Acute cardiovascular clinical events such as myocardial infarction and cerebral stroke represent the major cause of death in Western societies. These pathologies are primarily resulting from... Show moreAcute cardiovascular clinical events such as myocardial infarction and cerebral stroke represent the major cause of death in Western societies. These pathologies are primarily resulting from atherosclerosis, a progressive condition characterized by the accumulation of lipids, immune cells, and fibrous elements in large arteries. The pathogenesis of atherosclerosis involves complex interactions between a wide variety of cells, including monocytes, macrophages, neutrophils, and lymphocytes. It is essential to identify novel targets for therapeutic application in order to reduce the residual atherosclerotic cardiovascular disease risk in current and future patients. Recent studies have suggested that members of the protein arginine methyltransferase (PRMT) family can potentially serve as novel therapeutic targets for atherosclerosis because of their regulatory role in inflammation and metabolism. To validate the contribution of PRMTs in the progression of atherosclerosis, in the studies presented in this thesis we have investigated the effect of inhibition of PRMT functionality on atherosclerosis susceptibility in established atherosclerotic mouse models.To address the role of PRMTs in atherosclerosis, we therefore made use of specific PRMT inhibitors, i.e. TC-E 5003 for PRMT1 inhibition, TP-064 for PRMT4 inhibition, and GSK3326595 for PRMT5 inhibition, that thus far have primarily been applied in vivo in the context of cancer treatment. Show less
Herniation of the lumbar disc can cause severe pain radiating down the leg alongside a dermatome. This pain can be caused by compression of the nerve root, but recent evidence has indicated that a... Show moreHerniation of the lumbar disc can cause severe pain radiating down the leg alongside a dermatome. This pain can be caused by compression of the nerve root, but recent evidence has indicated that a local inflammation response may also play a role. This thesis focuses on how macrophages that infiltrate the herniated disc in patients with lumbar disc herniation, influence pain and recovery after discectomy. Our data shows that for most patients, if macrophages are present, they benefit the process of healing by leading to a quicker resorption of the herniated material which results in faster recovery. However, for patients with Modic changes, which indicates a degenerated endplate (structure between disc and vertebrae), the presence of macrophages is less beneficial, for they recover more slowly after surgery. The reason for this discrepency seems to be an altered differentiation profile in macrophages. Macrophages differentiate into different types with different behaviours: the M2 macrophages are known for its anti-inflammatory properties and tissue resorption. Our study found M2 macrophages in lower numbers in patients with degenerated endplates, which can explain their slower recovery. Together the data indicates that macrophage differentiation profiles in lumbar herniated discs are promising treatment targets. Show less
Relevant human in vitro culture models can contribute to a reduction in animal use for respiratory research and generate data that can be better translated to patients. In this thesis various... Show moreRelevant human in vitro culture models can contribute to a reduction in animal use for respiratory research and generate data that can be better translated to patients. In this thesis various improvements of different current human lung in vitro models were explored. Human induced pluripotent stem cells (hiPSC) were used to generate alveolar type 2 (AEC2)-like cells, that were subsequently cultured at the air-liquid interface (ALI) to create a model of alveolar wound healing at the ALI. Furthermore, a method to utilize organoids was developed for expansion of cells from human lungs to obtain sufficient numbers to reliably establish epithelial cultures. This was done for AEC2 isolated from resected lung tissue, as well as for cell populations from broncho-alveolar lavage fluid from adults and tracheal aspirates from preterm new-borns. Finally, the cellular complexity of the in vitro cultures was expanded by introducing polarized macrophages to the airway ALI cultures. Using these cultures, the cross-talk between macrophages and airway was studied in relation to wound repair. Collectively, the methods that have been developed are expected to contribute to contribute to the use of patient- and disease relevant models in respiratory research. Show less
