BLT Stages

Abstract Bachelor Project 1 FBT 2021-2022: NKT cell activation by ER-stressed hepatocytes in the pathogenesis of NASH

Endoplasmic Reticulum (ER) stress is widely acknowledged to contribute to the development of inflammatory and autoimmune diseases. Lipids synthesized in the ER have been shown to bind cluster of differentiation 1d (CD1d) expressed on the surface of the antigen presenting cells (APCs) and to induce CD1d-specific natural killer T (NKT) cell responses. Upon activation, NKT cells can produce cytokines that play a role in either the amelioration or exacerbation of a range of diseases including non-alcoholic steatohepatitis (NASH).

Previous research has shown that ER stress in APCs promotes NKT cell activation via endogenous neutral lipids. The aim of the current project is to investigate the direct mechanistic link between APCs undergoing ER stress during NASH, lipid biogenesis, and the activation of CD1d-restricted NKT cells.

To study this, primary APCs such as hepatocytes will be isolated and an ER stress-driven NASH model will be induced in vitro. Secondly, it will be assessed whether neutral and/or polar lipid species generated in the ER stress-driven NASH model are capable of binding CD1d and of activating murine NKT cells in vitro by means of enzyme-linked immunosorbent assay (ELISA). Specific immunogenic lipids in NASH derived from cells undergoing ER stress will be isolated and quantified.

First, primary murine APCs such as hepatocytes are isolated. Two methods are compared: mechanical isolation and enzymatic isolation. The enzymatic isolation, which involves an additional perfusion step with collagenase, is found to produce a higher yield and viability of APCs. Second, NASH is induced in the APCs by adding free fatty acids such as oleic acid and palmitic acid. Confirmation of a successful induction is still needed by analyzing the expression of genes involved in the ER stress response with quantitative polymerase chain reaction. The APCs are then co-cultured with the NKT cell hybridoma cell line N38-2C12 and interleukin-2 (IL-2) production is measured with an ELISA. IL-2 production of N38-2C12 appears to be significantly higher after treatment with a certain compound. However, treatment with another compound results in negligible IL-2 levels. The positive control with thapsigargin is also not significant, with a p-value of 0,0513. Finally, the endogenous lipids produced by the APCs under pathological conditions are extracted using the Bligh and Dyer method.

Altogether, the results suggest that ER stress in hepatocytes drives NKT cell activation in the NASH disease. This is a promising step toward better understanding of the role of NKT cells in the disease pathogenesis. However, additional research is needed to support these findings.

Abstract Bachelor Project 2 FBT 2021-2022: Role of BHLHE40 in inflammatory arthritis

In Western society, one in four adults suffer from inflammation of the joints, or arthritis. This chronic disease triggers immune attacks that result in irreversible joint damage. Since arthritis lasts for decades, it has a serious impact on the patient's quality of life. In addition, existing treatments are effective in only 60% of patients and do not provide a cure. Only when the pathogenesis is thoroughly understood can an effective treatment be found.

The aim of this project can be divided into two parts. The first part is to investigate how the transcription factor BHLHE40 (in human) is controlled by force placed on in joint fibroblasts, and what role Bhlhe40 (in mice) plays in controlling the phenotype of the fibroblast. The second part is to investigate whether Bhlhe40 contributes to experimental arthritis in mice. This is done by looking at histology of the ankles from arthritic mice deficient for Bhlhe40.  

In a first fibroblast experiment, short interfering ribonucleic acid (siRNA) was added to the fibroblasts, which caused knockdown of BHLHE40. These cells were then stretched and their RNA was extracted immediately after stretching. With this RNA, quantitative real time polymerase chain reaction (qPCR) and western blot were performed to check whether the knockdown works properly. In a second fibroblast experiment, chemical components were added to the fibroblasts to activate or inhibit certain mechanotransduction pathways. After the cells were stretched, the RNA was extracted to perform a qPCR. The results of the qPCR indicated whether certain pathways regulate BHLHE40. To see if there is a difference in arthritis in knockout (KO) or wild type (WT) mice they were given arthritis by passively immunization with autoantibody containing KBxN serum. Seven days after this immunisation, the mice were killed and the decalcification of the ankles started. This made it easy to cut sections that were later stained and scored. 

