Title Promoter Affiliations Abstract "Unraveling the enigmas in pulmonary and cerebral cryptococcosis pathogenesis by dynamic, in vivo monitoring of the pathogenesis and host response to fungal infection" "Greetje Vande Velde" "Biomedical MRI" "The subject of this research is imaging of fungal infectious disease and host immune cell interaction, bringing together preclinical imaging and mycology research, the latter being a subject in the domains of Microbiology and Immunology." "Dengue virus infection of endothelial cells: Pathogenesis and antiviral potential of glycosaminoglycan targeting" "Sandra Liekens" "Laboratory of Virology and Chemotherapy (Rega Institute)" "Dengue viruses (DENV) are single-stranded positive-sense RNA viruses that belong to the family of the Flaviviridae, together with other important human pathogens (e.g. yellow fever virus and West Nile virus). Dengue is currently considered to be the most important arthropod-borne vial disease in humans. Four genetically distinct, but serologically related serotypes (DENV 1-4) are transmitted to humans by mosquitoes of the Aedes genus in more than 100 countries in tropical and subtropical regions in South East Asia, the Americas and Africa. The principal dengue vector is Aedes aegypti, whereas Aedes albopictus functions as a secondary vector. Over 2.5 billion people live in areas that are affected by dengue and it is estimated that each year 390 million DENV infections occur. The DENV disease spectrum ranges from a self-limiting febrile illness called dengue fever (DF), to the severe and potentially life-threatening dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS). While DF symptoms include a sudden, high fever (>39°C), headache, nausea, vomiting, rash, and joint and muscle pains, DHF/DSS are characterized by increased vascular permeability, leading to plasma leakage, thrombocytopenia and coagulation disorders. Despite decades of extensive research, the mechanisms responsible for DENV-induced plasma leakage remain to be elucidated. The ease of collecting blood samples has prompted scientists to focus on the involvement of immune cells and soluble mediators. These studies have identified dendritic cells, monocytes and macrophages as the primary DENV target cells. Although endothelial cells (ECs) line the inner layer of the endothelium and regulate the exchange of gasses, fluid and solutes between the blood vessels and the surrounding tissues, an active role of the endothelium in the pathogenesis of DENV has not been considered until recently. Immunohistological studies and in situ hybridization performed on tissues from fatal DHF/DSS patients have demonstrated the presence of DENV antigens and viral RNA in ECs of various organs (e.g. lung, spleen, liver, heart and brain), indicative of viral infection and replication in ECs. Therefore the aims of this study were to (i) investigate the infection of microvascular endothelial cells by DENV; (ii) compare the response of different EC types to infection with DENV 1-4; and (iii) evaluate compounds for their ability to inhibit DENV infection of ECs. In Chapter 2 we investigated the susceptibility of primary microvascular ECs (HMVEC-d) and the immortalized EC line HMEC-1 to DENV infection, since DENV-induced plasma leakage is believed to occur at the level of the microvasculature. We showed for the first time that primary microvascular ECs are susceptible to DENV infection and replication in the absence of the well-described receptors DC-SIGN (on dendritic cells) or the mannose receptor and Fcγ-receptor (on monocytes/macrophages). The ability of natural glycosaminoglycans (GAGs), including heparin and heparan sulfate, to inhibit DENV-2 infection of ECs suggested the involvement of heparan sulfate-containing proteoglycans (HSPGs). Flow cytometric analysis revealed high expression levels of HSPGs on these cells and moreover, enzymatic removal of heparan sulfate moieties from the cellular surface resulted in a strong decrease in the number of infected cells. Taken together, these results indicated that HSPGs mediate DENV-2 infection of microvascular ECs. The clinical use of heparin and heparin analogues is limited because of their well-known anticoagulant activity. Therefore, we evaluated the ability of sulfated derivatives of the K5 capsular polysaccharide of Escherichia coli, which has the same structure as the natural precursor of heparin and is devoid of anticoagulant activity, to inhibit DENV-2 infection of HMEC-1 and HMVEC-d cells. The highly sulfated K5-OS(H) and K5-N,OS(H) proved to be very active compounds with EC50 values in the nanomolar range. Time-of-addition and attachment/entry assays revealed that these compounds inhibit the early steps in the DENV infection cycle. We showed that the K5 derivatives inhibit the fusion of DENV-infected C6/36 mosquito cells by interacting with the DENV envelope (E) protein. Using surface plasmon resonance (SPR) analysis we demonstrated that K5-OS(H) and K5-N,OS(H) bind to DENV envelope protein domain III (E DIII), which contains the putative receptor-binding domain. Furthermore, the highly sulfated K5 derivatives inhibited the binding of DENV E DIII to immobilized heparin, which mimics the infection process in microvascular ECs. We also demonstrated that K5-OS(H) and K5-N,OS(H) inhibit DENV-2 infection of dendritic cells. Together, these results indicate that highly sulfated K5 derivatives may provide a new class of anti-DENV compounds, although their antiviral activity against the other serotypes should still be confirmed. In Chapter 3 we evaluated the antiviral activity of chemokine peptides. As DENV infection of ECs is dependent on HSPGs, positively charged peptides may compete with DENV for binding to HS. Chemokines are dependent on GAG binding on ECs to create a chemokine gradient for the recruitment of immune cells to the site of production. Chemokine binding to GAGs is thought to be mediated by basic amino acid clusters in the carboxy-terminus and the negatively charged sulfated/carboxyl groups of HSPGs. The CXC chemokines, CXCL9 and CXCL12γ, have a characteristic basic COOH-terminus and contain multiple putative HS-binding motifs. Therefore, the binding of different synthetic COOH-terminal CXCL9 and CXCL12γ peptides to heparin was characterized using SPR analysis. These data revealed high binding affinities of the positively charged peptides to immobilized heparin, except for CXCL9(86-103) in which 2 putative HS-binding motifs are missing. Furthermore, the CXCL9 and CXCL12γ peptides were able to compete for heparin binding to CXCL8, a strong heparin binder. Since several viruses have been shown to use HSPGs as attachment receptors on target cells, the antiviral activity of the chemokine peptides was investigated against a range of such viruses.  