La Broncho-Pneumopathie Chronique Obstructive (BPCO) est une maladie pulmonaire inflammatoire consécutive à l'exposition chronique à la pollution atmosphérique et surtout au tabagisme dans environ 90% des cas. Cette maladie se caractérise par une obstruction des bronches due à une hypersécrétion de mucus, une hypertrophie des muscles lisses, ainsi qu’une destruction de la paroi des alvéoles respiratoires amenant le patient à l’emphysème. Le stress induit par la fumée de cigarette provoque une activation de la barrière épithéliale pulmonaire associée à une altération de la réponse immunitaire responsable d’une susceptibilité accrue aux infections pulmonaires. De ce fait, les patients atteints de cette maladie développent des exacerbations principalement liées à ces infections bactériennes en particulier à Non-Typable Haemophilus influenza (NTHi) et Streptoccocus pneumoniae (Sp).La cytokine IL-22 est un acteur très important des défenses antibactériennes et du maintien de la barrière épithéliale. Cette cytokine appartient à la grande famille de l’IL-10, et à la sous-famille des cytokines IL-20 composée de l’IL-19, l’IL-20 et l’IL-24. L’IL-22 se lie au récepteur formé par les sous-unités IL-10Rb et IL-22Ra, tandis que les cytokines IL-19, IL-20 et IL-24 utilisent deux récepteurs associant l’IL-20Rb avec l’IL-20Ra ou l’IL-22Ra. Il a été démontré que les cytokines de la famille IL-20 (IL-19, IL-20, IL-24) agissent sur la clairance bactérienne au cours d’une infection cutanée par Staphylococcus aureus (Myles et al., 2013), en inhibant la production des cytokines IL-17 et IL-22. De plus, des précédents travaux au laboratoire, ont montré un défaut de l’expression des cytokines IL-17 et IL-22 qui participaient à la susceptibilité à l’infection chez les souris atteintes de BPCO (Pichavant et al., 2015). Enfin, nos données actuelles montrent que l'exposition à la fumée de cigarette augmente l'expression des cytokines de la famille IL-20 et que l'inhibition de cette voie permet de bloquer le développement d'épisodes d'exacerbation chez des souris BPCO.L'objectif de cette thèse est de préciser le rôle des cytokines IL-20 dans la réponse à l'infection bactérienne (Sp, NTHi) tant dans un contexte physiologique qu'au cours d’un contexte mimant la BPCO. Pour cela, nous nous focaliserons sur le rôle de l’épithélium pulmonaire tant dans la production que dans la fonction de ces cytokines en contexte infectieux.Pour répondre à ces questions, nous avons analysé l’expression des cytokines IL-20 par l’épithélium pulmonaire in vitro et ex vivo dans un modèle murin mimant l’exacerbation de la BPCO ainsi que dans des biopsies pulmonaires de patients fumeurs atteints ou non de BPCO. Dans un second temps nous avons évalué la modulation par un anticorps bloquant le récepteur des cytokines IL-20 (anti-IL-20Rb) au cours de la réponse anti-infectieuse de l'épithélium dans nos modèles in vivo (souris infectées par Sp) et in vitro (cellules épithéliales de trachées murines). Nous avons en parallèle évalué l'implication des cytokines IL-20 dans la réparation épithéliale.L’ensemble des résultats acquis au cours de la thèse nous a permis de démontrer l'implication des cytokines IL-20 et de préciser leur rôle sur l’épithélium pulmonaire au cours de l'infection bactérienne ainsi que dans la pathologie de la BPCO. De plus, les résultats obtenus avec l’anticorps neutralisant anti-IL-20Rb dans ces contextes d’infections et de BPCO, font de celui-ci une potentielle piste thérapeutique pour le traitement des lésions dues à l’infection. / Chronic Obstructive Pulmonary Disease (COPD) is an inflammatory lung disease due to chronic exposure to air pollution and especially to cigarette smoke exposure in approximately 90% of the cases. This disease is characterized by obstruction of the bronchi due to hypersecretion of mucus, hypertrophy of the smooth muscles, and destruction of the alveolar wall leading the patient to emphysema. The stress induced by cigarette smoke exposure causes activation of resident cells including pulmonary epithelial cells and an alteration of the immune system responsible for an increased susceptibility to pulmonary infections. As a result, patients with this disease develop exacerbations especially du to Non-Typable Haemophilus influenza (NTHi) and Streptoccocus pneumoniae (Sp).The IL-22 cytokine plays a key role in antibacterial defenses and maintenance of the epithelial barrier. This cytokine belongs to the large IL-10 family, and to the IL-20 cytokine subfamily also including IL-19, IL-20 and IL-24. IL-22 binds to the receptor formed by the IL-10Rb and IL-22Ra subunits, while the IL-19, IL-20 and IL-24 cytokines binds to IL-20Rb