthma aufweisen, könnten davon profitieren. Material und Methoden Hierfür wurde Pferdevollblut, in den Verdünnungen zu 10%, 20% und 50% eingesetzt und mit Lipopolysaccharid (LPS), einer Kombination aus Phytohämagglutinin, Concanavalin A, Pokeweed-Mitogen und LPS (PCPwL) oder equinem rekombinantem TNF-a (erTNF-α) zur Zytokinfreisetzung stimuliert. Zur Erstellung der Zytokinkinetiken wurden Zellkulturüberstände zu verschiedenen Zeitpunkten gesammelt und die Konzentrationen von TNF-α, IL-1Ra und IFN-γ mit spezifischen enzyme-linked immunosorbent assays (ELISA) analysiert. In weiterführenden Versuchen wurden in der etablierten equinen Vollblutzellkultur DEX und HC in Konzentrationen von 10-12 - 10-5 M eingesetzt, um die LPS- oder PCPwL- induzierte Zytokinfreisetzung zu modulieren. Statistische Analysen erfolgten über die Berechnungen der Mittelwerte mit den dazugehörigen Standardfehlern. Signifikanzen wurden über ein- und zweifaktorielle ANOVA bestimmt. Ergebnisse Die durchgeführten Untersuchungen ergaben, dass die optimale Detektion der Zytokine in equinen Vollblutzellkulturen mit einem Blutanteil von 20% durchgeführt werden kann. TNF-α, IL-1Ra und IFN-γ wurden zeitabhängig freigesetzt und zeigten unterschiedliche Freisetzungskinetiken. Die PCPwL- induzierte TNF-α- und IL-1Ra-Freisetzung stiegen jeweils kontinuierlich über 24 - 48 Stunden an. In ähnlicher Weise erreichte die LPS- stimulierte TNF-α-Konzentration ein Maximum zu Zeitpunkten zwischen 8 - 12 Stunden und begann daraufhin abzufallen, wohingegen die Konzentration von IL-1Ra 24 Stunden später gipfelte und vielmehr fortgeführt über 48 Stunden hinaus akkumulierte. Equines rekombinantes TNF-α konnte ebenso die IL-1Ra-Freisetzung induzieren. Die PCPwL-induzierte IFN-γ-Freisetzung begann zeitversetzt und verlief kontinuierlich ansteigend über 48 - 72 Stunden. In weiterführenden konzentrationsabhängigen Untersuchungen konnte anhand der equinen Vollblutzellkultur eine stärkere Suppression der LPS-induzierten TNF-α- und IL-1Ra-Produktion sowie der PCPwL-induzierten IFN-γ-Produktion durch DEX als durch HC nachgewiesen werden. DEX hemmte die Zytokinfreisetzung mit einer mittleren inhibitorischen Konzentration (IC50) von 0,09 μM (TNF-α), 0,453 μM (IL-1Ra) und 0,001 μM (IFN-γ), während HC IC50 Werte von 1,45 μM (TNF-α), 2,96 μM (IL-1Ra) und 0,09 μM (IFN-γ) aufwies. Schlussfolgerungen Schlussfolgernd kann zusammengefasst werden, dass sich das Model der equinen Vollblutzellkultur hervorragend eignet, um nach erfolgreicher Mitogenstimulation den zeitabhängigen Freisetzungsverlauf von Zytokinen evaluieren zu können. Somit bietet das Model der equinen Vollblutzellkultur durch die Vorteile einer einfachen, günstigen Durchführung im in vivo-nahen, physiologischen Milieu, die Möglichkeit den Zytokinstatus gesunder sowie kranker Pferde zu beurteilen und stellt seinen Nutzen und die Verlässlichkeit unter Beweis potentielle Arzneimittel und immunologische Zusammenhänge des Pferdes untersuchen zu können.:INHALTSVERZEICHNIS I ABBILDUNGSVERZEICHNIS III TABELLENVERZEICHNIS III ABKÜRZUNGSVERZEICHNIS IV 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 3 2.1 Allgemeine wissenschaftliche Hintergründe 3 2.1.1 Das Blut und das Immunsystem des Pferdes 3 2.1.1.1 Zusammensetzung des equinen Blutes 3 2.1.1.2 Allgemeiner Aufbau des Immunsystems 4 2.1.2 Zytokine und Entzündungsreaktionen - Mediation der Immunantwort durch Zytokine und Chemokine 14 2.1.2.1 Pro-inflammatorische Zytokine: Tumornekrosefaktor-α und Interferon-γ 17 2.1.2.2 Anti-inflammatorische Zytokine: Interleukin-1 Rezeptor-Antagonist 19 2.2 Therapeutische Beeinflussung der Zytokin- und Mediator-Freisetzung 21 2.2.1 Inhibition der Zytokinfreisetzung durch Glukokortikoide 21 2.2.2 Inhibition der Zytokinfreisetzung durch weitere Pharmaka und Substanzen 23 2.2.2.1 NSAID 23 2.2.2.2 Small molecules und Anti-Zytokinantikörper 24 2.3 Equine Zellkulturmodelle zur Zytokindetektion 25 2.3.1 Stimulation der Zytokinfreisetzung 26 2.3.2 Vollblutzellkultursysteme und Systeme mit isolierten Zellen 27 2.4 Fragestellung der Dissertation 30 3 PUBLIKATIONEN 31 3.1 Freisetzungskinetik von TNF-α und IL-1Ra im equinen Vollblut 32 3.2 Modulation