BHLHE40 RNA was increased 1,5 times with stretching in vivo. The use of siRNA reduced the expression of BHLHE40 by 80 %. When using mechanotransduction chemical components, BHLHE40 levels under stretching and resting conditions could be changed. The biggest difference is visible with nuclear factor kappa B (NF-κB) inhibitors and a protein kinase C (PKC) activator, resulting in blocking of stretch-induced BHLHE40 expression and increasing of BHLHE40 expression in resting cells respectively. Furthermore, there is no significant difference between histological scores of arthritic KO and WT ankles as the p-value of the t-test is 0,1432. However, there is a strong correlation between the clinical and histological scores of the mice. The R-value determined using the Pearson r test is 0,7806. 

This study shows that the siRNA can effectively knockdown BHLHE40. This makes it possible to perform RNA sequencing to investigate whether BHLHE40 changes the phenotype of the fibroblast. It is also clear that NF-κB influences the stretch-induced expression of BHLHE40 and PKC the expression in resting cells. The histology shows that there is a difference between the KO mice and the WT mice, but this difference is not significant. There is a strong correlation between the histological and clinical scores.

Abstract Bachelor Project 1 FBT 2020-2021:  Validation of Endoplasmic Reticulum (ER) stress induced Natural Killer T-cell (NKT) ligand

Endoplasmic reticulum (ER) stress occurs when proteins are not properly folded or confirmed. To cope with the ER stress, the cell activates a signaling pathway called the unfolded protein response (UPR) to resolve the accumulation of misfolded proteins and restore homeostasis. The immune system utilizes distinct classes of lipids as antigens which binds glycoproteins called CD1d and activates specific immune cell called NKT cell. The aim of this experiment is to validate, this specific lipid antigen synthesized during ER stress.

In vitro assays are established wherein ER stress is induced in J774.2 cells and specific enzymes involved in synthesizing this lipid antigen are inhibited with Fumonisin B₁ or Myriocin in the presence of ER stress inducer Thapsigargin and then co-cultured with NKT cell hybridoma (2C12). The effects of  L-serine on this lipid antigen synthesis during ER stress is also addressed. The results are acquired through measurement of 2C12-secreted cytokines using ELISA. The effects of the inhibitors on CD1d surface expression in J774.2 are also tested. A quantitative Polymerase Chain Reaction (qPCR) is performed to determine the expression of genes involved in L-Serine synthesis during ER stress.

Acquired data suggests that the lipid antigen is synthesized during ER stress in J774.2 and can activate CD1d-restricted NKT cells. L-serine addition during ER stress resulted in no change in cytokine release and the qPCR results indicate that the tested genes involved in L-serine synthesis remain unaltered during ER stress. This suggests that L-serine is not involved in the lipid antigen synthesis during ER stress in J774.2.  

The pathway is thus validated leading to an insight into the creation of a novel NKT cell lipid antigen so that they can be used for modulating various diseases.

Abstract Bachelor Project 2 FBT 2020-2021:  The function of the protein of interest in IL-23 induced inflammation in mice

