Genome-Wide Screen for Differential DNA Methylation Associated with Neural Cell Differentiation in Mouse

Cellular differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Using Differential Methylation Hybridization (DMH) in combination with a custom DMH array containing 51,243 features covering more than 16,000 murine genes, we carried out a genome-wide screen for cell- and tissue-specific differentially methylated regions (tDMRs) in undifferentiated embryonic stem cells (ESCs), in in-vitro induced neural stem cells (NSCs) and 8 differentiated embryonic and adult tissues. Unsupervised clustering of the generated data showed distinct cell- and tissue-specific DNA methylation profiles, revealing 202 significant tDMRs (p<0.005) between ESCs and NSCs and a further 380 tDMRs (p<0.05) between NSCs/ESCs and embryonic brain tissue. We validated these tDMRs using direct bisulfite sequencing (DBS) and methylated DNA immunoprecipitation on chip (MeDIP-chip). Gene ontology (GO) analysis of the genes associated with these tDMRs showed significant (absolute Z score>1.96) enrichment for genes involved in neural differentiation, including, for example, Jag1 and Tcf4. Our results provide robust evidence for the relevance of DNA methylation in early neural development and identify novel marker candidates for neural cell differentiation

The cathelicidins LL-37 and rCRAMP are associated with pathogenic events of arthritis in humans and rats

Background: In rheumatoid arthritis (RA), neutrophil granulocytes fuel inflammation and damage tissue in the joint by releasing cytotoxic agents, antimicrobial peptides, proteases and other inflammatory mediators. The human cathelicidin LL-37 has recently been implicated in the development of systemic lupus erythematosus and psoriasis. Objective: To elucidate if antimicrobial peptides (AMPs) contribute to the pathogenesis of arthritis. Methods: Expression of LL-37 was determined in synovial membranes from patients with arthritis and control subjects. Expression of the rat cathelicidin rCRAMP and defensins was characterised in joints, blood and secondary lymphoid organs during pristane-induced arthritis (PIA) in rats and in a transfer model of PIA induced by CD4 T cells. Serum samples of rats with arthritis were tested for IgG and IgM autoantibodies against rCRAMP by immunoblot and for interferon (IFNα) by ELISA. Results: Cathelicidins are strongly upregulated in RA synovial membranes and in joints from rats with arthritis as compared with healthy joints. Expression was most prominent in neutrophil granulocytes and macrophages/osteoclasts. Cathelicidin expression is also upregulated in the blood and spleen of pristane-injected rats, with strongest expression detected in activated CD62L− cells coexpressing granulocyte and monocyte markers. Pristane injection caused accumulation of low-density granulocytes in the blood. After pristane injection, the increased expression of rCRAMP coincided with higher levels of cell death, raised levels of interferon (IFN)α and development of autoantibodies. Conclusions: Our results show strong upregulation of cathelicidins and β-defensins coinciding with pathological events of arthritis. Higher expression and release of AMPs might contribute to development and/or maintenance of disease by systemic or local mechanisms

Generation of a genomic tiling array of the human Major Histocompatibility Complex (MHC) and its application for DNA methylation analysis

Background: The major histocompatibility complex (MHC) is essential for human immunity and is highly associated with common diseases, including cancer. While the genetics of the MHC has been studied intensively for many decades, very little is known about the epigenetics of this most polymorphic and disease-associated region of the genome.Methods: To facilitate comprehensive epigenetic analyses of this region, we have generated a genomic tiling array of 2 Kb resolution covering the entire 4 Mb MHC region. The array has been designed to be compatible with chromatin immunoprecipitation (ChIP), methylated DNA immunoprecipitation (MeDIP), array comparative genomic hybridization (aCGH) and expression profiling, including of non-coding RNAs. The array comprises 7832 features, consisting of two replicates of both forward and reverse strands of MHC amplicons and appropriate controls.Results: Using MeDIP, we demonstrate the application of the MHC array for DNA methylation profiling and the identification of tissue-specific differentially methylated regions (tDMRs). Based on the analysis of two tissues and two cell types, we identified 90 tDMRs within the MHC and describe their characterisation.Conclusion: A tiling array covering the MHC region was developed and validated. Its successful application for DNA methylation profiling indicates that this array represents a useful tool for molecular analyses of the MHC in the context of medical genomics

