L'hépatite C pose un problème de santé publique majeur, dans la mesure où le risque de développer une infection chronique est relativement élevé (40 à 60%) et où la résistance au traitement de choix - l’interféron alpha pégylé et la ribavirine - touche près de la moitié des patients. Cette persistence virale repose avant tout sur de puissantes stratégies d’évasion du système immunitaire inné de l’hôte par le virus. Dans ce projet, nous nous sommes intéressés à la caractérisation de la réponse antivirale dans des hépatocytes primaires humains normaux et chroniquement infectés avec le VHC, un domaine encore largement inconnu dû à la difficulté d’obtenir ce type de matériel primaire. Nous avons étudié la fonctionnalité de deux voies majeures de détection des pathogènes viraux suite à l’exposition d’hépatocytes primaires humains à de l’ARNdb intracellulaire, via le récepteur et adaptateur RIG-I/MDA5-CARDIF, et extracellulaire via TLR3-TRIF, mimant ainsi les étapes précoces de la détection d’un virus par la cellule hôte. Nous avons établi par RT-PCR quantitatif et analyse transcriptomique par microarray, que ces deux voies de stimulation sont fonctionnelles dans des hépatocytes primaires normaux et que leur activation entraîne à la fois l’expression de gènes antiviraux communs (ISG56, ISG15, CXCL10, …) mais aussi spécifiques avec les gènes IL28A, IL28B et IL29 qui sont une signature de l’activation de la voie de détection de l’ARNdb intracellulaire. La protéine virale NS3/4A joue un rôle majeur à la fois dans le clivage de la polyprotéine virale initiale, mais aussi en interférant avec les cascades de signalisation engagées suite à la détection par la cellule hôte de l’ARN du VHC. Plus particulièrement, nous avons démontré que l’expression ectopique de NS3/4A dans des hépatocytes primaires humains normaux entraîne une diminution significative de l’induction des gènes antiviraux dûe au clivage de CARDIF au cours de l’activation de la voie de signalisation médiée par RIG-I. Nous avons également démontré que l’expression de la NS3/4A entraîne des modifications de l’expression de gènes-clé impliqués dans la régulation de l’apoptose et du programme de mort cellulaire, en particulier lorsque la voie TLR3 est induite. L’ensemble de ces effets sont abolis en présence de BILN2061, inhibiteur spécifique de NS3/4A. Malgré les stratégies de subversion de l’immunité innée par le VHC, nous avons démontré l’induction significative de plusieurs ISGs et chemokines dans des hepatocytes primaires provenant de patients chroniquement infectés avec le VHC, sans toutefois détecter d’interférons de type I, III ou certains gènes antiviraux précoces comme CCL5. Ces observations, concomitantes avec une diminution de l’expression de CARDIF et une correlation inverse entre les niveaux d’ARNm des ISGs et l’ARN viral révèlent une réponse antivirale partielle dûe à des mécanismes interférents sous-jacents. Cette réponse antivirale détectable mais inefficace est à mettre en lien avec l’échec du traitement classique PEG-IFN-ribavirine chez la moitié des patients traités, mais aussi en lien avec l’inflammation chronique et les dommages hépatiques qui mènent ultimement au développement d’une fibrose puis d’une cirrhose chez une grande proportion de patients chroniquement infectés. / Hepatitis C infection is a worldwide health problem since the risk to develop a persistent infection is relatively elevated (40 to 60%) and nearly half of the infected patients do not respond to the classical anti-HCV therapy based on a combination of PEG-IFNα and ribavirin. Viral persistence is based on powerful evasion strategies of the host’s innate immune system. In our study, we characterized antiviral response in primary human normal and chronically HCV-infected hepatocytes, a cutting-edge in our field due to the difficulty to isolate this particular cell type. In order to better define the antiviral response in freshly isolated human primary hepatocytes, we stimulated these cells with extracellular and intracellular dsRNA to trigger TLR3/TRIF and RIG-I-MDA5/CARDIF-mediated antiviral signaling pathways. By