Training at MIR
Medical Inflammation Research (MIR) at Karolinska Institutet, Sweden, will offer doctoral and post-doctoral training in using animal models for studies of rheumatoid arthritis and multiple sclerosis. This training is based on a broad integrated disciplinary approach with the aim of initiating a better understanding of the pathogenic pathways leading to autoimmune inflammatory disease in general. The long-term objectives are to reach a more fundamental basis for better diagnosis and treatment in these disabling and enigmatic diseases affecting a large part of the European population.
Courses
We also give the doctoral course "Multifactorial immune mediated diseases – etiology and pathogenesis"
The course includes an overview on chronic immune mediated multifactorial diseases and the challenge they represent to medicine. It covers topics such as epidemiology, clinical characteristics, suggested or known disease mechanisms and genetics including experimental animal models available to study such multifactorial and polygenetic diseases and bioinformatics tools available to analyze results. Key concepts and techniques within each topic will be introduced.
Student projects at Medical Inflammation Research
Available student projects:
I) Characterisation of MHC class III regulation in experimental arthritis
Background and Aim: A number of human genetic association studies have suggested that, in addition to the HLA-DRB1 locus within the major histocompatiblity complex (MHC), at least one other susceptibility gene for rheumatoid arthritis (RA) could also exist in the MHC class III region. This region consists of many genes with important immune functions; and polymorphisms in MHC class III have been associated to susceptibility to or severity of RA and different responses to drugs in RA patients in various studies. However, MHC class III has been difficult to study in human, mainly due to the strong linkage disequilibrium and high gene density of the region. This project takes an alternative approach to tackle this question by isolating congenic fragments covering part of MHC class III in rats and use these congenic fragments to study the association of MHC class III in animal models of RA, such as oil-induced arthritis and pristane-induced arthritis. By both minimizing the congenic fragments and performing different in vivo and in vitro experiments, we aim to identify the genes and pathways involved in arthritis development.
Methods: Disease phenotyping of congenic lines for different models of autoimmunity, High throughput genotyping using microsatellite markers, Sanger sequencing of candidate genes, Quantitative real-time PCR for gene expression, ELISA, Western blot, Flow cytometry, RNA extraction, cDNA synthesis, Bioinformatic analysis of sequence comparison.
II) Tracking antigen-specific T cells during the development of autoimmune collagen-induced arthritis using polychromatic flow cytometry (FACS)
Project: Collagen-induced arthritis (CIA) is an animal model of rheumatoid arthritis. This model is dependent on both B- and T cells, as mice lacking either population are resistant to CIA. T cells plays an important role in the early phase of disease development by giving help to B cells in producing antibodies that can initiate clinical symptoms. However, the role for T cells in the subsequent phases are less clear.
In the present project we will make use of our newly established models where we can identify and follow antigen-specific T cells by flow cytometry in order to determine the role of these at the different stages of disease development.
Methods: Polychromatic flow cytometry, animal models of rheumatoid arthritis, cell cultures, ELISA, ELISPOT, PCR.
III) Generation and Characterization of Rheumatoid Factors against ACPAs and Anti-Collagen antibodies
Background: A classical hallmark of arthritis is the development of rheumatoid factors. These are antibodies binding other antibodies, predominantly on the Fc part. Their origin and role is still not known but it has been shown that raised levels of RF precedes clinical onset of RA. In addition, it has recently been found that certain neo-epitopes, formed by citrullination of arginins, are common epitopes for autoantibodies in RA, these antibodies also precedes RA diagnosis. Another class of antibodies are those directed toward cartilage proteins and the most commonly studied antibodies are directed towards type II collagen (CII). These antibodies are occurring in approximately 30 % of RA patients.
Project: As mentioned above, pathogenic/protective role of Rheumatoid factors (RFs) in RA is not yet clearly ascertained. In this project, we will generate and characterize rheumatoid factors against anti-citrllinated protein/peptide monoclonal antibodies (ACPAs) and anti-collagen monoclonal antibodies to understand the pathogenic/protective role of RFs.
Methods: Monoclonal antibody generation and characterization, sequencing, antibody purification, immunoassays, collagen induced arthritis (CIA) and collagen antibody induced arthritis (CAIA).
IV) Epigenetic regulation of experimental arthritis (2 projects)
Recent successes of therapeutic intervention of chronic inflammatory diseases using epigenetic modifiers such as histone deacetylase inhibitors and inhibitors of DNA methylation suggest that epigenetic reprogramming plays a role in the aetiology of these diseases. However, the disease mechanisms driving epigenetic modulation in chronic inflammation are poorly understood. Therefore, identifying disease specific epigenetic marks in the genome would provide excellent clues to disease pathways as well as serve as potential therapeutic targets. We are focusing on an experimental form of rheumatoid arthritis and aim to identify such arthritis regulating epigenetic modifications in the rat genome and the genes that are affected.
Methods 1- differential gene expression:
The student will be using qRT-PCR to quantify altered gene expression in healthy and arthritic rat cells treated with epigenetic modifiers. Flowcytometry and ELISA will be used to identify cellular characteristics of the cells involved in disease development.
Methods: 2 -differential DNA methylation:
The student will use immune precipitation to enrich for epigenetic regulated genomic regions. qRT-PCR and bisulphite sequencing will be used to assess the levels of epigenetic modification of specific genomic regions.
V) Identification of Foxp3+ regulatory T cell suppressive mechanisms in arthritis.