associated with either IL-20Ra or IL-22Ra subunits. IL-20 cytokines (IL-19, IL-20, IL-24) have been shown to impair bacterial clearance during cutaneous infection with Staphylococcus aureus (Myles et al., 2013), by inhibiting the production of IL-17 and IL-22 cytokines. In addition, previous work in the laboratory showed a defect in the expression of IL-17 and IL-22 cytokines contributing to the susceptibility to infection in COPD mice (Pichavant et al., 2015). In fact, our current data show that exposure to cigarette smoke increases cytokine expression of the IL-20 family and that inhibition of this pathway blocks the development of exacerbation episodes in COPD mice.The aim of this thesis is to clarify the role of IL-20 cytokines in the response to bacterial infections (Sp, NTHi) both in a physiological context and in a context mimicking COPD. To do so, we will focus on the role of pulmonary epithelium both in the production and function of these cytokines in infectious context.To answer these questions, we analyzed the expression of IL-20 cytokines by pulmonary epithelium in vitro and ex vivo in a mouse model mimicking the COPD exacerbation as well as in pulmonary biopsies of smokers and non-smokers patients and of COPD patients. In a second step we evaluated the modulation by an IL-20 receptor blocking antibody (anti-IL-20Rb) of the anti-infectious response in our in vitro (murine tracheal epithelial cells) and in vivo models (Sp-infected mice). In parallel, we evaluated the involvement of IL-20 cytokines in the epithelial repair.All the results acquired during the thesis allowed us to demonstrate the expression of IL-20 cytokines and to demonstrate their role on the pulmonary epithelium during bacterial infection as well as in COPD. In addition, the results obtained with the anti-IL-20Rb neutralizing antibody in these contexts of infections and COPD, suggests a potential therapeutic application for respiratory infection.

Orientador: Mauro Pedrine [Unesp] Santamaria / Resumo: A destruição periodontal resulta principalmente da resposta inflamatória exacerbada do hospedeiro frente ao desafio bacteriano. Por isso, pesquisas envolvendo a modulação da resposta do hospedeiro têm sido desenvolvidas com o objetivo de facilitar a resolução da inflamação, bem como promover reparação tecidual e estabilidade periodontal. Recentemente, o uso de ácidos graxos poli-insaturados de ômega-3 (AGP Ω-3) e ácido acetilsalicílico (AAS) foi relacionado à produção de mediadores lipídicos mais bioativos e à melhores resultados clínicos no tratamento de periodontite crônica. Desse modo, pesquisas envolvendo modulação das respostas inflamatórias de portadores de periodontite agressiva (PAg) podem ser de grande valia. Assim, o objetivo dos presentes estudos clínicos controlados randomizados foi avaliar a utilização da suplementação de 900 mg AGP Ω-3 e 100 mg de AAS por 180 dias como adjuvantes ao tratamento de PAg generalizada (PAgG). (1) Selecionou-se 38 pacientes com PAgG os quais receberam debridamento subgengival associado a AGP Ω-3 e AAS (n=19) ou placebo (n=19). Ambos os grupos apresentaram diminuição (p<0,05) em todos os parâmetros clínicos avaliados, bem como em IL-1β, sem diferença entre os tratamentos (p>0,05). O nível de TIMP-2 diminuiu significantemente no grupo controle, porém se manteve estável no grupo teste. Concluiu-se que a nova terapia proposta não trouxe benefícios clínicos no tratamento não-cirúrgico de PAgG. (2) Selecionou-se 34 pacientes com PAgG previa... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Orientador: Luiz Henrique de Araújo Machado / Resumo: A dermatite atópica canina é uma importante alergopatia de ordem genética, inflamatória, pruriginosa e crônica. Os tratamentos existentes atualmente são sintomáticos, algumas vezes insatisfatórios no controle da doença e passíveis de efeitos colaterais. As células-tronco mesenquimais (CTM), pelas suas propriedades imunomoduladores e anti-inflamatórias, podem ser uma alternativa no tratamento da dermatite atópica. Foi realizado um estudo longitudinal, duplo-cego, com período inicial de tratamento placebo com três aplicações quinzenais de soro fisiológico, com posterior aplicação de CTM heterólogas, derivadas de tecido adiposo, em seis cães com dermatite atópica, totalizando três aplicações quinzenais na dose de 5x106 células por animal. A escala visual de prurido esclarecido (EVPE), o escore de lesões cutâneas (CADESI-4), a resposta inflamatória cutânea pela análise das citocinas IL4, IL6, IL10, IL31 e TNF-alfa e da proteína filagrina pela técnica de imunoistoquímica e a avaliação dos lipídeos epidérmicos pela coloração de Sudam III foram comparadas entre o tratamento placebo e com as CTM. O tratamento com CTM foi benéfico para a melhora das manifestações clínicas nos animais com dermatite atópica, diminuindo de forma significativa o escore CADESI-4 e EVPE, com redução consistente de mais de 50% destes dois parâmetros ao final do tratamento com CTM. A melhora clínica evidente nos animais com DA tratados com terapia celular aliadas à baixa frequência de efeitos colaterais, sina... