Comprehensive Proteomic Profiling of Aqueous Humor in Idiopathic Uveitis and Vogt-Koyanagi-Harada Syndrome.

Idiopathic uveitis (IU) and Vogt-Koyanagi-Harada (VKH) syndrome are common types of uveitis. However, the exact pathological mechanisms of IU and VKH remain unclear. Proteomic analysis of aqueous humor (AH), the most easily accessible intraocular fluid and a key site of uveitis development, may reveal potential biomarkers and elucidate uveitis pathogenesis. In this study, 44 AH samples, including 12 IU cases, 16 VKH cases, and 16 controls, were subjected to label-free quantitative proteomic analysis. We identified 557 proteins from a comprehensive spectral library of 634 proteins across all samples. The AH proteomic profiles of the IU and VKH groups were different from those of the control group. Differential analysis revealed a shared pattern of extracellular matrix disruption and downregulation of retinal cellular proteins in the IU and VKH groups. Enrichment analysis revealed a protein composition indicative of inflammation in the AH of the IU and VKH groups but not in that of the control group. In the IU and VKH groups, innate immunity played an important role, as indicated by complement cascade activation and overexpression of innate immune cell markers. Extreme gradient boosting (XGBoost), an efficient and robust machine learning algorithm, was subsequently used to screen potential biomarkers for classifying the IU, VKH, and control groups. Transferrin and complement factor B were deemed the most important and represent a promising biomarker panel. These proteins were validated by high-resolution multiple reaction monitoring (HR-MRM) in an independent validation cohort. A classification decision tree was subsequently built for the diagnosis. Our findings further the understanding of the underlying molecular mechanisms in IU and VKH and facilitate the development of potential therapeutic and diagnostic strategies.

Proteomic analysis of human aqueous humor from fuchs uveitis syndrome.

Fuchs uveitis syndrome (FUS) is a commonly misdiagnosed uveitis syndrome often presenting as an asymptomatic mild inflammatory condition until complications arise. The diagnosis of this disease remains clinical because of the lack of specific laboratory tests. The aqueous humor (AH) is a complex fluid containing nutrients and metabolic wastes from the eye. Changes in the AH protein provide important information for diagnosing intraocular diseases. This study aimed to analyze the proteomic profile of AH in individuals diagnosed with FUS and to identify potential biomarkers of the disease. We used liquid chromatography-tandem mass spectrometry-based proteomic methods to evaluate the AH protein profiles of all 37 samples, comprising 15 patients with FUS, six patients with Posner-Schlossman syndrome (PSS), and 16 patients with age-related cataract. A total of 538 proteins were identified from a comprehensive spectral library of 634 proteins. Subsequent differential expression analysis, enrichment analysis, and construction of key sub-networks revealed that the inflammatory response, complement activation and hypoxia might be crucial in mediating the process of FUS. The hypoxia inducible factor-1 may serve as a key regulator and therapeutic target. Additionally, the innate and adaptive immune responses are considered dominant in the patients with FUS. A diagnostic model was constructed using machine-learning algorithm to classify FUS, PSS, and normal controls. Two proteins, complement C1q subcomponent subunit B and secretogranin-1, were found to have the highest scores by the Extreme Gradient Boosting, suggesting their potential utility as a biomarker panel. Furthermore, these two proteins as biomarkers were validated in a cohort of 18 patients using high resolution multiple reaction monitoring assays. Therefore, this study contributes to advancing of the current knowledge of FUS pathogenesis and promotes the development of effective diagnostic strategies.

Plasma-Derived Small Extracellular Vesicles From VKH Patients Suppress T Cell Proliferation Via MicroRNA-410-3p Modulation of CXCL5 Axis.

Purpose: Circulating exosomes regulate immune responses and induce immune tolerance in immune-mediated diseases. This study aimed to investigate the role of circulating small extracellular vesicles (sEVs) derived from patients with Vogt-Koyanagi-Harada (VKH) syndrome, in T-cell responses. Methods: The sEVs were isolated from the plasma of healthy controls, patients with VKH, and other uveitis patients. The effects of autologous and allogeneic sEVs on the proliferation of circulating CD4+ T cells were evaluated. Microarray analysis of sEVs was performed to determine their differential miRNA expression profiles. The target genes of the candidate miRNA were predicted and verified. The role of both the candidate miRNA and target genes in T-cell proliferation was tested. Results: Plasma-derived sEVs from patients with VKH inhibited the proliferation of autologous CD4+ T cells. Among all the miRNAs that might be associated with inflammatory activity, we found that miR-410-3p had the largest number of T-cell proliferation target genes. MiR-410-3p mimics inhibited the proliferation of Jurkat cells and CD4+ T cells. C-X-C motif chemokine ligand 5 (CXCL5) was confirmed to be a potential target gene of miR-410-3p, and siRNA-mediated CXCL5 knockdown inhibited cell proliferation. Conclusions: Circulating sEVs exert an inhibitory effect on autologous CD4+ T cells mediated by miR-410-3p by targeting CXCL5, supporting the possibility of using autogenic sEVs to inhibit ocular inflammation.

