Pathogenesis of autoimmune and auto-inflammatory diseases is induced by auto-aggressive helper T (Th) cells (i.e. Th1 and Th17 cells), and can be controlled by regulatory T cells (Tregs) characterized by expression of the transcription factor Foxp3. Thus, development of autoimmunity is regulated by the balance of Tregs and Th1/Th17 cells. Prostaglandin E₂ (PGE₂) is a bioactive lipid mediator with immune-modulatory potential that acts through 4 receptors (EP1-4). It has been shown that PGE₂ facilitates Th1 and Th17 cell development and expansion, therefore promoting autoimmune inflammation. However, the role of PGE₂ in Treg development and function is largely unclear. The aim of this PhD was to test the hypothesis that PGE₂ regulates Treg development, function and subsequent immune response. I observed that in vivo inhibition of endogenous PGE₂ biosynthesis using a COX inhibitor resulted in increased Foxp3+ Tregs in various lymphoid organs. This response was prevented by addition of an EP4 agonist. PGE₂-EP4 signalling particularly inhibits RORγt+ Tregs in the intestine. This was not observed in either antibiotic-treated mice or MyD88/TRIF double-knockout mice, suggesting gut commensal microbiota involvement. In addition, PGE₂ has a role in microbiota-dependent regulation of intestinal CD11c+MHCII+CD11b+CD103- mononuclear phagocytes (MNPs) which drive intestinal Treg expansion through production of type 1 interferons. Consistent with these in vivo observations, gut microbial metabolites from indomethacin treated mice enhanced in vitro RORγt+ Treg differentiation in the dendritic cell- T cell co-culture system. Adoptive transfer of caecal microbiota from COX inhibitor- treated mice into naïve mice also provided protective benefits in a chemical (DSS)-induced colitis disease model. In summary, this work has demonstrated that PGE₂ affects intestinal Tregs, indicating a novel mechanism for interaction of PGE₂, the adaptive immune system and the gut microbiota in homeostasis within this environment. These findings increase our understanding of the role of PGE₂ in development of inflammatory bowel disease and offer potential therapeutic strategies for treating this disease.

Aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) are currently widely used as fever and pain relief in patients with arthritis and other inflammatory symptoms. NSAIDs effect by inhibiting cyclooxygenase-1 (COX-1) and/or cyclooxygenase-2 (COX-2). COX isozymes (COXs) are key enzymes in the biosynthesis of prostaglandin H2 (PGH2) from arachidonic acid (AA). It is now clear that prostaglandin E2 (PGE2), one of the downstream products of PGH2, is the main mediator in both chronic and acute inflammation. Microsomal prostaglandin E synthase (mPGES-1) is the terminal enzyme of COX-2 in the PGE2 biosynthesis pathway. Different from other two constitutively expressed PGE2 synthase (PGES), mPGES-2 and cPGES, mPGES-1 is induced by pro-inflammatory stimuli and responsible for the production of PGE2 related to inflammation, fever and pain. For these reasons, selective inhibition of mPGES-1 is expected to suppress inflammation induced PGE2 production and, therefore, will exert anti-inflammatory activity while avoid the side effects of COXs inhibitors, such as gastrointestinal (GI) toxicity, and cardiovascular events. A combination of computational and experimental approaches was used to discovery mPGES-1 inhibitors with new scaffolds. The methods used include molecular docking, molecular dynamic simulation, molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculation, and in vitro activity assays. Our large-scale structure-based virtual screening was performed on compounds in the NCI libraries, containing a total of ~260,000 compounds. 7 compounds have been determined for their IC50 values (about 300 nM to 8000 nM). What’s more, these new inhibitors of mPGES-1 identified from virtual screening did not shown significant inhibition against COX isozymes even at substantially high concentrations (e.g. 100 µM). Rational methodology for drug design and organic synthesis were applied to generate three series of mPGES-1 inhibitors with different scaffolds. In total, about 200 compounds were synthesized and tested for their in vitro inhibition against human mPGES-1. Compounds with high potency against human mPGES-1 were further screened for their inhibition against mouse mPGES-1 and selectivity of human mPGES-1 over COXs. Several compounds were identified as submicromolar inhibitors against human mPGES-1 with high selectivity over COXs. In general, we have successfully identified a library of compounds as potent mPGES-1 inhibitors without significant inhibition against COXs. Structure information and in vitro activity evaluation data generated from the virtual screening and the library of compounds will be used to guide future design and synthesis of the mPGES-1 inhibitors.

