| 1. |
- Bachmayer, Nora, 0061-
(författare)
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The role of natural killer cells and inflammatory mediators in preeclamptic pregnancies
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The maternal immune system must be able to adjust during pregnancy and accept the foetus that expresses paternal antigens. These changes are found both in placenta and circulation, including a mild inflammatory response. NK cells are abundant during the early part of pregnancy in placenta and are thought to be important for placental development. During preeclampsia the placenta is poorly developed, together with an escalated pro-inflammatory profile noticed in both placenta and circulation. We wanted to study NK cells in placenta and circulation from preeclamptic cases as well as levels of cytokines. HMGB1, an alarmin involved in inflammation, was also measured in preeclamptic placentae.When studying preeclamptic placentae in third trimester we found higher numbers of NK cells as well as a higher expression of CD94+ NK cells. We also found slightly elevated levels of HMGB1 together with significantly lower expression of IL-12 in preeclamptic placentae. Further, the NK cell activating cytokines IL-12/IL-23p40 and IL-15 in sera from preeclamptic women were increased compared to healthy pregnancies. The elevated levels of NK cell activating IL-12/IL-23p40 and IL-15 found in preeclamptic sera, made us investigate the circulating NK cells in preeclampsia. However, no differences were seen between healthy and preeclamptic pregnancies.The main immunological alterations in third trimester preeclamptic pregnancies with regard to NK cells were found in placenta. Altered maternal cytokine levels in placenta could influence decidual NK cells in preeclampsia, noticed by their higher numbers and altered receptor expression. If these alterations also exist during early pregnancy it could result in a poorly developed and dysfunctional placenta.
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| 2. |
- Björk, Emma, 1977-
(författare)
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Immunosuppressive mechanisms in endometriosis : a focus on the role of exosomes
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Endometriosis is defined as the presence of endometrial-like tissue outside the uterine cavity. It has been suggested that the aberrant immunological mechanisms that cause dysfunction of immune cells and mediators are involved in the pathogenesis of endometriosis. There is substantial evidence of downregulated NK cell cytotoxicity and changes in inflammatory mediators such as cytokines in endometriosis. This research aimed to elucidate the immunosuppressive mechanisms in endometriosis, focusing on NK cells, the role of cytokines, and exosomes derived from endometriotic tissue.Cytokines are small peptides/proteins used for intercellular communication, and regulate immune-effector functions in health and disease. In Paper I, real-time RT-qPCR and a set of primers and probes for 11 cytokines were used defining cytotoxic Th1, humoral Th2, regulatory Tr1/Th3, and inflammatory cytokine profiles. Cytokine mRNA expression in endometriotic tissue was compared with endometrium, and systemically with peripheral blood mononuclear cells (PBMC) from women with endometriosis and healthy controls. In addition, immunohistochemical staining with monoclonal antibodies was performed to investigate T-regulatory cells in endometriotic lesions. A downregulation of mRNA for cytokines that mediate cytotoxicity and antibody response was found in the endometriotic lesions. At the same time, there was an upregulation of inflammatory and T-regulatory cytokines in the endometriotic lesions, suggesting enhanced local inflammation and priming of an adaptive regulatory response. Consistent with these findings, T-regulatory cells were abundant in the endometriotic lesions. These findings suggest that the ectopic implantation seen in endometriosis may be a consequence of increased inflammation and priming of adaptive T regulatory cells, resulting in impaired cytotoxicity and enhanced immune suppression. Exosomes are nanometer-sized extracellular vesicles of endosomal origin; they are produced by most cells in the body, convey intercellular communication and participate in both normal and pathological processes. Paper II show that endometriotic lesions produce high amounts of exosomes. The exosomes expressed on their surfaces the NKG2D ligands MICA/B and ULBP1-3 and the proapoptotic molecules FasL and TRAIL. These molecules are known as immunosuppressive signatures. Functional experiments were performed to show that these exosomes can downregulate the main activating NK receptor NKG2D on CTL and NK cells, reduce the killing ability of PBMC from healthy donors, and induce apoptosis of activated lymphocytes through the FasL/Fas pathway. The production and secretion of exosomes from the endometriotic tissue may be further enhanced by the vigorous local inflammation at ectopic sites. The results show that endometriotic lesions secrete immunosuppressive exosomes that inhibit cytotoxicity and promote apoptosis of activated immune cells. The exosomes form a “protective shield” around the endometriotic