In this thesis the aim was to study immune cell interactions at the maternal-fetal interface to understand the role for immune cells during healthy pregnancy development an pregnancy complications.... Show moreIn this thesis the aim was to study immune cell interactions at the maternal-fetal interface to understand the role for immune cells during healthy pregnancy development an pregnancy complications. Specifically in cases of recurrent pregnancy loss and chronic histiocytic intervillositis. Show less
Work described in this thesis was aimed to further understand the role of macrophages in acute cellular rejection of transplanted kidneys, and perform a pre-clinical assessment of the localization... Show moreWork described in this thesis was aimed to further understand the role of macrophages in acute cellular rejection of transplanted kidneys, and perform a pre-clinical assessment of the localization and efficacy of liposomal prednisolone. First, we explored the role of anti-inflammatory macrophages in the onset of acute cellular rejection using human biopsy samples. Here, we discovered that the presence of high levels of CD163+ macrophages was associated with a lower risk of rejection, in a population of patients with a high rate of delayed graft function. Subsequently, we identified the ability of liposomal prednisolone to target macrophages in the kidney using in vitro models of human macrophages, and their ability to accumulate in inflamed kidney tissue in vivo rat models of ischemia reperfusion injury in the kidney. Finally, we evaluated the treatment efficacy of liposomal prednisolone in a mouse model of cellular rejection following kidney transplantation. Here, we were able to show an improved efficacy of liposomal prednisolone in treating rejection, when compared to treatment with free prednisolone. Show less
Elsas, M.J. van; Labrie, C.; Etzerodt, A.; Charoentong, P.; Thans, J.J.C.V.; Hall, T. van; Burg, S.H. van der 2023
BackgroundPrimary and secondary resistance is a major hurdle in cancer immunotherapy. Therefore, a better understanding of the underlying mechanisms involved in immunotherapy resistance is of... Show moreBackgroundPrimary and secondary resistance is a major hurdle in cancer immunotherapy. Therefore, a better understanding of the underlying mechanisms involved in immunotherapy resistance is of pivotal importance to improve therapy outcome.MethodHere, two mouse models with resistance against therapeutic vaccine-induced tumor regression were studied. Exploration of the tumor microenvironment by high dimensional flow cytometry in combination with therapeutic in vivo settings allowed for the identification of immunological factors driving immunotherapy resistance.ResultsComparison of the tumor immune infiltrate during early and late regression revealed a change from tumor-rejecting toward tumor-promoting macrophages. In concert, a rapid exhaustion of tumor-infiltrating T cells was observed. Perturbation studies identified a small but discernible CD163(hi) macrophage population, with high expression of several tumor-promoting macrophage markers and a functional anti-inflammatory transcriptome profile, but not other macrophages, to be responsible. In-depth analyses revealed that they localize at the tumor invasive margins and are more resistant to Csf1r inhibition when compared with other macrophages. In vivo studies validated the activity of heme oxygenase-1 as an underlying mechanism of immunotherapy resistance. The transcriptomic profile of CD163(hi) macrophages is highly similar to a human monocyte/macrophage population, indicating that they represent a target to improve immunotherapy efficacy.ConclusionsIn this study, a small population of CD163(hi) tissue-resident macrophages is identified to be responsible for primary and secondary resistance against T-cell-based immunotherapies. While these CD163(hi) M2 macrophages are resistant to Csf1r-targeted therapies, in-depth characterization and identification of the underlying mechanisms driving immunotherapy resistance allows the specific targeting of this subset of macrophages, thereby creating new opportunities for therapeutic intervention with the aim to overcome immunotherapy resistance. Show less
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