Cardiometabolic diseases including atherosclerotic cardiovascular disease (CVD) and non-alcoholic fatty liver disease (NAFLD) are the leading cause of preventable death worldwide.... Show moreCardiometabolic diseases including atherosclerotic cardiovascular disease (CVD) and non-alcoholic fatty liver disease (NAFLD) are the leading cause of preventable death worldwide. Hypercholesterolemia and inflammation are common major risk factors for atherosclerotic CVD as well as NAFLD. The studies described in this thesis aimed to get insight in strategies how to further improve cholesterol metabolism and inflammation, by exploring the therapeutic potential of brown fat activation and transcription factors involved in both processes. The results described in this thesis have increased our insight into regulation of cholesterol metabolism and inflammation by brown fat and nuclear receptors, respectively, and provided promising leads for innovative treatment of cardiometabolic diseases including brown fat activation, Δ24-dehydrocholesterol reductase inhibition, and farnesoid X receptor activation. Show less
Tuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm)... Show moreTuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm) infection in zebrafish larvae as an animal model for this disease to study the role of the myeloid differentiation factor 88 (Myd88), the key adapter protein of Toll-like receptors. Previously, Myd88 has been shown to enhance innate immune responses against bacterial infections, and in the present study, we have investigated the effect of Myd88 deficiency on the granuloma morphology and the intracellular distribution of bacteria during Mm infection. Our results show that granulomas formed in the tail fin from myd88 mutant larvae have a more compact structure and contain a reduced number of leukocytes compared to the granulomas observed in wild-type larvae. These morphological differences were associated with an increased bacterial burden in the myd88 mutant. Electron microscopy analysis showed that the majority of Mm in the myd88 mutant are located extracellularly, whereas in the wild type, most bacteria were intracellular. In the myd88 mutant, intracellular bacteria were mainly present in compartments that were not electron-dense, suggesting that these compartments had not undergone fusion with a lysosome. In contrast, approximately half of the intracellular bacteria in wild-type larvae were found in electron-dense compartments. These observations in a zebrafish model for tuberculosis suggest a role for Myd88-dependent signalling in two important phenomena that limit mycobacterial growth in the infected tissue. It reduces the number of leukocytes at the site of infection and the acidification of bacteria-containing compartments inside these cells. Show less
Diabetes mellitus type 2 (DM) is a major risk factor for developing active tuberculosis (TB) disease, yet the causal mechanisms driving this association remain largely elusive. As the incidence of... Show moreDiabetes mellitus type 2 (DM) is a major risk factor for developing active tuberculosis (TB) disease, yet the causal mechanisms driving this association remain largely elusive. As the incidence of DM is rising, especially in TB endemic countries, it is important to identify the relevant immunological and metabolic processes that underlie TB-DM comorbidity, because such insights will facilitate optimal treatment, diagnosis and prevention. In this thesis, we have started to unravel key factors underlying the association between TBand DM using two approaches. Firstly, we identified and analyzed human macrophage subsets and studied the interactions between these human cells and a major pathogen, Mycobacterium tuberculosis (Mtb), and the specific metabolic changes involved using well-controlled in vitro systems. Next, we employed metabolomics to determine the impact of concurrent TB-DM on circulating metabolites in patient cohorts ex vivo. In this thesis we present evidence derived from in vitro experiments and from ex vivo observational data which collectively suggest a pathogenic role of atherogenic lipid species during TB development. Show less
