Chronic energy surplus causes obesity and promotes insulin resistance and type 2 diabetes (T2D). A major contributor to insulin resistance is chronic, low-grade inflammation in metabolic tissues,... Show moreChronic energy surplus causes obesity and promotes insulin resistance and type 2 diabetes (T2D). A major contributor to insulin resistance is chronic, low-grade inflammation in metabolic tissues, also coined metaflammation. In this context, white adipose tissue and liver-resident innate and adaptive immune cells produce proinflammatory cytokines that exacerbate inflammation and inhibit canonical insulin signaling. Among them, macrophages and dendritic cells were shown to play central roles in metaflammation, although the environmental and cellular changes dictating proinflammatory activation in the context of obesity are not fully understood. This thesis describes novel mechanisms by which macrophages and dendritic cells control metabolic homeostasis in obese mice. In addition, we show that immunomodulatory molecules derived from parasitic worm eggs promote an immune response in metabolic tissues that maintains insulin sensitivity. Finally, we describe the pleiotropic beneficial effects of a novel plant-derived nutritional supplement on metaflammation and metabolic homeostasis in obese mice. Altogether, this work may provide new leads for interventions aimed at improving immunological control of metabolic dysfunctions. Show less
Dendritic cells are the canonical professional antigen-presenting cell and are therefore crucial in the generation of efficient adaptive T cell responses. It is now well described that immune cells... Show moreDendritic cells are the canonical professional antigen-presenting cell and are therefore crucial in the generation of efficient adaptive T cell responses. It is now well described that immune cells – including dendritic cells – make drastic changes to their biology to transition between different life stages and to deal efficiently with the threat of infection. However, an unanswered question was if DCs with different T cell polarizing properties - that is to say they preferentially skew T cells towards a specific specialization (for example T helper 1 cells over T helper 2 cells) - rely on distinct metabolic characteristics for their T cell polarizing ability. This thesis tries to address that question by studying the metabolism of dendritic cells after in vitro stimulation with antigens or immunomodulatory compounds that are known to prime either T helper 1 cells, T helper 2 cells, T helper 17 cells or regulatory T cells. In addition, we interrogate the role of liver kinase B1 (LKB1) and mechanistic target of rapamycin complex 1 (mTORC1) in DC biology. Show less
Type 1 Diabetes (T1D) is an auto-immune disease in which beta cells in the pancreas are killed by auto-reactive T-cells. Auto-reactive T-cells are activated by dendritic cells that present antigens... Show moreType 1 Diabetes (T1D) is an auto-immune disease in which beta cells in the pancreas are killed by auto-reactive T-cells. Auto-reactive T-cells are activated by dendritic cells that present antigens. Immunotherapy could reverse T1D, however. A case report of a T1D patient showed that after intravenous immunoglobulin treatment her insulin needs dropped completely. Similarly, the majority of T1D patients were insulin independent after autologous hematopoietic stem cell transplantation. As these therapies only showed incidental success or are a drastic reset of the immune system, respectively, other milder therapies were studied as well. Autologous tolerogenic dendritic cell therapy, for instance, is a reproducible, stable therapy and does not differ between T1D patients and healthy subjects. In addition, the author described that when mesenchymal stromal cells were activated, they were able to suppress an antigen-specific immune response, thereby potentiating them as an antigen-specific therapy besides their natural immunosuppressive nature. Activated mesenchymal stromal cells could also improve the islet of Langerhans’ microenvironment, as they secreted immunosuppressive and angiogenic factors. To conclude, the future of T1D therapies lies in finding a balance between suppressing the immune system and antigen-specific therapies combined with therapies that increase the vitality of beta cells. 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