Transplantation is the golden standard for the treatment of end-stage renal disease. During this process, the transplanted organ is often damaged. In this thesis, we investigated whether activation... Show moreTransplantation is the golden standard for the treatment of end-stage renal disease. During this process, the transplanted organ is often damaged. In this thesis, we investigated whether activation of the complement system, part of our innate immune system, plays a local role. We reviewed that properdin, the only known positive regulator of the complement system, was detected in serum, plasma and urine from patients with various complement-mediated renal diseases. In protocol biopsies obtained 10 days after transplantation, properdin was found deposited in addition to complement activation markers. Next, we showed that dendritic cells secrete properdin and a decrease in properdin levels during dendritic cell- T-cell interaction resulted in reduced T-cell proliferation and activation. We also showed that properdin is able to bind to surfaces of both viable and dead cells, contributing to complement activation. Macrophages can also produce properdin and negative regulators factor H and its splice variant FHL-1. Increasing knowledge on complement factor production by other cells than hepatocytes, including immune cells, hints towards a local role of the complement system in various processes. These findings contribute to a better understanding of the local role of the complement system and are important for the applications of (new) complement-inhibiting drugs. Show less
Extracellular vesicles (EVs) are promising drug carriers of photosensitizers for photodynamic therapy (PDT) in cancer treatment, due to their ability to circulate in blood and enter cells... Show moreExtracellular vesicles (EVs) are promising drug carriers of photosensitizers for photodynamic therapy (PDT) in cancer treatment, due to their ability to circulate in blood and enter cells efficiently. The therapeutic potential of EVs has been suggested to depend on the type and physiological state of their cell of origin. However, the effects of deriving EVs from various cells in different physiological states on their antitumor capacity are rarely evaluated. In the present study, we compared the antitumor efficacy of EV-mediated PDT by incorporating the photosensitizer Zinc Phthalocyanine (ZnPc) into EVs from multiple cells sources. ZnPc was incorporated by a direct incubation strategy into EVs derived from immune cells (M1-like macrophages and M2-like macrophages), cancer cells (B16F10 melanoma cancer cells) and external sources (milk). Our data show that all EVs are suitable carriers for ZnPc and enable efficient PDT in vitro in co-culture models and in vivo. We observed that EV-mediated PDT initiates immunogenic cell death through the release and exposure of damage associated molecular patterns (DAMPs) on cancer cells, which subsequently induced dendritic cell (DC) maturation. Importantly, of all ZnPc-EVs tested, in absence of light only M1-ZnPc displayed toxicity to MC38, but not to DC, in monoculture and in co-culture, indicating specificity for cancer over immune cells. In MC38 tumor-bearing mice, only M1-ZnPc induced a tumor growth delay compared to control in absence of light. Interestingly, M1- but not M2-mediated PDT, induced complete responses against MC38 tumors in murine models (100% versus 38% of cases, respectively), with survival of all animals up to at least 60 days post inoculation. Finally, we show that all cured animals are protected from a rechallenge with MC38 cells, suggesting the induction of immunological memory after EV-mediated PDT. Together, our data show the importance of the cell type from which the EVs are obtained and highlight the impact of the immunological state of these cells on the antitumor efficacy of EV-mediated PDT. Show less
The glioma microenvironment harbors a variety of immune cells including innate immune cells such as monocytes, macrophages and microglia. Microglia are the major innate immune cells present in the... Show moreThe glioma microenvironment harbors a variety of immune cells including innate immune cells such as monocytes, macrophages and microglia. Microglia are the major innate immune cells present in the glioma microenvironment. Communication between glioma and these immune cells is crucial to maintain a tumor-promoting environment. In this thesis the role of a specific type of communication is described. In detail, the consequence of extracellular communication from glioma to the innate immune cells is studied, this includes the transferring of messages (including miRNAs) through extracellular vesicles. In addition, the changes that these cells undergo in the presence of a tumor is documented. 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