der Freisetzung von TNF-α, IL-1Ra und IFN-γ in der equinen Vollblutzellkultur durch Glukokortikoide 40 4 DISKUSSION 45 4.1 Zytokinfreisetzung im equinen Vollblut 46 4.2 Der Einfluss von Glukokortikoiden auf die Zytokinfreisetzung 52 4.3 Schlussfolgerungen 56 4.4 Ausblick 56 5 ZUSAMMENFASSUNG 58 6 SUMMARY 60 7 LITERATURVERZEICHNIS 62 8 ANHANG 72 8.1 Freisetzungskinetik von IFN-γ 72 8.2 Konzentration von TNF-α, IL-1Ra und IFN-γ in der equinen Vollblutzellkultur 72 9 DANKSAGUNG 74 / Introduction The communication and coordination between the cellular and humoral effector compartments of the innate and adaptive immunity were mainly accomplished by cytokines. Immunocompetent cells themselves represent the main source for cytokines. Pro- and anti-inflammatory cytokines not only play a pivotal role within the cell signaling of expiring immune- and inflammatory reactions but also take part in the pathogenesis of several equine diseases. Despite various studies based on different experimental setups no uniform availability of data about validated, comparable in vivo cell culture systems exists which enables the description of the kinetically time course of cytokines as foundation of further investigations of cytokine interactions as well as the testing of potential drugs. These days especially glucocorticoids are still frequently used for treatment of equine diseases because of their anti-inflammatory and immunomodulatory, but with respect to cytokines unspecific properties. Objectives of the investigations The aim of the study was to develop an easy, quick, cheap and reproducible ex vivo method for measuring cytokines (tumor necrosis factor alpha [TNF-α], interleukin-1 receptor antagonist [IL-1Ra] and interferon gamma [IFN-γ]) and their time- and concentration-dependent release in the equine whole blood cell culture. Whereby the impact of the glucocorticoids dexamethasone (DEX) and hydrocortisone (HC) on production of TNF-α, IL-1Ra and IFN-γ should be investigated subsequently. Currently, cytokines, their release and eventuation of their mediated effects are objects of actual research with the aim to reveal specific interactions and thus be able to establish purposeful therapeutic agents. This could be beneficial especially for horses which display a susceptibility to septic diseases or equine asthma. Material and Methods Therefore horse whole blood diluted to 10%, 20% and 50% was stimulated with lipopolysaccharide (LPS), a combination of phytohemagglutinin, concanavalin A, pokeweed mitogen and LPS (PCPwL) or equine recombinant TNF-α (erTNF-α). To generate cytokine kinetics TNF-α, IL-1Ra and IFN-γ were analyzed in culture supernatants, which were collected at different time points using specific enzyme-linked immunosorbent assays (ELISA). In further investigations within the equine whole blood cell culture DEX and HC were applied with concentrations between 10-12 and 10-5 M to modulate LPS- or PCPwL-induced cytokine release. Statistics were performed by calculation of means with associated standard errors. Statistical significances were assessed by one- and two-way analysis of variance. Results The evaluations revealed that cytokines could be detected optimal in whole blood cell cultures with 20% blood volume. TNF-α, IL-1Ra and IFN-γ were released time-dependently and differing kinetics were displayed. PCPwL-induced TNF-α and IL-1Ra release was enhanced continuously over 24 - 48 hours, respectively. Similarly, LPS-stimulated TNF-α was at maximum at time points between 8 - 12 hours and started to decrease thereafter, whereas IL-1Ra peaked 24 hours later and rather continued to accumulate beyond 48 hours. ErTNF-α could induce also the IL-1Ra release. PCPwL- induced IFN-γ release started time displaced and showed a continuously enhanced course over 48 - 72 hours. In subsequent investigations within equine whole blood cell culture, LPS-induced TNF-α and IL-1Ra as well as PCPwL-induced IFN-γ production were more potently suppressed concentration-dependently by DEX than by HC. DEX inhibited cytokine release with the inhibition concentration (IC50) 0.09 μM (TNF-α), 0.453 μM (IL-1Ra) and 0.001 μM (IFN-γ), whereas HC with IC50 