Parasitic helminths are important organisms to study because their infections have both adverse and beneficial effects on the human host. Helminth infections are considered a burden, as these... Show moreParasitic helminths are important organisms to study because their infections have both adverse and beneficial effects on the human host. Helminth infections are considered a burden, as these infections cause significant morbidity in a large proportion worldwide. However, helminth infections, by means of their ability to modify host immune responses can also provide protection against inflammatory diseases (inflammatory bowel disease, diabetes, and asthma). It is important to better understand the underlying mechanisms of these Yin (positive) and Yang (negative) consequences of helminth infections. The general objective of this thesis is to track helminths at different levels. On the one hand to improve the detection of helminth infections, essential for the studying helminths and the interaction with their human host. Moreover, a more sensitive diagnostics is instrumental for monitoring the distribution of helminth infections and to evaluate the helminth infections elimination program. On the other hand, to understand the mechanistic insights of the interplay between helminths and the host immune system results in priming of Th2 and regulatory T cell responses. This could contribute to the identification of targeted pathways to manipulate immune responses, as part of developing therapeutics to treat inflammatory disorders characterized by deregulated Th2 and/or Treg responses. Show less
In this thesis, we have addressed aspects of two main arms of the adaptive immune system; the B cell and antibody arm and the T cell arm. This led to a division in the presentation of the... Show more In this thesis, we have addressed aspects of two main arms of the adaptive immune system; the B cell and antibody arm and the T cell arm. This led to a division in the presentation of the results described in this thesis into two sections. In the first section, we present the results regarding the characterization of ACPA responses, B cells and ACPA secreting plasmablasts/-cells in RA as well as autoantibody responses and their regulation by an effective anti-rheumatic drug, abatacept, in the arthritis mouse model; Collagen Induced Arthritis (CIA). The second section is compiled of results obtained from studies examining the regulatory and other aspects of CD49b+CD4+ T cells on proinflammatory responses involved in the pathogenesis of arthritis. Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by inflammation of the synovial membrane of the joints, culminating in destruction of cartilage and deformity of the joints if remains untreated. Infiltration of inflammatory immune cells such as B cells and T cells into the inflamed joints is a characteristic feature of RA. These immune cells are in continuous interaction with each other and create a viscous circle that sustains persistent synovitis and damage to articular cartilage. Show less
Synthetic long peptides (SLPs) derived from cancer antigens hold great promise as well-defined antigens for immunotherapy of cancer. However, the formulation of SLPs for in vivo administration... Show moreSynthetic long peptides (SLPs) derived from cancer antigens hold great promise as well-defined antigens for immunotherapy of cancer. However, the formulation of SLPs for in vivo administration still needs to be improved. So far, SLPs have been formulated in Montanide-based water-in-oil emulsions in (pre-)clinical trials. However, the use of Montanide as an adjuvant has some important limitations, such as: non-biodegradability; significant local side effects; poor control of release rate; lack of specific dentritic cell (DC)-activating capacity; and the presence of organic solvents (needed to dissolve the peptides prior to mixing with the adjuvant) in the final formulation. Therefore, alternative formulations containing an effective delivery system for peptide-based cancer vaccines are highly needed. Among the numerous vaccine delivery systems, poly(lactic-co-glycolic acid) (PLGA) biodegradable particulate delivery systems are particularly interesting because they are biocompatible; can protect soluble antigens from degradation and rapid clearance once administered; allow for co-encapsulation of (multiple) antigens and adjuvants; and mimic the size and structure of a pathogen, being more efficiently taken up by DCs than soluble antigen. This thesis describes fundamental studies on the design and applicability in a preclinical setting of PLGA-based particulate formulations for the delivery of SLP-based cancer vaccines. Show less