The protein of interest is highly expressed in the placenta and the prostate. It is elevated in blood in patients with diabetes and obesities, cancer, cardiovascular diseases, inflammation and pregnant women. It is already known that it induces weight loss by binding on its receptor, but not much in other pathologies. Interleukin (IL)-23 is a pro-inflammatory cytokine which will activate many immune cells and other cytokines to induce inflammation and auto-immune diseases. By injecting mice with mIL-23 Enhanced Episomal Vector (IL-23 EEV), it is possible to further elucidate the role of the protein of interest in inflammation. This research could contribute to finding new pathological pathways and treatments for auto-immune diseases. By injecting knock-out (KO) mice and wildtype (WT) littermates with IL-23 EEV, they will develop uveitis and dermatitis. Over 28 days the mice will be scored clinically for inflammation and weighed, after which they will be sacrificed for organs to be collected. The adipose tissues will be weighed to determine whether the mice burned fat to produce energy. The ear, colon, and visceral adipose tissue will be processed for histology to quantify inflammation. As a control, serum will be taken to quantify the IL-23 levels by using enzyme linked immuno sorbent assay (ELISA). Lastly, the end of the tail will be collected to double check the genotype of the mice by using polymerase chain reaction (PCR). The PCR results matched the genotype when done the first time. The results of the ELISA has shown that only group 1 has received the correct dose of IL-23 EEV which means that the IL-23 levels of group 2 and 3 were too low to develop a clear phenotype in all read-outs. Therefore there is no difference in scores of inflammation between the KO mice and the WT mice. However group 2 and 3 follow the same trend as group 1 but can’t be proven by aid of statistics, that there’s a significance and has a role in inflammation. There is less adipose tissue in the WT mice with IL-23 than KO-mice, which means that the protein of interest is responsible for the burning of fat to produce energy. Finally the WT-mice lose more weight than the KO-mice, which proves that the protein of interest is responsible for the weight loss and is induced in inflammation. The protein of interest appears to induce weight loss in mice during the inflammation process. Because of the low concentration of IL-23, the mice didn’t develop severe inflammation. That’s why this study will need to be repeated with the correct dose of IL-23 EEV.

Abstract Bachelor Project 1 FBT 2019-2020: Effect of modulation of gut microbiota in spondyloarthritis

The aim of this project was to unravel the role of the gut microbiota in spondyloarthritis disease. To achieve this, two different experiments were set up. For the first experiment, SKG mice were given either antimycotic (fluconazole) or antibiotic (amoxicillin) treatments to better understand their impact on SpA progression, as to validate the findings of a previous experiment using the TNFdare mouse line. By scoring the clinical manifestations during the growth of these mice as well as histological scoring, the influence of these microbial lifeforms on SpA is checked. Ankle and ileum samples were collected, and ankle samples were processed to slides for the scoring. The resulting raw data was statistically analysed. This analysis concluded that neither treatment proved to have a significant influence on disease progression for the SKG mice. The second experiment focused on Dialister invisus and Prevotella copri, two important biomarkers in human SpA disease. The aim of this second experiment was to try and colonise the gut of mice with D. invisus and P. copri to lead the way to future testing on these bacteria in vivo. A dysbiosis was created in the gut of the mice before administering the bacteria by gavage, and DNA extracted from stools was checked using qPCR to determine if the colonisation was successful. Primers used in the qPCR turned out to be insufficiently specific and precise to measure the colonisation if it did succeed in a small quantity. In conclusion, further testing is required to validate the findings of the SKG mice experiment, to find out if there are possible genetical differences or different gut microbiota that cause these mice to react to the treatment differently than the TNFdare mice. Further and more in depth sequencing of the bacterial DNA extracted from the stools used in experiment 2 will need to be performed to find out the influence of these bacteria on SpA disease in the mouse model.

Abstract Bachelor Project 2 FBT 2019-2020: Study of the evolution of a TGF-β superfamily member in serum from different mouse models for osteoarthritis and rheumatoid arthritis

Rheumatoid arthritis and osteoarthritis are most common in the Western society and are clearly on the rise. Therefore, it is important to unravel the mechanisms of inflammation and immunity for these diseases in order to offer better prevention and recovery for the patients. In a previous study it was shown that mice lacking a transforming growth factor beta (TGF-β) superfamily member become sicker in a cartilage model when compared to wildtype littermates. On the other hand, the same type of mice become less ill in an inflammation model. These findings show that this cytokine may play a role in both diseases. In this study, the course of the TGF-β superfamily member is studied in three different mouse models, namely a surgical model of destabilization of the medial meniscus, a collagen induced arthritis model and a collagen antibody induced arthritis model. The first model is a cartilage model and the latter two are inflammation models. To measure the concentration of the cytokine, an Enzyme Linked Immuno Sorbent Assay (ELISA) is used. On the obtained concentrations, a number of statistical tests are performed where the majority of the results are without significant difference. Because of this, it is not possible to assign a specific function to the TGF-β superfamily member. In the cartilage model, this cytokine may fulfill a protective role. In the two inflammation models, this member could still play a role in the synovial inflammation, as relevant trends are observed. As expected, the cytokine levels correlate with the age of the mice. 