tissue thus promoting their survival.NK cells are cytotoxic cells of the innate immune system. Human NK cells can be divided into two subsets: CD56+bright and CD56+dim. The CD56+dim subset is more naturally cytotoxic, whereas the CD56+bright subset produces more cytokines, but has low natural cytotoxicity. The majority (>90%) of circulating NK cells are CD56+dim, whereas very few (0-10 %) are CD56+bright. In Paper III a higher amount of CD56+bright cells in serum was observed in one third of endometriosis patients compared to healthy controls. The amount of these cells was normalized after treatment with surgery, with or without medical treatment. Untreated patients had a lower expression of NKG2D receptors on their NK cells and CTLs compared to treated patients and healthy controls, which could be due to endometriotic exosomes carrying the NKG2D ligands that downregulate the receptor. Thus, surgery might have a beneficial effect on cytotoxic NK-cell function in endometriosis.Endometriosis is considered a benign disease; however it has many features in common with tumors, and shares multiple microenvironmental hallmarks with cancer, including angiogenesis, immune dysregulation, inflammation, invasion, and metastasis. Paper II shows that endometriotic tissue secretes immunosuppressive exosomes. In Paper IV, exosomes in the peripheral blood of epithelial ovarian cancer (EOC) patients, and the impairment of the NKG2D receptor-ligand system in vivo before and after surgery, were studied. The serum exosomes isolated from the EOC patients carried the NKG2D ligands MICA/B and ULBP1-3. In functional experiments, the EOC exosomes downregulated the expression of the NKG2D receptor, and subdued NKG2D-mediated cytotoxicity in NK cells from healthy donors in a similar manner to the endometriotic exosomes studied in Paper II. In Paper IV, surgery of the primary EOC tumor had a beneficial effect, alleviating the exosome-mediated suppression of NKG2D-mediated cytotoxicity. Thus, exosome-mediated immunosuppression is revealed as a common mechanism of action for immune escape in endometriosis and cancer. The results presented in this thesis provide novel and important insights into the function of the immune system in endometriosis, and give new explanations for why ectopic endometrial tissue persists and proliferates outside the uterine cavity. Furthermore, the immunosuppression in the microenvironment of endometriosis, which has many similarities with the local tumor microenvironment (TME), was investigated with a focus on the role of endometriotic exosomes. Taken together, this thesis contributes to understanding of the pathogenesis of endometriosis, and might be useful in identifying biomarkers for endometriosis and developing new immunomodulatory therapies.
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| 3. |
- Hedlund, Malin, 1981-
(författare)
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Exosomes and the NKG2D receptor-ligand system in pregnancy and cancer : using stress for survival
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Although not obvious at first sight, several parallels can be drawn between pregnancy andcancer. Many proliferative, invasive and immune tolerance mechanisms that supportnormal pregnancy are also exploited by malignancies to establish a nutrient supply andevade or edit the immune response of the host. The human placenta, of crucial importancefor pregnancy success, and its main cells, the trophoblast, share several features withmalignant cells such as high cell proliferation rate, lack of cell-contact inhibition andinvasiveness. Both in cancer and in pregnancy, the immune defense mechanisms,potentially threatening the survival of the tumor or the fetus, are progressively blunted oreven turned into tumor- or pregnancy-promoting players. Amongst immune mechanisms that are meant to protect the host from cancer and can be apotential threat to the fetus, the NKG2D receptor-ligand system stands out as the mostpowerful, stress-inducible “danger detector” system that comprises the activating NK cellreceptor NKG2D and its ligands, the MIC (MHC class I Chain-related proteins A and B)and ULBP (UL-16 Binding Proteins) families. It is the major cytotoxic mechanism in thebody promoting surveillance and homeostasis. In the present thesis we investigate theNKG2D receptor-ligand system in human early normal pregnancy and in theleukemia/lymphoma cell lines Jurkat and Raji and ask the questions “How is the NKG2Dreceptor-ligand system functioning in pregnancy and tumor? How is the danger of cytotoxicattack of the fetus avoided? Why is the immunosurveillance function compromised incancer patients?” We developed a method to isolate and culture villous trophoblast from early human normalplacenta and used it to study the NKG2D receptor-ligand system. We discovered that theNKG2D ligand families of molecules MICA/B and ULBP1-5 are constitutively expressedby the syncytiotrophoblast of the chorionic villi. Using immnunoelectron microscopy, westudied the expression of these molecules at the subcellular level and could show for thefirst time that they are preferably expressed on microvesicles in multivesicular bodies(MVB) of the late endosomal compartment and are secreted as exosomes. Exosomes