This antiviral screen showed marked inhibitory activity of the heparin-binding peptides against herpes simplex virus type 1 (HSV-1), respiratory syncytial virus (RSV) and DENV-2 in the micromolar range. SPR analysis demonstrated that CXCL9(74-103) binds  faster to heparin than DENV E DIII and inhibits the interaction of DENV E DIII with heparin, hence explaining its anti-DENV-2 activity. These results indicate that the positively charged COOH-terminal CXCL9 and CXCL12γ chemokine peptides may be lead compounds for the development of a new class of antiviral agents targeting virus-HSPG interaction. In Chapter 4 we aimed to compare the effects of DENV 1-4 infection in ECs from different origin. Contradictory findings have been reported regarding the DENV infection rate and EC response. The use of ECs from different origin further complicates the comparison of the obtained results. Furthermore, most data involve DENV-2 and to a lesser extent serotype 4, whereas very few studies reported the effects of EC infection with DENV-1 and -3. Therefore, we infected the microvascular EC line HMEC-1 and primary microvascular (HMVEC-d) and macrovascular (HUVEC) ECs with four DENV serotype strains: DENV-1 Djibouti, DENV-2 NGC, DENV-3 H87 and DENV-4 Dak. DENV-4 proved to be the most infectious serotype, followed by DENV-2, DENV-1 and DENV-3. We found the highest percentage of DENV infection in HMEC-1 cells, whereas infection of primary HMVEC-d and HUVEC cells resulted in a comparable, lower infection rate. The observed differences in the infection rate between the EC types were found to correlate with the expression of HS on the surface of these cells. Using a custom-made Bio-Plex assay, we simultaneously quantified the levels of 18 cytokines involved in the regulation of EC permeability or the recruitment and/or stimulation of immune cells. DENV 1-4 infection of ECs resulted in the strong upregulation of IL-6, MCP-1, RANTES, CXCL9, CXCL10 and CXCL11. Furthermore, we observed a time-dependent cytopathogenic effect in all EC types after DENV 1-4 infection. Specific apoptosis assays revealed the translocation of phosphatidylserine to the extracellular side of the plasma membrane in HMEC-1 cells infected with DENV 1-4 and the activation of caspase-3 in DENV-4 infected HMEC-1 and HUVEC cells. To investigate the effect of direct infection of ECs on vascular permeability, we monitored the integrity of a HUVEC monolayer during DENV-4 infection in real-time. We found that DENV-4 infection alone did not alter the cell index in comparison to uninfected control cells. However, when DENV-4 infected ECs were stimulated with TNF-α 24 h after infection, a strong decrease in the cell index was observed, when compared to uninfected ECs treated with TNF-α. These results suggest that DENV infection sensitizes ECs to the permeability-enhancing effects of TNF-α, although the underlying mechanisms remain to be elucidated." "The role of the adipose tissue in the pathogenesis of insulin resistance and increased disease susceptibility in high yielding dairy cows" "Geert Opsomer" "Department of Internal Medicine, Reproduction and Population Medicine" "Obesity around the moment of calving increases the risk to develop metabolic, infectious and reproductive diseases in high yielding dairy cows. In this project, the role of the adipose tissue in the parthogenesis of these diseases, will be examined macroscopically, microscopically and molecularly." "Molecular pathogenesis in head and neck cancer: role tyrosine kinases." "Sandra Nuyts" "Laboratory of Experimental Radiotherapy, Laboratory of Molecular Biology of Leukemia (VIB-KU Leuven)" "Head-and-neck squamous cell carcinoma (HNSCC) is the fifth most common malignancy worldwide,  responsible for approximately half a millionnew cases every year. The treatment of this disease is challenging and characterized by high rates of therapy failure and toxicity, stressing the need for new innovative treatment strategies. In this work we primarily aimed to identify potential starting points for new therapeutic targeting strategies. We therefore performed a high throughput shRNAmir based screen on HNSCC cells with the aim to identify tyrosine kinases that are mediating radiotherapy resistance. Using this screen setup we identified the receptor tyrosine kinase FLT1 (VEGFR1) as an important driver of cell survival and radioresistance, and demonstrate receptoractivation through autocrine production of FLT1 ligands. Immunohistochemistry on HNSCC patient samples demonstrated FLT1 and ligands to be uniformly expressed. Interestingly, FLT1 was selectively overexpressed in tumour tissue as compared to non-cancerous epithelium. Remarkably, we found only membrane permeable FLT1 kinase inhibitors to be effective, which was in agreement with the intracellular localization of FLT1. Taken together, we document expression of FLT1 in HNSCC and demonstrate this kinase to modulate radioresistance and cancer cell survival. Given the fact that FLT1 kinase is selectively upregulated in tumour tissue and that itskinase function seems expendable for normal life and development, this kinase holds great promise as a new potential therapeutic target. Nonetheless several scientific questions still need to be resolved before FLT1targeted therapy can be transferred from bench to bedside. Most crucialis the development of a good FLT1 targeting drug, which is able to shutdown FLT1 activity at concentrations which are maintainable in a clinical setting. Also further validation of such a FLT1 targeting strategy isrecommended in different patient derived HNSCC xenograft models to evaluate therapy responses as well as to validate prognostic