Abstract Bachelor Project 1 FBT 2018-2019: Research into the role of a TGFb superfamily member in osteoporosis

Preliminary studies indicate that a transforming growth factor beta (TGFb) superfamily member might play a role in cartilage and bone erosion in the context of arthritis, yet it remains unknown whether these effects are also related to osteoporosis. Some mice studies have suggested that osteoporosis has an inhibitory effect on osteoclasts which goes against the conclusions of other studies that suggest it has an activating effect on osteoclasts. The purpose of this research is to determine the potential role of the transforming growth factor beta (TGFb) superfamily member in the context of osteoporosis using the ovariectomy osteoporosis mouse model. Knock out (KO) mice and control wild type (WT) mice underwent ovariectomy or SHAM surgery to induce postmenopausal osteoporosis. A successful surgery was confirmed by histology of the ovaries and a weight follow-up to evaluate menopause. Six weeks post-surgery, bone strength was tested using a femur bending test. Also, bone geometry was analyzed using computed tomography (CT) scans. Lastly, two bone resorption regulating cytokines were measured using Enzyme Linked Immuno Sorbent Assay (ELISA). The femur bending test indicates that the bone, originating from the osteoporosis mouse model (OVX), breaks at a significantly lower force compared to the SHAM mice. Moreover, the µCT-analysis indicates that cortex volume, trabeculae volume and bone density are significantly higher among the OVX groups compared to the SHAM groups. In both experiments, the KO mice and the WT mice show no significant difference. Beyond that, the ELISA quantification of two bone resorption regulating cytokines in serum shows a not significant trend. For this reason the ELISA experiment still needs extra optimization. In general, the osteoporosis mouse model shows a recurring trend in which both bone geometry and bone strength are significantly reduced compared to the control mice. Additionally, KO mice show no significant difference compared to the control mice.

Abstract Bachelor Project 2 FBT 2018-2019: Optimization of the in vitro activation of primary synovial fibroblasts using Western blot

Rheumatoid arthritis (RA) is a chronic autoimmune disease that affects approximately 1% of the world population. In this disease, many signaling pathways are aberrantly activated, leading to auto-reactive T-cells and autoantibodies. One of these pathways is the nuclear factor-kappa B (NF-kB) pathway, which is of high interest in the study for new therapies for RA. Besides T-cells and autoantibodies, synovial fibroblasts are thought to be central players in the disease. This cell type has a destructive potential that is boosted by inflammatory cytokines such as tumor necrosis factor-a (TNF-a), interleukin (IL)-1b and IL-17.

The ultimate goal of this project is to study the role of specific regulatory mechanisms of the NF-

kB pathway in synovial fibroblasts. More specifically, NF-kB activation will be compared between synovial fibroblasts of wild type (WT) mice and those of transgenic mice of which a regulatory signaling protein is mutated. However, the optimal conditions for NF-kB activation needed to be determined first and a pilot experiment was performed. The latter was done using synovial fibroblasts of WT mice that were stimulated with several RA-associated cytokines such as TNF-a, IL-1b and IL-17. The activation of the NF-kB pathway was then assessed via Western blotting.

Analysis of the immunoblots for several signaling proteins showed no clear activation of the NF-

kB pathway for the chosen conditions. Although some degradation was observed for the signaling protein IkBa, an indication for NF-kB activation, it could not be confirmed by the presence of its phosphorylated form. It was not clear whether the detection of the phosphorylated proteins was unsuccessful or whether no activation had taken place. Overall, no conclusions could be made since there was no positive control to compare to. Therefore, the experiment needs to be repeated with a positive control and an optimized protocol for the detection of phosphorylated proteins.