arenanometer sized microvesicles of endosomal origin, produced and secreted by a great7variety of normal and tumor cells. The exosomes are packages of proteins and ribonucleicacids that function as “mail” or “messengers” between cells conveying different biologicalinformation. We isolated and studied exosomes from placental explant cultures. We foundthat they carry NKG2D ligands on their surface and are able to bind and down-regulate thecognate receptor on NK-, CD8+ and T cells. The down-regulation selectively causedimpairment of the cytotoxic response of the cells but did not affect their lytic ability asmeasured by perforin content and gene transcription. Thus, the NKG2D ligand-bearingexosomes suppress the cytotoxic activity of the cells in the vicinity of the placenta, leavingtheir cytolytic machinery intact, ready to function when the cognate receptor isrestored/recycled. These findings highlight the role of placental exosomes in the fetalmaternalimmune escape and support the view of placenta as an unique immunomodulatoryorgan. Next, we studied the expression and exosomal release of NKG2D ligands by tumor cellsusing the leukemia cell lines Jurkat and Raji as a tumor model. We found that NKG2Dligand-bearing exosomes with similar immunosuppressive properties as placental exosomesare constitutively secreted by the tumor cells, as a mechanism to blunt the cytotoxicresponse of the immune cells and thus protect themselves from cytotoxic attack by the host.Interestingly, we found that thermal- and oxidative stress up-regulates the exosomesecretion and the amount of exosome-secreted NKG2D ligands. Our results imply thattumor therapies that cause stress-induced damage, such as thermotherapy and stripping ofoxygen supply to the tumor, might have a previously unrecognized side effect causingenhanced exosome production and secretion, which in turn suppresses the natural antitumorimmune response and thus should be taken into account when designing an optimaltherapy of cancer patients. In conclusion, we describe a novel stress-inducible mechanism shared by placenta andtumors as an immune escape strategy. We found that placenta- and tumor-derived NKG2Dligand-bearing exosomes can suppress immune responses to promote the survival and wellbeing of the fetus or the tumor. Our work comprises an important contribution to theelucidation of the NKG2D ligand-receptor system and its mode of operation in the humanbody and opens new perspectives for designing novel therapies for infertility and cancer.
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| 4. |
- Israelsson, Pernilla, 1984-
(författare)
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Mechanisms for immune escape in epithelial ovarian cancer
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Tumors develop mechanisms to subvert the immune system, constituting immune escape. Epithelial ovarian cancer (EOC), the deadliest of all gynecological malignancies, uses a variety of mechanisms to undermine immune surveillance, aiding its establishment and metastatic spreading. Despite progress in oncoimmunology, a lot remains unknown about the cancer-immune system interplay. The aim of this thesis was to study tumor-mediated mechanisms for immune escape in EOC patients, focusing on the role of cytokines and EOC- derived exosomes. Cytokines are key molecules regulating immune effector functions in health and disease. We used real-time RT-qPCR and a set of primers and probes for 12 cytokines, discriminating between different immune responses and compared the cytokine mRNA expression profiles locally in the TME and systemically in peripheral blood immune cells of EOC patients, to women with benign ovarian conditions and women with normal ovaries. The cytokine mRNA expression was in general most prominent in EOC patients, confirming the immunogenicity of EOC. We found significant dominance of inflammatory and immunosuppressive/ regulatory cytokines, known to promote tumor progression by priming and activating T regulatory cell-mediated immune suppression. In contrast, IFN-γ, crucially important for evoking a cytotoxic anti-tumor response, was not upregulated. Instead, a systemic increase of IL-4 prevailed, deviating the immune defense towards humoral immunity. With regard to our cytokine study, we performed comparative analyses of cytokine mRNA versus protein expression in the EOC cell lines OVCAR-3 and SKOV-3. We found that cytokine mRNA signals were universally detected, and in some instances translated into proteins, but the protein expression levels depended on the material analyzed and the method used. Due to the high sensitivity of real-time RT-qPCR, we suggest that cytokine mRNA expression profiles can be used for some instances, such as in studies of mechanistic pathways and in comparisons between patient groups, but cannot replace expression at the protein level. Exosomes are nanometer-sized vesicles of endosomal origin, released by virtually all cells, participating in normal and pathological processes. Like many tumors, EOC is a great exosome producer. We isolated exosomes from EOC ascitic fluid and supernatant from tumor explant cultures to study their effect on the NK cell receptors NKG2D and DNAM-1, involved in tumor killing. We found that EOC