using qRT-PCR and microarray analysis, we report that both detection pathways are functional in normal human hepatocytes, their activation leading to the expression of both common (IFIT1, OASL, ISG15 and CXCL10) and specific genes (IL28A, IL28B and IL29), these last ones being a signature of the intracellular dsRNA-mediated pathway. HCV NS3/4A plays a key role in the viral polyprotein processing and upon viral RNA detection by interfering with the host’s antiviral signalling cascades. We report that major antiviral genes induction following activation of RIG-I mediated pathway are severely impaired in ectopically NS3/4A expressing normal hepatocytes due to CARDIF cleavage, but can be restored by specific NS3/4A inhibitor BILN2061. Our microarray analysis also revealed a role for NS3/4A following TRL3-mediated pathway activation on regulation of apoptosis and programmed cell death, which could be linked to strategies for the virus to persist in its host. Despite HCV strategies to circumvent the host’s immune defense system, we observed significant upregulation of ISGs and chemokines in liver biopsies and corresponding isolated hepatocytes from chronically HCV-infected patients. However, no type I and III interferon, neither key-antiviral genes (e.g., CCL5) were detected, underlying an ongoing –but inefficient- antiviral response unable to eradicate the virus. Moreover, we obtained significant inverse correlations between ISGs mRNAs and viral RNA in addition to CARDIF decrease, clearly unravelling efficient viral interfering strategies in a context of chronic HCV infection. This sustained -albeit incomplete- hepatic innate immune response is certainly associated to the failure of the classical IFN-based therapy in half of the infected patients and to the chronic inflammation causing liver damages and eventually leading to hepatocarcinoma which is often observed at late stage of the disease.

Les cellules dendritiques plasmacytoïdes (pDC) sont des cellules dendritiques spécialisées, aussi connues sous le nom de cellules productrices d’interféron-α (IFN-α). Les pDC jouent un rôle essentiel dans l’induction de la réponse immunitaire antivirale, en reconnaissant les antigènes viraux via les Toll-like receptors (TLR) 7 et 9. Toutefois, les pDC du sang de cordon sont incapables de produire de l’IFN-α en réponse à une stimulation du TLR9, mais leur maturation en cellules présentatrices d’antigènes est normale. Dans le cadre de ce travail, nous nous sommes intéressés aux effets des facteurs immuno-régulateurs sécrétés par le placenta sur la différenciation et la fonction des pDC. Nous avons analysé l’effet, seules ou en combinaison, de la progestérone (PG), de l'interleukine (IL)-10 et du tumor growth factor (TGF)-β sur la différenciation et la fonction des pDC. Nous démontrons qu’à des niveaux supra-physiologiques ces trois facteurs modulent la différenciation et la production d’IFN-α des pDC. À des niveaux observés dans le sang de cordon, ces facteurs ont peu d’impact sur les pDC lorsque utilisés individuellement. Toutefois lorsque utilisés en combinaison, ils diminuent la production d’IFN-α. Nous avons aussi démontré que la PG, l’IL-10 et le TGF-β n’induisent pas l’expression des micro-ARN 146a et 155 par les pDC. Finalement nous démontrons que les niveaux de ces molécules sont plus élevés dans le sang de cordon que dans le sang d’adulte. Nos résultats révèlent le rôle important des facteurs immuno-régulateurs sécrétés par le placenta sur la fonction des pDC et en conséquence, sur la réponse immunitaire fœtale et néonatale. / Plasmacytoid dendritic cells (pDC) are specialized dendritic cells, also known as IFN-α producing cells. pDC are key inducers of immune responses following viral infections, by detecting viral antigens through the Toll-like-receptors (TLR) 7 and 9. Although cord blood pDC are unable to produce IFN-α after stimulation, they retain their capacity to mature into antigen presenting cells. To gain insights into the mechanisms of pDC regulation in cord blood, we investigated the effects of immune regulators secreted by the placenta on