Type I interferons (IFN-I) are pleiotropic cytokines that were originally identified for their antiviral properties and are now recognized for playing key roles in the defense against a range of... Show moreType I interferons (IFN-I) are pleiotropic cytokines that were originally identified for their antiviral properties and are now recognized for playing key roles in the defense against a range of other microorganisms as well as cancer. Their production should be well-controlled to be of benefit to the host, as excessive or chronic IFN-I expression leads to adverse effects such as immunosuppression or the induction of severe immunopathology.The studies presented in this thesis are aimed at uncovering mechanisms that regulate the production of IFN-I. The obtained knowledge on the involved molecular processes, may aid the development of targeted therapies that enhance or intercept IFN-I responses for maximum host protection while minimizing damage. Show less
The aim of this thesis was to better understand the underlying biology of tumor-immune interactions, especially in the circulation of CRC patients. The focus was primarily on the innate immune... Show moreThe aim of this thesis was to better understand the underlying biology of tumor-immune interactions, especially in the circulation of CRC patients. The focus was primarily on the innate immune system including NK cells, NKT cells, and macrophages. Show less
Faria, B.; Costa, M.G. da; Lima, C.; Willems, L.; Brandwijk, R.; Berger, S.P.; ... ; Poppelaars, F. 2020
Introduction Various studies have reported the importance of complement regulators in preventing mesothelial damage during peritoneal dialysis (PD). Its assessment, however, is limited in clinical... Show moreIntroduction Various studies have reported the importance of complement regulators in preventing mesothelial damage during peritoneal dialysis (PD). Its assessment, however, is limited in clinical practice due to the lack of easy access to the peritoneal membrane. Recently, a soluble form of the complement regulatory protein CD59 (sCD59) has been described. We therefore aimed to investigate the role of sCD59 in PD.Methods Plasma sCD59 was measured in 48 PD patients, 41 hemodialysis patients, 15 non-dialysis patients with chronic kidney disease and 14 healthy controls by ELISA (Hycult; HK374-02). Additionally, sCD59 and sC5b-9 were assessed in the peritoneal dialysate.Results sCD59 and sC5b-9 were detectable in the peritoneal dialysate of all patients, and marginally correlated (r = 0.27, P = 0.06). Plasma sCD59 levels were significantly higher in PD patients than in patients with chronic kidney disease and healthy controls, but did not differ from hemodialysis patients. During follow-up, 19% of PD patients developed peritoneal membrane failure and 27% of PD patients developed loss of residual renal function. In adjusted models, increased sCD59 levels in the dialysate (HR 3.44, 95% CI 1.04-11.40, P = 0.04) and in plasma (HR 1.08, 95% CI 1.01-1.17, P = 0.04) were independently associated with the occurrence of peritoneal membrane failure. Higher plasma levels of sCD59 were also associated with loss of residual renal function (HR 1.10, 95% CI 1.04-1.17, P < 0.001).Conclusions Our study suggests that sCD59 has potential as a biomarker to predict peritoneal membrane function and loss of residual renal function in PD, thereby offering a tool to improve patient management. Show less
Hoogen, B. van den; Santoni, A.; Sciume, G.; Bowie, A.; O'Farrelly, C.; O'Neill, L.; ... ; Hiscott, J. 2020
The past century has witnessed major advances in the control of many infectious diseases, yet outbreaks and epidemics caused by (re-) emerging RNA viruses continue to pose a global threat to human... Show moreThe past century has witnessed major advances in the control of many infectious diseases, yet outbreaks and epidemics caused by (re-) emerging RNA viruses continue to pose a global threat to human health. As illustrated by the global COVID19 pandemic, high healthcare costs, economic disruption and loss of productivity reinforce the unmet medical need to develop new antiviral strategies to combat not only the current pandemic but also future viral outbreaks.Pivotal for effective anti-viral defense is the innate immune system, a first line host response that senses and responds to virus infection. While molecular details of the innate immune response are well characterized, this research field is now being revolutionized with the recognition that cell metabolism has a major impact on the antiviral and inflammatory responses to virus infections. A detailed understanding of the role of metabolic regulation with respect to antiviral and inflammatory responses, together with knowledge of the