Autophagy is a fundamental degradative process, maintaining cellular homeostasis and functions in host defense against intracellular pathogens, including mycobacteria and Salmonella. The thesis... Show moreAutophagy is a fundamental degradative process, maintaining cellular homeostasis and functions in host defense against intracellular pathogens, including mycobacteria and Salmonella. The thesis investigated the function of an regulator of antibacterial autophagy, Damage Regulated Autophagy Modulator 1 (DRAM1) against infection and shows that DRAM1 restricts bacterial growth not only through canonical antibacterial autophagy (xenophagy) but also promotes an autophagy-related pathway, named LC3-associated phagocytosis (LAP). The function of DRAM1 in restricting bacterial proliferation is independent from the recognition of bacteria by xenophagy receptors. Mechanistically, DRAM1 promotes the infection-induced activation of autophagy and LAP as well as the maturation of bacteria-containing vesicles in both pathways. This maturation process, stimulated by DRAM1, involves multiple vesicle fusion steps directing bacteria to lysosomes. Through this maturation process, DRAM1 delivers the cytosolic protein Fau to bacteria-containing vesicles, where it serves as a precursor for antimicrobial peptides. The underlying mechanism may be explained by the discovery of an interaction between DRAM1 and the SNARE protein VTI1B. Overall, the work in this thesis contributes to ongoing research into the potential application of autophagy modulation as a host-directed therapy against infectious diseases. Show less
Replication stress, caused by Rev1 deficiency, is associated with mitochondrial dysfunction, and metabolic stress. However, the overall metabolic alterations and possible interventions to rescue... Show moreReplication stress, caused by Rev1 deficiency, is associated with mitochondrial dysfunction, and metabolic stress. However, the overall metabolic alterations and possible interventions to rescue the deficits due to Rev1 loss remain unclear. Here, we report that loss of Rev1 leads to intense changes in metabolites and that this can be manipulated by NAD + supplementation. Autophagy decreases in Rev1-/- mouse embryonic fibroblasts (MEFs) and can be restored by supplementing the NAD+ precursor nicotinamide riboside (NR). The abnormal mitochondrial morphology in Rev1-/-MEFs can be partially reversed by NR supplementation, which also protects the mito-chondrial cristae from rotenone-induced degeneration. In nematodes rev-1 deficiency causes sensitivity to oxidative stress but this cannot be rescued by NR supplementation. In conclusion, Rev1 deficiency leads to metabolic dysregulation of especially lipid and nucleotide metabolism, impaired autophagy, and mitochondrial anomalies, and all of these phenotypes can be improved by NR replenishment in MEFs. Show less
Exposure to cigarette smoke (CS) is the primary risk factor for developing chronic obstructive pulmonary disease. The impact of CS exposure on the molecular mechanisms involved in mitochondrial... Show moreExposure to cigarette smoke (CS) is the primary risk factor for developing chronic obstructive pulmonary disease. The impact of CS exposure on the molecular mechanisms involved in mitochondrial quality control in airway epithelial cells is incompletely understood. Undifferentiated or differentiated primary bronchial epithelial cells were acutely/chronically exposed to whole CS (WCS) or CS extract (CSE) in submerged or air-liquid interface conditions. Abundance of key regulators controlling mitochondrial biogenesis, mitophagy and mitochondrial dynamics was assessed. Acute exposure to WCS or CSE increased the abundance of components of autophagy and receptor-mediated mitophagy in all models. Although mitochondrial content and dynamics appeared to be unaltered in response to CS, changes in both the molecular control of mitochondrial biogenesis and a shift toward an increased glycolytic metabolism were observed in particular in differentiated cultures. These alterations persisted, at least in part, after chronic exposure to WCS during differentiation and upon subsequent discontinuation of WCS exposure. In conclusion, smoke exposure alters the regulation of mitochondrial metabolism in airway epithelial cells, but observed alterations may differ between various culture models used. Show less
Grijmans, B.J.M.; Kooij, S.B. van der; Varela Alvarez, M.; Meijer, A.H. 2022
