Neuroretinal degeneration in a mouse model of systemic chronic immune activation observed by proteomics.

Blindness or vision loss due to neuroretinal and photoreceptor degeneration affects millions of individuals worldwide. In numerous neurodegenerative diseases, including age-related macular degeneration, dysregulated immune response-mediated retinal degeneration has been found to play a critical role in the disease pathogenesis. To better understand the pathogenic mechanisms underlying the retinal degeneration, we used a mouse model of systemic immune activation where we infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13. Here, we evaluated the effects of LCMV infection and present a comprehensive discovery-based proteomic investigation using tandem mass tag (TMT) labeling and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in protein regulation in the posterior part of the eye, neuroretina, and RPE/choroid were compared to those in the spleen as a secondary lymphoid organ and to the kidney as a non-lymphoid but encapsulated organ at 1, 8, and 28 weeks of infection. Using bioinformatic tools, we found several proteins responsible for maintaining normal tissue homeostasis to be differentially regulated in the neuroretina and the RPE/choroid during the degenerative process. Additionally, in the organs we observed, several important protein pathways contributing to cellular homeostasis and tissue development were perturbed and associated with LCMV-mediated inflammation, promoting disease progression. Our findings suggest that the response to a systemic chronic infection differs between the neuroretina and the RPE/choroid, and the processes induced by chronic systemic infection in the RPE/choroid are not unlike those induced in non-immune-privileged organs such as the kidney and spleen. Overall, our data provide detailed insight into several molecular mechanisms of neuroretinal degeneration and highlight various novel protein pathways that further suggest that the posterior part of the eye is not an isolated immunological entity despite the existence of neuroretinal immune privilege.

Tear film proteome changes following Tobradex® therapy in anterior blepharitis.

PURPOSE: The management of blepharitis continues to challenge clinicians due to the poorly understood aetiology of the condition. We recently identified the family of intracellular plakin proteins as essential driving forces underlying anterior blepharitis. A large-scale protein analysis was used to study if a topical dexamethasone/tobramycin solution could be used to reverse the expression of plakin proteins. METHODS: Tear film samples from treatment naïve patients with anterior blepharitis (n = 15) were collected with Schirmer filtration paper. A subgroup of the patients (n = 10) received treatment with a dexamethasone/tobramycin 1 + 3 mg/mL ophthalmic suspension (Tobradex® ) for 3 weeks and collection of tear film samples was repeated. The samples were analysed with label-free quantification nano liquid chromatography-tandem mass spectrometry requiring quantification in at least 70% of the samples in each group. Proteins were considered differentially expressed if p < 0.05. RESULTS: Following Tobradex® intervention, 27 proteins were upregulated while 61 proteins were downregulated. Regulated proteins after Tobradex® treatment were involved in intermediate filament cytoskeleton organization including downregulation of the plakin proteins envoplakin, epiplakin and periplakin. Plectin, a protein of the plakin family, remained unchanged after Tobradex® therapy. Tobradex® treatment resulted in the regulation of proteins involved in translation including a cluster of downregulated ribosomal proteins. Tobradex® intervention was associated with the regulation of proteins involved in fructose metabolism and glycolytic processes including fructose-1.6-bisphosphatase 1, fructose-bisphosphate aldolases A and B, pyruvate kinase PKM and transketolase. Ig lambda chain V-I region, prominin-1, and protein Niban were upregulated after Tobradex® treatment. CONCLUSIONS: Tobradex treatment reversed the expression of plakin proteins in anterior blepharitis. Topical solutions which inhibit the expression of plakin proteins may have the potential to restore the ocular surface integrity in anterior blepharitis and should be explored further.

Meibomian Gland Dysfunction Is Associated with Low Levels of Immunoglobulin Chains and Cystatin-SN.

Meibomian gland dysfunction (MGD) is a highly prevalent condition and the most common cause of evaporative dry eye disease. Studying the proteome of MGD can result in important advances in the management of the condition. Here, we collected tear film samples from treatment naïve patients with MGD (n = 10) and age-matched controls (n = 11) with Schirmer filtration paper. The samples were analyzed with label-free quantification nano liquid chromatography-tandem mass spectrometry. The proteins were considered differentially expressed if p < 0.05. A total of 88 proteins were significantly regulated. The largest change was observed in cystatin-SN, which was downregulated in MGD and correlated negatively with tear meniscus height. The downregulation of cystatin-SN was confirmed with targeted mass spectrometry by single reaction monitoring (SRM). Eighteen immunoglobulin components involved in B cell activation, phagocytosis, and complement activation were downregulated in MGD including Ig alpha-1 chain C region, immunoglobulin J chain, immunoglobulin heavy variable 3-15, and Ig mu chain C region. The changes in cystatin-SN and immunoglobulin chains are likely to result from the inflammatory changes related to tear film evaporation, and future studies may assess their association with the meibum quality.

Anterior blepharitis is associated with elevated plectin levels consistent with a pronounced intracellular response.

PURPOSE: Anterior blepharitis is a frequent ocular condition which may result in severe ocular surface disease. In this study, advanced proteome analysis was performed to elucidate biological mechanisms underlying anterior blepharitis. METHODS: All patients underwent full ophthalmological examination including Ocular Surface Disease Index score (OSDI). Measurement of non-invasive break-up time (NBUT), Oxford score, and meibography were performed. Tear film samples from treatment naïve patients with anterior blepharitis (n = 15) and age-matched controls (n = 11) were collected with Schirmer filtration paper. The samples were analyzed with label-free quantification nano liquid chromatography - tandem mass spectrometry (LFQ nLC-MS/MS). Significantly regulated proteins were identified with a permutation-based calculation with a false discovery rate at 0.05. RESULTS: Among the 927 proteins detected, a total of 162 proteins were significantly changed. Regulated proteins were involved in cytoplasmic translation, positive regulation of B cell activation, complement activation and phagocytosis. High levels of plakin proteins, a group of proteins involved in cytoskeleton organization, were observed in anterior blepharitis, including plectin, desmoplakin, envoplakin, epiplakin, periplakin, and vimentin. The upregulation of plectin was confirmed with single reaction monitoring. Patients with anterior blepharitis had lower levels of immunoglobulin chains, VEGF coregulated chemokine 1 (CXCL17), and platelet-derived growth factor C. CONCLUSIONS: Anterior blepharitis was associated with a high level of plectin indicating a pronounced intracellular response with cytoskeletal reorganization. Our data suggest a lack of immunoglobulin chains and CXCL17 in anterior blepharitis with potential alterations in the ocular surface immune response.

Human herpesvirus-6B protein U19 contains a p53 BOX I homology motif for HDM2 binding and p53 stabilization.

In order to establish a successful infection, it is of crucial importance for invading viruses to alter the activities of the regulatory protein p53. Beta-herpesviruses stabilize p53 and likely direct its activities towards generation of a replication-friendly environment. We here study the mechanisms behind HHV-6B-induced stabilization and inactivation of p53. Stable transgene expression of the HHV-6B protein U19 was sufficient to achieve upregulation of p53. U19 bound directly to the p53-regulating protein HDM2 in vitro, co-precipitated together with HDM2 in lysates, and co-localized with HDM2 in the nucleus when overexpressed. U19 contained a sequence with a putative p53 BOX I-motif for HDM2 binding. Mutation of the two key amino acids within this motif was sufficient to inhibit all the described U19 functions. Our study provides further insight into p53-modulating strategies used by herpesviruses and elucidates a mechanism used by HHV-6B to circumvent the antiviral response.