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The atopic canine is an inflammatory allergic dermatitis frequent and important. The distinct treatment available is symptomatic, currently unsatisfactory in the control of the disease and susceptible to collateral effects. The mesenchymal Stem Cells (MSC) by their immunomodulators and anti-inflammatory characteristics can be an alternative in the treatment of the atopic dermatitis. A longitudinal study was performed, blind-double, with an initial period of placebo treatment within saline solution, after the application of 5x106 heterologous CTM, derived from adipose tissue, in six dogs with atopic dermatitis. Each treatment constituted of three applications in by-weekly intervals. The visual analog scale of pruritis, the score of skin lesions (CADESI-4), the skin inflammatory response and filaggrin through the immunohistochemistry technique and the evaluation of the skin lipids by the Sudam III coloration were compared between the placebo treatment and the CTM treatment. The treatment with CTM with the used protocol was beneficial to the improvement of the clinical responses of the animals with atopic dermatitis, decreasing significantly the CADESI-4 and EVDP score, with a significant reduction of more than 50% of these two parameters at the end of the treatment with CTM while compared to the placebo. The clinical improvement was evident in animals with DA treated with cellular therapy allied to the low frequency of the collateral effects, indicates that with CTM can be cons... (Complete abstract click electronic access below) / Doutor

Importance: Immunological response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination is important, especially in people with multiple sclerosis (pwMS) on immunosuppressive therapies. Objective: This study aims to determine whether adjuvanted protein-based vaccine NVX-CoV2373 is able to induce an immune response to SARS-CoV-2 in pwMS with inadequate responses to prior triple mRNA/viral vector vaccination. Design, setting, and participants: We conducted a single-center, prospective longitudinal cohort study at the MS Center in Dresden, Germany. In total, 65 participants were included in the study in accordance with the following eligibility criteria: age > 18 years, immunomodulatory treatment, and insufficient T-cellular and humoral response to prior vaccination with at least two doses of SARS-CoV-2 mRNA (BNT162b2, mRNA-1273) or viral vector vaccines (AZD1222, Ad26.COV2.S). Interventions: Intramuscular vaccination with two doses of NVX-CoV2373 at baseline and 3 weeks of follow-up. Main outcomes and measures: The development of SARS-CoV-2-specific antibodies and T-cell responses was evaluated. Results: For the final analysis, data from 47 patients on stable treatment with sphingosine-1-phosphate receptor (S1PR) modulators and 17 on ocrelizumab were available. The tolerability of the NVX-CoV2373 vaccination was overall good and comparable to the one reported for the general population. After the second NVX-CoV2373 vaccination, 59% of S1PR-modulated patients developed antispike IgG antibodies above the predefined cutoff of 200 binding antibody units (BAU)/ml (mean, 1,204.37 [95% CI, 693.15, 2,092.65] BAU/ml), whereas no clinically significant T-cell response was found. In the subgroup of the patients on ocrelizumab treatment, 23.5% developed antispike IgG > 200 BAU/ml (mean, 116.3 [95% CI, 47.04, 287.51] BAU/ml) and 53% showed positive spike-specific T-cellular responses (IFN-gamma release to antigen 1: mean, 0.2 [95% CI, 0.11, 0.31] IU/ml; antigen 2: mean, 0.24 [95% CI, 0.14, 0.37]) after the second vaccination. Conclusions: Vaccination with two doses of NVX-CoV2373 was able to elicit a SARS-CoV-2-specific immune response in pwMS lacking adequate immune responses to previous mRNA/viral vector vaccination. For patients receiving S1PR modulators, an increase in anti-SARS-CoV-2 IgG antibodies was detected after NVX-CoV2373 vaccination, whereas in ocrelizumab-treated patients, the increase of antiviral T-cell responses was more pronounced. Our data may impact clinical decision-making by influencing the preference for NVX-CoV2373 vaccination in pwMS receiving treatment with S1PR modulation or anti-CD20 treatment.