the differentiation and function of pDC. Using in vitro differentiated pDC, we analyzed the effect of progesterone (PG), IL-10 and TGF-β, separately or in combination, on pDC differentiation and activation. The data revealed that supra-physiological concentrations of these three factors could individually impair pDC differentiation and IFN-α production. Physiological concentrations of PG, IL-10 or TGF-β, individually, had low impact on pDC while in combination they were able to modulate IFN-α production. We also showed that PG, IL-10 or TGF-β failed to induce the expression of micro-RNA 146a and 155 in pDC. Lastly, our results indicated that PG, IL-10 and TGF-β were present at high levels in cord blood as compared to adult blood. These results, therefore, shed new light on the synergy of immune regulators secreted by the placenta and their impact on foetal and neonatal immune responses.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

L’immunité innée est notre premier mécanisme de défense contre l’invasion des pathogènes. Cette défense est basée sur la reconnaissance d’éléments invariables des pathogènes par des récepteurs encodés dans les lignées germinales. Dans la réponse anti-virale, le facteur de transcription Interferon Regulatory Factor 3 (IRF3) joue un rôle clé dans la réponse interféron de type I, combattant ainsi la réplication virale et conférant un état anti-viral aux cellules infectées ainsi qu’aux cellules avoisinantes. IRF3 est une protéine dont l’activation et la phosphorylation sont régulées par les kinases TBK1 et IKKi. Nous proposons ici que l’acétylation est une modification post-traductionnelle importante dans la régulation de l’activité d’IRF3. Nous avons observé par immunobuvardage qu’IRF3 est acétylé de façon basale et que cette acétylation est induite par la présence du co-facteur CBP et est inhibée par la présence de la kinase TBK1. Par spectrométrie de masse, nous avons ensuite identifié huit lysines sujettes à l’acétylation sur IRF3. Aussi, par mutagénèse dirigée, nous avons muté de façon ponctuelle chacun de ces sites et avons déterminé que la mutation de la lysine 87 inhibe la capacité d’IRF3 à s’attacher à l’ADN en EMSA et à transactiver son élément de réponse en essai luciférase. Aussi, nous proposons que l’acétylation masque la charge positive de la lysine 87 et contrôle de façon négative l’activité du facteur de transcription IRF3. Notre groupe démontre ainsi pour la première fois l’acétylation du facteur de transcription dans un modèle cellulaire et propose que ce processus joue un rôle inhibiteur dans la régulation de la protéine. / Innate immunity is our first line of defense against invading pathogens. This process is based on the recognition of invariable molecular patterns present on different pathogens by germ-line encoded receptors. In the innate immune response against invading viruses, the transcription factor Interferon Regulatory Factor 3 (IRF3) plays a major role in the regulation of type I interferons, priming the defense of infected and surrounding cells against viral infection. The phosphorylation and activation of IRF3 is regulated by the kinases TBK1 and IKKi. Here we suggest that acetylation is also an important post-translational modification in the regulation of this transcription factor. We have observed by immunoblot analysis a basal acetylation of IRF3, which is increased in the presence of its co-factor CBP and inhibited in the presence of its kinase TBK1. Also, we have identified on IRF3 eight different lysine residues subjected to acetylation using mass spectrometry and we have mutated these sites using sitedirected mutagenesis. We found that the K87R mutation inhibits IRF3-DNA binding in EMSA and leads to the transactivation of its promoter in luciferase assays. We also suggest that by masking the positive charge of the lysine 87 residue, acetylation negatively controls the activity of IRF3. Our group thus demonstrates for the first time the acetylation of IRF3 in a cellular model and suggests that this modification plays a role in the inhibition of the IRF3 transcription factor.

