Altered expression of talin 1 in peripheral immune cells points to a significant role of the innate immune system in spontaneous autoimmune uveitis

The molecular mechanism which enables activated immune cells to cross the blood-retinal barrier in spontaneous autoimmune uveitis is yet to be unraveled. Equine recurrent uveitis is the only spontaneous animal model allowing us to investigate the autoimmune mediated transformation of leukocytes in the course of this sight threatening disease. Hypothesizing that peripheral blood immune cells change their protein expression pattern in spontaneous autoimmune uveitis, we used DIGE to detect proteins with altered abundance comparing peripheral immune cells of healthy and ERU diseased horses. Among others, we found a significant downregulation of talin 1 in peripheral blood granulocytes of ERU specimen, pointing to changes in β integrin activation and indicating a significant role of the innate immune system in spontaneous autoimmune diseases.     

Label-free LC-MSMS analysis of vitreous from autoimmune uveitis reveals a significant decrease in secreted Wnt signalling inhibitors DKK3 and SFRP2

Equine recurrent uveitis is a severe and frequent blinding disease in horses which presents with auto-reactive invading T-cells, resulting in the destruction of the inner eye. Infiltration of inflammatory cells into the retina and vitreous is driven by currently unknown guidance cues, however surgical removal of the vitreous (vitrectomy) has proven therapeutically successful. Therefore, proteomic analyses of vitrectomy samples are likely to result in detection of proteins contributing to disease pathogenesis. Vitreous from healthy and ERU diseased horses were directly compared by quantitative mass spectrometry based on label-free quantification of peak intensities across samples. We found a significant upregulation of complement and coagulation cascades and downregulation of negative paracrine regulators of canonical Wnt signalling including the Wnt signalling inhibitors DKK3 and SFRP2. Based on immunohistochemistry, both proteins are expressed in equine retina and suggest localisation to retinal Müller glial cells (RMG), which may be the source cells for these proteins. Furthermore, retinal expression levels and patterns of DKK3 change in response to ERU. Since many other regulated proteins identified here are associated with RMG cells, these cells qualify as the prime responders to autoimmune triggers.     

Osteopontin and fibronectin levels are decreased in vitreous of autoimmune uveitis and retinal expression of both proteins indicates ECM re-modeling

Autoimmune uveitis is an intraocular inflammation that arises through autoreactive T-cells attacking the inner eye, eventually leading to blindness. However, the contributing molecular pathomechanisms within the affected tissues remain as yet elusive. The extracellular matrix (ECM) is a highly dynamic structure that varies tremendously and influences the encompassing tissue. In order to assess ECM re-modeling in autoimmune uveitis, we investigated the expression of ECM molecules fibronectin and osteopontin in vitreous and retina samples. This was carried out in the only spontaneous animal model for human autoimmue uveitis, namely equine recurrent uveitis (ERU) that resembles the human disease in clinical as well as in immunopathological aspects. ERU is a naturally occurring autoimmune disease in horses that develops frequently and has already proved its value to study disease-related pathomechanisms. Western blot analysis of fibronectin and osteopontin in healthy and uveitic vitreous revealed significant reduction of both proteins in uveitis. Immunohistochemical expression of fibronectin in healthy retinas was restricted to the inner limiting membrane abutting vimentin positive Müller cell endfeet, while in uveitic sections, a disintegration of the ILM was observed changing the fibronectin expression to a dispersed pattern extending toward the vitreous. Retinal expression of osteopontin in control tissue was found in a characteristic Müller cell pattern illustrated by co-localization with vimentin. In uveitic retinas, the immunoreactivity of osteopontin in gliotic Müller cells was almost absent. The ability of Müller cells to express fibronectin and osteopontin was additionally shown by immunocytochemistry of primary cultured equine Müller cells and the equine Müller cell line eqMC-7. In conclusion, severe ECM re-modeling in autoimmune uveitis reported here, might affect the adhesive function of fibronectin and thus the anchoring of Müller cell endfeet to the ILM. Furthermore, the absence of osteopontin in gliotic Müller cells might represent reduced neuroprotection, an osteopontin attribute that is intensively discussed.     