Background: Evidence indicates that inflammatory processes are involved in radicular pain as well as in resorption of herniated disc tissue. Furthermore there are indications that the presence of... Show moreBackground: Evidence indicates that inflammatory processes are involved in radicular pain as well as in resorption of herniated disc tissue. Furthermore there are indications that the presence of vertebral end plate pathology (Modic changes; MC) is associated with a negative effect on inflammation. It is hypothesized that in patients with MC, the (possibly bacterial induced) inflammation will be accompanied by pro inflammatory cytokines that worsen the outcome, and that in patients without MC, the inflammation is accompanied by cytokines that induce a resorption process to accelerate recovery.Methods: This prospective cohort study will include 160 lumbar and 160 cervical patients (total of 320), which are scheduled for surgery for either a lumbar or cervical herniated disc with ages between 18 and 75. The main and interaction effects of local bacterial infection (culture), inflammatory cells in disc material (immunohistology), MC (MRI), and blood biomarkers indicating inflammation or infection (blood sample evaluation) will be evaluated. Clinical parameters to be evaluated are leg pain on the 11 point NRS pain scale, Oswestry (lumbar spine) or Neck (cervical spine) Disability Index, Global Perceived Recovery, Womac Questionnaire, and medication status, at baseline, and after 6, 16, 26 and 52 weeks.Discussion: Gaining insight in the aetiology of pain and discomfort in radiculopathy caused by a herniated disc could lead to more effective management of patients. If the type of inflammatory cells shows to be of major influence on the rate of recovery, new immunomodulating treatment strategies can be developed to decrease the duration and intensity of symptoms. Moreover, identifying a beneficial inflammatory response in the disc through a biomarker in blood could lead to early identification of patients whose herniations will resorb spontaneously versus those that require surgery. Show less
Korporaal, S.J.A.; Sluis, R.J. van der; Eck, M. van; Hoekstra, M. 2021
The genetic disorder Down syndrome is associated with a decreased susceptibility for atherosclerotic cardiovascular disease. Hematological and immune abnormalities occur frequently in Down syndrome... Show moreThe genetic disorder Down syndrome is associated with a decreased susceptibility for atherosclerotic cardiovascular disease. Hematological and immune abnormalities occur frequently in Down syndrome patients. We evaluated, in a preclinical setting, the impact of a Down syndrome-like hematological/immune phenotype on atherosclerosis susceptibility. Hereto, hypercholesterolemic low-density lipoprotein receptor knockout mice were transplanted with bone marrow from either a trisomic Ts65Dn mouse or euploid wild-type control and subsequently fed a Western-type diet to induce the development of atherosclerotic lesions. T and B cell concentrations were markedly reduced in blood of Ts65Dn bone marrow recipients (p < 0.001). Expression levels of the pro-atherogenic scavenger receptor CD36 were respectively 37% and 59% lower (p < 0.001) in trisomic monocytes and macrophages. However, these combined effects did not translate into an altered atherosclerosis susceptibility. Notably, blood platelet numbers were elevated in Ts65Dn bone marrow recipients (+57%; p < 0.001), which was paralleled by higher platelet GPVI protein expression (+35%; p < 0.001) and an enhanced collagen-induced platelet activation (p < 0.001). In conclusion, we have shown that providing mice with a Down syndrome-like hematological profile does not change the susceptibility to atherosclerosis. Furthermore, our studies have uncovered a novel effect of the trisomy on platelet functionality that may be relevant in human clinical settings. Show less
For many years, cancer has been described as the accumulation of germinal and somatic mutations of the genome, impairing the function of tumor suppressor genes and stimulating oncogenes. Nowadays,... Show moreFor many years, cancer has been described as the accumulation of germinal and somatic mutations of the genome, impairing the function of tumor suppressor genes and stimulating oncogenes. Nowadays, it is commonly accepted that the tumor is not only a mass of malignant cells, rather than the result of a delicate network of interactions between tumor and stromal cells. Indeed, bidirectional communications between cancer cells and the surrounding microenvironment can strongly influence tumor development and progression. Stromal cells might support tumorigenesis, either via direct cell-cell contact mechanisms with tumor cells, or by releasing specific factors, including cytokines and growth factors in the surrounding extracellular matrix (ECM), with remodeling of the tumor microenvironment (TME) as a result.The aim of this thesis is to elucidate the delicate network of interactions between different TME components and tumor cells in prostate cancer (PCa) and oropharyngeal squamous cell carcinoma (OPSCC). Show less