Nach einer Gewebeschädigung stellt die Entzündungsreaktion den ersten essentiellen Schritt für die Wundheilung dar, deren anschließendes Auflösen den Übergang zur Phase der Gewebeneubildung einleitet. Voraussetzung für diesen Ablauf ist die Herunterregulierung der Aktivität proinflammatorischer M1-Makrophagen (M1-Ma) sowie die Induktion antiinflammatorischer M2-Makrophagen (M2-Ma). Zwischen diesen beiden Phänotypen steht ein breites Spektrum unterschiedlich aktivierter Ma, die in der Wundheilung aktiv sind. Die Auslöser für diesen wichtigen Übergang sind dabei weitgehend unbekannt. Es ist in in vitro Versuchen beschrieben, dass entzündlich aktivierte humane dermale Fibroblasten (dFb) die inflammatorische Aktivität von M1-Ma reduzieren und zusätzlich die Polarisierung von inflammatorisch aktivierten Monozyten zu M2-Ma fördern. Diese Effekte vermitteln sie über die Freisetzung immunmodulierender Mediatoren, insbesondere von TSG-6 und PGE2, einem Produkt der Cyclooxygenase 2 (COX 2). Bisher wurden diese Faktoren noch nicht im zeitlichen Verlauf der Entzündungsreaktion während der Wundheilung in einem in vivo Tiermodell untersucht. In dieser Arbeit konnte festgestellt werden, dass in dem angewendeten murinen in vivo Wundheilungsmodell die initiale Entzündungsreaktion nach 24 Stunden ihren Höhepunkt erreicht. Synchron dazu konnte erstmals gezeigt werden, dass das Auftreten von TSG-6 und COX-2 ebenfalls die höchste Expression am 1. Tag nach der Wundsetzung aufzeigt. In der Analyse der aufgetrennten Zellschichten der Haut wurde nachgewiesen, dass COX-2 in der epidermalen und dermalen Schicht exprimiert wird. Die Synthese von TSG-6 hingegen ist auf die Zellen in der dermalen Schicht beschränkt. Die Isolierung und Untersuchung von dFb aus dem restlichen Zellverband des Wundgewebes bestätigte dFb als Quelle für die TSG-6 Synthese. In einem weiteren Versuchsansatz mit entzündlich aktivierten humanen Keratinozyten wurde gezeigt, dass sie kein TSG-6 bilden können. Somit stellen die gewonnenen Erkenntnisse die Grundlagen zukünftiger Untersuchungen zum funktionalen Ablauf der Wundheilung dar, in welchem die dermalen Fibroblasten als zentrale Schlüsselrolle im Entzündungsgeschehen betrachtet werden müssen. Daraus können sich neue therapeutische Ansätze zur Modulation einer gestörten Wundheilung ergeben.:1 Einleitung 1.1 Aufbau und Funktion der Haut 1.2 Wundheilung 1.2.1 Phasen der Wundheilung 1.2.1 Bedeutung der Makrophagen in der Wundheilung 1.3 Beeinflussung der Entzündungsauflösung 1.4 Einfluss von dFb auf die Ma-Differenzierung 1.5 Aufgabenstellung 2 Materialien und Methoden 2.1 Materialien 2.1.1 Maus 2.1.2 Geräte und Verbrauchsmaterialien 2.1.3 Software 2.1.4 Chemikalien und molekularbiologische Reagenzien 2.1.5 Antikörper und Primer 2.2 Methoden 2.2.1 Zellkultur 2.2.2 In vivo Wundheilungsmodell 2.2.3 Aufbereitung der Gewebeproben 2.2.3.1 Gesamtwundgewebe 2.2.3.2 Auftrennung der dermalen und epidermalen Schicht 2.2.3.3 Isolierung von dermalen Fibroblasten aus Wund- und Hautbiopsien 2.2.4 Histologische Analyse 2.2.5 Zellzahlbestimmung 2.2.6 Durchflusszytometrie 2.2.7 Genexpressionsanalysen 2.2.7.1 RNA- Isolierung/Konzentrations- und Reinheitsbestimmung 2.2.7.2 Herstellung von cDNA 2.2.7.3 Quantitative Echtzeit-PCR 2.2.8 Proteinbiochemische Analysen 2.2.8.1 Proteingewinnung aus Zellkulturen 2.2.8.2 Proteinisolation aus den Wund- und Hautbiopsien 2.2.8.3 Immunoassays 2.2.8.4 Analytische Auswertung der Proteinmessungen aus den Wund- und Hautbiopsien 2.2.9 Statistische Auswertung 3 Ergebnisse 3.1 Charakterisierung der