biomarkers likefor example (semi)quantitative VEGFA expression or Human Papilloma Virus (HPV) status. A subgroup of HNSCC is etiologically related to HPV infection. These HPV related tumors show an intriguing clinicalbehavior as compared to classical HPV-unrelated HNSCC: they tend to have a better prognosis and show a better response to radio/chemotherapy although these tumors are undifferentiated and present themselves at more advanced clinical stages. Additionally, in contrast to HPV unrelated HNSCC they seem not responsive to hypoxia targeted therapy although the level of hypoxia between these tumors is comparable. To date these characteristics are unexplained on a molecular level. As a starting point to shed light on this remarkable behavior, we started from observations done on breast cancer cells where it was shown that p16 suppresses the expression of VEGFA (a FLT1 activating ligand), through inhibition of HIF1a, the key regulator of hypoxic metabolism. If we could show that p16 is suppressing HIF1a in HPV positive HNSCC, a resulting reduction of VEGFA expression could maybe explain part of the increased radiosensitivity through impaired FLT1 activity. Additionally, an impaired HIF1a function couldlead to hypoxia intolerance, owing to impaired adaptation to cellular hypoxia related metabolic stress. This hypoxia intolerance could then severely impact cellular survival under oxygen deprivation. This would thenlead to increased radiosensitivity in vivo, since these cells would be unable to rely on hypoxic niches within a tumor to protect them from radiation. We clearly demonstrated p16 related HIF1a suppressive activity in HPV positive HNSCC resulting in suppressed VEGFA expression. However we also showed that this suppressive activity did not suffice to prevent a HIF1a mediated metabolic shift nor to induce hypoxia intolerance in these cells. The reason why HPV positive HNSCC is not responsive on hypoxia targeted therapy therefore remains elusive. HPV associated HNSCC is currently becoming a global epidemic. According to American SEER data, the incidence of this type of cancer is increasing with 2-3% each year. American studies have shown that 40 to 65% of HNSCC arising from the base of tongue or tonsils could be attributed to HPV16. As already mentioned these tumors are more radiocurable but not responsive to hypoxia targeted therapy. These factors could probably have an impact on treatment selection. Currently, locally advanced stage oropharyngeal cancers are being treated in the same way as the classical HNSCC with radio-chemotherapy schedules. These intense treatment regimens come however at the cost of severe acute and late toxicity (like dysphagia and PEG-tube dependency). Patients with HPV-associated HNSCC might not need these intense treatment regimens, and perhaps more function sparing therapies would lead to equal survival numbers in this population. To set up new clinical studies, we needed to have an idea about the prevalence of HPV in oropharyngeal cancer in our Flemish (Belgian) patient population, certainly because large geographical differences are existing. Therefore a multicenter cooperative study was undertaken between the radiation-oncology departments of the Flemish universities.  We found indeed an increasing incidence of oropharyngeal carcinoma in males as well as females.  The prevalence of HPV(+) oropharyngeal carcinoma was found to be 24.78% (19.93-30.36%). HPV status remained a strong predictor of better locoregional control after multivariate analysis. Regarding locoregional control we found the addition of concurrent chemotherapy to be of equal benefit in HPV(+) and HPV(-) patients suggesting that treatment de-escalation by omitting concurrent chemotherapy is probably not a good idea." "Can high-resolution optical projection tomography of the interplay between cryptococci and macrophages unravel the enigmas in pulmonary and cerebral cryptococosis pathogenesis?" "Greetje Vande Velde" "Biomedical MRI" " Scientific state-of-the-art & aims of this proposal: Cryptococcus neoformans and C. gattii are encapsulated yeasts that can cause life-threatening disease in both immune-competent and immune-suppressed individuals (e.g. cancer therapy patients or patients with AIDS). Cryptococcosis affects the lung and may spread to the brain, manifesting itself by meningitis or pseudocystic brain lesions. It remains unknown why, how and when the cryptococci are able to cross an apparently intact blood-brain barrier (BBB). The role of the host immune cells in the course of the disease and in particular in the process of crossing the BBB is known to be important, but is still largely enigmatic. Imaging techniques are therefore indispensable to monitor dynamic processes in vivo, to define the relevant time frames for detailed cellular analysis and this for each animal individually.The overall objective of our research is to dynamically monitor immune cells and cryptococci in individual animals in vivo to resolve their interplay during the course of the initial lung infection and eventually their interaction with the BBB. By using different, complementary imaging modalities, we aim to track the different types of cells involved and correlate this to the progression of the infection in a mouse model for pulmonary and cerebral cryptococcosis, thereby defining the key events in cryptococcosis pathogenesis, which is essential for its diagnosis and therapy. However, imaging techniques specific for fungal infections are currently very limited as fungi pose some particular challenges1,2. We have been able to successfully develop non-invasive imaging methods to visualize and quantify the overall cryptococcal lung disease progression, using lung MRI and micro-CT. However, a method to image fungal load in vivo is currently unavailable. Using GFP-positive C. gattii, we were able to image the cryptococci in the lung with intravital confocal fluorescence microscopy – however, the field-of-view (FOV) is very limited and there is no reference to the surrounding tissue context, essential for mapping the cryptococcal cell density and spatial progression across the entire lung, as there is evidence that these parameters change during infection, depending on