Cells of the innate immune system continuously patrol the extracellular environment for potential microbial threats that are to be neutralized by phagocytosis and delivery to lysosomes. In addition... Show moreCells of the innate immune system continuously patrol the extracellular environment for potential microbial threats that are to be neutralized by phagocytosis and delivery to lysosomes. In addition, phagocytes employ autophagy as an innate immune mechanism against pathogens that succeed to escape the phagolysosomal pathway and invade the cytosol. In recent years, LC3-associated phagocytosis (LAP) has emerged as an intermediate between phagocytosis and autophagy. During LAP, phagocytes target extracellular microbes while using parts of the autophagic machinery to label the cargo-containing phagosomes for lysosomal degradation. LAP contributes greatly to host immunity against a multitude of bacterial pathogens. In the pursuit of survival, bacteria have developed elaborate strategies to disarm or circumvent the LAP process. In this review, we will outline the nature of the LAP mechanism and discuss recent insights into its interplay with bacterial pathogens. Show less
The zebrafish has earned its place among animal models to study tuberculosis and other infections caused by pathogenic mycobacteria. This model host is especially useful to study the role of... Show moreThe zebrafish has earned its place among animal models to study tuberculosis and other infections caused by pathogenic mycobacteria. This model host is especially useful to study the role of granulomas, the inflammatory lesions characteristic of mycobacterial disease. The optically transparent zebrafish larvae provide a window on the initial stages of granuloma development in the context of innate immunity. Application of fluorescent dyes and transgenic markers enabled real-time visualization of how innate immune mechanisms, such as autophagy and inflammasomes, are activated in infected macrophages and how propagating calcium signals drive communication between macrophages during granuloma formation. A combination of imaging, genetic, and chemical approaches has revealed that the interplay between macrophages and mycobacteria is the main driver of tissue dissemination and granuloma development, while neutrophils have a protective function in early granulomas. Different chemokine signaling axes, conserved between humans and zebrafish, have been shown to recruit macrophages permissive to mycobacterial growth, control their microbicidal capacity, drive their spreading and aggregation, and mediate granuloma vascularization. Finally, zebrafish larvae are now exploited to explore cell death processes, emerging as crucial factors in granuloma expansion. In this review, we discuss recent advances in the understanding of mycobacterial pathogenesis contributed by zebrafish models. Show less
Autopaghy is an intracellular degradation system which targets cytosolic components to lytic compartments for degradation and recycling of the building blocks of the cell. The process is highly... Show moreAutopaghy is an intracellular degradation system which targets cytosolic components to lytic compartments for degradation and recycling of the building blocks of the cell. The process is highly induced by carbon starvation conditions. In this thesis, different roles of autophagy in the filamentous fungus Aspergillus niger were studied, focusing on its role during carbon starvation, endoplasmic reticulum (ER) stress and unconventional protein secretion. Show less
Purpose Non-medullary thyroid cancer (NMTC) treatment is based on the ability of thyroid follicular cells to accumulate radioactive iodide (RAI). However, in a subset of NMTC patients tumor... Show morePurpose Non-medullary thyroid cancer (NMTC) treatment is based on the ability of thyroid follicular cells to accumulate radioactive iodide (RAI). However, in a subset of NMTC patients tumor dedifferentiation occurs, leading to RAI resistance. Digoxin has been demonstrated to restore iodide uptake capacity in vitro in poorly differentiated and anaplastic NMTC cells, termed redifferentiation. The aim of the present study was to investigate the in vivo effects of digoxin in TPO-Cre/LSL-Braf(V600E) mice and digoxin-treated NMTC patients. Methods Mice with thyroid cancer were subjected to 3D ultrasound for monitoring tumor growth and I-124 PET/CT for measurement of intratumoral iodide uptake. Post-mortem analyses on tumor tissues comprised gene expression profiling and measurement of intratumoral autophagy activity. Through PALGA (Dutch Pathology Registry), archived tumor material was obtained from 11 non-anaplastic NMTC patients who were using digoxin. Clinical characteristics and tumor material of these patients were compared to 11 matched control NMTC patients never treated with digoxin. Results We found that in mice, tumor growth was inhibited and I-124 accumulation was sustainably increased after short-course digoxin treatment. Post-mortem analyses revealed that digoxin treatment increased autophagy activity and enhanced expression of thyroid-specific genes in mouse tumors compared to vehicle-treated mice. Digoxin-treated NMTC patients exhibited significantly higher autophagy activity and a higher differentiation status as compared to matched control NMTC patients, and were associated with favourable clinical outcome. Conclusions These in vivo data support the hypothesis that digoxin may represent a repositioned adjunctive treatment modality that suppresses tumor growth and improves RAI sensitivity in patients with RAI-refractory NMTC. Show less