Nearly one quarter of the world__s population is infected with helminth parasites. A common feature of helminth infections is the manifestation of a type 2 immune response, characterized by T... Show moreNearly one quarter of the world__s population is infected with helminth parasites. A common feature of helminth infections is the manifestation of a type 2 immune response, characterized by T helper 2 (Th2) cells. In addition to their involvement in anti-helminth immunity, recent studies have shown that components of the type 2 immune responses can have additional functions. For example, recent evidence indicates that multiple facets of the type 2 immune response can regulate tissue-specific metabolic processes and whole-body nutrient homeostasis, and protect against insulin resistance. In this work we use omega-1, a glycosylated RNase excreted from Schistsoma mansoni eggs with strong Th2-inducing capacities, to study the requirements that equip DCs for Th2 skewing. In addition, we analyse the effect of chronic S. mansoni infection and administration of S. mansoni-derived egg antigens on metabolic homeostasis in diet-induced obese mice. Elucidating how helminths generate Th2 responses and contribute to metabolic homeostasis will not only shed light on the mechanisms that promote control of parasite infection, but may provide valuable leads for the development of pharmaceutical agents for the treatment of metabolic disorders. Show less
Atherosclerosis is one of the primary causes of cardiovascular disease; the number one cause of death in the western society. Atherosclerotic plaque formation is a dynamic multi-cellular process... Show moreAtherosclerosis is one of the primary causes of cardiovascular disease; the number one cause of death in the western society. Atherosclerotic plaque formation is a dynamic multi-cellular process where regulation of different genes essentially determines the activity of the different cell types involved. Gene expression is regulated, amongst others, by epigenetic processes. Epigenetic mechanisms change the accessibility of the DNA sequence and is thought to form a link between environmental factors and gene expression. Epigenetics may therefor play an important role in atherosclerosis pathology. The research described in this thesis evaluated the role of epigenetic regulation on various aspects of atherosclerosis pathology. It was found that the epigenetic H3K27Me3-mark was reduced in later stages of the disease. Monocytes differentiating into dendritic cells and macrophages (an important process in atherosclerosis pathology) showed higher transcription of the epigenetic regulatory gene KMT1c. Specifically blocking this gene resulted in reduction of DC-SIGN (a dendritic cell specific molecule) expression. By specifically blocking other epigenetic proteins, CCR5 (a molecule important to monocyte migration) was re-expressed on cells which did not express CCR5. This shows that epigenetic regulation is an important process in atherosclerosis pathology and might prove to be novel pharmacological target for treatment of atherosclerosis. Show less
In this thesis we describe our studies aimed at optimizing the efficacy of synthetic long peptide (SLP) vaccines via the encapsulation in Poly-(lactic-co-glycolic acid) (PLGA)particles.... Show moreIn this thesis we describe our studies aimed at optimizing the efficacy of synthetic long peptide (SLP) vaccines via the encapsulation in Poly-(lactic-co-glycolic acid) (PLGA)particles. Immunotherapy based on SLP-vaccines has resulted in strong tumor specific immune response and importantly, improved clinical benefit in patients with pre-malignant lesions. One important drawback associated with SLP-vaccines is their current form of administration in Montanide, a clinical grade water-in-oil emulsion. The aim of this Ph.D project was to device an alternative method of delivery which overcomes the drawbacks associated with the use of Montanide. For this purpose we explored the use of PLGA (nano)particles (NP) as a delivery vehicle for SLP. Several important aspects for vaccination were assessed in this thesis; from the pharmaceutical formulation to the immunological characterization of different PLGA-SLP preparations. Together, the data presented in this thesis show that PLGA-NP mediated delivery of SLP is a very efficient method to target, load and mature Dendritic cells (DCs) as immune stimulatory compounds can be co-encapsulated with the vaccine Ag Show less