In recent years there have been major advances in our understanding of the role of free fatty acids (FAs) and their metabolism in shaping the functional properties of macrophages and DCs. This... Show moreIn recent years there have been major advances in our understanding of the role of free fatty acids (FAs) and their metabolism in shaping the functional properties of macrophages and DCs. This review presents the most recent insights into how cell intrinsic FA metabolism controls DC and macrophage function, as well as the current evidence of the importance of various exogenous FAs (such as polyunsaturated FAs and their oxidation products-prostaglandins, leukotrienes, and proresolving lipid mediators) in affecting DC and macrophage biology, by modulating their metabolic properties. Finally, we explore whether targeted modulation of FA metabolism of myeloid cells to steer their function could hold promise in therapeutic settings. Show less
Heparanase is the predominant enzyme that cleaves heparan sulfate, the main polysaccharide in the extracellular matrix. While the role of heparanase in sustaining the pathology of autoimmune... Show moreHeparanase is the predominant enzyme that cleaves heparan sulfate, the main polysaccharide in the extracellular matrix. While the role of heparanase in sustaining the pathology of autoimmune diabetes is well documented, its association with metabolic syndrome/type 2 diabetes attracted less attention. Our research was undertaken to elucidate the significance of heparanase in impaired glucose metabolism in metabolic syndrome and early type 2 diabetes. Here, we report that heparanase exerts opposite effects in insulin-producing (i.e., islets) vs. insulin-target (i.e., skeletal muscle) compartments, sustaining or hampering proper regulation of glucose homeostasis depending on the site of action. We observed that the enzyme promotes macrophage infiltration into islets in a murine model of metabolic syndrome, and fosters beta-cell-damaging properties of macrophages activated in vitro by components of diabetogenic/obese milieu (i.e., fatty acids). On the other hand, in skeletal muscle (prototypic insulin-target tissue), heparanase is essential to ensure insulin sensitivity. Thus, despite a deleterious effect of heparanase on macrophage infiltration in islets, the enzyme appears to have beneficial role in glucose homeostasis in metabolic syndrome. The dichotomic action of the enzyme in the maintenance of glycemic control should be taken into account when considering heparanase-targeting strategies for the treatment of diabetes. Show less
Abnormal vascular physiology and precipitating inflammatory pathways underlie many different diseases, including hemorrhage, stroke, vascular dementia and even cancer. Pluripotent stem cells (PSCs)... Show moreAbnormal vascular physiology and precipitating inflammatory pathways underlie many different diseases, including hemorrhage, stroke, vascular dementia and even cancer. Pluripotent stem cells (PSCs) can now be derived by reprogramming from any individual so that it is possible in principle to derive all somatic cells of the human body that would normally be difficult to access. In this thesis, I studied the derivation of myeloid cells from human induced pluripotent stem cells (hiPSCs) to model the inflammatory component of vascular disease and characterized the development path of hiPSC-derived endothelial cells (hiPSC-ECs) which form the vascular walls. Functional defects in either of these cell types can cause or exacerbate vascular disease. I then used these cell types to gain insight into the mechanisms underlying two genetic diseases: Hereditary Hemorrhagic Telangiectasia (HHT) which is caused by mutations in a gene called Endoglin expressed on cells of the vascular wall and inflammatory macrophages, and a vascular tumor called Pseudomyogenic hemangioendothelioma (PHE) in which endothelial cells are thought to be the tumor cell of origin. I developed new differentiation protocols to generate inflammatory cells from hiPSC, characterized these cells functionally and used Next-Generation Sequencing and bioinformatic analysis to gain insight into the molecular pathways controlling development of one particular type of endothelial cells from hiPSC and the underlying tumorigenic mechanisms of PHE. Show less
Riet, S. van; Schadewijk, A. van; Vos, S. de; Vandeghinste, N.; Rottier, R.J.; Stolk, J.; ... ; Khedoe, P. 2020
Airway epithelial cells and macrophages participate in inflammatory responses to external noxious stimuli, which can cause epithelial injury. Upon injury, epithelial cells and macrophages act in... Show moreAirway epithelial cells and macrophages participate in inflammatory responses to external noxious stimuli, which can cause epithelial injury. Upon injury, epithelial cells and macrophages act in concert to ensure rapid restoration of epithelial integrity. The nature of the interactions between these cell types during epithelial repair is incompletely understood. We used an in vitro human coculture model of primary bronchial epithelial cells cultured at the air-liquid interface (ALI-PBEC) and polarized primary monocyte-derived