Retinal Glycoprotein Enrichment by Concanavalin A Enabled Identification of Novel Membrane Autoantigen Synaptotagmin-1 in Equine Recurrent Uveitis

Complete knowledge of autoantigen spectra is crucial for understanding pathomechanisms of autoimmune diseases like equine recurrent uveitis (ERU), a spontaneous model for human autoimmune uveitis. While several ERU autoantigens were identified previously, no membrane protein was found so far. As there is a great overlap between glycoproteins and membrane proteins, the aim of this study was to test whether pre-enrichment of retinal glycoproteins by ConA affinity is an effective tool to detect autoantigen candidates among membrane proteins. In 1D Western blots, the glycoprotein preparation allowed detection of IgG reactions to low abundant proteins in sera of ERU patients. Synaptotagmin-1, a Ca2+-sensing protein in synaptic vesicles, was identified as autoantigen candidate from the pre-enriched glycoprotein fraction by mass spectrometry and was validated as a highly prevalent autoantigen by enzyme-linked immunosorbent assay. Analysis of Syt1 expression in retinas of ERU cases showed a downregulation in the majority of ERU affected retinas to 24%. Results pointed to a dysregulation of retinal neurotransmitter release in ERU. Identification of synaptotagmin-1, the first cell membrane associated autoantigen in this spontaneous autoimmune disease, demonstrated that examination of tissue fractions can lead to the discovery of previously undetected novel autoantigens. Further experiments will address its role in ERU pathology.     

Genetic risk factors for insidious equine recurrent uveitis in Appaloosa horses

Appaloosa horses are predisposed to equine recurrent uveitis (ERU), an immune‐mediated disease characterized by recurring inflammation of the uveal tract in the eye, which is the leading cause of blindness in horses. Nine genetic markers from the ECA1 region responsible for the spotted coat color of Appaloosa horses, and 13 microsatellites spanning the equine major histocompatibility complex (ELA) on ECA20, were evaluated for association with ERU in a group of 53 Appaloosa ERU cases and 43 healthy Appaloosa controls. Three markers were significantly associated (corrected P‐value <0.05): a SNP within intron 11 of the TRPM1 gene on ECA1, an ELA class I microsatellite located near the boundary of the ELA class III and class II regions and an ELA class II microsatellite located in intron 1 of the DRA gene. Association between these three genetic markers and the ERU phenotype was confirmed in a second population of 24 insidious ERU Appaloosa cases and 16 Appaloosa controls. The relative odds of being an ERU case for each allele of these three markers were estimated by fitting a logistic mixed model with each of the associated markers independently and with all three markers simultaneously. The risk model using these markers classified ~80% of ERU cases and 75% of controls in the second population as moderate or high risk, and low risk respectively. Future studies to refine the associations at ECA1 and ELA loci and identify functional variants could uncover alleles conferring susceptibility to ERU in Appaloosa horses.     

Retinal Mueller glial cells trigger the hallmark inflammatory process in autoimmune uveitis

Spontaneous equine recurrent uveitis (ERU) is an incurable autoimmune disease affecting the eye. Although retinal-autoantigen specific T-helper 1 cells have been demonstrated to trigger disease progression and relapses, the molecular processes leading to retinal degeneration and consequent blindness remain unknown. To elucidate such processes, we studied changes in the total retinal proteome of ERU-diseased horses compared to healthy controls. Severe changes in the retinal proteome were found for several markers for blood-retinal barrier breakdown and whose emergence depended upon disease severity. Additionally, uveitic changes in the retina were accompanied by upregulation of aldose 1-epimerase, selenium-binding protein 1, alpha crystallin A chain, phosphatase 2A inhibitor (SET), and glial fibrillary acidic protein (GFAP), the latter indicating an involvement of retinal Mueller glial cells (RMG) in disease process. To confirm this, we screened for additional RMG-specific markers and could demonstrate that, in uveitic retinas, RMG concomitantly upregulate vimentin and GFAP and downregulate glutamine synthetase. These expression patterns suggest for an activated state of RMG, which further downregulate the expression of pigment epithelium-derived factor (PEDF) and begin expressing interferon-gamma, a pro-inflammatory cytokine typical for T-helper 1 cells. We thus propose that RMG may play a fatal role in uveitic disease progression by directly triggering inflammatory processes through the expression and secretion of interferon-gamma.     