values of 1.45 μM (TNF-α), 2.96 μM (IL-1Ra) and 0.09 μM (IFN-γ). Conclusion In conclusion our results could suggest the eminent suitability of equine whole blood cell culture to assess the release of a variety of cytokines following successful mitogen stimulation. Therefore the model of the equine whole blood cell culture provides, because of its advantages including simple and cheap performance in an in vivo close physiological ambient, the opportunity to evaluate the cytokine status of healthy and diseased horses. Furthermore it could give the proof of its benefit and reliability to evaluate potential equine drugs and immunological coherences of the horse.:INHALTSVERZEICHNIS I ABBILDUNGSVERZEICHNIS III TABELLENVERZEICHNIS III ABKÜRZUNGSVERZEICHNIS IV 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 3 2.1 Allgemeine wissenschaftliche Hintergründe 3 2.1.1 Das Blut und das Immunsystem des Pferdes 3 2.1.1.1 Zusammensetzung des equinen Blutes 3 2.1.1.2 Allgemeiner Aufbau des Immunsystems 4 2.1.2 Zytokine und Entzündungsreaktionen - Mediation der Immunantwort durch Zytokine und Chemokine 14 2.1.2.1 Pro-inflammatorische Zytokine: Tumornekrosefaktor-α und Interferon-γ 17 2.1.2.2 Anti-inflammatorische Zytokine: Interleukin-1 Rezeptor-Antagonist 19 2.2 Therapeutische Beeinflussung der Zytokin- und Mediator-Freisetzung 21 2.2.1 Inhibition der Zytokinfreisetzung durch Glukokortikoide 21 2.2.2 Inhibition der Zytokinfreisetzung durch weitere Pharmaka und Substanzen 23 2.2.2.1 NSAID 23 2.2.2.2 Small molecules und Anti-Zytokinantikörper 24 2.3 Equine Zellkulturmodelle zur Zytokindetektion 25 2.3.1 Stimulation der Zytokinfreisetzung 26 2.3.2 Vollblutzellkultursysteme und Systeme mit isolierten Zellen 27 2.4 Fragestellung der Dissertation 30 3 PUBLIKATIONEN 31 3.1 Freisetzungskinetik von TNF-α und IL-1Ra im equinen Vollblut 32 3.2 Modulation der Freisetzung von TNF-α, IL-1Ra und IFN-γ in der equinen Vollblutzellkultur durch Glukokortikoide 40 4 DISKUSSION 45 4.1 Zytokinfreisetzung im equinen Vollblut 46 4.2 Der Einfluss von Glukokortikoiden auf die Zytokinfreisetzung 52 4.3 Schlussfolgerungen 56 4.4 Ausblick 56 5 ZUSAMMENFASSUNG 58 6 SUMMARY 60 7 LITERATURVERZEICHNIS 62 8 ANHANG 72 8.1 Freisetzungskinetik von IFN-γ 72 8.2 Konzentration von TNF-α, IL-1Ra und IFN-γ in der equinen Vollblutzellkultur 72 9 DANKSAGUNG 74

The goal of this thesis was to develop a new drug-delivering material to deliver anti-inflammatory protein for treating OA. Our central hypothesis for this work is that a controlled release/presentation system will more effectively deliver anti-inflammatory protein therapies to the OA joint. The primary goal of this work was to synthesize a block copolymer that could self-assemble into injectable, sub-micron-scale particles and would allow an anti-inflammatory protein, IL-1ra, to be tethered to its surface for efficient protein delivery. The block copolymer incorporated an oligo-ethylene monomer for tissue compatibility and non-fouling behavior, a 4-nitrophenol group for efficient protein tethering, and cyclohexyl methacrylate, a hydrophobic monomer, for particle stability. We engineered the copolymer and tested it in both in vitro culture experiments and an in vivo model to evaluate protein retention in the knee joint. The rationale for this project was that the rational design and synthesis of a new drug- and protein-delivering material can create a modular polymer particle that can deliver multi-faceted therapies to treat OA. This work characterizes the in vitro and in vivo behavior of our polymer particle system. The protein tethering strategy allows IL-1ra protein to be tethered to the surface of these particles. Once tethered, IL-1ra maintains its bioactivity and actively targets synoviocytes, cells crucial to the OA pathology. This binding happens in an IL-1-dependent manner. Furthermore, IL-1ra-tethered particles are able to inhibit IL-1beta-induced NF-kappaB activation. These studies show that this particle system has the potential to deliver IL-1ra to arthritic joints and that it has potential for localizing/targeting drugs to inflammatory cells of interest as a new way to target OA drug treatments.