Sekundäre bakterielle Infektionen im Verlauf oder in Folge einer Infektion mit Influenza A Viren (IAV) steigern oftmals die Schwere des Krankheitsverlaufes, was besonders während der IAV Pandemien von 1918, 1968 und 2009 deutlich wurde. Genaue mechanistische Ursachen, welche dieser gesteigerten Kopathogenität zugrunde liegen wurden überwiegend in Tierversuchsmodellen adressiert und sind immunologisch unvollständig. Aufgrund organstruktureller und immunfunktioneller Speziesunterschiede ist ungewiss, inwieweit eine Übertragbarkeit der Daten zwischen Mensch und Maus besteht. Fokus der Arbeit bildete die Analyse potentieller IAV assoziierter Änderungen der angeborenen Immunität, welche sekundäre Pneumokokkeninfektionen in humanem ex vivo Lungengewebe begünstigen. Dafür wurden zentrale Zyto - bzw. Chemokine als Reaktion auf Einzelinfektionen mit dem saisonalen IAV Pan/99(H3N2) sowie Streptococcus pneumoniae D39 mit denen subsequenter viral-bakteriellen Koinfektion verglichen. Ausgelöst durch die antivirale Interferonantwort erfolgte die Reduktion der pneumokokkeninduzierten Bildung von IL-1β und GM-CSF auf translationaler und transkriptioneller Ebene. Vermutlich beeinflussen Typ I und II Interferone die IL-1β Bildung, welches über parakrine Wechselwirkungen an der GM-CSF Regulation beteiligt ist. Auf zellulärer Ebene verursachte IAV die Freisetzung von Typ I, II und III Interferonen aus primären humanen Alveolarepithelzellen vom Typ II. In humanen Alveolarmakrophagen unterdrückten Typ I und II Interferone die pneumokokkeninduzierte IL-1β Freisetzung. Folglich unterblieb die IL-1β-regulierte GM-CSF Sekretion aus Alveolarepithelzellen vom Typ II. Die Ergebnisse zeigen, dass influenzainduzierte Interferone durch die Unterdrückung der IL-1β regulierten Bildung von GM-CSF in humanem Lungengewebe beitragen. Damit unterstützt sie das Verständnis immunologischer Faktoren, welche diesem Krankheitsbild im Menschen pathophysiologisch zugrunde liegen können. / Secondary bacterial infections, which occur during or following an IAV infection, exaggerate the severity of the course of disease up to a lethal outcome, clearly recognizable during the fatal IAV pandemics from 1918, 1968 or 2009. Particular mechanisms underlying this exaggerated viral-bacterial copathogenity were almost solely addressed using animal models and are immunologically incomplete. Due to structural and immunofunctional interspecies differences the transferability of data between human and mice remains indeterminate. The study mainly purposed to investigate IAV associated modulations of innate immunity, which potentially facilitates secondary pneumococcal pneumonia in primary human ex vivo lung tissue. Hence secretion of central cyto- and chemokines initiated by single infection with the seasonal IAV Pan/99(H3N2) or the bacterium Streptococcus pneumoniae D39 were compared to subsequent viral-bacterial coinfection. In context of an antiviral interferon response the pneumococcal induced translation and transcription of IL-1β and GM-CSF were reduced. Probably type I and type II interferons affect generation of IL-1β, which participates in the regulation of GM-CSF by paracrine interactions. On a cellular basis the infection of primary human alveolar epithelial cells type II (AECII) with IAV triggered the release of interferon type I, II and III. In human alveolar macrophages type I and II interferons suppressed the pneumococcal induced release of IL-1β. Consequently, the IL-1β regulated generation of GM-CSF in AECII was impeded. The present study indicates, that influenza related induction of interferons suppresses the IL-1β related release of GM-CSF in human lung tissue. Thereby it takes part in contributing to pathophysiological comprehension of immunological factors underlying this copathogenity.