A Genome-Wide Association Study Identifies Risk Loci to Equine Recurrent Uveitis in German Warmblood Horses

Equine recurrent uveitis (ERU) is a common eye disease affecting up to 3–15% of the horse population. A genome-wide association study (GWAS) using the Illumina equine SNP50 bead chip was performed to identify loci conferring risk to ERU. The sample included a total of 144 German warmblood horses. A GWAS showed a significant single nucleotide polymorphism (SNP) on horse chromosome (ECA) 20 at 49.3 Mb, with IL-17A and IL-17F being the closest genes. This locus explained a fraction of 23% of the phenotypic variance for ERU. A GWAS taking into account the severity of ERU, revealed a SNP on ECA18 nearby to the crystalline gene cluster CRYGA-CRYGF. For both genomic regions on ECA18 and 20, significantly associated haplotypes containing the genome-wide significant SNPs could be demonstrated. In conclusion, our results are indicative for a genetic component regulating the possible critical role of IL-17A and IL-17F in the pathogenesis of ERU. The associated SNP on ECA18 may be indicative for cataract formation in the course of ERU.     

Mercury-induced inflammation and autoimmunity

BackgroundHuman exposure to mercury leads to a variety of pathologies involving numerous organ systems including the immune system. A paucity of epidemiological studies and suitable diagnostic criteria, however, has hampered collection of sufficient data to support a causative role for mercury in autoimmune diseases. Nevertheless, there is evidence that mercury exposure in humans is linked to markers of inflammation and autoimmunity. This is supported by experimental animal model studies, which convincingly demonstrate the biological plausibility of mercury as a factor in the pathogenesis of autoimmune disease.Scope of the reviewIn this review, we focus on ability of mercury to elicit inflammatory and autoimmune responses in both humans and experimental animal models.Major conclusionsAlthough subtle differences exist, the inflammatory and autoimmune responses elicited by mercury exposure in humans and experimental animal models show many similarities. Proinflammatory cytokine expression, lymphoproliferation, autoantibody production, and nephropathy are common outcomes. Animal studies have revealed significant strain dependent differences in inflammation and autoimmunity suggesting genetic regulation. This has been confirmed by the requirement for individual genes as well as genome wide association studies. Importantly, many of the genes required for mercury-induced inflammation and autoimmunity are also required for idiopathic systemic autoimmunity. A notable difference is that mercury-induced autoimmunity does not require type I IFN. This observation suggests that mercury-induced autoimmunity may arise by both common and specific pathways, thereby raising the possibility of devising criteria for environmentally associated autoimmunity.General significanceMercury exposure likely contributes to the pathogenesis of autoimmunity.     

Sex differences in autoimmune diseases

Women are more susceptible to a variety of autoimmune diseases including systemic lupus erythematosus (SLE), multiple sclerosis (MS), primary biliary cirrhosis, rheumatoid arthritis and Hashimoto's thyroiditis. This increased susceptibility in females compared to males is also present in animal models of autoimmune diseases such as spontaneous SLE in (NZBxNZW)F1 and NZM.2328 mice, experimental autoimmune encephalomyelitis (EAE) in SJL mice, thyroiditis, Sjogren's syndrome in MRL/Mp-lpr/lpr mice and diabetes in non-obese diabetic mice. Indeed, being female confers a greater risk of developing these diseases than any single genetic or environmental risk factor discovered to date. Understanding how the state of being female so profoundly affects autoimmune disease susceptibility would accomplish two major goals. First, it would lead to an insight into the major pathways of disease pathogenesis and, secondly, it would likely lead to novel treatments which would disrupt such pathways.     