In the current thesis, we provide novel insights in antigen uptake, storage, processing, and sustained cross-presentation mechanisms in dendritic cells (DCs) in vitro and in vivo. We have studied... Show moreIn the current thesis, we provide novel insights in antigen uptake, storage, processing, and sustained cross-presentation mechanisms in dendritic cells (DCs) in vitro and in vivo. We have studied antigen handling functions by dendritic cells in three different antigen delivery routes: antibody targeting involving Fcγ receptors and complement factor C1q, C-type lectin receptor targeting, and toll-like receptor ligand targeting systems. Our data highlights that antigen storage in specialized compartments in DCs, despite the chosen uptake route, is beneficial for prolonged antigen cross-presentation by DCs and sustained T cell activation. Further in vivo studies in different antigen presenting cell (APC) subsets confirmed the presence of antigen storage compartments by isolating APC subsets after in vivo antigen uptake. Besides, we revealed a dominant role of C1q in antigen-antibody immune complex uptake and cross-presentation in vivo in contrast to the crucial role of Fcγ receptors in vitro. Furthermore, we demonstrated that autophagosomes have a negative impact on the storage of antigen in those specialized compartments and thereby affecting DC cross-presentation efficiency. With the current studies, we unraveled some mechanics of antigen processing in DCs which contribute to future vaccine designs against diseases such as cancer. Show less
Oncolytic reovirus has the natural preference to kill transformed cells while sparing normal cells. Its administration has been shown to be safe in clinical trials, but the anti-cancer efficacy... Show moreOncolytic reovirus has the natural preference to kill transformed cells while sparing normal cells. Its administration has been shown to be safe in clinical trials, but the anti-cancer efficacy remains to be improved. In this thesis, we discuss several aspects that are important for the design of a potent anti-cancer therapeutic strategy using reovirus. We explored which cellular factors and pathways are important for efficient reovirus replication. Furthermore, we genetically modified the reovirus genome to encode potentially therapeutic transgenes. We tested the potency of these recombinant reoviruses, and describe what we believe is the most promising strategy to move forward. Moreover, we discuss the stability issues that we encountered during the generation of recombinant reoviruses. Finally, we discuss the various challenges and opportunities in how to proceed. Show less
Atherosclerosis is the main underlying pathology of cardiovascular disease. Atherosclerosis is caused by an immune response which is directed against (modified) lipoproteins which accumulate in the... Show moreAtherosclerosis is the main underlying pathology of cardiovascular disease. Atherosclerosis is caused by an immune response which is directed against (modified) lipoproteins which accumulate in the vessel wall. Over time, this accumulation of lipids and immune cells induce morphological abnormalities in the vessel wall which cause the vessel lumen to narrow. This narrowing of the lumen (stenosis) causes ischemia in the downstream tissue. Prolonged ischemia causes myocardial ischemia and/or stroke. The research described in my thesis examines a well-recognized risk factor of atherosclerosis, being dyslipidemia, from an entirely new perspective. More specifically, it describes how dyslipidemia affects intrinsic metabolic processes in T cells, the conductors of the immune response characterizing atherosclerosis, and how this affects their function. My research has contributed to knowledge on the pathophysiology of atherosclerosis and might one day pave the way for the development of novel therapeutic approaches to treat cardiovascular disease. Show less