Interferons (IFNs) are a class of structurally related proteins first discovered to be produced by virus-infected cells. By now, several other inducing agents have been described. IFNs exert multiple effects on cells exemplified by the establishment of an antiviral state, inhibition of cell proliferation and alteration of different immune reactions. In the present thesis the inhibition of cellular growth concentrated on effects in the early cell cycle have been studied. The human glioma cell line 251 MG was found to be blocked in the S phase of the cell cycle upon addition of IFN both to exponentially growing and growth-factor depleted, synchronized cells. Thymidine kinase and DNA-polymerase activities were reduced in parallel with the S phase effect. 2-5 oligo Anucleotides transfected into glioma cells lead to inhibition of cell growth, exponentially growing cells being blocked in the S phase as during IFN treatment. In contrast, synchronized, restimulated cells were blocked in the cellcycle phase where they resided at the time of transfection. As 2-5 oligo A synthetase activity was induced in the middle of the Gl phase, these results might indicate that the kinetics of expression of oligonucleotides after IFN additiondetermines the type of cell cycle block obtained in differenttumor cells. IFN inhibited preferentially proteins originating from newly synthesized mRNA in Sw 3T3 cells, c-mvc did not seem to be included among these proteins. In both cell systems c-myc expression was unaltered after IFN treatment. In clone T1 selected from the the Sw 3T3 cell line , c-mvc expression was uncoupled to growth and seemed to be growth factor independent. The change in c-myc expression in clone T1 compared to SW 3T3 cells did not render the cells sensitive to IFN. Hence, c-myc regulation does not seem to be the mechanism by which IFN regulates cell growth in this system. The proliferation marker KI-67 antigen was shown not to be causatively involved in growth inhibition of IFN. The reduced levels of the antigen was proposed to be a secondary effect caused by the G0/G1 arrest. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1991, härtill 6 uppsatser</p> / digitalisering@umu

Throughout history, human populations have continuously been challenged by new and emerging infectious diseases. For the past 26 years, sub-Saharan Africa and other countries around the world have been dealing with a pandemic caused by a relatively new pathogen called the human immunodeficiency virus (HIV). Although antiretroviral (ARV) therapies effectively reduce morbidity and mortality rates, the long term use in those who have access to treatment inevitably leads to drug-related toxicity and resistance. Even with a strong commitment from governments to expand and finance prevention and treatment programs, transmission rates continue to outpace the benefits of these efforts. Therefore to effectively eradicate the disease, research is focusing on the design of protective and therapeutic vaccines. The first major step in designing these alternative therapies is to define correlates of immune protection. / The research presented in this thesis focuses on characterizing the quantitative and qualitative features of T cell immune responses in individuals who spontaneously control viral replication and exhibit a benign course of disease while remaining off ARV therapy. A comprehensive analysis of HIV-specific IFN-gamma secreting immune responses revealed that neither the breadth nor the magnitude of responses directed against the entire HIV proteome accurately predicts the viral load or rate of CD4 decline. Subsequent analyses showed that the preferential targeting of Gag was associated with reduced rates of CD4 decline and was later confirmed in a cohort of individuals in primary infection whereby the relative breadth and magnitude of Gag p24 was inversely correlated with viral load set point. / The maintenance of polyfunctional immune responses in HIV-infected subjects with a benign course of disease prompted us to develop a method that could comprehensively assess the breadth, magnitude and specificity of three functionally distinct subsets of HIV-specific lymphocytes (single IFN-gamma, single IL-2 and dual IFN-gamma/IL-2 secretors). Survey of immune responses in chronically infected individuals revealed that only the breadth and magnitude of dual IFN-gamma/IL-2 secreting lymphocytes correlated with reduced viral loads and increased CD4 counts suggesting that secretion of IFN-gamma alone was a poor correlate of protection. We also showed that the contribution of polyfunctional lymphocytes to the total response was greater for epitopes restricted by major histocompatibility complex (MHC) class I alleles associated with slow disease progression compared to those restricted by alleles associated with rapid or neutral rates of HIV disease progression. / Taken together, this work supports the view that immune monitoring of infected and vaccinated individuals should include methodologies capable of detecting both IFN-gamma and IL-2 secretion from responding T lymphocytes. The studies presented here have furthered our understanding of what constitutes protection from disease progression emphasizing that both specificity and polyfunctionality are features of effective control of viral replication.