exosomes constitutively expressed NKG2D ligands on their surface while DNAM-1 ligand expression was rare and not associated with the exosomal membrane. Consistently, the major cytotoxic pathway of NKG2D-mediated killing was dysregulated by EOC exosomes while the accessory DNAM-1- mediated pathway remained unchanged. Our results provide a mechanistic explanation to the previously made observation that in EOC patients, tumor killing is only dependent on the accessory DNAM-1 pathway. Following these iii iv results, we studied NKG2D-mediated cytotoxicity in vivo in EOC patients before and after surgery. We found that the serum exosomes isolated from EOC patients were able to downregulate the NKG2D receptor and suppress NKG2D-mediated cytotoxicity in NK cells from healthy donors, in a similar way as exosomes from EOC ascites. We also found that surgery of the primary EOC tumor has a beneficial effect on the patients’ anti-tumor cytotoxic immune response. One mechanistic explanation could be a decrease in circulating NKG2D ligand- expressing exosomes, thus improving the cytotoxic NK cell function. In conclusion, our results contribute to the understanding of the mechanisms responsible for tumor immune escape in general, and in EOC patients in particular, and might be useful in developing novel antitumor therapies. Our studies highlight the prevailing immunosuppression in the local TME and the immunosuppressive role of EOC exosomes. Furthermore, they support the notion that cancer surgery is also a way of removing exosome-producing cells and reducing the serum concentration of immunosuppressive exosomes, thus boosting the patients’ cytotoxic anti-tumor response.
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| 5. |
- Jonsson, Yvonne, 1974-
(författare)
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Cytokines and immune balance in preeclampsia : a survey of some immunological variables and methods in the study of preeclampsia
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Preeclampsia is one of the most feared pregnancy complications, with a risk of maternal and fetal death and with no ideal therapy readily available. The cause of this strictly pregnancyrelated disease is still unknown and is therefore a great challenge to all researchers in the field of pregnancy-related pathophysiology.Today, the dominating theory of the origin of preeclampsia is defective initial placentation with insufficient penetration of the trophoblasts, leading to impaired maternal blood flow through narrow spiral arteries. However, the cause of this defective trophoblast behavior is not known. The maternal immune system has been proposed to have an influence on both the placentation and the subsequent systemic reactions. Therefore, it is very interesting to study the maternal immune system during preeclampsia, in hope of achieving a better understanding of this puzzling disease.Earlier studies have suggested that normal pregnancy requires a shift to a Th2/antiinflammatory type of immunity, at least directed towards the fetus and placenta, while some pregnancy complications, such as preeclampsia, could be due to a skewed Th1/proinflammatory type of immunity. However, the results from earlier studies designed to test the Th1/Th2 hypothesis in preeclampsia have not been consistent. Therefore, the aim of this thesis was to examine if established preeclampsia is associated with increased innate inflammatory responses and a deviation of adaptive responses towards Th1 when compared with normal pregnancy.Enumerations of cytokine-producing cells from peripheral blood did not show any difference in the production of IFN-γ, IL-4, IL-10 and IL-12 between women with preeclampsia and normal pregnancies. However, a decrease in the spontaneously produced levels of IL-5 was detected in cell cultures on peripheral blood mononuclear cells in women with preeclampsia. Furthermore, a decreased production of IL-10 in response to paternal antigens, believed to represent the fetus, was also detected for the preeclamptic women.Serum analysis showed increased levels of the pro-inflammatory mediators IL-6 and IL-8 during preeclampsia. Also, preeclamptic women displayed increased serum levels of the soluble IL-4 receptor, but no difference in the levels of IL-4 compared to normal pregnant women. This was an elusive finding, since the receptor was originally thought to reflect the levels of IL-4, but has recently been shown to have both agonistic and antagonistic properties on the IL-4 levels. Further studies of the local immune responses in the placenta showed no difference in the immunohistochemical staining of IL-4 and TNF-α between women with preeclampsia and women with normal pregnancies. In general, there were no hallmarks of abnormal morphology in the placental sections examined, regardless of diagnosis.In conclusion, the decreased levels of IL-10 in response to paternal antigens and the systemically increased levels of IL-6 and IL-8 suggest a specific decrease in antiinflammatory responses towards fetal antigens, together with a systemic activation of proinflammatory mediators during preeclampsia. Furthermore, the decreased production of IL-5 also indicates, at least partly, decreased Th2 responses in the established preeclampsia.