Detection of antinuclear and antilaminin antibodies in autistic children who received thimerosal-containing vaccines

Autism, a neurodevelopmental disorder, may involve autoimmune pathogenesis. Since mercury is potentially a risk factor for autoimmunity, we conducted a study of mercury-induced antinuclear and antilaminin antibodies in autistic and normal children who had been pre-administered with thimerosal-containing vaccines. Laboratory analysis by different immunoassays showed that the serum level of these two autoimmune markers did not significantly differ between autistic and normal children. This finding suggests that the mercury as in thimerosal-containing vaccines is likely not related to autoimmune phenomenon in autism.     

Integration of expression profiles and genetic mapping data to identify candidate genes in intracranial aneurysm.

Intracranial aneurysm (IA) is a complex genetic disease for which, to date, 10 loci have been identified by linkage. Identification of the risk-conferring genes in the loci has proven difficult, since the regions often contain several hundreds of genes. An approach to prioritize positional candidate genes for further studies is to use gene expression data from diseased and nondiseased tissue. Genes that are not expressed, either in diseased or nondiseased tissue, are ranked as unlikely to contribute to the disease. We demonstrate an approach for integrating expression and genetic mapping data to identify likely pathways involved in the pathogenesis of a disease. We used expression profiles for IAs and nonaneurysmal intracranial arteries (IVs) together with the 10 reported linkage intervals for IA. Expressed genes were analyzed for membership in Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathways. The 10 IA loci harbor 1,858 candidate genes, of which 1,561 (84%) were represented on the microarrays. We identified 810 positional candidate genes for IA that were expressed in IVs or IAs. Pathway information was available for 294 of these genes and involved 32 KEGG biological function pathways represented on at least 2 loci. A likelihood-based score was calculated to rank pathways for involvement in the pathogenesis of IA. Adherens junction, MAPK, and Notch signaling pathways ranked high. Integration of gene expression profiles with genetic mapping data for IA provides an approach to identify candidate genes that are more likely to function in the pathology of IA.     

The genetics of generalized vitiligo and associated autoimmune diseases

Vitiligo is an acquired disorder in which patches of depigmented skin and often overlying hair, and mucous membranes, are the result of progressive autoimmune loss of melanocytes from the involved areas. Considered the most common pigmentary disorder, vitiligo involves complex interaction of environmental and genetic factors that ultimately contribute to melanocyte destruction, resulting in the characteristic depigmented lesions. In the past few years, studies of the genetic epidemiology of vitiligo have led to the recognition that generalized vitiligo is part of a broader autoimmune disease diathesis. Attempts to identify genes involved in susceptibility to generalized vitiligo have involved gene expression studies, genetic association studies of candidate genes, and genome-wide linkage analyses to discover new genes. These studies have begun to yield results that shed light on the mechanisms of vitiligo pathogenesis. It is anticipated that the discovery of biological pathways of vitiligo pathogenesis will provide novel targets for future approaches to the treatment and prevention of vitiligo and its associated autoimmune diseases.     