Entzündungsphase 3.1.1 Wundverschluss 3.1.2 Zeitliche Expression proinflammatorischer Mediatoren 3.1.3 Zeitliche Expression antiinflammatorischer Mediatoren 3.2 Zeitliche Expression von TSG-6 und COX-2 und deren Produkte in der Gesamtwunde 3.3 Bestimmung des Ursprungs von TSG-6 und COX-2 in der Wundheilung 3.3.1 Auftrennung des Wundgewebes in Epidermis und Dermis 3.3.1.1 Charakterisierung der Schichten 3.3.1.2 Nachweis von TSG-6 und COX-2 3.3.2 Isolierung von dFb aus dem Wundrand 3.3.2.1 Charakterisierung der separierten Zellfraktionen 3.3.2.2 Nachweis von TSG-6 und COX-2 3.4 Humanes Modell: in vitro Kultur hudFb und huKC 3.4.1 Nachweis von TSG-6 und COX-2 und deren Produkte 4 Diskussion 5 Zusammenfassung der Arbeit 6 Literaturverzeichnis A Appendix A1 Erklärung über die eigenständige Abfassung der Arbeit A2 Erklärung über die Vorbehaltlichkeit der Verfahrenseröffnung zur Verleihung des Titels Dr. med. A3 Publikationen A4 Danksagung

In der vorliegenden Arbeit wurde die bisher kaum in der Literatur beschriebene Stoffklasse der Prostaglandin-Glycerolester (PG-G) in Hinblick ihrer biochemischen Wirkmechanismen genauer charakterisiert. Einige Studien beschreiben PG-G als relevante Mediatoren im Rahmen sowohl inflammatorischer als auch nozizeptiver Prozesse. Dieser Sachverhalt macht diese Stoffklasse zum interessanten Gegenstand aktueller pharmakologischer Forschung, dessen Grundlage die Identifizierung und Charakterisierung entsprechender Rezeptoren darstellt. Im Rahmen unserer Arbeit, identifizierten wir als ersten Schritt das Target des PGE2-G. Weder Prostaglandinrezeptoren noch bekannte orphane GPCR konnten durch die Stimulation mit PGE2-G aktiviert werden. Jedoch konnten in bestimmen Zelllinien wie RAW264.7 und H1819 Effekte durch PGE2-G erzielt werden, während andere Zelllinien keine Reaktionen zeigten. Dies machten wir uns zu Nutzen und sequenzierten das Transkriptom dieser Zelllinien, um so durch subtraktive Analysen Rezeptoren einzugrenzen, welche voraussichtlich als Target von PGE2-G fungieren. Als vielversprechender Kandidat exprimierten wir den UDP-Rezeptor P2Y6 heterolog in HEK293-Zellen und wiesen mittels Zellkultur-basierter Assays den Agonismus von PGE2-G am P2Y6 nach. Weitere Untersuchungen bestätigten diese hochspezifische Bindung zwischen beiden Interaktionspartnern. Auffällig war jedoch die ausordentlich hohe Potenz von PGE2-G, welche schon bei picomolaren Stoffkonzentrationen eine Aktivierung von P2Y6 initiiert. Insbesondere in Hinblick auf den deutlich höheren EC50-Wert von UDP stellt sich die Frage, inwiefern dies die Signaltransduktionsmechanismen und die damit einhergehenden physiologischen Effekte moduliert. Es stellte sich weiterhin die Frage, wie der P2Y6 die Bindung zweier chemisch unterschiedlichen Substanzen realisieren kann. Ziel dieser Arbeit war es nun, die Struktur von P2Y6 auf molekularbiologischer Ebene zu untersuchen, um ein Verständnis über die Art und Weise der Bindung der unterschiedlichen Agonisten zu erhalten.:Entzündungsreaktionen ................................................................................................................. 6 Prostaglandine ............................................................................................................................... 7 Prostaglandin‐Glycerolester und die Identifizierung ihrer Rezeptoren ......................................... 9 Prostaglandin‐Glycerolester ........................................................................................................9 G‐Protein‐gekoppelte Rezeptoren (GPCR) ............................................................................... 