UZ Gent afdeling reumatologie (labo voor moleculaire immunologie)
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 A20myeloid-KO- and/or A20stromal-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 A20stromal-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, A20stromal-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.
De Pintelaan 185
Prof. Dr. D. Elewaut
Renée Van der Cruyssen