Im medizinischen Alltag steht Gewebeersatz oftmals nicht ausreichend zur Verfügung. Tissue Engineering (TE) bietet eine wertvolle Methode, um aus wenigen Zellen des gewünschten Gewebes größere Strukturen herzustellen. Aber selbst autologe über Tissue Engineering gebildete Gewebe können einer Abstoßung unterliegen. Diese Arbeit befaßt sich daher mit der Immunmodulation von TE-Transplantaten. Chondrocyten wurden aus Ohrknorpel von Neuseeland-Kaninchen enzymatisch isoliert, in Zellkultur vermehrt und wurden a) in allogenem Fibrinkleber oder b) in Agarose suspendiert, dann in ein Scaffold eingebracht. 24 Transplantate wurden zusätzlich mit einer Polyelectrolyt Kapsel versehen. Nativer Ohrknorpel diente als Kontrolle. So entstanden n=84 Transplantate, 14 autologe Transplantate pro Kaninchen: jeweils 2 native, 4 Fibrin-, 4 Agarose- und 4 Kapsel-Transplantate. Die 1cm x 1cm x 0.2 cm großen Transplantate wurden subkutan auf dem Rücken der Kaninchen implantiert. Eine der in zwei Gruppen aufgeteilten Kaninchen erhielt eine 3 wöchige i. m. Gabe von Methylprednisolon. 70 Transplantate wurden in 5 Versuchstiere implantiert. 14 Transplantate wurden in vitro ernährt. 2 Kaninchen wurden nach 6 Wochen getötet und die Transplantate entnommen. 3 Kaninchen wurden nach 12 Wochen getötet. Im Anschluß wurden die Transplantate histologisch untersucht. Die Transplantate der nicht immunmodulierten Tiere konnten zu den Entnahmezeitpunkten 6 und 12 Wochen kaum aus den Implantationsorten entnommen werden: sie waren nur schwer vom umliegenden Gewebe zu unterscheiden. Die Transplantate der immunmodulierten Tiere blieben in ihrer Größe konstant. Histologisch zeigte sich bei den nicht immunmodulierten Tieren nach 6 Wochen massive zelluläre Infiltration, nach 12 Wochen die Einwachsung von Fibrozyten und kaum noch Knorpel- oder Knochengewebe. In der Gruppe der immunmodulierten Tiere konnte keine bis eine geringe Inflammation festgestellt werden. Bis auf die nativen Transplantate, war in dieser Gruppe bei allen Transplantaten vitaler trabekulärer Knochen mit hämatopoetisch aktivem Knochenmark zu beobachten. Der Vergleich der Gruppen Nicht immunmoduliert und Immunmoduliert war hinsichtlich der Immunreaktionen statistisch signifikant (Chi-Quadrat Test nach Pearson). / Tissue replacement is a common need in clinical medicine. And often there is too less tissue available. Tissue Engineering (TE) is a valuable measure to solve this problem: only a few cells of the origin tissue are cultivated and new threedimensional structures are built. But even autologeously built tissues can be rejected by the host. Therefore, this investigation is about immunomodulation of TE-transplants. Chondrocytes of ear cartilage of New Zealand rabbits were enzymatically isolated, amplified and were solved in a) allogenic fibrin glue or b) agaraose. Then they were taken into scaffolds. 24 transplants were encapsulated by a polyelectrolyte-complex membrane. Native cartilage served as control. There were formed n=84 transplants, 14 autologeous transplant per rabbit: each with 2 native, 4 fibrin-, 4 agarose- and 4 capsule-transplants. The tranplants size was 1 cm x 1 cm x 0.2 cm. The rabbits were divided into two groups one of the group has been treated with methylprednisolone IM for 3 weeks. 70 transplants were taken into the ridge of 5 rabbits. 14 transplants were cultivated in vitro. 2 rabbits were sacrificed after 6 weeks and 3 rabbits after 12 weeks. Afterwards the transplants were investigated histologically. The transplants of the non-immunomodulated group could hardly be separated from the surrounding tissue, whereas the transplants of the immunomodulated group remained constant in seize and shape. Histologically, the non-immunomodulated tranplants underwent cellular (granulocytes) infiltration after 6 weeks, respectively ingrowth of fibrocytes after 12 weeks. No cartilage or bone could was evident. In the immunomodulated group no signs of inflammation were identifiable after 6 and 12 weeks. In all transplants of this group bone formation with hematopoietically-active bone marrow was detectable. The native control-cartilage had not become bone, inflammation wasnot evident there. The difference of the two groups non-immunomodulated and immunomodulated was statistically significant concerning th degree of inflammation. (Chi-square-test).