Genetic dissection of experimental arthritis in the DA rat

Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting peripheral joints. Persistent inflammation causes cartilage deterioration with severe joint deformations as a consequence. The etiology is largely unknown but complex interactions between genetic and environmental factors contribute to the disease. The disease is also clinically heterogeneous which further hampers etiological investigations. Analogs of certain disease pathways can be studied in experimental models mimicking RA. A more thorough characterization of such disease pathways may tell us of discrete disease subsets of RA. Numerous experimental models for RA exists, both spontaneous and induced. The DA rat is remarkably arthritis prone, an intradermal injection of a mineral oil is sufficient to induce arthritis. The difference in arthritogenicity between the arthritis inducers depends on the genetic susceptibility threshold of each rat strain. The general aim of this thesis was to by a genome - inducer approach identify a suitable experimental system to use for mapping arthritis susceptibility genes in the rat, and through this approach identify genes of equal importance in humans, ultimately to provide new insights for pathway characterization of RA. I characterized the arthritis susceptibility in a set of recombinant congenic strains overlapping several arthritis regulating regions on rat chromosome 4, in five arthritis models; collagen type H-induced arthritis, pristane-induced arthritis, mycobacteria-induced arthritis, squalene-induced arthritis and oil-induced arthritis. All five induced arthritis-models were regulated by chromosome 4 genes. A 10 cM fragment that harbor the Oia2 locus mediated arthritis down-regulation in collagen type H induced arthritis and squalene-induced arthritis. Oil-induced arthritis was completely prevented. Further fine mapping was continued in oilinduced arthritis. By using 18 Oia2 intra-recombinant congenic strains the arthritis regulating interval was fine-mapped to 1.2Mb. The arthritis regulating interval was further mapped to 600 kb that only harbor a Ctype lectin gene complex denoted Aplec. Comparison of gene sequences identified a nonsense mutation in Dcar1 in the DA strain as the most possible arthritis-regulating rat gene. The human homolog to rat Dcir, one of the other genes in the complex, was tested in a patient 1 control material. One SNP showed significant association to RA. Association was pronounced in RF-negative patients The genome - inducer approach was also applied in mapping of the arthritis regulating region, Oia3, on rat chromosome 10 in the F7 generation of an advanced intercross (AIL) between the arthritis susceptible rat strain DA, and the arthritis resistant PM I AV I. To chose the most appropriate arthritis model for linkage mapping in the AIL pristane-induced arthritis, squaleneinduced arthritis and oil-induced arthritis was induced. Pristane-induced arthritis was the most appropriate for the population. Aplec and the newly identified arthritis regulating gene Ncf1 was also mapped to determine the mapping resolution. Linkage mapping of Oia3 identified two distinct quantitative trait loci (QTL), one at D10Rat13 at 97.2Mb, and an other at D10Got158 at 105.2Mb. The placement for the Ncf1 gene, and the Aplec were both less than 100kb, 200kb surrounding the Oia3 peak marker was considered as the confidence interval. The proximal Oia3 QTL contains the Protein kinase C alpha gene together with a set of calcium channel voltage-dependent gamma subunit genes. The distal Oia3 QTL contain a cluster of dendritic cell derived Ig-like receptors, among them the homolog to the human CMRF35 gene previously associated to psoriasis. In conclusion the search for appropriate experimental systems to map arthritis susceptibility regions, subsequent congenic mapping in oil induced arthritis, lead to the identification of Aplec, a C-type lectin complex, that codes for genes important in a number of immunological processes. The human homolog to rat showed association to RA in a patient/control material

Identification of Clec4b as a novel regulator of bystander activation of auto-reactive T cells and autoimmune disease.

The control of chronic inflammation is dependent on the possibility of limiting bystander activation of autoreactive and potentially pathogenic T cells. We have identified a non-sense loss of function single nucleotide polymorphism in the C-type lectin receptor, Clec4b, and have shown that it controls chronic autoimmune arthritis in rat models of rheumatoid arthritis. Clec4b is specifically expressed in CD4+ myeloid cells, mainly classical dendritic cells (DCs), and is defined by the markers CD4+/MHCIIhi/CD11b/c+. We found that Clec4b limited the activation of arthritogenic CD4+αβT cells and the absence of Clec4b allowed development of arthritis already 5 days after adjuvant injection. Clec4b sufficient CD4+ myeloid dendritic cells successfully limited the arthritogenic T cell expansion immediately after activation both in vitro and in vivo. We conclude that Clec4b expressed on CD4+ myeloid dendritic cells regulate the expansion of auto-reactive and potentially pathogenic T cells during an immune response, demonstrating an early checkpoint control mechanism to avoid autoimmunity leading to chronic inflammation

