Abstract
Autoimmune uveitis is a blinding disease presenting with autoantibodies against eye-specific proteins as well as autoagressive T cells invading and attacking the immune-privileged target tissue retina. The molecular events enabling T cells to invade and attack the tissue have remained elusive. Changes in membrane protein expression patterns between diseased and healthy stages are especially interesting because initiating events of disease will most likely occur at membranes. Since disease progression is accompanied with a break-down of the blood-retinal barrier, serum-derived proteins mask the potential target tissue-related changes. To overcome this limitation, we used membrane-enriched fractions derived from retinas of the only available spontaneous animal model for the disease equine recurrent uveitis, and compared expression levels by a label-free LC-MSMS-based strategy to healthy control samples. We could readily identify a total of 893 equine proteins with 57% attributed to the Gene Ontology project term "membrane." Of these, 179 proteins were found differentially expressed in equine recurrent uveitis tissue. Pathway enrichment analyses indicated an increase in proteins related to antigen processing and presentation, TNF receptor signaling, integrin cell surface interactions and focal adhesions. Additionally, loss of retina-specific proteins reflecting decrease of vision was observed as well as an increase in Müller glial cell-specific proteins indicating glial reactivity. Selected protein candidates (caveolin 1, integrin alpha 1 and focal adhesion kinase) were validated by immunohistochemistry and tissue staining pattern pointed to a significant increase of these proteins at the level of the outer limiting membrane which is part of the outer blood-retinal barrier. Taken together, the membrane enrichment in combination with LC-MSMS-based label-free quantification greatly increased the sensitivity of the comparative tissue profiling and resulted in detection of novel molecular pathways related to equine recurrent uveitis.
Highlights
Autoimmune uveitis is a blinding disease presenting with autoantibodies against eye-specific proteins as well as autoagressive T cells invading and attacking the immuneprivileged target tissue retina
In line with these qualitative data, we found that a major proportion (57%) of proteins identified were annotated to the GO term “membrane” (Fig. 1C) and prediction of transmembrane domains by TMHMM resulted in 297 proteins with one or more transmembrane domains (TMD) predicted
Since we have studied protein expression in retina of equine recurrent uveitis (ERU) cases before using conventional 2D gel-based proteomics, we know that this disease presents with activation of retinal Muller glial cells and with a break-down of the blood-retinal barrier (BRB) with variable severity [5]
Summary
Autoimmune uveitis is a blinding disease presenting with autoantibodies against eye-specific proteins as well as autoagressive T cells invading and attacking the immuneprivileged target tissue retina. Since disease progression is accompanied with a break-down of the blood-retinal barrier, serum-derived proteins mask the potential target tissue-related changes To overcome this limitation, we used membrane-enriched fractions derived from retinas of the only available spontaneous animal model for the disease equine recurrent uveitis, and compared expression levels by a label-free LC-MSMS-based strategy to healthy control samples. We have shown that retinal Mueller cells are players in the pathogenesis of the disease as they transform into a gliotic phenotype-decreasing expression of glutamine synthetase and pigment epithelium– derived factor (PEDF) and simultaneously start expressing interferon gamma [5] This observation is complemented by a quantitative comparison of serum samples with twodimensional–DIGE, where PEDF was significantly downregulated in ERU cases [6]. Changes in the membrane proteome containing cellular and tissue surface proteins are especially interesting for understanding early events in the pathogenesis of this autoimmune disease because it involves auto-aggressive T cells entering and attacking the retinal structures and surface changes can potentially trigger these processes [7]
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