the disease stage3,4. This is thought to be directly related to virulence and therefore most probably to important events during pathogenesis including cryptococcal invasion of the brain. However, not much is known about this phenomenon as currently no methods are available to investigate the cryptococcal cellular distribution in intact infected mouse lungs, in 3D and with cellular resolution.We have the ambition to provide the missing link between whole-body imaging methods that visualize overall lung disease progress in 3D, and microscopic techniques that give information with high, cellular resolution in 2D and with a very small FOV. Optical projection tomography (OPT) is a relatively new optical imaging technology that was developed to bridge this imaging gap by providing high-resolution 3D imaging and quantification of morphological features in intact mouse organs5,6. It has been successfully applied in embryology and in diabetes research7-9, but its use for adult mouse lung and brain imaging has not been explored before. Nonetheless, OPT of Cryptococcus-infected lungs would be the perfect tool to give us the missing information on fungal load, to correlate disease parameters such as cryptococcal distribution and density with infection stages (as assessed by in vivo lung MRI and CT) and eventually, with the time of crossing the BBB and invasion of the brain. As OPT offers the possibility of multiplexing different fluorescent signals in an entire organ of the mouse7,10, we plan to combine OPT for GFP-expressing cryptococci with OPT looking at the spatial distribution and co-localization with fluorescently labeled macrophages. Macrophages are very important for cryptococcosis pathogenesis. This is illustrated by the fact that cryptococci can survive inside macrophages after being phagocytized11. This observation has led to the hypothesis that cryptococci might be shuttled over the BBB inside macrophages like a Trojan horse - but the exact mechanism is not known 12. To unravel this is one of our ultimate goals, by studying the interaction between cryptococci and macrophages during lung infection, hereby defining key events during pathogenesis of cryptococcosis.We expect that the introduction of the here developed novel complementary imaging approaches will greatly push the cryptococcosis research forward as it will give us the tools to answer the still enigmatic key phases in the pathogenesis of cryptococcosis, thereby contributing to the identification of potential targets for therapy that may result in new diagnostic approaches. These imaging tools will have further applications in enabling rapid in vivo screening and ex vivo validation of different (pathogenic) strains, including mutant strains in order to identify key virulence factors and potential therapeutic targets in mouse models of infection. The research on other life-threatening fungal infections will benefit from the research we will conduct, as our approach will be readily or with minor adaptations translatable to other (fungal) infectious disease models, such as invasive aspergillosis.Description of the research method: As it is currently unknown if lungs of adult mice can be imaged with OPT, a first step will be a proof-of-concept for lung imaging with OPT of normal, adult mouse lungs, optimizing the sample preparation starting from the currently available protocols for optical clearing and making the sample compatible for OPT. Contrary to embryonic lungs, adult mouse lungs contain air which is detrimental for OPT imaging and one of the challenges that need to be tackled. Therefore, careful organ preparation by tying off the trachea after inflating the lungs with OPT-compatible liquid and adaptation of the sample processing steps (clearing, embedding, imaging, data processing) will be undertaken. Possible proof-of-principle (PPoP): OPT of healthy adult mouse lung. We have an ongoing collaboration with Prof. Sharpe’s lab, co-inventor of OPT, and a shared interest in validating OPT for lung and brain imaging. We agreed to collaborate on validating lung OPT and I am very welcome in their lab to share our mutual expertise for optimal feasibility.A next challenge will be the immunostaining of a large organ such as the lungs, as reagents have to diffuse throughout the whole lung which would require specific optimization (such as tissue permeabilization) and long incubation times. For stainings using primary (anti-GFP) and secondary (Alexa488-labeled) antibodies, we will try an approach whereby the immunostaining reagents will be intratracheally applied to ensure optimal distribution and facilitate diffusion throughout the entire lung tissue while keeping the trachea closed (tied off) to avoid reagents leaking out of the lung and air from entering the lung. PPoP: OPT of crypto-infected adult mouse lung. In a next step, we would cross-validate our established in vivo imaging protocols (MRI, CT) with high-resolution OPT of the 3D-distribution of fungal load in the intact lung during pulmonary cryptococcosis. Infected mice will be scanned at baseline and weekly up to 5 weeks post infection with MRI and CT to monitor overall disease progression, after every imaging time point sacrificing three mice for OPT. Signal intensities and lung parameters will be quantified and cross-correlated with OPT results, with parallel ‘traditional’ histology and fungal load quantification. PPoP: cross-validated OPT and in vivo MRI & CT, validated against standard histology and fungal load quantification. A second OPT-imaging mode that is available and very promising to answer our research questions, is selective plane illumination microscopy (SPIM). This imaging mode works best with smaller samples (up to a few millimeters) such as individual cryptococcomas, but can resolve individual cells. The sample preparation protocol is essentially the same as for OPT apart from the smaller sample size. SPIM would be the perfect modality to test the hypothesis that different capsule sizes and therefore density of the cryptococcal distribution in cryptococcomas reflect virulence and correlate with the severity of infection, and possibly with cryptococci crossing the BBB. PPoP: time line of cryptococcal density over the course