strategies used by viruses to exploit immunometabolic pathways, will ultimately change our understanding and treatment of pathogenic viral diseases.INITIATE is a Marie Sklodowska-Curie Actions Innovative Training Network (MSCA-ITN), with the goal to train 15 early stage PhD researchers (ESRs) to become experts in antiviral immunometabolism (https://initiateitn.eu/). To this end, INITIATE brings together a highly complementary international team of academic and corporate leaders from 7 European countries, with outstanding track records in the historically distinct research fields of virology, immunology and metabolism. The ESRs of INITIATE are trained in these interdisciplinary research fields through individual investigator-driven research projects, specialized scientific training events, workshops on academia-industry interactions, outreach & communication. INITIATE will deliver a new generation of creative and entrepreneurial researchers who will be able to face the inevitable future challenges in combating viral diseases. Show less
Landman, S.L.; Ressing, M.E.; Veen, A.G. van der 2020
Rapid detection of microbes is crucial for eliciting an effective immune response. Innate immune receptors survey the intracellular and extracellular environment for signs of a microbial infection.... Show moreRapid detection of microbes is crucial for eliciting an effective immune response. Innate immune receptors survey the intracellular and extracellular environment for signs of a microbial infection. When they detect a pathogen-associated molecular pattern (PAMP), such as viral DNA, they alarm the cell about the ongoing infection. The central signaling hub in sensing of viral DNA is the stimulator of interferon genes (STING). Upon activation, STING induces downstream signaling events that ultimately result in the production of type I interferons (IFN I), important cytokines in antimicrobial defense, in particular towards viruses. In this review, we describe the molecular features of STING, including its upstream sensors and ligands, its sequence and structural conservation, common polymorphisms, and its localization. We further highlight how STING activation requires a careful balance: its activity is essential for antiviral defense, but unwanted activation through mutations or accidental recognition of self-derived DNA causes autoinflammatory diseases. Several mechanisms, such as post-translational modifications, ensure this balance by fine-tuning STING activation. Finally, we discuss how viruses evade detection of their genomes by either exploiting cells that lack a functional DNA sensing pathway as a niche or by interfering with STING activation through viral evasion molecules. Insight into STING's exact mechanisms in health and disease will guide the development of novel clinical interventions for microbial infections, autoinflammatory diseases, and beyond. Show less
Background and aims: Following an infection, cytokines not only regulate the acute immune response, but also contribute to symptoms such as inflammatory hyperalgesia. We aimed to characterize the... Show moreBackground and aims: Following an infection, cytokines not only regulate the acute immune response, but also contribute to symptoms such as inflammatory hyperalgesia. We aimed to characterize the acute inflammatory response induced by a human endotoxemia model, and its effect on pain perception using evoked pain tests in two different dose levels. We also attempted to determine whether combining a human endotoxemia challenge with measurement of pain thresholds in healthy subjects could serve as a model to study drug effects on inflammatory pain.Methods and results: This was a placebo-controlled, randomized, cross-over study in 24 healthy males. Twelve subjects were administered a bolus of 1 ng/kg LPS intravenously, and twelve 2 ng/kg LPS. Before days of placebo/LPS administration, subjects completed a full study day without study drug administration, but with identical pain threshold testing. Blood sampling and evoked pain tests (electrical burst and -stair, heat, pressure, and cold pressor test) were performed pre-dose and at frequent intervals up to 10hr post-dose. Data were analysed with a repeated-measures ANCOVA. For both dose levels, LPS induced an evident acute inflammatory response, but did not significantly affect any of the pain modalities. In a post-hoc analysis, lowering of pain thresholds was observed in the first 3 h after dosing, corresponding with the peak of the acute inflammatory response around 1-3 h post-dose.Conclusion: Mild acute systemic inflammation, as induced by 1 ng/kg and 2 ng/kg LPS intravenous administration, did not significantly change pain thresholds in this study. The endotoxemia model in combination with evoked pain tests is not suitable to study acute inflammatory hyperalgesia in healthy males. Show less