Genetic investigation of equine recurrent uveitis in Appaloosa horses

Equine recurrent uveitis (ERU) is characterized by intraocular inflammation that often leads to blindness in horses. Appaloosas are more likely than any other breed to develop insidious ERU, distinguished by low-grade chronic intraocular inflammation, suggesting a genetic predisposition. Appaloosas are known for their white coat spotting patterns caused by the leopard complex spotting allele (LP) and the modifier PATN1. A marker linked to LP on ECA1 and markers near MHC on ECA20 were previously associated with increased ERU risk. This study aims to further investigate these loci and identify additional genetic risk factors. A GWAS was performed using the Illumina Equine SNP70 BeadChip in 91 horses. Additive mixed model approaches were used to correct for relatedness. Although they do not reach a strict Bonferroni genome-wide significance threshold, two SNPs on ECA1 and one SNP each on ECA12 and ECA29 were among the highest ranking SNPs and thus warranted further analysis (P = 1.20 × 10⁻⁵, P = 5.91 × 10⁻⁶, P = 4.91 × 10⁻⁵, P = 6.46 × 10⁻⁵). In a second cohort (n = 98), only an association with the LP allele on ECA1 was replicated (P = 5.33 × 10⁻⁵). Modeling disease risk with LP, age and additional depigmentation factors (PATN1 genotype and extent of roaning) supports an additive role for LP and suggests an additive role for PATN1. Genotyping for LP and PATN1 may help predict ERU risk (AUC = 0.83). The functional role of LP and PATN1 in ERU development requires further investigation. Testing samples across breeds with leopard complex spotting patterns and a denser set of markers is warranted to further refine the genetic components of ERU.     

Post-translational modification of crystallins in vitreous body from experimental autoimmune uveitis of rats

Experimental autoimmune uveitis (EAU) is a well-known animal model of posterior uveitis that is one of the major causes of blindness. EAU could be induced in susceptible animals (i.e., Lewis rat) by immune reactions using evolutionarily conserved retinal proteins, such as interphoto-receptor retinoid binding protein (IRBP), or epitaphs of the protein. First, we prepared the following four test groups that subsequently increased or decreased inflammation. (1) Normal control group, (2) IRBP-induced uveitis group, (3) Hemin-treated uveitis group, and (4) Sn(IV) protoporphyrin IX dichloride (SnPP)-treated uveitis group. Second, in the vitreous bodies of Lewis rats, the infiltrated proteins were analyzed using two-dimensional electrophoresis (2-DE), MALDI-TOF/MS, and Micro LC/LC-MS/MS analysis. Finally, Western blotting was applied to confirm the relative amount of crystallins and phosphorylation sites of alphaB-crystallin. Thirty spots were identified in vitreous bodies, and 27 of these spots were members of the crystallin family. Unlike betaA4- and B2-crystallins (that were significantly increased without truncation), alphaA- and B-crystallins were only truncated in EAU vitreous body. Taken as a whole, in the rat EAU model, we suggest that post-translational truncations of alphaA- and alphaB-crystallins, phosphorylation of alphaB-crystallin, and new production of betaA4- and betaB2-crystallins are intercorrelated with uveitis progression and inflammatory responses.     

The Identification of Pathway Markers in Intracranial Aneurysm Using Genome-Wide Association Data from Two Different Populations

The identification of significant individual factors causing complex diseases is challenging in genome-wide association studies (GWAS) since each factor has only a modest effect on the disease development mechanism. In this study, we hypothesize that the biological pathways that are targeted by these individual factors show higher conservation within and across populations. To test this hypothesis, we searched for the disease related pathways on two intracranial aneurysm GWAS in European and Japanese case–control cohorts. Even though there were a few significantly conserved SNPs within and between populations, seven of the top ten affected pathways were found significant in both populations. The probability of random occurrence of such an event is 2.44E−36. We therefore claim that even though each individual has a unique combination of factors involved in the mechanism of disease development, most targeted pathways that need to be altered by these factors are, for the most part, the same. These pathways can serve as disease markers. Individuals, for example, can be scanned for factors affecting the genes in marker pathways. Hence, individual factors of disease development can be determined; and this knowledge can be exploited for drug development and personalized therapeutic applications. Here, we discuss the potential avenues of pathway markers in medicine and their translation to preventive and individualized health care.     