Dendritic cells (DCs) are antigen-presenting cells (APCs) which play a key role in the regulation of immune responses. DCs are often referred to as __professional__ APCs, since their primary... Show moreDendritic cells (DCs) are antigen-presenting cells (APCs) which play a key role in the regulation of immune responses. DCs are often referred to as __professional__ APCs, since their primary function is to present antigens from pathogens or malignant cells. Consequently, there is a great deal of interest in how DCs might be exploited as a form of immunotherapy e.g. to induce immunity to cancers. However, DCs are also thought to play an important role in directing regulatory immune responses to innocuous antigens, which are targeted in autoimmune disease or during transplantation. Soluble factors secreted by DCs are crucial mediators in determining this balance between the immunogenic and regulatory arms of the immune system. One such group of factors is cytokines and one family which is gaining increasing attention is the IL-12 family. It is composed of four members; two are immunogenic and their expression has been very well characterised in DCs. The other two are regulatory, but relatively little is known about their regulation and expression in DC populations. In this thesis we aim to give a comprehensive overview of the expression and regulation of IL-12 family members in human DCs, with a particularly emphasis on IL-12, IL-27 and IL-35. Show less
Parasitic worms of the genus, Schistosoma, infect millions of people mainly in the tropics and can cause serious morbidity. Human schistosoma infection is often associated with strong T helper 2... Show moreParasitic worms of the genus, Schistosoma, infect millions of people mainly in the tropics and can cause serious morbidity. Human schistosoma infection is often associated with strong T helper 2 polarized immune responses as well as immunehyporesponsiveness. Dendritic cells play a central role in sensing of pathogens and generation of appropriate immune responses against these pathogens. This thesis describes that human schistosoma infection suppresses phenotype and T cell polarizing capacity of dendritic cells present in blood of these subjects. Furthermore, in vitro studies identified molecular markers in dendritic cells that can be used to predict whether these cells will induce T helper 1 or 2 responses following exposure to Th1-polarizing bacterial extracts or Th2- skewing lipids derived from schistosoma worms. Finally, the identification of the major Th2-polarizing component secreted by schistosoma eggs and the molecular mechanisms through which this factor instructs dendritic cells to drive this response is described. Taken together, these studies provide new insights in the molecular interplay between dendritic cells and schistosomes and as such in the cellular and molecular mechanisms behind shaping of T helper 2 immune responses and/or immunehyporesponsiveness observed during these parasitic worm infections. Show less
Atherosclerosis is a chronic inflammatory disease of the vasculature in which both a disturbed lipid metabolism and inflammatory immune responses against several self-antigens are involved. In this... Show moreAtherosclerosis is a chronic inflammatory disease of the vasculature in which both a disturbed lipid metabolism and inflammatory immune responses against several self-antigens are involved. In this thesis we have explored the effectiveness of DC-immunotherapy in atherosclerosis. We have used different strategies to target the immune component in different stages of atherosclerosis. First we used DCs as a vaccination strategy to induce a protective antibody response trough the injection of oxLDL-pulsed DCs or to target NKT cells by the injection of OCH-pulsed DCs. Next we assessed the potential of DC-immunotherapy in a model of established atherosclerosis. We also evaluated the effects of a disturbed TGF-_ signaling in DCs and the subsequent effects on atherosclerosis by using ApoE-/- which have a dysfunctional TGF-__ Receptor II under the CD11c promoter. Next, we were interested in the effect of foam-cell formation on the antigen-presenting capacity of DCs and macrophages. Therefore we studied the effect of oxLDL-loading on antigen uptake and antigen presentation by DCs and macrophages. Finally, by depleting or inducing Tregs we investigated the potential role of regulatory T cells in a mouse model for aneurysm formation. Show less
Following allograft transplantation, the immune system is triggered to induce an immunogenic response against the non-self organ. To prevent the induction of this immunogenic response, recipients... Show moreFollowing allograft transplantation, the immune system is triggered to induce an immunogenic response against the non-self organ. To prevent the induction of this immunogenic response, recipients are treated with immunosuppressive medication. The majority of these medications target T cells, which play a key role in the rejection process, and thereby prevent acute rejection in most of the recipients. Non-specific targeting of these T cells not only prevents acute rejection, it also prevents responses against pathogens or tumor growth. In addition, long-term use of immunosuppressive agents may cause organ failure due to toxic effects on the organ [1]. Therefore, the ultimate goal is to develop a therapy, which targets alloreactive T cells, allowing a normal response against pathogens and tumors, in