Background: To facilitate better discrimination between patients with active tuberculosis (TB) and latent TB infection (LTBI), whole blood transcriptomic studies have been performed to identify... Show moreBackground: To facilitate better discrimination between patients with active tuberculosis (TB) and latent TB infection (LTBI), whole blood transcriptomic studies have been performed to identify novel candidate host biomarkers. SERPING1, which encodes C1-inhibitor (C1-INH), the natural inhibitor of the C1-complex has emerged as candidate biomarker. Here we collated and analysed SERPING1 expression data and subsequently determined C1-INH protein levels in four cohorts of patients with TB.Methods: SERPING1 expression data were extracted from online deposited datasets. C1-INH protein levels were determined by ELISA in sera from individuals with active TB, LTBI as well as other disease controls in geographically diverse cohorts.Findings: SERPING1 expression was increased in patients with active TB compared to healthy controls (8/11 cohorts), LTBI (13/14 cohorts) and patients with other (non-TB) lung-diseases (7/7 cohorts). Serum levels of C1-INH were significantly increased in The Gambia and Italy in patients with active TB relative to the endemic controls but not in South Africa or Korea. In the largest cohort (n = 50), with samples collected longitudinally, normalization of C1-INH levels following successful TB treatment was observed. This cohort, also showed the most abundant increase in C1-INH, and a positive correlation between C1q and C1-INH levels. Combined presence of increased levels of both C1q and C1-INH had high specificity for active TB (96 %) but only very modest sensitivity 38 % compared to the endemic controls.Interpretation: SERPING1 transcript expression is increased in TB patients, while serum protein levels of C1-INH were increased in half of the cohorts analysed. Show less
Bot, I.; Velden, D. van der; Bouwman, M.; Kröner, M.J.; Kuiper, J.; Quax, P.H.A.; Vries, M.R. de 2020
Mast cells have been associated with arteriogenesis and collateral formation. In advancedhuman atherosclerotic plaques, mast cells have been shown to colocalize with plaque neovessels,and mast... Show moreMast cells have been associated with arteriogenesis and collateral formation. In advancedhuman atherosclerotic plaques, mast cells have been shown to colocalize with plaque neovessels,and mast cells have also been associated with tumor vascularization. Based on these associations,we hypothesize that mast cells promote angiogenesis during ischemia. In human ischemic muscletissue from patients with end-stage peripheral artery disease, we observed activated mast cells,predominantly located around capillaries. Also, in mouse ischemic muscles, mast cells were detectedduring the revascularization process and interestingly, mast cell activation status was enhanced up to10 days after ischemia induction. To determine whether mast cells contribute to both arteriogenesisand angiogenesis, mast cells were locally activated immediately upon hind limb ischemia in C57Bl/6mice. At day 9, we observed a 3-fold increase in activated mast cell numbers in the inguinal lymphnodes. This was accompanied by an increase in the amount of Ly6Chigh inflammatory monocytes.Interestingly, local mast cell activation increased blood flow through the hind limb (46% at day 9)compared to that in non-activated control mice. Histological analysis of the muscle tissue revealedthat mast cell activation did not aect the number of collaterals, but increased the collateral diameter,as well as the number of CD31+ capillaries. Together, these data illustrate that locally activated mastcell contribute to arteriogenesis and angiogenesis. Show less
Replication of positive-stranded RNA viruses requires the activity of proteases that cleave the viral replicase polyproteins. For Middle East respiratory coronavirus (MERS-CoV), the virus-encoded... Show moreReplication of positive-stranded RNA viruses requires the activity of proteases that cleave the viral replicase polyproteins. For Middle East respiratory coronavirus (MERS-CoV), the virus-encoded papain-like protease (PLpro) is one of such proteases. This protease also functions as a deubiquitinating enzyme (DUB) that removes ubiquitin from substrates, most likely to suppress the ubiquitin-dependent activation of the innate immune response. The work described in this thesis provides novel insights in the interaction between PLpro and ubiquitin. The crystal structure of the PLpro-ubiquitin complex facilitated the design of substitutions in PLpro that selectively disrupted its DUB activity. DUB-negative