The Fas pathway is critical for the maintenance of normal T cell homeostasis. Humans and mice with defects in this pathway exhibit the accumulation of large numbers of peripheral lymphocytes and lupus-like autoimmunity. A major feature of these organisms is the accumulation of non-NK TCRαβ+CD4-CD8- “double negative” (DN) T cells. While regulatory T cells (Tregs) with the DN phenotype have been extensively characterized in Fas-sufficient mice and humans, limited data exist on the role of DN T cells as Tregs in Fas-deficient animals. In fact, most of the literature suggests that the DN T cells accumulating in Fas-deficiency states are pathogenic, contributing to secondary lymph node enlargement and autoimmune disease. In this body of work, data are presented that illustrate that Fas-deficient lymphoproliferative (LPR) DN T cells can act as Tregs in an interferon γ (IFNγ)- and Fas ligand (FasL)-dependent fashion toward Fas-sufficient T cells. LPR DN T cells needed to be able to secrete and respond to IFNγ in order to upregulate surface FasL, in order to ameliorate GVHD mediated by CD4+ T cells in vivo and to suppress the proliferation of and kill activated CD4+ T cells in vitro. FcRγ, a key molecule involved in innate immune responses, can substitute for CD3ζ in the T cell receptor (TCR) of mouse and human T cells in certain circumstances; in doing so, it is essential for the regulatory function of TCR transgenic DN Tregs. FcRγ-deficient LPR mice were found to have exacerbated T cell accumulation and early mortality. We show that while FcRγ expression was required for LPR DN T cells to regulate CD4+ and CD8+ T cells responding to alloantigens in vitro and in vivo, it does not control autologous lymphoproliferation in LPR mice by supporting the function of a regulatory cell, nor does it affect the rate of proliferation of LPR T cells in vivo. Instead, FcRγ-expressing LPR CD4+, CD8+ and DN T cells were found to be undergoing apoptosis at a high rate in vivo, and in contrast to their FcRγ-deficient counterparts, FcRγ+ LPR DN T cells were capable of undergoing TCR restimulation-induced cell death (RICD). The data presented in this thesis therefore show that LPR DN T cells can exhibit IFNγ-, FasL- and FcRγ-dependent regulatory function, and also illustrate a previously unknown function for FcRγ in controlling the expansion of Fas-deficient T cells. The implications of these data for autoimmune lymphoproliferative syndromes, and normal T cell homeostasis, are discussed.

L’idée qu’une cellule puisse effectuer la cytolyse de cellules transformées, comme une cellule Natural Killer (NK), tout en ayant la capacité de présenter des antigènes, comme une cellule dendritique (DC), peut sembler fantaisiste. Cependant, de telles cellules furent bel et bien identifiées chez la souris en 2006. Ces cellules, nommées Interferon-producing Killer Dendritic Cells (IKDC), furent l’objet d’une caractérisation extensive qui révéla leur énorme potentiel immunologique. La combinaison de fonctions associées à des cellules NK et à des DC a doté les IKDC d’un pouvoir antitumoral remarquable. D’ailleurs, il a été démontré que les IKDC sont plus efficaces que les cellules NK pour limiter la croissance tumorale. Ainsi, suite à leur découverte, les IKDC ont suscité beaucoup d’intérêt. Cependant, une controverse émergea sur la nature des IKDC. Plusieurs groupes indépendants tentèrent de reproduire les expériences attestant les fonctions de DC des IKDC, sans y parvenir. De plus, des études additionnelles révélèrent que les IKDC possèdent des similitudes très importantes avec les cellules NK. Ces observations ont mené la communauté scientifique à suggérer que les IKDC sont des cellules NK en état d’activation (aNK). Malgré cette controverse, les caractéristiques antitumorales des IKDC sont si uniques et considérables qu’il est primordial de poursuivre l’étude de ces cellules. Pour y arriver, il est essentiel de déterminer la nature des IKDC et de mettre fin à ce débat. Par la suite, il sera important d’identifier des façons de cibler spécifiquement les IKDC pour permettre leur usage dans le cadre de thérapies antitumorales. Ainsi, l’objectif de cette thèse est de définir l’identité des IKDC, puis de déterminer les facteurs génétiques responsables de la régulation de ces cellules. Nous