the absence of chronic use of immunosuppressive agents. Various strategies have been employed to induce such a donor-specific tolerance, amongst which treatment with immature DC [2]. These immature DC have, in contrast to mature DC, the capacity to induce tolerogenic responses and are therefore an attractive candidate for cellular therapy. The studies presented in this thesis demonstrate that in fully mismatched kidney transplantation models, administration of modulated donor-derived DC to recipient__s results in regulation of recipient__s immune response. Both the donor-specific hyporesponsiveness of recipient T cells and the reduced influx of CD8+ T cells into the graft of LPS-DexDC treated recipients indicate a positive effect of this treatment. However, optimization of this treatment is necessary, since no prolonged allograft survival was induced. Several mechanisms, which are not regulated by LPS-DexDC, may be responsible for the observed rejection, amongst which the preformed alloantibodies, increased levels of C3 in the graft and the increased influx of NK cells. Additional studies are required to explore the modulating effects of antibodies which block co-stimulation and/or short courses of immunosuppressive drugs as a co-treatment in these settings. Show less
IgA nephropathy (IgAN) is the most common form of glomerulonephritis worldwide. The hallmark of the disease is depositions of polymeric IgA1 in the mesangium of the glomeuli. These depositions will... Show moreIgA nephropathy (IgAN) is the most common form of glomerulonephritis worldwide. The hallmark of the disease is depositions of polymeric IgA1 in the mesangium of the glomeuli. These depositions will lead to inflammation in the kidneys and eventually to deterioration of renal function. The pathogenesis of IgAN is not clear, but it is generally accepted that disturbances in the immune system of IgAN patients are responsible for this disease. In the current thesis we have investigated the immune response of IgAN patients in comparison with control persons. We have shown that IgAN patients have a hampered primary IgA immune response upon mucosal vaccination with a neoantigen, whereas a systemic vaccination with a neoantigen resulted in a similar immune response in both groups. We hypothesized that dendritic cells (DC), as professional antigen presenting cells could have an impaired function , or that less DC are present in the nasal mucosa. We were able to show that the number of DC present in the nasal mucosa of IgAN patients was not reduced as compared with controls. Using an in vitro model we studied the function of DC in the primary immune response and showed that DC of IgAN patients induced less IgA production in na_ve B cells than DC of control persons. Furthermore we studied the size distribution of the antigen specific IgA molecules in IgAN patients. In summary we showed that patients with IgAN have an impaired IgA production upon mucosal vaccination with a neoantigen and that at least part of this IgA hypo response is due to an impaired capacity of DC to induce IgA production, whereas the number of mucosal DC in IgAN patients is not reduced. Show less
The major targets in Systemic lupus erythematosus (SLE, a systemic autoimmune disease) are nuclear components (DNA, histones, ribonucleoproteins), which are mainly derived from dying cells ... Show moreThe major targets in Systemic lupus erythematosus (SLE, a systemic autoimmune disease) are nuclear components (DNA, histones, ribonucleoproteins), which are mainly derived from dying cells (apoptotic and necrotic cells). Defective clearance of dying cells by phagocytes may lead to the breakdown of peripheral tolerance and initiation of autoimmune SLE. I have investigated the role of the innate immune system in the processing of dying cells and its immunological consequences. I found that a subset of macrophages driven by M-CSF have intrinsic anti-inflammatory properties and are potent phagocytes that have the unique capacity to preferentially bind and ingest early apoptotic cells in a silent manner. I also identified that human peritoneal macrophages freshly isolated from patients on peritoneal dialysis resemble functionally the in vitro-generated M-CSF-driven macrophages. I further showed that the anti-inflammatory and pro-inflammatory macrophages co-exist but can re-differentiate towards opposing phenotype depending on the local cytokine environment. Next to the phagocyte system, I investigated the role of components of the innate immune system in the processing of dying cells. I found that one of the complement regulators called properdin, binds predominantly to late apoptotic and necrotic cells independently of C3b, resulting alternative pathway complement activation. Show less