Abstract Bachelor Project 3 FBT 2018-2019: Role of hypoxia in rheumatoid arthritis

Rheumatoid arthritis (RA) is one of the most prevalent autoimmune condition that affects nearly 1 % of the world population. RA is an autoimmune disease that manifests itself by inflammation in various joints. Studies showed that the absence of oxygen plays an important role within the disease. Hypoxia is involved in different processes that also characterize RA such as inflammation, cartilage degradation and angiogenesis. The precise mechanism of hypoxia within RA is still to be defined. Recent studies have already revealed the importance of hypoxic genes that could be part of causing RA. In this thesis, ELISA and flow cytometry techniques were used to determine the function of specific immune cells. These techniques will give information about the cytokine secreting activity of cells: the number of inflammatory cytokines producing positive cells was measured by flow cytometry and the amount of secreted cytokine by ELISA. The immune cells and secretion capacity of inflammatory cytokines by these specific immune cells were analysed from wild type mice and specific hypoxic gene knock-out mice. The obtained results showed a possible link between the hypoxic genes and RA. Both experiments conducted to the same conclusion namely that the hypoxic gene could have an impact in developing RA. Further research is still needed to confirm this statement.

Spondyloarthritis (SpA) consists of a varied group of chronic immune-mediated diseases in which inflammation occurs mainly axially and/or peripherally, particularly in the larger joints of lower limbs, e.g. Achilles tendon in the ankle. The origin of the inflammation is the enthesis, the attachment site of tendon to bone, which can cause development of SpA in predisposed persons.

The aim of this research project is to optimize different techniques, necessary to investigate the role of biomechanical stress in the development of SpA and to further validate one of the mouse models used to study the effect of biomechanical stress in SpA.

One of the techniques used within the larger research project are running wheel and tail suspension, were mice are either subjected to higher or lower biomechanical load than in normal housing conditions. Until now we made use of voluntary running wheels combined with tail suspensions, however, certain transgene strains run significantly less compared to their wild littermate controls. Therefore, optimization of forced running wheel will be performed and evaluated A20^myeloid-KO- and/or A20^stromal-KO-mice, strains used in SpA-research.

Results showed that both strains perform well on the running band and that alleviation of biomechanical strain (tail suspension) seems to lower arthritis score in A20^stromal-KO-mice. Chondrocyte cultures are optimized to be used in in vitro stretch-experiments that mimic the continuous force chondrocytes undergo at high impact sites, e.g. ankle joint.

Next to that, one of the mouse models used, A20^stromal-KO, needs further validation to ensure that A20-deletion only happens in stromal cells of the joint and not in the hematopoietic compartment. For this, bone marrow transplants are optimized and western blot for the detection of A20 is tested. Both techniques still need further optimization.

Spondyloarthritis (SpA) is a group of rheumatic diseases. Ankylosing spondylitis, as prototype, is an auto-inflammatory immune disease. Inflammation occurs in the spine and/or the larger joints of the lower body such as knees, ankle,…. In addition to arthritis, extra-articular symptoms can occur: acute anterior uveitis, psoriasis and inflammatory bowel disease (IBD). About 50% of the SpA patients develop subclinical intestinal inflammation and a part of the patients with Crohn’s disease develop arthritis. Clearly, there is a link between joint and gut inflammation, however, up till now no mechanistic explanation has been found.

The aim of this project is to optimise different techniques that will enable the research into this joint-gut axis. As a model for combined joint-gut inflammation, a human TNF-transgene mouse model will be used. These mice overexpress hTNF in the gut and as a result develop inflammation of the sacro-iliac joint. This model provides a nice research tool to study the anatomical and functional link between joint and gut. More specifically, how the lymphatic and immunological compartment might contribute to development of disease. 

In the first part of this study, digestion of sacro-iliac joints for cell isolation for flow cytometry was optimised. Since the use of different enzymes in the digestion mix might result in ‘shaving’ of markers, i.e. the digestion of markers necessary for antibodies to bind, this was tested on spleen cells from which we know certain cell populations have to be present. Afterwards, a try-out to compare transgene and wild type mice was performed.

To determine the presence, amount and anatomical variation of lymph vessels in transgene versus wild type mice,  optimization of immunohistochemically staining of Prox-1 and Lyve-1 and visualization of the mesentery was performed. Additionally, primers were designed to determine the relative gene expression of Flt4, Prox-1 and VEGF-C in different tissues.

To be sure hTNF is present on the protein level in the gut, digestion of gut samples and western blotting for hTNF was optimized.