This review discusses the relevant metabolic pathways and their regulators which show potential for T cell metabolism-based immunotherapy in diseases hallmarked by both metabolic disease and autoim...Show moreThis review discusses the relevant metabolic pathways and their regulators which show potential for T cell metabolism-based immunotherapy in diseases hallmarked by both metabolic disease and autoimmunity. Multiple therapeutic approaches using existing pharmaceuticals are possible from a rationale in which T cell metabolism forms the hub in dampening the T cell component of autoimmunity in metabolic diseases. Future research into the effects of a metabolically aberrant micro-environment on T cell metabolism and its potential as a therapeutic target for immunomodulation could lead to novel treatment strategies for metabolic disease-associated autoimmunity. Show less
This review discusses the relevant metabolic pathways and their regulators which show potential for T cell metabolism-based immunotherapy in diseases hallmarked by both metabolic disease and... Show moreThis review discusses the relevant metabolic pathways and their regulators which show potential for T cell metabolism-based immunotherapy in diseases hallmarked by both metabolic disease and autoimmunity. Multiple therapeutic approaches using existing pharmaceuticals are possible from a rationale in which T cell metabolism forms the hub in dampening the T cell component of autoimmunity in metabolic diseases. Future research into the effects of a metabolically aberrant micro-environment on T cell metabolism and its potential as a therapeutic target for immunomodulation could lead to novel treatment strategies for metabolic disease-associated autoimmunity. Show less
This review discusses the relevant metabolic pathways and their regulators which show potential for T cell metabolism-based immunotherapy in diseases hallmarked by both metabolic disease and... Show moreThis review discusses the relevant metabolic pathways and their regulators which show potential for T cell metabolism-based immunotherapy in diseases hallmarked by both metabolic disease and autoimmunity. Multiple therapeutic approaches using existing pharmaceuticals are possible from a rationale in which T cell metabolism forms the hub in dampening the T cell component of autoimmunity in metabolic diseases. Future research into the effects of a metabolically aberrant micro-environment on T cell metabolism and its potential as a therapeutic target for immunomodulation could lead to novel treatment strategies for metabolic disease-associated autoimmunity. Show less
This review discusses the relevant metabolic pathways and their regulators which show potential for T cell metabolism-based immunotherapy in diseases hallmarked by both metabolic disease and... Show moreThis review discusses the relevant metabolic pathways and their regulators which show potential for T cell metabolism-based immunotherapy in diseases hallmarked by both metabolic disease and autoimmunity. Multiple therapeutic approaches using existing pharmaceuticals are possible from a rationale in which T cell metabolism forms the hub in dampening the T cell component of autoimmunity in metabolic diseases. Future research into the effects of a metabolically aberrant micro-environment on T cell metabolism and its potential as a therapeutic target for immunomodulation could lead to novel treatment strategies for metabolic disease-associated autoimmunity. Show less
This review discusses the relevant metabolic pathways and their regulators which show potential for T cell metabolism-based immunotherapy in diseases hallmarked by both metabolic disease and... Show moreThis review discusses the relevant metabolic pathways and their regulators which show potential for T cell metabolism-based immunotherapy in diseases hallmarked by both metabolic disease and autoimmunity. Multiple therapeutic approaches using existing pharmaceuticals are possible from a rationale in which T cell metabolism forms the hub in dampening the T cell component of autoimmunity in metabolic diseases. Future research into the effects of a metabolically aberrant micro-environment on T cell metabolism and its potential as a therapeutic target for immunomodulation could lead to novel treatment strategies for metabolic disease-associated autoimmunity. Show less
In this thesis the zebrafish tail fin infection model is presented, which enables the study of a complex immune response towards (myco)bacterial infection using a combination of light and electron... Show moreIn this thesis the zebrafish tail fin infection model is presented, which enables the study of a complex immune response towards (myco)bacterial infection using a combination of light and electron microscopy. The induction of autophagy upon a mycobacterial infection as an important innate immune response was visualized using correlative light and electron microscopy. Studying the role of leukocyte dynamics and function during the course of infection provided new insights into the complex host-pathogen interactions. Using a myd88 mutant zebrafish line it was shown that the recruitment of leukocytes towards the site of infection and subsequent phagocytosis of bacteria is dependent on MyD88-mediated signaling. With the advancement of medical translational studies using zebrafish disease models, the tail fin infection model may 104 5 provide new opportunities to develop novel therapies against pathogenic infections like tuberculosis. Show less