avons démontré que les IKDC ne sont pas des cellules aNK, contrairement à ce qui avait été suggéré. Nous avons constaté que les IKDC prolifèrent activement et possèdent un phénotype unique, des caractéristiques associées à des cellules NK très immatures. Afin de déterminer si les IKDC peuvent acquérir un phénotype mature, nous avons effectué des expériences de transfert adoptif. Suite à leur injection in vivo, les IKDC acquièrent un phénotype de cellules matures, mais étonnamment, elles se différencient aussi en cellules NK. Ainsi, nous avons révélé que les IKDC sont un intermédiaire dans la différenciation des cellules NK. En parallèle, nous avons démontré que la proportion d’IKDC varie grandement entre des souris de fond génétique différent, indiquant que des facteurs génétiques sont impliqués dans la régulation de ces cellules. Nous avons alors effectué une analyse génétique qui a révélé que les IKDC sont régulées par des facteurs génétiques compris dans une région distale du chromosome 7. Les résultats présentés dans cette thèse constituent une avancée importante pour la recherche sur les IKDC. Ils ont permis de définir la nature des IKDC et d’identifier un intervalle génétique impliqué dans la régulation de ces cellules. Ces découvertes sont des connaissances précieuses pour l’identification des IKDC chez l’Homme et la création de nouvelles thérapies dans la lutte contre le cancer. / The idea that a cell could kill transformed cells, like a Natural Killer (NK) cell, all the while exhibiting also the capacity to present antigens to T cells, like a Dendritic Cell (DC), may seem farfetched. However, in mice, a cell presenting these specific properties was identified in 2006. These cells were named Interferon-producing Killer Dendritic Cells (IKDC) and extensive studies revealed that they were endowed with an important immunological potential. Indeed, the fact that IKDCs exhibit properties of both DC and NK cells conferred them with an exceptional anti-tumor potential. Notably, on a per cell basis, the in vivo anti-tumor activity of IKDCs is more efficient than NK cells. Therefore, following their identification, IKDCs showed great therapeutic promise. However, a debate on the cell lineage origin of IKDCs emerged. Several independent groups could not replicate the finding that IKDCs showed functional antigen-presentation properties similar to DCs. Also, additional studies revealed that IKDCs are very similar to NK cells. These and other observations led the scientific community to believe that IKDCs were activated NK cells. Despite this controversy, IKDCs clearly exhibit a unique and outstanding anti-tumor potential, highlighting the relevance to further explore these cells. We must first close the debate regarding the lineage origin of IKDCs. We subsequently need to identify a means to specifically target IKDCs to facilitate their use in novel anti-tumor therapies. Thus, the objective of my thesis is first, to define the identity of IKDCs and second, to determine the genetic factors implicated in the regulation of these cells. For the first objective, we demonstrated that IKDCs do not represent activated NK cells, as previously suggested. We show that IKDCs are highly proliferative and exhibit a unique phenotype associated with very immature NK cells. In an attempt to verify if IKDCs could acquire a mature phenotype, we conducted an adoptive transfer experiment. We found that, after adoptive transfer, IKDCs adopt a mature phenotype, but also surprisingly differentiate into NK cells. These findings indicate that IKDCs represent an intermediate in NK-cell differentiation. For the second objective, we demonstrated that the IKDC proportion was highly variable between strains of different background origins, indicating that these cells are regulated by genetic factors. A genetic study revealed that genetic factors in distal arm of chromosome 7 associate with the proportion of IKDCs. The results presented in this thesis represent an important breakthrough for the research on IKDCs. They allowed to define the cell lineage origin of IKDCs and to identify a genetic region involved in the regulation of this cell type. These discoveries are valuable knowledge for the identification of human IKDCs and the development of novel anti-tumor therapies.