of the infection, with 3D cellular density within lung cryptococcomas, in order to be correlated with cryptococci detected in the brain (with non-invasive optical imaging approaches and/or OPT of brain (see further), or in the worst case, with standard histological techniques).It is not known how and when cryptococci are able to cross an intact BBB to infect the brain, therefore we aim to image 3D cryptococcal distribution in the brain with OPT in order to help unraveling the process. As a first step, we would optimize the sample preparation for OPT of the intact mouse brain. PPoP: OPT of healthy adult mouse brain. Imaging cryptococci invading the brain with OPT involves immunostaining of an intact, large organ, which is again highly challenging. PPoP: OPT of cryptococci-infected intact adult mouse brain. A next step would be to use OPT and SPIM to image the interplay between cryptococci and immune cells, i.e. host macrophages. They are known to be important for spread of the infection, but it is not known how and when this happens. Cryptococci can survive intracellularly after being phagocytized by macrophages. Therefore the hypothesis has been formulated that cryptococci are being shuttled over the BBB by macrophages, as in a Trojan horse. PPOP: It is our ultimate aim to verify this hypothesis by imaging this interaction. A challenging aspect here will be to identify the exact time frame during the course of infection when the cryptococci will cross the BBB. As the process of crossing the BBB probably involves few cells, it will be challenging to track the cells dynamically in vivo. Therefore, we expect that a complimentary approach involving multi-modality (MRI, CT and optical) imaging approach will be necessary to define the time frame, combining overall infectionimaging (MRI, CT) with in vivo BLI of cryptococci. We will engineer cryptococci to express luciferase, the reporter gene for BLI. This will hopefully enable us to quantify viable cryptococci in the lung during initial lung infection and to non-invasively track cryptococcal cells that are spreading from the lungs to other organs and to the brain. PPOP: BLI of luciferase-expressing cryptococci; established time profile for the spreading of cryptococcosis (theoretical time resolution of one day). A potential challenge is that the BLI signal intensity needs to be high enough to reach optimal sensitivity. If needed, we could use the Nanoluc® luciferase gene (higher BLI signal for lower intracellular substrate concentrations; the cDNA is available in the lab) or engineer cryptococci to express an extracellularly located Gaussia luciferase, as shown to improve BLI of other yeast infections by overcoming limitations regarding the intracellular uptake of the substrate13,14. BLI is particularly challenging for fungal pathogens2,15 as also illustrated by the limited applications of fungal BLI in the literature. However, experience in our and other laboratories has shown that these challenges can be overcome13,14.In general, we will validate OPT as a generic cross-validation tool for our in vivo experiments. This will also form the basis for future development and validation of probes for in vivo fluorescence imaging, having also applications in particular for easier clinical diagnosis of fungal infections. We will also build up expertise for a possible future implementation of OPT at KU Leuven.Proposed staffing: postdoc (myself, for protocol development, OPT imaging) working together with a full-time technician (mainly for performing established in vivo imaging (MRI, CT, optical), sample preparation and immunostaining, mouse model induction, fungal cell culture,… during one year. The financing would be used for a full-time technician, animals and housing, consumables such as 1° and 2° Abs, scanning costs incl. anesthesia and contrast agents, open access publication costs (detailed calculations have been made)." "Mucus, the missing link in the pathogenesis of necrotic enteritis" "Filip Van Immerseel" "Department of Pathobiology, Pharmacology and Zoological Medicine" "C. perfringens (CP) is the causative agent of many enteric diseases in both animals and humans, including necrotic enteritis (NE) in broilers, a disease characterized by necrosis and inflammation of the small intestine. In addition to NetB toxin, other virulence factors essential for NE disease development have been identified, although their function remains unknown. This project focusses on the early stages of NE pathogenesis with great interest in CP colonization. The expression of two virulence genes, encoding putative chitinases, is upregulated during the first stages of the pathogenesis. In WP1, the importance of these enzymes during colonization and infection will be assessed using in vivo models. Since chitin is not present in the broiler intestine, these chitinases have to carry out an alternative function. We hypothesize that intestinal mucus layer is key in facilitating CP colonization. The mucus-altering potential of chitinases will be assessed in WP2, together with its impact on CP proliferation and binding. Furthermore, we hypothesize that the parasite Eimeria, a predisposing factor to NE, is able to induce mucus secretion or alter mucus composition. This mucus-altering potential and its impact on CP colonization will be assessed in WP3. In summary, a thorough knowledge of the early stages of NE pathogenesis, with regard to the impact of both CP and Eimeria spp. on the mucus layer, is key in better understanding the disease, eventually guiding novel research." "Vector-free transmission of Japanese encehalitis virus (JEV) in pigs - increased risk associated with a potential emergence: Study of JEV pathogenesis in pigs upon infection via different inoculation route" "Herman Favoreel" "Department of Translational Physiology, Infectiology and Public Health" "The goals of this project are: - to finalize the administrative procedures necessary to obtain the permission to work with Japanese encephalitis virus (JEV) at CODA-CERVA. - to perform an experimental infection study whereby pigs will be infected with JEV via three inoculation routes (oronasal inoculation to mimic the vector-free transmission and intradermal inoculation of