INTRODUCTION : Il a été démontré que le nombre de lipoprotéines apolipoprotéine B (apoB) est un prédicteur du développement du diabète de type 2 (DT2), mais le mécanisme est inconnu. La résistance à l'insuline (RI) et l'hyperinsulinémie compensatoire (HI) entraînent l’épuisement des cellules β et la progression vers le DT2. De plus, l'activation du système de l'interleukine -1β (IL- 1β) est impliquée dans la pathophysiologie du DT2. Notre objectif était donc d'étudier si l’apoB est associé à la RI et à l’HI chez les humains et si cette corrélation est médiée par l’activation du système IL-1β. MÉTHODOLOGIE : 47 femmes ménopausées, non diabétiques, obèses ou en surpoids et 28 hommes, âgés de 45 à 74 ans ont été recrutés. La sécrétion d'insuline (SI) et la sensibilité à l'insuline ont été mesurées par un clamp Botnia modifié. La 1ère et 2ème phase de SI furent mesurées lors d'un test de tolérance au glucose intraveineux (IVGTT) d’une heure, suivi d’un clamp hyperinsulinémique euglycémique (HEIC) de 3 heures (taux de perfusion d'insuline de 75 mU/m2/min) pour mesurer la sensibilité à l'insuline lors des 30 dernières minutes du clamp (état d'équilibre). La sensibilité à l'insuline est exprimée comme étant le taux de perfusion de glucose (GIR) seul ou divisé par le taux d’insuline à l’état d’équilibre (M/I). RÉSULTATS : Chez les femmes, l’apoB à jeun corrélait avec une augmentation de la 2e phase de SI, la SI totale et la sécrétion totale de C-peptide (r=0,202; r=0,168; r=0,204) et avec une diminution de la sensibilité à l'insuline (GIR r=-0,299; M/I r=-0,180) indépendamment de l'adiposité. L’IL-1Ra à jeun (indicateur de l’activation du système IL-1β) corrélait positivement avec la 2e phase, la SI totale et la sécrétion totale de C-peptide (r=0,217; r=0,154; r=0,198) et négativement avec la sensibilité à l'insuline (GIR r=-0,304; M/I r=-0,214). L’IL-1Ra était également corrélée avec l'apoB (r=0,352). Une fois corrigé pour l'IL-1Ra, toutes les associations entre l'apoB et les indices de sensibilité à l'insuline et de SI ont été perdues. Malgré des glycémies similaires, il n’y avait pas de corrélation de l’apoB avec les indices mesurés chez les hommes. CONCLUSION : L’apoB est associé à l’HI et la RI chez les femmes non diabétiques obèses et en surpoids, potentiellement via l'activation du système IL-1β. Ces différences sexuelles doivent être prises en compte dans l'exploration de la physiopathologie du DT2. / INTRODUCTION: The number of plasma apolipoprotein B lipoproteins (apoB) is reported to predict the development of type 2 diabetes (T2D); however the underlying mechanism is unknown. Insulin resistance (IR) and compensatory hyperinsulinemia (HI) are believed to promote β-cell exhaustion and progression to T2D. Moreover, the activation of the interleukin-1β (IL-1β) system is implicated in the pathophysiology of T2D. Our aim was thus to investigate whether plasma apoB associates with IR and HI in humans and whether this is mediated through the IL-1β system. METHODOLOGY: 47 non-diabetic overweight and obese postmenopausal women and 28 men, 45-74 years old were recruited. Insulin secretion (IS) and insulin sensitivity were examined by a modified Botnia clamp. 1st and 2nd phase IS were measured during a 1 hour intravenous glucose tolerance test (IVGTT), followed by a 3 hour hyperinsulinemic-euglycemic clamp (HEIC, insulin infusion rate of 75 mU/m2/min) to measure insulin sensitivity during the last 30 minutes of the clamp (steady state). Insulin sensitivity was expressed as steady state glucose infusion rate (GIR) alone or divided over steady state plasma insulin (M/I). RESULTS: In women, fasting plasma apoB correlated positively with increased 2nd phase and total IS and with total C-peptide secretion (r=0.202; r=0.168; r=0.204 respectively) and negatively with insulin sensitivity (r: GIR= -0.299 and M/I =-0.180) independent of adiposity. Similar to plasma apoB, fasting plasma IL-Ra (indicator of activated IL-1β system) correlated positively with 2nd phase and total IS and with total C-peptide secretion (r=0.217; r=0.154; r=0.198 respectively) and negatively with insulin sensitivity (GIR r=-0.304; M/I r=-0.214). Fasting plasma IL-Ra also correlated with apoB r=0.352). Once corrected for IL-1Ra, the associations between apoB and the indexes of insulin sensitivity and IS were all lost. Despite similar fasting glucose, plasma apoB did not correlate with any indices of insulin secretion or sensitivity in men. CONCLUSION: ApoB is associated with HI and IR in non-diabetic overweight and obese women, which may be mediated through activation of the IL-1β system. Gender differences may need to be considered in exploring the pathophysiology of T2D in humans.