MERS-CoV induced enhanced immune responses compared to wild-type virus, while showing similar replication in infected cells. Relative to wild-type virus, the virulence of DUB-negative MERS-CoV was reduced in mice and earlier, better-regulated immune responses were measured in their lungs. In the search for novel antivirals, ubiquitin sequence variants were selected that bound with very high affinity to MERS-CoV PLpro. Expression of those ubiquitin variants affected the activity of PLpro and concomitantly inhibited virus replication resulting in severely less virus progeny. Collectively, the gained knowledge can be used to design novel coronavirus vaccines or further develop ubiquitin variants as antiviral agents against viruses that encode DUBs. Show less
The incidence of bacterial infections and sepsis, as well as the mortality risk from sepsis, is sex specific. These clinical findings have been attributed to sex differences in immune... Show moreThe incidence of bacterial infections and sepsis, as well as the mortality risk from sepsis, is sex specific. These clinical findings have been attributed to sex differences in immune responsiveness. The aim of the present study was to investigate sex differences in monocyte-derived cytokine production response upon stimulation with the gram-negative stimulus lipopolysaccharide (LPS) using cytokine data from 15 study populations. Individual data on ex vivo cytokine production response upon stimulation with LPS in whole blood were available for 4,020 subjects originating from these 15 study populations, either from the general population or from patient populations with specific diseases. Men had a stronger cytokine production response than women to LPS for tumour necrosis factor-α, interleukin (IL)-6, IL-12, IL-1β, IL-1RA, and IL-10, but not for interferon-γ. The granulocyte-macrophage colony-stimulating factor production response was lower in men than in women. These sex differences were independent of chronological age. As men had higher monocyte concentrations, we normalized the cytokine production responses for monocyte concentration. After normalization, the sex differences in cytokine production response to LPS disappeared, except for IL-10, for which the production response was lower in men than in women. A sex-based approach to interpreting immune responsiveness is crucial. Show less
The incidence of bacterial infections and sepsis, as well as the mortality risk from sepsis, is sex specific. These clinical findings have been attributed to sex differences in immune... Show moreThe incidence of bacterial infections and sepsis, as well as the mortality risk from sepsis, is sex specific. These clinical findings have been attributed to sex differences in immune responsiveness. The aim of the present study was to investigate sex differences in monocyte-derived cytokine production response upon stimulation with the gram-negative stimulus lipopolysaccharide (LPS) using cytokine data from 15 study populations. Individual data on ex vivo cytokine production response upon stimulation with LPS in whole blood were available for 4,020 subjects originating from these 15 study populations, either from the general population or from patient populations with specific diseases. Men had a stronger cytokine production response than women to LPS for tumour necrosis factor-alpha, interleukin (IL)-6, IL-12, IL-1 beta, IL-1RA, and IL-10, but not for interferon-gamma. The granulocyte-macrophage colony-stimulating factor production response was lower in men than in women. These sex differences were independent of chronological age. As men had higher monocyte concentrations, we normalized the cytokine production responses for monocyte concentration. After normalization, the sex differences in cytokine production response to LPS disappeared, except for IL-10, for which the production response was lower in men than in women. A sex-based approach to interpreting immune responsiveness is crucial. Show less
The disease is characterized by a progressive and largely irreversible decline in lung function, which is associated with long-term airway exposures to cytotoxic particles and gasses, such as... Show moreThe disease is characterized by a progressive and largely irreversible decline in lung function, which is associated with long-term airway exposures to cytotoxic particles and gasses, such as cigarette smoke. Microbial colonization and infections are an important pathophysiological aspect in COPD patients. However, the underlying mechanisms linking smoking with microbial colonization and infections in COPD are incompletely understood. The airway epithelium is the first target of inhaled cigarette smoke. Furthermore, epithelial cells are the first defense lining of the respiratory tract that prevents microbial colonization and infections. Therefore, alterations in host defense and airway epithelial remodeling may contribute to COPD development and progression. In this thesis, studies are presented in which the impact of cigarette smoke exposure and COPD disease status on the innate host defense functions of the airway epithelium are explored. This was done by using cell culture experiments in which the effect of cigarette smoke was examined, or in which epithelial cultures of COPD patients and non-COPD (ex)smokers were compared. Show less