Genetic dissection of a major haplotype associated with arthritis reveal FcγR2b and FcγR3 to act additively

A haplotype with tightly linked Fc gamma receptor (FcγR) genes is known as a major locus controlling immune responses and autoimmune diseases, including arthritis. Here, we split a congenic fragment derived from the NOD mouse (Cia9) to study its effect on immune response and arthritis in mice. We found that arthritis susceptibility was indeed controlled by the FcγR gene cluster and a recombination between the FcγR2b and FcγR3 loci gave us the opportunity to separately study their impact. We identified the NOD-derived FcγR2b and FcγR3 alleles as disease-promoting for arthritis development without impact on antibody secretion. We further found that macrophage-mediated phagocytosis was directly correlated to FcγR3 expression in the congenic mice. In conclusion, we positioned FcγR2b and FcγR3 alleles as disease regulatory and showed that their genetic polymorphisms independently and additively control innate immune cell activation and arthritis

Genetic dissection of a major haplotype associated with autoimmune disease, FcγR2b and FcγR3 act additively

A haplotype with tightly linked Fc gamma receptor (FcγR) genes is known as a major locus controlling immune responses and autoimmune diseases, including arthritis. Here, we split a congenic fragment derived from the NOD mouse (Cia9) to study its effect on immune response and arthritis in mice. We found that arthritis susceptibility was indeed controlled by the FcγR gene cluster and a recombination between the FcγR2b and FcγR3 loci gave us the opportunity to separately study their impact. We identified the NOD‐derived FcγR2b and FcγR3 alleles as disease‐promoting for arthritis development without impact on antibody secretion. We further found that macrophage‐mediated phagocytosis was directly correlated to FcγR3 expression in the congenic mice. In conclusion, we positioned FcγR2b and FcγR3 alleles as disease regulatory and showed that their genetic polymorphisms independently and additively control innate immune cell activation and arthritis

Glycan Activation of Clec4b Induces Reactive Oxygen Species Protecting against Neutrophilia and Arthritis

Animal models for complex diseases are needed to position and analyze the function of interacting genes. Previous positional cloning identified Ncf1 and Clec4b to be major regulators of arthritis models in rats. Here, we investigate epistasis between Ncf1 and Clec4b, two major regulators of arthritis in rats. We find that Clec4b and Ncf1 exert an additive effect on arthritis given by their joint ability to regulate neutrophils. Both genes are highly expressed in neutrophils, together regulating neutrophil availability and their capacity to generate reactive oxygen species. Using a glycan array, we identify key ligands of Clec4b and demonstrate that Clec4b-specific stimulation triggers neutrophils into oxidative burst. Our observations highlight Clec4b as an important regulator of neutrophils and demonstrate how epistatic interactions affect the susceptibility to, and severity of, autoimmune arthritis

Neuroendocrine profile in a rat model of psychosocial stress: relation to oxidative stress

Psychosocial stress alters the hypothalamic-pituitary-adrenal axis (HPA-axis). Increasing evidence shows a link between these alterations and oxidant elevation. Oxidative stress is implicated in the stress response and in the pathogenesis of neurologic and psychiatric diseases. NADPH oxidases (NOXs) are a major source of reactive oxygen species (ROS) in the central nervous system. Here, we investigated the contributory role of NOX2-derived ROS to the development of neuroendocrine alterations in a rat model of chronic psychosocial stress, the social isolation

Neuroendocrine profile in a rat model of psychosocial stress: relation to oxidative stress

Psychosocial stress alters the hypothalamic-pituitary-adrenal axis (HPA-axis). Increasing evidence shows a link between these alterations and oxidant elevation. Oxidative stress is implicated in the stress response and in the pathogenesis of neurologic and psychiatric diseases. NADPH oxidases (NOXs) are a major source of reactive oxygen species (ROS) in the central nervous system. Here, we investigated the contributory role of NOX2-derived ROS to the development of neuroendocrine alterations in a rat model of chronic psychosocial stress, the social isolation