Comprehensive profiling of extracellular vesicles in uveitis and scleritis enables biomarker discovery and mechanism exploration.

BACKGROUND: Uveitis and posterior scleritis are sight-threatening diseases with undefined pathogenesis and accurate diagnosis remains challenging. METHODS: Two plasma-derived extracellular vesicle (EV) subpopulations, small and large EVs, obtained from patients with ankylosing spondylitis-related uveitis, Behcet's disease uveitis, Vogt-Koyanagi-Harada syndrome, and posterior scleritis were subjected to proteomics analysis alongside plasma using SWATH-MS. A comprehensive bioinformatics analysis was performed on the proteomic profiles of sEVs, lEVs, and plasma. Candidate biomarkers were validated in a new cohort using ELISA. Pearson correlation analysis was performed to analyze the relationship between clinical parameters and proteomic data. Connectivity map database was used to predict therapeutic agents. RESULTS: In total, 3,668 proteins were identified and over 3000 proteins were quantified from 278 samples. When comparing diseased group to healthy control, the proteomic profiles of the two EV subgroups were more correlated with disease than plasma. Comprehensive bioinformatics analysis highlighted potential pathogenic mechanisms for these diseases. Potential biomarker panels for four diseases were identified and validated. We found a negative correlation between plasma endothelin-converting enzyme 1 level and mean retinal thickness. Potential therapeutic drugs were proposed, and their targets were identified. CONCLUSIONS: This study provides a proteomic landscape of plasma and EVs involved in ankylosing spondylitis-related uveitis, Behcet's disease uveitis, Vogt-Koyanagi-Harada syndrome, and posterior scleritis, offers insights into disease pathogenesis, identifies valuable biomarker candidates, and proposes promising therapeutic agents.

Overexpressing Kallistatin Aggravates Experimental Autoimmune Uveitis Through Promoting Th17 Differentiation.

Kallistatin or kallikrein-binding protein (KBP) has been reported to regulate angiogenesis, inflammation and tumor progression. Autoimmune uveitis is a common, sight-threatening inflammatory intraocular disease. However, the roles of kallistatin in autoimmunity and autoreactive T cells are poorly investigated. Compared to non-uveitis controls, we found that plasma levels of kallistatin were significantly upregulated in patients with Vogt-Koyanagi-Harada (VKH) disease, one of the non-infectious uveitis. Using an experimental autoimmune uveitis (EAU) model induced by human interphotoreceptor retinoid-binding protein peptide 651-670 (hIRBP651-670), we examined the effects of kallistatin on the pathogenesis of autoimmune diseases. Compared to wild type (WT) mice, kallistatin transgenic (KS) mice developed severe uveitis with dominant Th17 infiltrates in the eye. In addition, the proliferative antigen-specific T cells isolated from KS EAU mice produced increased levels of IL-17A, but not IFN-γ or IL-10 cytokines. Moreover, splenic CD4+ T cells from naïve KS mice expressed higher levels of Il17a mRNA compared to WT naïve mice. Under Th17 polarization conditions, KS mice exhibited enhanced differentiation of naïve CD4+ T cells into Th17 cells compared to WT controls. Together, our results indicate that kallistatin promotes Th17 differentiation and is a key regulator of aggravating autoinflammation in EAU. Targeting kallistatin might be a potential to treat autoimmune disease.

Proteomics Profiling of Plasma Exosomes in VKH Patients.

OBJECTIVES: Vogt-Koyanagi-Harada syndrome is common autoimmune uveitis that can cause blindness. Recent studies have shown that plasma exosomes carry disease-related proteins that may serve as biomarkers. Here, we aimed to find candidate biomarkers of Vogt-Koyanagi-Harada disease using proteomic analysis of plasma exosomes. METHODS: Exosomes were isolated from the plasma of normal controls and Vogt- Koyanagi-Harada patients in the following groups: a) initial inflammatory attack (active stage), b) remission after one month of treatment (unstable stage), and c) stationary phase after three months of treatment (stable stage). Groups were analyzed by mass spectrometry using isobaric tags for relative and absolute quantitation. After functional analysis, proteins of interest were verified by ELISA. RESULTS: 463 proteins were identified in the exosomes. Forty-three were upregulated at the active inflammation stage, including inflammation-associated proteins. Thirty-one were downregulated. Gene ontology and pathway analyses revealed differential proteins related to cell adhesion, cell phagocytosis, cytoskeleton movement, leukocyte migration across endothelial cells, and platelet activation. By ELISA, Carbonic anhydrase 2 and Ras-related protein Rap-1b were verified as more plentiful at the active stage compared to the normal control and stationary phase in exosomes, but not, however, in microvesicles or plasma. CONCLUSION: Plasma exosomes of Vogt-Koyanagi-Harada patients contain many proteins related to the degree of inflammation. The levels of Carbonic anhydrase 2 and Ras-related protein Rap-1b in exosomes can be used as biomarkers for active inflammation in Vogt-Koyanagi-Harada disease. Further investigation could help study the pathogenesis of Vogt-Koyanagi-Harada disease and identify therapeutic targets.