macrophages. Using this coculture, we studied the contribution of macrophages to epithelial innate immunity, wound healing capacity, and epithelial exposure to whole cigarette smoke (WCS). Coculture of ALI-PBEC with lipopolysaccharide (LPS)-activated M(GM-CSF) macrophages increased the expression ofDEFB4A,CXCL8, andIL6at 24 h in the ALI-PBEC, whereas LPS-activated M(M-CSF) macrophages only increased epithelialIL6expression. Furthermore, wound repair was accelerated by coculture with both activated M(GM-CSF) and M(M-CSF) macrophages, also following WCS exposure. Coculture of ALI-PBEC and M(GM-CSF) macrophages resulted in increasedCAMPexpression in M(GM-CSF) macrophages, which was absent in M(M-CSF) macrophages.CAMPencodes LL-37, an antimicrobial peptide with immune-modulating and repair-enhancing activities. In conclusion, dynamic crosstalk between ALI-PBEC and macrophages enhances epithelial innate immunity and wound repair, even upon concomitant cigarette smoke exposure. Show less
Hegedus, J.H. von; Kahnt, A.S.; Ebert, R.; Heijink, M.; Toes, R.E.M.; Giera, M.; Ioan-Facsinay, A. 2020
Inflammation is a tightly regulated process. During the past decade it has become clear that the resolution of inflammation is an active process and its dysregulation can contribute to chronic... Show moreInflammation is a tightly regulated process. During the past decade it has become clear that the resolution of inflammation is an active process and its dysregulation can contribute to chronic inflammation. Several cells and soluble mediators, including lipid mediators, regulate the course of inflammation and its resolution. It is, however, unclear which signals and cells are involved in initiating the resolution process. Macrophages are tissue resident cells and key players in regulating tissue inflammation through secretion of soluble mediators, including lipids. We hypothesize that persistent inflammatory stimuli can initiate resolution pathways in macrophages.In this study, we detected 21 lipids in LPS-stimulated human monocyte-derived macrophages by liquid chromatography coupled to tandem mass spectrometry. Cyclooxygenase-derived Prostaglandins were observed in the first six hours of stimulation. Interestingly, a switch towards 15-lipoxygenase products, such as the proresolving lipid precursors 15-HEPE and 17-HDHA was observed after 24 h. The RNA and protein expression of cyclooxygenase and 15-lipoxygenase were in line with this trend. Treatment with 17-HDHA increased IL-10 production of monocyte-derived macrophages and decreased LTB4 production by neutrophils, indicating the anti-inflammatory property of this lipid.These data reveal that monocyte-derived macrophages contribute to the resolution of inflammation in time by the production of pro-resolving lipids after an initial inflammatory stimulus. Show less
Verwilligen, R.A.F.; Bussmann, J.; Eck, M. van 2020
Background and aims: Neuroimmune guidance cues have been shown to play a role in atherosclerosis, but their exact role in human pathophysiology is largely unknown. In the current study, we... Show moreBackground and aims: Neuroimmune guidance cues have been shown to play a role in atherosclerosis, but their exact role in human pathophysiology is largely unknown. In the current study, we investigated the role of a c.1769G > T variant in Netrin-1 in (premature) atherosclerosis.Methods: To determine the effect of the genetic variation, purified Netrin-1, either wild type (wtNetrin-1) or the patient observed variation (mutNetrin-1), was used for migration, adhesion, endothelial barrier function and bindings assays. Expression of adhesion molecules and transcription proteins was analyzed by RT-PCR, Western blot or ELISA. To further delineate how mutNetrin-1 mediates its effect on cell migration, lenti-viral knockdown of UNC5B or DCC was used.Results: Bindings assays revealed a decreased binding capacity of mutNetrin-1 to the receptors UNC5B, DCC and beta 3-integrin and an increased binding capacity to neogenin, heparin and heparan sulfate compared to wtNetrin-1. Exposure of endothelial cells to mutNetrin-1 resulted in enhanced monocyte adhesion and expression of IL-6, CCL2 and ICAM-1 compared to wtNetrin-1. In addition, mutNetrin-1 lacks the inhibitory effect on the NF-kappa B pathway that is observed for wtNetrin-1. Moreover, the presence of mutNetrin-1 diminished migration of macrophages and smooth muscle cells. Importantly, UNC5B or DCC specific knockdown showed that mutNetrin-1 is unable to act through DCC resulting in enhanced inhibition of migration.Conclusions: Our data demonstrates that mutNetrin-1 fails to exert anti-inflammatory effects on endothelial cells and more strongly blocks macrophage migration compared to wtNetrin-1, suggesting that the carriers of this genetic molecular variant may well be at risk for premature atherosclerosis. Show less