Helminthen besitzen effektive Strategien um das Immunsytem ihrer Wirte zu modulieren. Sie sekretieren Moleküle mit deren Hilfe Immunzellen unterdrückt werden und verhindern dadurch Immunantworten, die ihnen schaden. AvCystatin ist ein Cystein-Protease-Inhibitor der Filarie Acanthocheilonema viteae. Die vorliegende Arbeit zeigt, dass dieses Molekül in Makrophagen MAPK (p38, ERK) sowie Transkriptionsfaktoren (CREB, STAT3) anspricht und zur Expression von spezifischen Markergenen führt, die eine M2a/M2b Makrophagen-Aktivierung repräsentieren. Ein intravenöser Transfer solcher Zellen 18-20 Stunden nach Stimulation mit AvCystatin führte in Mäusen zu einer signifikanten Reduktion von wichtigen Merkmalen der Ovalbumin-induzierten Atemwegshyperreaktivität wie Schleimproduktion, Th2 Zytokinen, IgE und Eosinophilen. Die Linderung von Krankheitsparametern war mit einem lokalen und systemischen Anstieg von IL-10 assoziiert. Unter Verwendung eines in vitro Systems zeigte sich, dass AvCystatin induzierte Makrophagen einen löslichen Faktor sekretieren, der eine IL-10-Produktion durch CD4+ T-Zellen induziert und parallel die Produktion von IL-13, IL-2 und IFN-gamma unterdrückt. Um zu untersuchen, ob die suppressive Aktivität der AvCystatin-Makrophagen auf Entzündungsprozesse der Atemwege beschränkt ist, wurde ein Makrophagen-Transfer in Tiere durchgeführt, die unter DSS-induzierter Kolitis litten. Eine einmalige Gabe von Zellen verhinderte den Verlust von Körpergewicht, eine Verkürzung des Kolons und verminderte die Migration entzündungsvermittelnder Zellen in die Lamina Propria. Im Gegensatz zum Krankheitsmodell der Ovalbumin-induzierten Atemwegshyperaktivität korrelierten die Effekte nicht mit erhöhten Mengen an IL-10. Zusammenfassend zeigen die Ergebnisse, dass AvCystatin Makrophagen adressiert und eine immunsuppressive Population generiert, welche Mäuse im Kontext von Atemwegsentzündung und Kolitis gegen überschießende Immunantworten schützen kann. / Helminth parasites possess effective strategies to modulate the immune system of their hosts. They release molecules which target immune cells and prevent reactions directed against them. AvCystatin is a cysteine protease inhibitor secreted by the nematode Acanthocheilonema viteae. The present thesis shows that this molecule modulates signaling pathways in murine macrophages through activation of MAPK (p38, ERK) and transcription factors (CREB, STAT3). Such macrophages express specific marker genes reflecting M2a/M2b activation. A single intravenious transfer of macrophages 18-20 hours after treatment with AvCystatin significantly reduced major parameters of ovalbumin-induced airway hyperreactivity in mice such as mucus production, Th2 cytokines, IgE and recruitment of eosinophils to the airways. The amelioration of inflammation was associated with significantly increased levels of local and systemic IL-10. By using an in vitro co-culture assay AvCystatin-induced macrophages were shown to secrete a soluble factor which induces IL-10 in CD4+ T cells and in parallel to suppress production of IL-13, IL-2 and IFN-gamma. To evaluate whether the suppressive potential of AvCystatin-macrophages is restricted to airway inflammation, macrophages were administered to animals suffering from DSS-induced colitis. A single application of cells into the tail vein sufficiently prevented body weight loss, colon shortening and suppressed the influx of inflammatory cells to the lamina propria. In contrast to the model of ovalbumin-induced airway inflammation the effects were not associated with increased levels of IL-10. On the whole these findings show that the helminth immunomodulator AvCystatin targets macrophages thereby inducing a distinct immunosuppressive population which protects mice against overshooting immune responses in disease models for airway and intestinal inflammation.