11 Orphane GPCR .......................................................................................................................... 12 Screening potentieller Targets von Prostaglandin‐E2‐Glycerolester ........................................ 13 Der Pyrimidinrezeptor P2Y6 .......................................................................................................... 14 Nucleotidrezeptoren ................................................................................................................ 14 Pharmakologische Bedeutung von Nucleotidrezeptoren ........................................................ 15 P2Y6 .......................................................................................................................................... 15 Fragestellung ................................................................................................................................ 18 Publikationen ................................................................................................................................... 20 Zusammenfassung der Ergebnisse und Diskussion .......................................................................... 50 Prostaglandin‐E2‐Glycerolester ist ein endogener Agonist am P2Y6 .......................................... 50 Die Bindungstasche des P2Y6 ....................................................................................................... 50 Evolutionäre Konservierung der Agonisten‐Promiskuität........................................................ 50 Simulation der Ligandenbindung im Homologiemodell........................................................... 51 Experimentelle Prüfung der Interaktionspartner ......................................................................... 52 Interaktionspartner beider Agonisten: R103, Y107, R287 ....................................................... 53 Interaktionpartner mit UDP: Y262 ........................................................................................... 53 Interaktionspartner mit PGE2‐G: Y75, N109, S291, N293 ........................................................ 53 Korrelation der experimentellen Daten mit einem Homologiemodell ........................................ 53 Ausblick ........................................................................................................................................ 54 Literatur ............................................................................................................................................ 56 Anhang ............................................................................................................................................. 62 Darstellung des eigenen Beitrags ..................................................................................................... 85 Erklärung über die eigenständige Abfassung der Arbeit ................................................................. 87 Lebenslauf ........................................................................................................................................ 88 Publikationen und Vorträge ............................................................................................................. 90 Danksagung ...................................................................................................................................... 91

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

No description available.