In this thesis, we will utilize embryonic zebrafish tumour models to understand the interaction between engrafted human cancer cells and macrophages from the host, test drug administration... Show moreIn this thesis, we will utilize embryonic zebrafish tumour models to understand the interaction between engrafted human cancer cells and macrophages from the host, test drug administration modalities and anti-cancer efficacies of newly-developed PDT and PACT compounds, and test a light-triggered liposomal system for targeted drug delivery specifically to cancer cells in vivo. In chapter 2, we investigate the role of macrophages in tumour-induced angiogenesis. We show that macrophage-dependent angiogenesis is driven by macrophage recruitment to lactic acid secreted by glycolytic B16 melanoma cells. Chemical inhibition of macrophages and glycolysis blocks the initiation of angiogenesis in these models, suggesting that macrophages attracted to glycolytic melanoma cells contribute to the tumour-induced angiogenesis process.In chapters 3 and 4, we explore novel PDT and PACT compounds, respectively, for treatment of conjunctival melanoma in zebrafish. We inject conjunctival melanoma cells into the retro-orbital site to establish an orthotopic model and into the Duct of Cuvier to generate an ectopic model. Our results prove that zebrafish provides a fast vertebrate cancer model to test the optimal administration regimen of drugs, conditions of light irradiation, host toxicity and anti-cancer efficacy of PDT and PACT drugs against conjunctival melanoma.In chapter 5, we focus on modifying liposomes to be light triggered in order to deliver drugs specifically to cancer cells. We inject MDA231 breast cancer cells into the Duct of Cuvier at 2 days post fertilization (dpf) to initiate metastasis to the CHT. We successfully demonstrate that light-triggered, cell-specific delivery of liposome-encapsulated doxorubicin reduces the xenograft cancer cell burden without enhanced cytotoxicity of the zebrafish embryos. In chapter 6, we summarize the novel anti-cancer strategies, which we have developed using zebrafish xenograft models. In the same chapter, we frame our findings in the current scientific landscape and discuss future perspectives. Show less
Purpose Sciatic symptoms due to lumbar disc herniation are likely to be caused not solely by mechanical compression of the nerve root, but also by pain-inducing elements from inflammatory processes... Show morePurpose Sciatic symptoms due to lumbar disc herniation are likely to be caused not solely by mechanical compression of the nerve root, but also by pain-inducing elements from inflammatory processes. Key components in the inflammatory reaction are M1 and M2 macrophages, with the M1 type being associated with pro-inflammatory processes and M2 with anti-inflammatory-processes. Method The present systematic review summarizes all studies on associations between M1 and M2 macrophages and their related inflammation factors and pain symptoms in lumbar disc herniations. Literature search was performed using an optimally sensitive search string. Studies were selected for inclusion by means of predefined inclusion and exclusion criteria and subsequently graded for risk of bias. A total of 14 studies were included. Overall risk of bias was moderate (8/14), and three studies had high risk of bias and three has low risk of bias. Results Regarding M1-related cytokines, high levels of TNF-alpha, TNFR1, IL-6, IL-8, and IFN-gamma were all associated high VAS scores. In contrast, high levels of TNFR2 were associated with lower VAS scores. Moreover, no associations were found for IL-1a and IL-1 beta. Results regarding M2-related cytokines revealed the opposite: high levels of both IL-4 and IL-10 were associated with lower VAS scores. No associations were established for TGF-beta. Moreover, the presence of macrophages (CD68) was negatively associated with VAS scores. Conclusion While M1-related pro-inflammatory cytokines worsen pain symptoms, M2-related anti-inflammatory cytokines alleviate pain symptoms. Nevertheless, the present evidence is limited, and further research on the underlying pathophysiological mechanism in sciatica is required. Graphic abstract These slides can be retrieved under Electronic Supplementary Material. Show less
The effective treatment of tuberculosis (TB) remains a major challenge to global health. Drug-resistant Mycobacterium tuberculosis (Mtb) strains and co-infection with HIV further increase the... Show moreThe effective treatment of tuberculosis (TB) remains a major challenge to global health. Drug-resistant Mycobacterium tuberculosis (Mtb) strains and co-infection with HIV further increase the difficulty of controlling TB. Thus, under the current situation, it is essential to develop effective treatment strategies for Mtb infections. Autophagy is a lysosomal degradation process and substantial experimental evidence has demonstrated that autophagy is an important host immune defense mechanism against mycobacterial infection. However, the development of effective therapies requires a better understanding of the interaction