virus alone or of virus combined with mosquito salivary gland extract to mimic vector mediated transmission). A maximum of samples will be collected during the trial that will allow to study the pathogenesis and immune response in detail. - to study the pathogenesis of JEV in pigs infected via 3 inoculation routes by performing qPCRs and virus isolations on blood, swabs and orgnas collected during the infection experiment. - to study the development of the humoral immune response via PRNT tests. - to perform a preliminary analysis of infiltrating immune cells in the tonsils upon JEV infection via IHC. Besides the implementation of diagnostic techniques for JEV at CODA-CERVA and acquiring knowledge on JEV pathogenesis, this research will allow to collect samples that can be used in the future to closely study the role of tonsils in JEV pathogenesis and to evaluate the importance of the cellular immunity upon JEV infection. It will also allow to identify an appropriate challenge model that can in the future be used for comparative vaccine trials." "The role of the innate immune system in the pathogenesis of systemic juvenile idiopathic arthritis: advances in understanding" "Patrick Matthys" "Molecular Immunology (Rega Institute), Immunobiology (Rega Institute)" "Systemic juvenile idiopathic arthritis (sJIA) is a severe autoinflammatory childhood-onset immune disorder, classified as one of the JIA subtypes. Patients clinically present with fever, skin rash, enlarged lymph nodes (LNs), spleen and/or liver, and develop arthritis. Haematologically, neutrophilia (including immature neutrophils), leukocytosis, anaemia, and thrombocytosis is noticed. Patients have high plasma cytokine levels of interleukin-6 (IL-6), IL-18, C-reactive proteins, and S100 proteins (S100A8/A9 and S100A12). Among all JIA subtypes, sJIA has the highest mortality rate since the disease is associated with a severe life-threatening complication macrophage activation syndrome (MAS). MAS is a hyperinflammatory syndrome with excessive activation of macrophages and T cells resulting in a cytokine storm and is associated with hemophagocytosis. More recently sJIA was also associated with pulmonary complications that include interstitial lung disease (ILD), and pulmonary alveolar proteinosis (PAP). In this doctoral thesis, we aimed to understand the role of the innate immune system in the pathogenesis of the sJIA. More specifically we aimed to gain insights into the successful use of IL-1 antagonists in patients considering that IL-1β blood levels are barely detectable. Next, we aimed to understand the effects of IL-1 on both the neutrophils and the γδ T cells. Considering the high numbers of neutrophils in blood of patients and the splenomegaly as a characteristic of sJIA, we studied extramedullary granulopoiesis and the role of neutrophils in the disease pathogenesis. Finally, we aimed to answer why sJIA patients show lung disorders, and more specifically investigated whether this is rather an epiphenomenon, or whether lung inflammation participates in the disease initiation. To address these aims, we took advantage of a described mouse model of sJIA that is based on the injection of complete Freund’s adjuvant (CFA) in interferon-gamma (IFN-γ) knock-out (KO) mice. These mice develop all clinical and haematological features reminiscent of sJIA, whereas CFA-immunisation in wild-type (WT) animals only results in a partial, self-resolving immune response. We demonstrated increased inflammasome activation in immunised IFN-γ KO mice, as evident by increased levels of active caspase-1 and IL-1β in the lymphoid organs and the blood. This could in part explain their increased susceptibility to develop sJIA. Using neutralising anti-IL-1β antibodies, the sJIA-like features could partially be prohibited and treated mice showed reduced weight loss, rash, granulocytosis, and thrombocytosis, and had reduced plasma IL-6 levels. Interestingly, the number of IL-17- and IL-22-producing CD27 negative γδ T cells and CD4+ T cells were decreased upon treatment. Beneficial effects of IL-17-depletion were reported in the initial description of the mouse model. In this doctoral thesis, we explored the role of IL-22 in the pathogenesis of sJIA. Increased IL-22 plasma levels were measured in the CFA-immunised IFN-γ KO mice. Using IL-22 neutralising antibodies we showed a partial role of IL-22 in the mouse model since neutralisation was associated with reduced weight loss, tail damage, neutrophilia, and thrombocytosis. In sJIA patients with active disease increased plasma levels of active IL-22 were measured. Using healthy donor γδ T cells or CD4+ cells, we could show a synergistic effect of IL-22 and IL-1β in the production of IL-17. To understand the role of neutrophils in the sJIA pathophysiology, we made use of anti-Ly6G (1A8) depleting antibodies. Anti-Ly6G can be used to study the in vivo role of neutrophils in murine models and became the gold standard considering its neutrophil-specific depletion. In the CFA-challenged BALB/C model, we demonstrated an incomplete anti-Ly6G-mediated neutrophil depletion. Furthermore, using BrdU incorporation, we demonstrated an increased proliferation in the bone marrow (BM) upon depletion, and a massive neutrophilic proliferation in the spleen, showing that a temporary depletion is followed by a fast rebound of new immature neutrophils that cannot be depleted. Therefore, we used an alternative approach in which we targeted granulocyte-colony stimulating factor (G-CSF) signalling. G-CSF is the main growth factor involved in the proliferation, maturation, release, and functionality of neutrophils. G-CSF release can be stimulated by IL-17. During excessive myelopoiesis, it can drive the formation of myeloid-derived suppressor cells (MDSCs), having an immune-tempering capacity. Increased plasma levels of G-CSF were measure in the CFA-immunised IFN-γ KO mice. Also, in patients with active disease increased plasma G-CSF levels were reported. Interestingly, these levels were correlating with some inflammatory mediators including the number of neutrophils and plasma levels of IL-6 or IL-1β. The role of G-CSF was studied in our mouse model using G-CSF receptor (G-CSFR)-blocking antibodies. We