The replication of all positive-stranded RNA viruses of eukaryotes is thought to take place at cytoplasmic membranous replication organelles. One of the most prominent types of viral ROs induced by... Show moreThe replication of all positive-stranded RNA viruses of eukaryotes is thought to take place at cytoplasmic membranous replication organelles. One of the most prominent types of viral ROs induced by a number of these viruses, including coronaviruses and arteriviruses, are double-membrane vesicles (DMVs) that contain viral double-stranded RNA. This thesis discusses the formation of these replication organelles by arteri- and coronaviruses and provides new insights in the viral proteins involved. The ultrastructure of the replication organelles was unravelled using both electron microscopy and tomography. Furthermore, this research described in this thesis also shows that the innate immune system in infected cells can prevent the formation of the membrane structures. Show less
Control of infectious diseases poses continuous challenges for human health. Salmonella bacteria are a major cause of gastrointestinal infections and systemic disease like typhoid fever. We used... Show moreControl of infectious diseases poses continuous challenges for human health. Salmonella bacteria are a major cause of gastrointestinal infections and systemic disease like typhoid fever. We used zebrafish-Salmonella infection models to study host immune responses to Salmonella, particularly focusing on the role of the autophagy machinery. Autophagy and a related process known as Lc3-associated phagocytosis (LAP) trap invading microbes in intracellular vesicles and eventually eliminate them through the lysosomal degradation pathway. We show that macrophages and neutrophils trap Salmonella in Lc3-decorated phagosomes by a process dependent on the host factors Rubicon and NADPH oxidase, which generates anti-bacterial reactive oxygen species. This process could be defined as LAP as it requires some but not all components of the autophagy machinery. Genetic inhibition of LAP and ablation of macrophages resulted in hypersusceptibility to infections with both wild type and attenuated strains, revealing that LAP is an essential line of defense against Salmonella during systemic disease, when macrophages are the main carriers of the infection. Our studies also revealed a novel functional link between the autophagy modulator Dram1 and induction of LAP. This work encourages further studies aimed at the identification of autophagy modulating drugs for host-directed therapy of antibiotic-resistant Salmonella infections. Show less
Persistent infections with high-risk type human papillomaviruses (hrHPVs) can progress to cancer. HrHPVs infect keratinocytes (KCs) and successfully suppress host immunity for up to two years... Show morePersistent infections with high-risk type human papillomaviruses (hrHPVs) can progress to cancer. HrHPVs infect keratinocytes (KCs) and successfully suppress host immunity for up to two years despite the fact that KCs are well equipped to detect and initiate immune responses to invading pathogens. HrHPV interferes with the innate immune response by affecting several signaling pathways that otherwise would prompt anti-viral mechanisms in the host cell. Furthermore, hrHPV interferes with the production of cytokines that are involved in the attraction of immune cells to the infected epithelium. In addition, hrHPV hides itself from the immune system by suppressing the antigen presentation machinery and employs means to hamper the response of KC__s to signals from adaptive immune cells. In this thesis we show that hrHPV attenuates innate immune signaling (Chapter 2) and CD40-mediated (Chapter 3) and IFN_ and/or TNF_-induced (Chapter 4) adaptive immune signaling. HrHPV exploits the cellular proteins UCHL1 (Chapter 2) and IFRD1 (Chapter 4) that act on multiple points in the IRF and NF_B signaling pathways. Moreover, hrHPV downregulates cellular IFITM1 to resist the growth inhibitory effects of IFN_ and/or TNF_ (Chapter 5). Our data provide important new insights on how hrHPV can persist in the face of host immunity. Show less