between the host and invading pathogens to identify host processes that can be targeted. A useful tool for such studies is the zebrafish model for TB. Zebrafish can be infected with Mycobacterium marinum (Mm), which is closely related to Mtb and causes similar disease characteristics. Taking advantage of the zebrafish TB model, this thesis presents new in vivo evidence for the important function of autophagy to inhibit mycobacterial proliferation inside macrophages. Furthermore, this study supports that stimulating the innate host defense processes that are dependent on the autophagy modulator, Dram1, and the selective autophagy receptors, p62 and Optineurin, could be a useful strategy to explore for adjunctive treatment of antibiotic-resistant TB infections. Show less
The aim of this thesis was to better understand the cellular origin of cholesteryl ester transfer protein (CETP) and to investigate the effects of the CETP inhibitor anacetrapib on the... Show moreThe aim of this thesis was to better understand the cellular origin of cholesteryl ester transfer protein (CETP) and to investigate the effects of the CETP inhibitor anacetrapib on the development of atherosclerosis. First, we investigated the specific characteristics of hepatic macrophages that express CETP. Our data clearly indicated that in the liver, CETP is exclusively expressed by F4/80+Ly6C-Clec4f+Vsig4+ macrophages that represent resident, rather than immature macrophages. Next, we showed that the HDL response to the inflammatory stimulus lipopolysaccharide is mediated by hepatic macrophages via down regulation of CETP expression in the liver that causes an increase in the level of HDL-C. In the second part of this thesis, we examined the effects and mechanism of pharmacological inhibition of CETP by anacetrapib on the development of atherosclerosis. We concluded that anacetrapib mainly decreases atherosclerotic lesion development via a reduction of non-HDL-C. Finally, we concluded that anacetrapib reduces (V)LDL-C by increasing hepatic remnant clearance via two mechanisms: 1) inhibition of CETP activity, resulting in remodelled VLDL particles that are more susceptible to hepatic clearance, and 2) a CETP-independent reduction in plasma PCSK9 level that has the potential to increase LDL receptor-mediated hepatic remnant clearance. Show less
Current therapies for patients with atherosclerosis are targeted primarily on reducing blood cholesterol, but this fails to prevent a large number of cardiovascular events.95 In order to identify... Show moreCurrent therapies for patients with atherosclerosis are targeted primarily on reducing blood cholesterol, but this fails to prevent a large number of cardiovascular events.95 In order to identify new therapeutic strategies for treating atherosclerosis, it is important to study the interactions between the cells that make up the atherosclerotic lesion. This thesis describes the role of endothelial cells and macrophages during lesion formation, as well as the expression patterns of sterol sensors in vascular smooth muscle cells present in the lesion. These new insights contribute to our understanding of atherosclerosis open up new avenues of atherosclerosis research Show less
Anti-inflammatory Mph have a high plasticity. Both in vivo and in vitro different types of Mph2 have been observed, all presenting an overlapping phenotype, but each having specific characteristics... Show moreAnti-inflammatory Mph have a high plasticity. Both in vivo and in vitro different types of Mph2 have been observed, all presenting an overlapping phenotype, but each having specific characteristics. Classifying Mph2 is difficult, especially since no specific markers for human Mph2 have yet been identified. At present, the full mechanism by which Mph-derived ROS affect T cell activation and signaling is unknown. We observed that Mph2 activate T cells in a ROS-dependent manner but also that the suppression of activated T cells by Mph2 was ROS-dependent. We observed that some cytokines were affected by DPI: the IL-12p40 and IL-10 production were suppressed by DPI, whereas no effect was observed with IL-6, suggesting that ROS is specifically involved in the regulation of some cytokines that may exert its effect on T cells. These results can be a new start point for further research in unraveling the mechanism, but how ROS affects T cell activation will need further investigations. In summary, T cells are activated by Mph-derived ROS and regulation of the cytokine production by the APC is a potential mechanism how ROS can affect T cell functions Show less