could demonstrate that G-CSF is responsible for the observed neutrophilia and extramedullary myelopoiesis. Next, by blocking the G-CSFR, we could inhibit the development of arthritis. However, treated mice showed a mild aggravation of some of the disease symptoms including weight loss, tail damage, number of immature RBCs, and had slightly increased plasma levels of IL-6, IL-17, and TNF-α. Single-cell sequencing revealed that neutrophil development in the spleen follows a similar developmental path as described in the BM and also includes stages of pre-neutrophils, immature neutrophils, and mature neutrophils. When we compared CFA-immunised WT and IFN-γ KO mice, the latter were characterised by the development of MDSCs, having a mature phenotype with high levels of CEBP/β. Administration of the G-CSFR-antagonising antibody could block the maturation and differentiation of neutrophils in CFA-immunised mice. The reduced numbers of MDSCs could in part explain their aggravated phenotype. In the final part of this doctoral thesis, lung abnormalities in our sJIA mouse model were the subject of research considering the recent lung complications that were described in patients. Using microcomputer tomography, a moderate enlargement of the lungs was measured upon CFA-immunisation as well as a decrease in aerated lung density. In the IFN-γ KO mice only, an increased non-aerated lung density was found. Next, functional alterations were noticed in the CFA-immunised IFN-γ KO mice, with male mice showing an increased tissue hysteresivity and tissue damping, female mice presented with increased airway hyperactivity. Histopathology revealed subpleural and parenchymal cellular infiltrates associated with the formation of granulomas. Using flow cytometry and quantitative PCR, we demonstrated an excessive innate immune activation with infiltration of mainly immature and mature neutrophils or monocytes, increased γδ T cells, activated macrophages, and increased expression of both IL-1β, IL-6, TNF-α, IL-17, and IL-22. Associated with increased neutrophilic infiltrates, high levels of G-CSF and neutrophil-attracting chemokines were measured in the CFA-injected IFN-γ KO mice. Although the pulmonary manifestations in mice were distinct to those described in sJIA patients, the work demonstrates an important innate immune activation in our model. It underlines that lung inflammation should not be neglected when the pathophysiology of sJIA is studied. Expanding our knowledge on neutrophils in autoinflammatory diseases, we studied neutrophils in both pyrin-associated inflammation with neutrophilic dermatosis (PAAND) and familial Mediterranean fever (FMF). PAAND and FMF are two IL-1β-driven autoinflammatory syndromes caused by distinct mutations in the same MEFV gene. Considering their high numbers of neutrophils in the blood and affected tissues, the transcriptional, functional, and phenotypical characteristics of neutrophils were extensively analysed. At the transcriptional level patients showed a partially overlapping activated phenotype with increased expression of S100 proteins, IL-4R, CD48, F5, MMP9, and NF-κB. Increased levels of MMP9 and S100A8/A9 were validated in plasma. Flow cytometric analysis demonstrated an immature character, an increased expression of TLR4 and TLR9, whereas a decreased expression of CXCR2, C5aR, and BLTR1 was observed on neutrophils from FMF patients only. PAAND patients showed an enhanced granularity and a functional increased random, but reduced CXCL8-induced migration. Similarly, in FMF patients a tendency for enhanced random migration and reduced chemoattractant-induced migration was noticed. Neutrophils from both FMF and PAAND patients showed an increased basal and TNF-α-induced ROS production and had an elevated release of the azurophilic granule protein myeloperoxidase (MPO). Analysing the phagocytic capacity, we showed enhanced activity in PAAND patients but reduced activity in FMF patients. Together our data describe the neutrophil abnormalities in two pyrin-associated diseases and underlines that FMF and PAAND are two distinct diseases." "The molecular pathogenesis of Fuchs' endothelial corneal dystrophy." "Joost van den Oord" "Translational Cell & Tissue Research" "In this doctoral thesis, we have investigated the pathogenesis of late-onset Fuchs’ endothelial corneal dystrophy (FECD). FECD is a degenerative disorder of the corneal endothelium (CE). The CE is the inner layer of the cornea (the cornea is the transparent front part of the eye). The cornea does not contain blood vessels, but receives water and nutrients from the anterior eye chamber fluid (a.k.a. aqueous humor) and the tear film. To prevent the cornea from swelling (edema), the CE constantly pumps out excess fluid and waste products, safeguarding the normal corneal thickness and transparency.FECD is characterized by a loss of CE cells (CECs), resulting in clouded vision, painful ulcerations and even blindness. Usually both eyes are affected. The prevalence of FECD is estimated at 4% of people older than 40. The only curative treatment consists of replacing the diseased CE layer with a healthy donor layer. FECD is currently the most common indication for this kind of surgery in the Western world. However, as an estimated 10 million people worldwide suffer from corneal blindness (awaiting corneal transplantation), and the prevalence of FECD is expected to rise with aging of the population, there is a need to develop alternative therapies. Therefore, we need a better understanding of the underlying molecular mechanisms of FECD. Significant efforts have been made to improve our understanding of the pathophysiology of FECD, but a unifying theory and tangible drug targets are lacking. This doctoral thesis aimed to gain new insights into the molecular pathogenesis of FECD, and to differentiate between processes that are specific for FECD (primary, causative factors) and processes that are nonspecific (e.g., secondary to corneal edema), to direct the future development of targeted drug therapy. To this end, we have analyzed the gene expression profile in CECs from patients with late-onset FECD, in comparison with normal controls (normal corneas from donors and eyes with malignant