Infektionen mit dem intestinalen Parasiten Giardia duodenalis, verursachen weltweit eine der häufigsten humanen Parasitosen. Bislang konnten keine eindeutigen Virulenz- oder Pathogenitätsmarker des Erregers beschrieben werden. Es wird allerdings vermutet, dass potentielle G. duodenalis Virulenzfaktoren Enzyme sind, die während des Kontaktes des Erregers mit den Dünndarmepithelzellen sezerniert werden. Eines dieser Enzyme ist die Arginin Deiminase (ADI), die Arginin zu Citrullin umwandelt. Ziel dieser Arbeit war es Merkmale zu identifizieren, die für die ADI als Virulenzfaktor sprechen. Dazu wurde das Enzym zunächst hinsichtlich seiner Bedeutung für die Wirt-Pathogen-Interaktion untersucht. Die mit rekombinanter, katalytisch aktiver ADI (Assemblage A) behandelten LPS-stimulierten humanen moDC zeigten eine Veränderung in ihrem Phänotyp als auch in ihrer Cytokinsekretion. Diese ließ sich auf die durch das Enzym hervorgerufene Arginindepletion und/oder auf die Bildung der Metabolite, Citrullin und NH4+, zurückführen. Weiterhin konnte gezeigt werden, dass Parasitenisolate verschiedener G. duodenalis Assemblage A-Subtypen, vermutlich durch die katalytische Aktivität der ADI, die Stickstoffmonoxid-Bildung einer intestinalen Epithelzelllinie inhibiert. Neben dem Einfluss auf die Wirtsimmunantwort wurde auch die Variabilität in der kodierenden Sequenz des Enzyms in verschiedenen Parasitenisolaten analysiert. Anschließend erfolgte die funktionelle Charakterisierung des nativen (verschiedene Assemblage A-Subtypen) als auch des rekombinant aufgereinigten Enzyms (Assemblage A, B und E). Dabei zeigten sich Unterschiede in der Substrataffinität der ADI für Arginin, sowohl zwischen unterschiedlichen Assemblage A-Subtypen als auch unterschiedlichen Assemblage-Klassen. Zusammenfassend wurde gezeigt, dass die G. duodenalis ADI immunmodulatorische Effekte hat und das vermutlich eine Korrelation zwischen der Variation in der Primärstruktur und der Funktion des Enzyms besteht. / Giardia duodenalis (G. duodenalis) is an intestinal protozoan parasite that causes giardiasis, one of the most prevalent parasitic diseases worldwide. So far, little is known concerning host-parasite interaction, in particular what determines the parasite’s pathogenicity. Several potential virulence factors, among them the arginine deiminase (ADI) that hydrolyzes arginine into citrulline and NH4+, are discussed. The ADI was identified to be released upon contact with intestinal epithelium by Giardia trophozoites and was recognized as an immunoreactive protein during acute human giardiasis. Aim of the study was to identify hints for G. duodenalis ADI to be a virulence factor. First, to analyze the enzyme’s impact on host-parasite interplay, its influence on human monocyte-derived dendritic cells (moDC) was investigated. Treatment of LPS-stimulated cells with recombinant ADI of assemblage A changed DC phenotype (CD83, CD86) and cytokine secretion (TNF-α, IL-10, IL-12p40). These immunomodulatory changes in DC response were due to arginine depletion and the formation of reaction products, in particular, ammonium ions. Furthermore, trophozoites of different assemblage subtypes were shown, probably due to consumption of arginine by ADI, to reduce nitric oxide formation by intestinal epithelial cells in vitro. Second, variation in the ADI coding sequence of different G. duodenalis isolates being collected in a Giardia biobank was analyzed by sequencing. Subsequently, functional genetics were performed with native ADI of different assemblage A subtypes expressed by these strains as well as with purified, recombinant ADI of assemblage A, B and E. It was recognized that enzymes of the same subtype as well as of different assemblages types had different substrate affinities for arginine. To sum up, this report identified G. duodenalis ADI to be immunomodulatory and gives first indications of a correlation between enzyme function and variation of the protein primary structure.