Many of the recognition molecules and mechanisms involved in immune responses have no bias towards external stimuli, but also sense and respond to pathological and physiological changes of non... Show moreMany of the recognition molecules and mechanisms involved in immune responses have no bias towards external stimuli, but also sense and respond to pathological and physiological changes of non infectious origin taking place within the body. Aiming at defining potential immuno-therapeutic strategies to treat human atherosclerosis, the focus of this work was the modulation of immune processes determinant of atherosclerosis lesion progression or cessation in mice, such as hematopoiesis, diapedesis and intravasation, leukocyte differentiation, cholesterol uptake apoptosis and cell survival. Modulation of these processes, by using bone marrow transplantation of hematopoietic stem cells with genetic deficiencies or over-expressing human or mouse engineered genes, demonstrated to alter the fate of atherosclerotic lesions at the balance between macrophage accumulation and lesion vulnerability versus resolution of inflammation and wound healing. This thesis demonstrates that processes responsible for the development and progression of atherosclerosis are dynamic and can be modulated to induce lesion stabilization and disease resolution. These results are promising for the development of novel therapeutics and challenge the current notion that atherosclerosis has a predetermined fate towards lesion vulnerability to rupture, which in humans results in thrombosis and clinical manifestations such myocardial infarction or stroke and sudden death. Show less
High-grade osteosarcoma is a malignant bone tumor with the highest incidence in young patients. In chapter 2, we studied MSCs of osteosarcoma patients and found downregulation of HCLS1 in... Show moreHigh-grade osteosarcoma is a malignant bone tumor with the highest incidence in young patients. In chapter 2, we studied MSCs of osteosarcoma patients and found downregulation of HCLS1 in osteosarcoma patient derived MSCs as compared to healthy donor derived MSCs. Despite almost two years in culture, none of the samples underwent spontaneous transformation. An increase in binucleation was noted upon increasing passage in both osteosarcoma patient and healthy donor derived MSCs. In chapter 3, prognostic factors related to the survival of patients with pulmonary metastasized high-grade osteosarcoma were studied. Higher metastatic tumor burden (i.e. larger number of pulmonary nodules), presence of vital metastases upon resection and male sex were associated with an increased risk of death. In chapter 4 we show that osteosarcoma metastasis seems to be inhibited by the presence of macrophages in the tumor microenvironm ent. In chapters 5 and 6 we show that osteosarcoma cells are sensitive to lysis by both autologous and allogeneic NK cells. Patient derived NK cells can be adequately activated by cytokine treatment with IL-15 (chapter 5) or IFN-_ (chapter 6). Therefore, activation of autologous NK cells (either in vivo or ex vivo) may be efficacious. In conclusion, the activation of innate immune cells such as macrophages and NK cells is a promising new adjuvant treatment strategy to treat patients with high-grade osteosarcoma Show less
Osteosarcoma and Ewing sarcoma are the most common bone cancers in children and young adults. Despite advanced surgical techniques and multi-drug chemotherapy, patients with recurrent, metastatic... Show moreOsteosarcoma and Ewing sarcoma are the most common bone cancers in children and young adults. Despite advanced surgical techniques and multi-drug chemotherapy, patients with recurrent, metastatic or chemotherapy-resistant disease have a poor outcome. Thus, novel targeted therapies are needed that combine potent and specific anti-cancer activity with limited toxicity toward normal tissues. The thesis is introduced by an outline of the biological properties of osteosarcoma and Ewing sarcoma, followed by an overview of cancer immunology and immunotherapy with the primary focus on innate immunity of human natural killer (NK) cells and macrophages. In the research chapters, cellular interactions of NK cells and macrophages with bone tumor cells are characterized in order to achieve favorable effects on anti-cancer immune cell functions. It is demonstrated that the anti-cancer potential of especially NK cells but also macrophages can be enhanced and directed to the bone tumor cells. It is discussed that the modulation of tumor__immune cell interactions may help to design novel immunotherapeutic approaches to harness anti-cancer functions of innate immune cells against osteosarcoma and Ewing sarcoma. Show less