This thesis focuses on the recognition of pathogenic bacteria and the defense mechanisms that are activated during the innate immune response to infection. Detection of pathogens, such as bacteria,... Show moreThis thesis focuses on the recognition of pathogenic bacteria and the defense mechanisms that are activated during the innate immune response to infection. Detection of pathogens, such as bacteria, viruses, and parasites, depends on receptors that bind to evolutionary conserved structures on their surface. The most extensively studied class of immune receptors is the Toll-like receptor (TLR) family, which signals via adaptor molecules such as myeloid differentiation factor 88 (MyD88) to initiate gene expression and activate the appropriate response upon recognition of a pathogen. We have used the zebrafish as a model organism to study how MyD88 orchestrates the immune response against intracellular bacterial pathogens like Mycobacterium marinum, the causative agent of tuberculosis disease (TB) in fish. We found that several defense mechanisms against TB are highly dependent on MyD88, including autophagy, cytokine and chemokine production, and the generation of microbe killing radicals. These findings in the zebrafish model will hopefully aid in the development of new therapeutic strategies against multi-drug resistant tuberculosis infections. Show less
This study investigated carbon starvation in the filamentous fungus Aspergillus niger during submerged cultivation in bioreactor batch cultures. The work described in this thesis can be discussed... Show moreThis study investigated carbon starvation in the filamentous fungus Aspergillus niger during submerged cultivation in bioreactor batch cultures. The work described in this thesis can be discussed as follows: (I) Establishment of computational resources for omics data analysis and interpretation in chapters 2 and 3; (II) Cultivation of A. niger, data generation, analysis and interpretation in chapter 4; (III) Investigation of a candidate pathway with strong transcriptional induction during carbon starvation by molecular genetic approaches in chapter 5. Show less
De resultaten beschreven in dit proefschrift verschaffen nieuwe inzichten in verschillende aspecten van de behandeling van inflammatoire darmziekten. Uit grote studies is gebleken dat mutaties in... Show moreDe resultaten beschreven in dit proefschrift verschaffen nieuwe inzichten in verschillende aspecten van de behandeling van inflammatoire darmziekten. Uit grote studies is gebleken dat mutaties in genen die betrokken zijn bij het autofagie proces (autophagos: __zelf-eten__), geassocieerd zijn met de ziekte van Crohn. In dit proefschrift wordt beschreven hoe deze mutatie in dendritische cellen leidt tot hyperactivatie van het immuunsysteem. Dit mechanisme kan mogelijk een nieuw aangrijpingspunt verschaffen voor nieuwe therapie voor pati_nten die een mutatie hebben in dit gen. Anti-tumor-necrosis-factor-alpha (anti-TNF_) therapie neemt een steeds belangrijkere plaats in in de behandeling van IBD; het werkingsmechanisme is echter niet geheel duidelijk. In dit proefschrift wordt beschreven hoe regulatoire macrophagen met immuunsuppressieve en wondgenezende eigenschappen ge_nduceerd worden door anti-TNF_ therapie. Dit werkingsmechanisme kan nieuwe aangrijpingspunten verschaffen voor toekomstige therapie_n. Tevens wordt in dit proefschrift een experimentele behandeling van Crohn met mesenchymale stamcellen (MSCs) beschreven. Deze behandeling is haalbaar en veilig gebleken, hetgeen aanleiding geeft tot verder onderzoek naar de effectiviteit van MSCs in de behandeling van Crohn. Tot slot werd het v__rkomen van kwaadaardige lymfomen onderzocht in IBD pati_nten in Nederland. Deze lymfomen bleken niet vaker voor te komen in de totale groep IBD pati_nten vergeleken met gezonde mensen, wel werd een verhoogde incidentie gezien in bepaalde leeftijdsgroepen. Daarnaast bleek het ontstaan van een EBV positief lymfoom (epstein Barr virus) sterk geassocieerd te zijn met het gebruik van azathioprine, een ander immuunsuppressieve therapie. Samenvattend beschrijft dit proefschrift verschillende aspecten van IBD therapie, en worden nieuwe inzichten en aangrijpinspunten verschaft Show less