CTLA-4 is a negative regulator of T cell activation that is constitutively expressed by Foxp3+ regulatory T cells (Tregs) and is found to be a critical mediator of Treg-mediated suppression. Treg-specific deletion of CTLA-4 in wild type BALB/c mice resulted in fatal myocarditis and gastritis. However, the role of CTLA-4 on other genetic backgrounds and in other diseases could be different and should be tested. This project will investigate the role of CTLA-4 expression on T cells and Tregs in collagen induced arthritis in an antigen-specific manner and a time-specific manner.
Methods: In vitro and in vivo assays characterizing and comparing CTLA-4 deficient Tregs with wild type. This work will include sterile bench work, cell purifications, flow cytometry, ELISA, histology, immunization of mice and disease scoring. This project involves work with advanced models of genetically modified mice and includes screening with PCR and Taqman probes.
VI) Identification of pathogenic pathways controlled by a spontaneous mutation controlling arthritis in the rat
Background: A spontaneous mutation on chromosome 9 in the DA rat strain has been discovered and we now aim to identify this gene and study its pathogenic importance
Aim: The aim of the project is to narrow down the size of the congenic fragment and eventually identify the gene(s) causing the phenotype in these rats. Several candidate genes involved in immunological processes have been suggested and the goal is to sequence them and identify the genetic polymorphism leading to complete protection to arthritis induction.
Methods: Analysis and recombinational selection of already established congenic rats with fragments of variable length on chromosome 9. Genotyping with microcapillary electrophoresis, PCR, DNA purification, DNA sequencing with microcapillary electrophoresis, SNP typing using TaqMan.
VII) Identification and phenotyping of T cells undergoing tolerance induction towards self-type II collagen in an animal model of rheumatoid arthritis
Project: T cells should recognize and respond to invading organisms and other pathogens, but should not respond to the body’s own tissues. The process by which T cells learn to discriminate between self and non-self is known as immunological tolerance, and failure to establish T cell tolerance to self-tissue may lead development of autoimmune diseases.
In the present model we will use a new animal model where it will be possible to investigate in detail how T cells interact with its own cartilage in order to establish immunological tolerance to self. We will also investigate mechanisms whereby these processes can fail and how this can lead to development of autoimmune disease.
Methods: Polychromatic flow cytometry, adoptive transfer experiments, labeling of cells, animal models of rheumatoid arthritis, cell cultures, ELISA, ELISPOT, PCR.
VIII) Identification of the arthritis-regulating gene in APLEC
Liselotte Bäckdahl (liselotte.backdahl @ ki.se)
Previous genetic studies in experimental arthritis have identified a complex of C-type lectin receptors that are expressed on antigen presenting cells to be regulating arthritis development in the rat. The complex, called APLEC, contains 7 genes and is ~500 mega bases in size. In order to identify the disease regulating gene in the complex a number of already established APLEC recombinant congenic strains will initially be tested for in vivo arthritis development. Following disease evaluation the recombinants will be assessed for quantitative expression of the APLEC encoded genes to ascertain the expression levels associated to disease development. The APLEC recombinants will also be analysed for characteristic cellular immune phenotypes.
Methods: The student will use qRT-PCR to quantify the expression levels of the 7 APLEC genes before and after arthritis induction in the APLEC recombinants. Flowcytometry will be used to identify cellular phenotypes associated with disease regulation in the APLEC complex.
IX) The influence of oxidation on regulatory T cells in arthritis.
It has been recently found that oxidative burst is a regulator of arthritis but the mechanism has not been elucidated. Mice that are mutated in the Ncf1 gene (a gene coding for the activator protein in the NADPH oxidation complex) have a lowered oxidative burst but a more severe collagen-induced arthritis (CIA). This project will be investigating if regulatory T cells are affected by this mutation. Furthermore, the project will be involved in investigating the role of regulatory T cells in established arthritis.
Methods: In vitro generation, expansion or depletion of regulatory T cells from Ncf1 mutated and wild type mice. In vitro and in vivo assays comparing these regulatory T cells. The project will therefore involve sterile bench work purifying different cell types, analysis by Flow cytometry, proliferation assays and ELISA as well as in vivo cell transfers and immunization of mice and scoring of arthritis. Furthermore, mice will be breed and screened for Ncf1 mutation by PCR and Taqman probes.
X) Studies of the role of rheumatoid factor in the rat
Project: We have recently identified a strong candidate gene associated with production of rheumatoid factor. Thus, for the first time we will have the possibility to investigate the origin and role of rheumatoid factors using relevant models for rheumatoid arthritis. Already established congenic strains will be tested for various diseases (arthritis, allergy) and various immune tests. We will also identify the B cells producing the rheumatoid factors.
Methods: Analysis and recombinational selection of already established congenic rats. Testing of arthritis and asthma models in the rats. Cellular immunologic assays and subphenotyping using flow cytometry (FACS). Analysis of antibody responses with ELISA and western blot. Sequence analysis of antibody V genes.
XI) Genetic polymorphism in the DA.E3-Pia67 congenic rat leads to delayed migration of melanocytes and mast cells.
Project: We have identified SNPs in the c-kit gene that leads to a delayed migration of melanocytes in the E3 rat causing the hooded coat colour phenotype. Recent studies using c-kit mutated mice have now lead us to believe that this delay is also affecting migration of mast cells. So far we have identified 200 SNPs in the introns of the c-kit gene. To locate the causative SNP we now aim to sequence closely related rat strains.
Methods: Testing asthma models in the rats, PCR, DNA purification, DNA sequencing with microcapillary electrophoresis, SNP typing using TaqMan.
Medical Inflammation Research (MIR) 