Les récepteurs Toll-like 7 et 8 jouent un rôle important dans l’activation de la réponse immunitaire innée et adaptative. Leur stimulation conduit à la production des cytokines pro-inflammatoires et d’interférons de type I. L’imiquimod et son dérivé le résiquimod sont les premières molécules de faible poids moléculaire décrites comme agonistes du TLR7 et TLR8. Ces deux molécules ont montré des activités anticancéreuses et adjuvantes très importantes. Récemment, les TLR 7 et 8 ont fait l’objet de plusieurs publications visant à développer de nouveaux agonistes TLR7 et/ou TLR8 dans la perspective d’être utilisés comme adjuvants vaccinaux. Malgré les rôles essentiels de TLR7 et TLR8 dans la stimulation du système immunitaire, une activation immunitaire chronique peut être responsable de plusieurs maladies infectieuses et auto-immunes. D’où l’importance de développer également des antagonistes TLR7 et/ou TLR8.Ce travail de thèse est consacré à la synthèse et le développement de nouvelles molécules hétérocycliques, analogues de l’imiquimod et de résiquimod, dans le but d’identifier de nouveaux ligands TLR7 et/ou TLR8. Des voies de synthèse innovantes, permettant une modulation chimique importante grâce à des couplages croisés pallado-catalysés, ont été mises au point et ont permis d’obtenir une cinquantaine de molécules appartenant à trois séries chimiques différentes de type imidazo[1,2-a]pyrazine, imidazo[1,5-a]quinoxaline et pyrazolo[1,5-a]quinoxaline. De nombreux essais d’alkylation ont été tentés sur ces trois séries chimiques afin d’introduire une large variété de substituants sur le cycle à cinq sommets. L’application du couplage croisé de Sonogashira nous a permis d’établir une liaison C-C et introduire diverses chaines alkyles. Ces composés ont été testés pour leur activité agoniste et antagoniste TLR7 et 8. Aucun des composés cibles n'a présenté d’activité agoniste TLR7 et TLR8, dans l'intervalle des concentrations testées. Par contre, tous les composés ont montré une activité antagoniste sélective du TLR7. Les composés les plus actifs, 5.35a et 5.35b, membres de la série pyrazolo[1,5-a]quinoxaline ont montré des IC50 de l’ordre de 10 μM. Ces résultats prometteurs nous ont permis la découverte d’une activité antagoniste TLR7 importante pour la série pyrazolo[1,5-a]quinoxaline, une série très peu développée dans la littérature. La modulation chimique des molécules actives nous permet de donner naissance à de nouveaux leaders, qui peuvent jouer un rôle important dans la thérapie de plusieurs maladies infectieuses et auto-immunes. / Toll-like receptors 7 and 8 play an important role in immune system activation. Their stimulation leads to the production of pro-inflammatory cytokines and type I interferons. Both receptors recognize viral ssRNA, as well as synthetic tricyclic imidazoquinoline derivatives such as imiquimod (TLR7 agonist) and resiquimod (TLR7/8 agonist). These two molecules showed significative anti-cancer and adjuvant activities. Many reports in the literature have been focused on the development of new TLR7/8 agonists belonging to different chemical series. These agonists strongly induce the production of T helper 1-polarizing cytokines and may therefore serve as promising candidate vaccine adjuvants. Despite the essential roles of TLR7 and TLR8 in the immune system stimulation, chronic immune activation may be responsible for several infectious and autoimmune diseases. Consequently, the development of TLR7 inhibitors may play an important role in the therapy of these diseases.In this study, we are interested in the synthesis and development of new heterocyclic molecules, analogs of imiquimod and resiquimod, in order to identify new TLR7 and/or TLR8 ligands. Different synthetic pathways have been developed, using cross coupling reactions, in order to obtain a wide variety of molecules belonging to three chemical series: imidazo[1,2-a]pyrazine, imidazo[1,5-a]quinoxaline et pyrazolo[1,5-a]quinoxaline. Various alkylation reactions were attempted on these three chemical series in order to introduce a wide variety of substituents on the five-membered ring. The application of Sonogashira's cross-coupling allowed us to establish a C-C bond and introduce various alkyl chains. All compounds have been tested for their TLR7/8 agonistic and antagonistic activity using HEK-Blue™-hTLR7/8 cells. The synthesized compounds are completely inactive as TLR7/8 agonists and are selective TLR7 antagonists. Two compounds of the pyrazolo[1,5-a]quinoxaline series, compound 5.35a and 5.35b, bearing butyl and isobutyl chain respectively, are potent and selective TLR7 antagonists with low micromolar IC50. Results allowed us to discover significative activity for the pyrazolo[1,5-a]quinoxaline series as selective TLR7 antagonists, which may therefore play an important role in the therapy of several infectious or autoimmune diseases.