We reported previously that prostaglandin E2 (PGE2) upregulates IL-23 in vitro in bone marrow-derived dendritic cells (DC), and in vivo in models of collagen-induced arthritis and inflammatory bowel disease, leading to preferential Th17 development and activity. There is very little information on the molecular mechanisms involved in the PGE2-induced upregulation of Il23a gene expression. In the present study we investigated the signaling pathways and transcription factors involved in the stimulatory effect of PGE2. Although PGE2 does not induce IL-23p19 expression by itself, it synergizes with both extra- and intracellular TLR ligands and with inflammatory cytokines such as TNFα. We established that the effect of PGE2 in conjunction with either LPS or TNFα is mediated through the EP4 receptor and the cAMP-dependent activation of both PKA and EPAC. Using the EP4 agonist PGE1OH in conjunction with TNFα, we found that PKA-induced PCREB and EPAC-induced PC/EBPβ mediate the stimulatory effect of PGE2 on IL-23p19 expression. This is the first report of CREB and C/EBPβ involvement in Il23a promoter activation. Mutation within the putative CREB and C/EBP sites combined with in vivo DNA binding (ChIP) assays identified the distal CREB site (-1125) and the two proximal C/EBP sites (-274 and -232) as essential for PKA-activated CREB and EPAC-activated C/EBPβ induced IL-23p19 expression. / Microbiology and Immunology

No / Dendritic cells (DCs) are central players in coordinating immune responses, both innate and adaptive. While the role of lipid mediators in the immune response has been the subject of many investigations, the precise role of prostaglandins has often been plagued by contradictory studies. In this study, we examined the role of PGE2 on human DC function. Although studies have suggested that PGE2 specifically plays a role in DC motility and cytokine release profile, the precise receptor usage and signaling pathways involved remain unclear. In this report we found that irrespective of the human donor, monocyte-derived dendritic cells (MoDCs) express three of the four PGE2 receptor subtypes (EP2–4), although only EP2 and EP4 were active with respect to cytokine production. Using selective EP receptor antagonists and agonists, we demonstrate that PGE2 coordinates control of IL-23 release (a promoter of Th17, an autoimmune associated T cell subset) in a dose-dependent manner by differential use of EP2 and EP4 receptors in LPS-activated MoDCs. This is in contrast to IL-12, which is dose dependently inhibited by PGE2 through both receptor subtypes. Low concentrations (∼1–10 nM) of PGE2 promoted IL-23 production via EP4 receptors, while at higher (>50 nM), but still physiologically relevant concentrations, IL-23 is suppressed by an EP2 dependent mechanism. These results can be explained by differential regulation of the common subunit, IL-12p40, and IL-23p19, by EP2 and EP4. By these means, PGE2 can act as a regulatory switch of immune responses depending on its concentration in the microenvironment. In addition, we believe these results may also explain why seemingly conflicting biological functions assigned to PGE2 have been reported in the literature, as the concentration of ligand (PGE2) fundamentally alters the nature of the response. This finding also highlights the potential of designing therapeutics which differentially target these receptors.

In the studies this thesis is based upon, microarrays were applied to profilemRNA populations in biological samples to gain insights into transcriptionalpatterns and their relation to inflammatory disease.Rheumatoid arthritis (RA) is a chronic inflammatory disease, which leads todegradation of cartilage and bone. RA is characterized by synovial inflammationwith varying levels of tissue heterogeneity. This was confirmed by microarrayanalyses of multiple biopsies from the joints of 13 patients, which showed interindividualvariation in transcript populations to be higher than intra‐individualvariationTherapeutic antibodies targeting TNF‐α have revolutionized treatment of RA,although some patients do not respond well. Identification of non‐responders isimportant, not only because anti‐TNF treatment elevates the risk of infections,but also because of the cost of treatment. A proof‐of‐concept study to investigatetranscriptional effects of anti‐TNF treatment demonstrated that differencesbetween response groups could be identified and that these differences revealedbiological themes related to inflammatory disease.A subsequent study was therefore initiated with a larger cohort of 62 patients toinvestigate