The retinoid-related orphan receptor alpha is essential for the end-stage effector phase of experimental epidermolysis bullosa acquisita.

Genetic studies have added to the understanding of complex diseases. Here, we used a combined genetic approach for risk-loci identification in a prototypic, organ-specific, autoimmune disease, namely experimental epidermolysis bullosa acquisita (EBA), in which autoantibodies to type VII collagen (COL7) and neutrophil activation cause mucocutaneous blisters. Anti-COL7 IgG induced moderate blistering in most inbred mouse strains, while some showed severe disease or were completely protected. Using publicly available genotyping data, we identified haplotype blocks that control blistering and confirmed two haplotype blocks in outbred mice. To identify the blistering-associated genes, haplotype blocks encoding genes that are differentially expressed in EBA-affected skin were considered. This procedure identified nine genes, including retinoid-related orphan receptor alpha (RORα), known to be involved in neurological development and function. After anti-COL7 IgG injection, RORα+/- mice showed reduced blistering and homozygous mice were completely resistant to EBA induction. Furthermore, pharmacological RORα inhibition dose-dependently blocked reactive oxygen species (ROS) release from activated neutrophils but did not affect migration or phagocytosis. Thus, forward genomics combined with multiple validation steps identifies RORα to be essential to drive inflammation in experimental EBA.

Immunomodulatory effects of heat shock protein 90 inhibition on humoral immune responses.

Heat shock protein 90 (Hsp90) inhibition blocks T-cell-linked inflammatory disease pathways and exhibits therapeutic activity in autoimmune disease mouse models, including the blistering disease epidermolysis bullosa acquisita. Although we previously showed that preformed autoreactive plasma cells do not seem to be directly affected by anti-Hsp90 treatment, immunomodulatory effects of Hsp90 inhibition on (auto-)antibody responses are not yet fully understood. In this study, the Hsp90 blocker 17-DMAG inhibited proliferation of activated total B cells and their IgG secretion in cultures of human peripheral B cells from healthy subjects, but IgG production was no longer affected when these activated B cells were allowed to differentiate prior to a deferred application of the inhibitor. 17-DMAG treatment was associated with induction of nuclear and cytoplasmic heat shock factor 1 and Hsp70 in stimulated human B cells, respectively. Type VII collagen (epidermolysis bullosa acquisita)-immunized mice early treated with 17-DMAG had reduced total B cells in spleens, a relative increase in splenic regulatory B cell fractions, higher serum IL-10 concentrations, and lower levels of circulating autoantibodies (paralleled by less pronounced disease induction) compared with vehicle-treated immunized mice. Autoantibody production was blunted in isolated and autoantigen-restimulated lymph node cells from immunized mice by either 17-DMAG or purified autologous splenic regulatory B cells. Thus, in addition to the previously described T cell inhibitory effects of Hsp90 blockade, this treatment potently modulates humoral immune responses at the B cell level, further supporting the introduction of Hsp90 inhibitors into the clinical setting for treatment of autoantibody-mediated disorders.

Genetic control of spontaneous arthritis in a four-way advanced intercross line.

Identifying the genetic basis of complex diseases, such as rheumatoid arthritis, remains a challenge that requires experimental models to reduce the genetic and environmental variability. Numerous loci for arthritis have been identified in induced animal models; however, few spontaneous models have been genetically studied. Therefore, we generated a four-way advanced intercross line (AIL) from four inbred strains, including BXD2/TyJ which spontaneously develops autoimmune arthritis. A genome-wide scan for spontaneous arthritis was performed in a cohort of 366 mice of the fourth generation (G4) of this cross. Five loci contributing to clinical phenotypes were identified in chromosomes 3, 7, 13, 18, and X. Three of the loci found in this study, confirm previously identified loci; whereas two of them are novel loci. Interesting candidate genes for the loci are highlighted. This study provides a genetic overview of spontaneous arthritis in mice and aids to solve the genetic etiology of rheumatoid arthritis and to gain a better understanding of the disease.

Identification of quantitative trait loci for murine autoimmune pancreatitis.

BACKGROUND AND AIMS: Autoimmune pancreatitis (AIP) represents a rare but clinically relevant cause of pancreatic inflammation. Using MRL/Mp mice as a model of spontaneous AIP, the genetic basis of the disease was studied. METHODS: To identify quantitative trait loci (QTL) of AIP, an advanced intercross line was studied, originating from MRL/MpJ parental mice and the following three mouse strains: Cast (healthy controls), BXD2 (susceptible to collagen induced arthritis), and NZM (a model of lupus erythematosus). This concept was chosen to identify both general autoimmune disease associated loci and AIP specific QTL. Therefore, generation G4 of outbred intercross mice was characterised phenotypically by scoring histopathological changes of the pancreas and genotyped with single nucleotide polymorphism (SNP) arrays. Data were analysed with the R implementation of HAPPY. RESULTS: Five QTLs, correlating with the severity of AIP, were identified. Two of them mapped to chromosome 4 and one to chromosomes 2, 5, and 6, respectively. The QTL on chromosome 6 displays the highest LOD score (5.4) and contains the C-type lectin domain family 4 member a2 in its peak region, which encodes a receptor protein of dendritic cells that has previously been implicated in autoimmune diseases such as Sjogren's syndrome. AIP candidate genes of other QTL's include heterogeneous nuclear ribonucleoprotein A3; nuclear factor, erythroid derived 2, like 2; Sjogren syndrome antigen B; and ubiquitin protein ligase E3 component n-recognin 3. CONCLUSIONS: This study has identified QTLs and putative candidate genes of murine AIP. Their functional role and relevance to human AIP will be studied further.

Ccdc66 null mutation causes retinal degeneration and dysfunction.

Retinitis pigmentosa (RP) is a group of human retinal disorders, with more than 100 genes involved in retinal degeneration. Canine and murine models are useful for investigating human RP based on known, naturally occurring mutations. In Schapendoes dogs, for example, a mutation in the CCDC66 gene has been shown to cause autosomal recessively inherited, generalized progressive retinal atrophy (gPRA), the canine counterpart to RP. Here, a novel mouse model with a disrupted Ccdc66 gene was investigated to reveal the function of protein CCDC66 and the pathogenesis of this form of gPRA. Homozygous Ccdc66 mutant mice lack retinal Ccdc66 RNA and protein expression. Light and electron microscopy reveal an initial degeneration of photoreceptors already at 13 days of age, followed by a slow, progressive retinal degeneration over months. Retinal dysfunction causes reduced scotopic a-wave amplitudes, declining from 1 to 7 months of age as well as an early reduction of the photopic b-wave at 1 month, improving slightly at 7 months, as evidenced by electroretinography. In the retina of the wild-type (WT) mouse, protein CCDC66 is present at highest levels after birth, followed by a decline until adulthood, suggesting a crucial role in early development. Protein CCDC66 is expressed predominantly in the developing rod outer segments as confirmed by subcellular analyses. These findings illustrate that the lack of protein CCDC66 causes early, slow progressive rod-cone dysplasia in the novel Ccdc66 mutant mouse model, thus providing a sound foundation for the development of therapeutic strategies.