Recent progress in the genetics of generalized vitiligo

Vitiligo is an acquired disease characterized principally by patchy depigmentation of skin and overlying hair. Generalized vitiligo (GV), the predominant form of the disorder, results from autoimmune loss of melanocytes from affected regions. GV is a “complex trait”, inherited in a non-Mendelian polygenic, multifactorial manner. GV is epidemiologically associated with other autoimmune diseases, both in GV patients and in their close relatives, suggesting that shared genes underlie susceptibility to this group of diseases. Early candidate gene association studies yielded a few successes, such as PTPN22, but most such reports now appear to be false-positives. Subsequent genomewide linkage studies identified NLRPI and XBPI, apparent true GV susceptibility genes involved in immune regulation, and recent genome-wide association studies (GWAS) of GV in Caucasian and Chinese populations have yielded a large number of additional validated GV susceptibility genes.Together, these genes highlight biological systems and pathways that reach from the immune cells to the melanocyte, and provide insights into both disease pathogenesis and potential new targets for both treatment and even prevention of GV and other autoimmune diseases in genetically susceptible individuals.     

Growth factor receptor trafficking as a potential therapeutic target in pediatric cancer

Growth factor receptors (GFRs) are often aberrantly expressed in tumor cells,and altered GFR expression and activity contribute to the pathogenesis of many types of cancer.A variety of mechanisms have ...     

Platelet-activating factor (PAF)-like oxidized phospholipids: relevance to atherosclerosis

Lipid peroxidation is involved in the pathogenesis of chronic diseases including atherosclerosis. Oxidized lipoprotein has diverse biological activities and is believed to initiate atheroma formation and maturate fatty plaque. The active components of oxidized lipoproteins still remain to be clarified, but a likely candidate is the phosphatidylcholine (PC) having an sn-2-short-chain acyl group with a methyl, hydroxyl, aldehydic or carboxylic terminal. These unique PCs, formed by oxidative fragmentation of the polyunsaturated acyl group of the parent PC in liposomes, low density lipoproteins and blood plasma, induce platelet aggregation through the activation of the receptor for platelet-activating factor (PAF), due to their resemblance in structure with PAF. We have found that PAF-like lipids regulate DNA synthesis and production of nitric oxide independently of the activation of the PAF receptor in vascular smooth muscle cells. Regulation of vascular cell function through two distinct signaling pathways mediated by PAF-like lipids provides new insight into the mechanism of induction of atherosclerosis.     

Identification and functional validation of novel autoantigens in equine uveitis

The development, progression, and recurrence of autoimmune diseases are frequently driven by a group of participatory autoantigens. We identified and characterized novel autoantigens by analyzing the autoantibody binding pattern from horses affected by spontaneous equine recurrent uveitis to the retinal proteome. Cellular retinaldehyde-binding protein (cRALBP) had not been described previously as autoantigen, but subsequent characterization in equine recurrent uveitis horses revealed B and T cell autoreactivity to this protein and established a link to epitope spreading. We further immunized healthy rats and horses with cRALBP and observed uveitis in both species with typical tissue lesions at cRALBP expression sites. The autoantibody profiling outlined here could be used in various autoimmune diseases to detect autoantigens involved in the dynamic spreading cascade or serve as predictive markers.     

Identification of crystallin family proteins in vitreous body in rat endotoxin-induced uveitis: Involvement of crystallin truncation in uveitis pathogenesis

Endotoxin-induced uveitis (EIU) is an animal model of acute ocular inflammation. To characterize the mechanism of EIU, we analyzed the infiltration of proteins in the vitreous bodies of rats with EIU and normal rats using 2-DE and micro LC/LC-MS/MS. Twenty spots were identified in vitreous bodies of rats. Eighteen of these spots were members of the crystallin family. The truncated form of beta A4- and beta B2-crystallin were predominant in normal vitreous bodies, but there were intact form of crystallins in lipopolysaccharide-injected rats with EIU. These results suggest that crystallin family proteins are the major group of proteins involved in uveitic vitreous and that C-terminal truncation of beta-crystallins may play a role in EIU-related disease progression.