gene expression patterns in the synovium prior to anti‐TNFtreatment. Here, the heterogeneity was even more pronounced, thetranscriptional patterns were confounded by the presence of synovial aggregatesand only a weak therapy‐correlated signature was detected. The presence oflymphocyte aggregates was found to correlate to response to therapy, which isconsistent with previous findings indicating a higher level of inflammation ingood responding patients.Periodontitis is an inflammatory disease with many similarities to RA. Both areincurable chronic auto‐immune diseases, characterized by tissue destructionwith common genetic associations. Individuals with RA are at higher risk ofaccumulating significant periodontal problems than the general population. PGE2(prostaglandin E2) is known to stimulate inflammation and bone resorption inperiodontitis. In further studies, microarrays were applied in a time seriesdesign on human gingival fibroblats to explore the signal transduction pathwayscontrolling TNF‐α induced PGE2 synthesis in order to identify novel therapeutictargets. The JNK and NF‐kb pathways were identified as being differentiallyaffected by TNF‐a treatment. The transcriptional patterns were further verifiedusing antibodies against phosphorylated JNK/NF‐kb molecules and specificinhibitors of the JNK and NF‐kb signaling cascades. / QC 20100820

Capsaicin, prostaglandin E2 (PGE2) and polydopamine (PDA) were used to target macrophage and myofibroblast activity to reduce biomaterial-induced fibrosis. The lifetime and efficacy of implantable biomedical devices are determined by the foreign body response. Immediately after implantation, proteins nonspecifically adsorb onto the material and initiate inflammation. Macrophages recruited to the site can differentiate into M1 and M2 phenotypes and upregulate inflammation and fibrosis which interferes with the intended function. M1 macrophages secrete pro-inflammatory mediators that induce chronic inflammation and promote myofibroblast differentiation while M2 macrophages are wound healing cells that suppress inflammation and regulate fibroblast activity. The fibrotic tissue is developed by myofibroblasts which produce collagen in an unregulated fashion. Collagen thickening and biomaterial encapsulation decreases efficacy and sensitive of biomedical devices. We investigated the in vitro and in vivo effects of capsaicin, PGE2 and polydopamine surface modification on macrophages and myofibroblasts. Capsaicin and PGE2 reduced poly(lactic-co-glycolic) acid (PLGA)-induced fibrosis by promoting M2 macrophage phenotype to secrete anti-inflammatory IL-10 and suppressing myofibroblast marker α-smooth muscle actin (α-SMA). Capsaicin decreased collagen by 40% and upregulated IL-10 secretion by 35% while PGE2 reduced collagen by 55% after 14 days of implantation and 40% less collagen after 42 days. PDA was used to bind an anti-fibrotic compound to the surface of a poly(dimethyl siloxane) (PDMS-PDA) to reduce fibrosis. However, PDMS-PDA controls gave an unexpected result by reducing fibrosis to the same extent as anti-fibrotic compound bound PDMS- v PDA. PDA modification reduced cellularity by 50% and significantly decreased collagen thickness by 30%. Overall, our results showed that biomaterial-induced fibrosis can be reduced by promoting M2 macrophage activity and inhibiting myofibroblast differentiation. This research demonstrates three compounds that have potential to reduce fibrosis and extend the lifetime and efficacy of implantable biomedical devices. / Thesis / Master of Applied Science (MASc) / Capsaicin, prostaglandin E2 (PGE2) and polydopamine were used to reduce scar tissue development around implanted polymers. Biomedical devices implanted in the body can undergo severe scar tissue formation, or fibrosis, and fail. Fibrosis is described by the accumulation of collagen and encapsulation of an implanted polymer. Macrophages regulate fibrosis by secreting pro-fibrotic compounds and myofibroblasts produce unregulated amounts of collagen. In this thesis, capsaicin, PGE2 and polydopamine were incorporated into implants to target macrophage and myofibroblast activity and reduce fibrosis in mice. Capsaicin and PGE2, released from a degradable polymer, altered macrophages to secrete anti-fibrotic compounds and decreased collagen by 40% and 55%, respectively. Polydopamine surface modified implants gave an unexpected result and suppressed overall cell activity to reduce fibrosis by 30%. The research conducted shows the potential of these compounds to reduce fibrosis and extend the lifetime of implantable devices.