melanoma), and non-FECD edematous (NFE) corneas (including pseudophakic bullous keratopathy and decompensated corneal grafts). To study these gene expression profiles, we used microarray expression analysis, reverse transcriptase quantitative polymerase chain reaction, and RNA-sequencing technology. Subsequently, we have examined interesting findings at the protein level, using immunohistochemistry (IHC) and immunofluorescence on CE whole mount specimens and cross-sections of full-thickness corneas. Finally, we have measured the concentration of molecules of interest in the aqueous humor. Novel methodological elements in this project are: 1) the use of RNA-sequencing to study the gene expression profile of CECs with FECD, 2) the comparison with a third group of NFE corneas to differentiate between specific and nonspecific findings in FECD, 3) the development of a semi-automated computer algorithm to analyze IHC staining. The main achievement of this work is the identification of circulating fibrocytes and dendritic derivatives as a consistent finding in CE layers of patients with symptomatic late-onset FECD. This is novel, because, formerly, it was thought that the CE layer from patients with FECD contained only CECs. Circulating fibrocytes are bone marrow-derived, and play a role in normal wound healing, but also in pathogenic fibrosis.Furthermore, our data indicate that oxidative phosphorylation, mitochondrial dysfunction, decreased unfolded protein response, inhibition of the oxidative stress response, and downregulation of complement system genes play a specific role in the pathogenesis of FECD, whereas other processes, such as premature cellular senescence, extracellular matrix deposition, epithelial-mesenchymal transition, and the newly identified circulating fibrocytes are nonspecific elements (which also play a role in NFE corneas).Moreover, we have selected a set of 20 genes that are specifically differentially expressed in FECD versus normal and FECD versus NFE, including cochlin (COCH), olfactomedin like 1 (OLFML1) and scm-like with four mbt domains 2 (SFMBT2). These results will impact the research field, as they lay the foundations for a novel branch of research into the role of circulating fibrocytes in FECD and other CE disorders. Further research is needed to characterize the recruitment, differentiation, and function of circulating fibrocytes in FECD.Furthermore, patients with FECD might benefit from antifibrotic treatments that are being developed to treat fibrosing disorders of other organs, in which circulating fibrocytes are involved.Moreover, the new insights into primary and secondary processes in the pathogenesis of FECD, will help direct the future development of non-surgical therapies (for prevention, treatment, and prevention of progression of FECD). In the long run, this should alleviate the burden that FECD imposes on corneal tissue banks, and help reduce corneal blindness worldwide." "Immuno-sequencing of T-cells receptors in paediatric patients' synovial fluid to understand the role of HLA-B27 and T-cells in the pathogenesis of enthesitis-related juvenile idiopathic arthritis" "My Ha" "Centre for Health Economics Research and Modelling of Infectious Diseases (CHERMID)" "Juvenile idiopathic arthritis (JIA) is a heterogeneous group of autoimmune diseases that mainly affect the joints in children under the age of 16-18 years. It is the most common chronic arthritis in childhood with a prevalence of 16-150 in 100,000 children, in which enthesitis-related JIA (JIA-ERA) represents up to 20% of JIA cases. JIA-ERA has long been characterised by a strong association with HLA-B27, which is a major histocompatibility complex class I on the cell surface that is responsible for presenting antigenic peptides (derived from self and nonself antigens) to T-cells. However, it remains unclear how HLA-B27 contributes to the onset and pathogenesis of JIA-ERA. In cell-mediated autoimmunity, T-cells (including CD4+ and CD8+ T-cells) have been implicated in mediating many aspects of autoimmune inflammation. Among CD4+ T-cells, CD4+CD25+FOXP3+ regulatory T-cells (Tregs), which are crucial in preserving immune homeostasis, are believed to be dysfunctional in autoimmunity. Although Tregs were identified and analysed in different rheumatic diseases, their role is incompletely understood. There are also very few publications available that investigate the heterogeneous phenotypes and functions of T-cells at the inflamed site across different paediatric rheumatic diseases. Stemming from this dilemma, there are two research questions that I want to answer in this proposed study: (1) by which mechanism HLA-B27 confers its effects in JIA-ERA (i.e., molecular mimicry/arthritogenic peptide theory, free heavy chain theory, or protein misfolding theory) and how does HLA-B27 participate distinctly in JIA-ERA compared to other adult or juvenile arthritis; (2) what are the roles of different subsets of synovial T-cells in JIA-ERA pathogenesis and how does the T-cell receptor (TCR) profile of Tregs differ from those of conventional CD4+ and CD8+ T-cells in HLA-B27+ JIA-ERA patients' synovial fluid. To my knowledge, this will be the first (pilot) study that analyses the roles of HLA-B27 and T-cells in different types of JIA and compares JIA with other adult and juvenile arthritis. Modern (paediatric) rheumatology is increasingly evolving in a direction of pathology-specific and patient-specific diagnostics and therapy (i.e., personalised medicine). Better molecular stratification is thus an absolute necessity to improve patient care. A key transforming factor in (paediatric) rheumatology has been the application of sequencing techniques (e.g., microarray, Sanger sequencing, and next-generation sequencing) in assessing the TCR repertoire in blood cells or synovial fluid cells. Characterisation of TCR profiles is rapidly becoming an important complement to conventional immunophenotyping in understanding disease pathogenesis, prognosis, and response to treatment. Considering the research questions and state-of-the-art mentioned above, I believe that studying the TCR repertoire of synovial T-cells in HLA-B27+ patients will provide important knowledge of JIA-ERA."