In ruminants, prostaglandin F2 alpha (PGF2α) is released from the endometrium in a pulsatile pattern at the time of luteolysis. The luteolytic PGF2α pulses are transported from the uterus to the corpus luteum (CL) through the utero-ovarian plexus (UOP) to cause luteolysis. At the time of establishment of pregnancy, interferon tau (IFNT) secreted by the conceptus suppresses the pulsatile release of PGF2α and thereby rescues the CL and maintains its secretion of progesterone. However, basal concentrations of PGF2α are higher in pregnant ewes than in cyclic ewes. The pulsatile release of PGF2α likely requires selective carrier-mediated transport and cannot be supported by a simple diffusion mechanism. The molecular and functional aspects of carrier mediated transport of PGF2α from the uterus to the ovary through the utero- ovarian plexus (UOP) at the time of luteolysis and recognition/establishment of pregnancy are largely unknown ruminants. Results indicate that intrauterine inhibition of (PGT) prevents the pulsatile release of PGF2α independently of spatial expressions of estrogen receptor (ESR-1) and oxytocin receptor (OXTR) proteins by the endometrium at the time of luteolysis in sheep. PGT protein is expressed in the UOP during the estrous cycle and pharmacological inhibition of PGT prevents transport of luteolytic PGF2α pulse through the UOP in sheep. IFNT activates novel JAK-SRC-EGFR-RAS-RAF-ERK1/2-EGR-1 signaling modules in endometrial luminal epithelial (LE) cells and regulates PGT- mediated release of PGF2α through these novel cell-signaling pathways. IFNT stimulates ERK1/2 pathways in endometrial LE cells and inhibition of ERK1/2 inhibits IFNT action and restores spatial expression of OXTR and ESR-1 proteins in endometrial LE cells and restores endometrial luteolytic pulses of PGF2α in sheep. Collectively, the results of the present study provide the first evidence to indicate that transport of endometrial luteolytic PGF2α pulses from the uterus to the ovary through the UOP is controlled by a PGT-mediated mechanism in sheep, new mechanistic insight into molecular mechanisms regulating cellular and compartmental transport of PGF2α at the time of luteolysis, and new mechanistic understanding of IFNT action and release of PGF2α from the endometrial LE cells and thus opens a new arena of research in IFNT signaling and PGT function.

Cytokines are soluble signalling mediators within the immune system, and have been shown to be of importance in the development of type 1 diabetes (T1D). This thesis studied the production of cytokines in experimental models of T1D and during transplantation of insulin-producing islets of Langerhans. We have demonstrated that the transcriptional TNFα-inhibitor MDL 201,449A, previously shown to reduce immune-mediated diabetes induced in mice by multiple low doses of streptozotocin, was not TNFα-specific, but also inhibited IFNγ and IL-10 in spleen cells. Furthermore, when the inhibitor was removed from in vitro cultures, a rebound phenomenon of increased cytokine secretion occurred. The thesis also investigated whether plastic adhesion, a method generally employed to deplete macrophages, influenced cytokine production in spleen cells. We observed that plastic adhesion increased TNFα, IFNγ and IL-10 release, and decreased IL-4 secretion. Plastic adhesion depleted only ~30% of the macrophages, but as much as ~60% of the regulatory T cells. Thirdly, we found that “control” treatments for islet transplantations, i.e. syngeneic and sham transplantations, exerted a clear effect on cytokine production from spleen cells, possibly due to a decrease in regulatory T cells that may be caused by the surgery and/or anaesthesia. Moreover, spleen cells from mice exposed to surgery exhibited a decreased proliferative capacity to concanavalin A stimulation. We also perceived a marked difference in cytokine response depending on the mouse strain used in the experiments. Finally, we aimed to elucidate if, besides autoimmune activities, also high glucose- and free fatty acid concentrations as seen in diabetes could cause changes in cytokine production. We observed that spleen cells cultured in varying glucose concentrations had different cytokine production profiles. The free fatty acid palmitate might also influence cytokine release, but this effect was obscured by the cytokine-suppressive action of the ethanol used to dissolve the palmitate.