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| 6. |
- Neumann Andersen, Grethe, 1951-
(författare)
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Systemic sclerosis : vascular, pulmonary and immunological aspects
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In systemic sclerosis (SSc), interstitial lung disease (ILD) and engagement of the vascular system lead to increased morbidity and mortality. The aim of this thesis was to elucidate, in a consecutively included cohort of SSc (limited and diffuse) patients (n = 33), the T cell cytokine profile driving the disease in ILD and to explore the role of matrix metalloproteinase 9 (MMP-9) and its inhibitor: tissue inhibitor of metalloproteinase 1 (TIMP-1) in the extracellular matrix (ECM) degrading process leading to fibrous scarring and honey combing. Moreover, to characterize the role of nitric oxide (NO) in vascular engagement. Peripheral arterial changes cause Raynaud’s phenomenon and digital ulcers. Nitric oxide (NO) a main inducer of vasodilation is produced by endothelial nitric oxide synthase (eNOS) in response to changes in blood flow or by inflammatory cytokine inducible (i) NOS. In the vascular smooth muscle cell (VSMC) NO activates guanylate cyclase to produce cGMP, causing relaxation. We showed elevated plasma nitrate, a degradation product of NO, and increased urinary excretion of nitrate and cGMP. Plasma nitrate correlated with elevated levels of endothelial adhesion molecules: endothelial (E) selectin and vascular adhesion molecule 1, indicating that the activated endothelium is the site of NO synthesis by iNOS. Endothelial staining for E-selectin and the finding of iNOS and eNOS in SSc skin biopsies supported this notion. In SSc increased vascular stiffness may limit the NO vasodilatory effects. We found normal endothelium-dependent (i.e. flow mediated (FMD%)) and endothelium-independent (i.e. nitroglycerin-induced (NTG%)) vasodilation in the brachial artery. Radial arterial wall stiffness measured as maximum increase in pulse pressure (dP/dtmax) was increased. FMD% and especially NTG% correlated negatively and dP/dtmax positively to measures of endothelial inflammation: plasma- nitrate and adhesion molecule levels. Thus inflammatory vascular wall changes may interfere with dilation as may the presence of nitrate tolerance. We found elevated alveolar MMP-9 in both its pro- and active form in ILD. The levels correlated to decline in lung capacity, pointing at a causal relation. We suggest that neutrophils secrete MMP-9, which may degrade collagen IV, (the main constituent of basal membranes), collagen V, gelatins, proteoglycans and elastin. MMP-9 activity is partly regulated by the binding of pro- and active form to TIMP-1. Alveolar TIMP-1, which even stimulates fibroblast ECM synthesis, was increased independent of ILD. The inflammatory process in ILD is orchestrated by activated T helper (h) lymphocytes. We found a mixed Th1/Th2 reaction in SSc alveolar T cells expressing messenger for interferon gamma (Th1), IL-6 and IL-10 (both Th2). No particular cytokine mRNA profile distinguished alveolar T cells in ILD. Neutrophils invaded the bronchial epithelium, which seemed otherwise inert as levels of inflammatory cytokine sensitive transcription factors and their nuclear translocation tended to be low. The neutrophil recruitment pathway is uncertain as chemoattractants and endothelial adhesion molecules were normally expressed. In conclusion, MMP-9 probably causes degradation of lung tissue in ILD and may represent a future therapeutic target. Alveolar T cells show a mixed Th1/Th2 cytokine profile independent of ILD. Neutrophils invade the bronchial epithelium. Activated endothelium produces increased amounts of NO and adhesion molecules and the level of activation influences brachial arterial FMD% and NTG% and radial arterial compliance. Nitrate tolerance may be present.
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