Introduction:Increasing evidence suggests that autoimmune responses are involved in the pathogenesis of age-related macular degeneration (AMD). This study aimed to characterize changes in serum autoantibody-captured retinal antigens in patients with different AMD subtypes, in order to gain further insight into the immunological mechanisms associated with AMD progression. Methods:Serum samples were collected from healthy controls (CTRL, n = 15) and patients with early AMD (n = 15), intermediate AMD (Int. AMD, n = 15), late AMD (n = 15), neovascular AMD with type 1 macular neovascularization (nAMD type 1, n = 10), and neovascular AMD with type 2 macular neovascularization (nAMD type 2, n = 10). Serum antibodies were isolated using Protein G magnetic beads and incubated with porcine retinal lysates to capture antigen-antibody complexes. After elution, the captured retinal antigens were subjected to proteomic analysis. Results:Mass spectrometry identified 91 differentially captured retinal antigen targets across AMD subtypes. Compared with controls, 11, 9, 11, 18, and 22 retinal antigen targets were significantly increased in serum samples from patients with early AMD, intermediate AMD, late AMD, nAMD type 1, and nAMD type 2, respectively. Conversely, 14, 11, 9, 10, and 20 retinal antigen targets were decreased in the corresponding AMD subtypes, respectively (FDR < 0.05; p < 0.05). Selected mass spectrometryderived retinal antigen targets were further evaluated in serum and tear samples using antigen microarray analysis. Consistent with the proteomics results, anti-ATP5A1, anti-HIST2H2AA3, and anti-PFKM autoantibody levels were significantly increased in tears from patients with nAMD type 2 (p = 0.03, p = 0.01, and p = 0.04, respectively). In addition, anti-FTH1 autoantibody levels were decreased in serum from patients with nAMD type 1. Discussion:Comprehensive screening of serum autoantibody-captured retinal antigens across AMD stages, together with pathway enrichment and STRING network analyses, revealed subtype-specific immune alterations. These findings provide new insights into the immunopathological features of AMD.

Glaucoma is a progressive neurodegenerative disorder that leads to irreversible vision loss, with neuroinflammation recognized as a key factor. Overexpression of glial fibrillary acidic protein (GFAP) is linked to glaucoma pathogenesis and plays a pivotal role in astrocyte-driven neuroinflammation. This study aimed to assess the neuroprotective effects of a monoclonal antibody (mAb) targeting GFAP in glaucoma and to elucidate the underlying mechanisms. An ocular hypertension (OHT) glaucoma model was established in female Sprague Dawley rats using episcleral vein occlusion. Three doses of GFAP mAb (2.5, 25, 50 µg) or vehicle were administered via intravitreal injection. Retinal nerve fiber layer (RNFL) thickness and photopic electroretinogram were monitored longitudinally. Retinal ganglion cell (RGC) survival and glial responses were evaluated with immunostaining. Western blot and microarray analyses were performed to investigate molecular and pathway alterations. Additionally, a cobalt chloride (CoCl2)-induced degenerative R28 cell model was used to validate the protective effects of GFAP mAb in vitro. A bioinformatics re-analysis of a public glaucomatous retina protein dataset was conducted using GSEA, GO, and Cytoscape with GENEMANIA. OHT resulted in a significant loss of RNFL thickness, PhNR amplitude, and RGC survival, all of which were preserved by GFAP mAb treatment. Retinal astrocyte reactivity was inhibited by GFAPmAb in a dose-dependent manner by suppressing GFAP protein overexpression. Notably, 25 µg GFAP mAb effectively regulated both astrocyte and microglial reactivity, leading to a substantial attenuation of neuroinflammation. Mechanistically, GFAP mAb inhibited the p38 MAPK and NF-κB pathways and the NLRP3/Caspase-1/GSDMD axis. In vitro, GFAP mAb improved R28 cell viability under CoCl2 exposure while reducing cell death via inhibition of pyroptosis. Bioinformatic re-analysis highlighted gliosis as a prominent pathway in the glaucomatous retina and indicated GFAP and Caspase1 as central nodes in the putative mechanism network modulated by GFAP mAb. This study demonstrates that GFAP mAb inhibits astrogliosis and glial-glial activation, exerting neuroprotection through the inhibition of inflammation and pyroptosis. The findings suggest that targeting GFAP represents a promising immunotherapeutic strategy for glaucoma treatment.

Aims/Purpose: Dry eye syndrome (DES) is a complex pathological condition of the ocular surface characterized by a disturbance of tear film due to lack of tears or excessive tear evaporation. Topical application of medicated eye drops has been shown to be an effective treatment for this disease. In a previous study, Thealoz® Duo eye drops (Laboratoires Thea, France) were demonstrated to significantly enhance vision‐related functions and to inhibit inflammation in patients with DES. The objective of this study was to provide further insight into the efficacy of Thealoz® Duo eye drops by analyzing the expression profiles of marker proteins in the tears of dry eye patients using microarray technology. Methods: A total of 58 candidates were divided into two groups: those with dry eye (DES, N = 35) and those with healthy eyes (CTRL, N = 23). Thealoz® Duo (hyaluronic acid 0.15% and trehalose 3%) was administered only to the DES patients. Tear samples were collected from each individual in both groups at three distinct time points: prior to treatment (day 0, designated T1), on day 28 ± 4 (T2) and on day 56 ± 4 (T3) following the administration of Thealoz® Duo. Thereafter, all samples underwent analysis of the expression of targeted proteins (e.g., APOA1, S100A8, TNFa, FBP1, IL2, IL8, IL17A, IL1RN, SERPINA1 and MMP9) using microarray technology. Results: The data analysis revealed a significant increase in all maker proteins in DES vs. CTRL at T1 ( p < 0.05). Furthermore, the data demonstrated that the application of Thealoz® Duo resulted in a decrease in the expression of inflammatory markers in DES vs. CTRL at T2 and T3. Conclusions: The present findings provide further evidence supporting the potential of Thealoz® Duo to improve the visual outcomes by mitigating the inflammation on the ocular surface in patients with DES.

Objectives: Topical lubricants are the fundamental treatment for dry eye disease (DED). However, the molecular mechanisms underlying their efficacy remain unknown. Here, the protective effects of Thealoz® Duo with 3% trehalose and 0.15% hyaluronic acid are investigated in DED patients by a longitudinal clinical study and subsequent elucidation of the tear proteome and cell signaling changes. Methods: Participants were classified as moderate to severe DED (DRY, n = 35) and healthy (CTRL, n = 23) groups. Specific DED subgroups comprising evaporative (DRYlip) and aqueous-deficient with DRYlip (DRYaqlip) were also classified. Only DED patients received Thealoz® Duo. All participants were clinically examined before (day 0, T1) and after the application of Thealoz® Duo at day 28 (T2) and day 56 (T3). Next, 174 individual tear samples from all groups at three time-points were subjected to proteomics analysis. Results: Clinically, Thealoz® Duo significantly improved the ocular surface disease index at T2 vs. T1 (DRY, p = 1.4 × 10−2; DRYlip, p = 9.2 × 10−3) and T3 vs. T1 (DRY, p = 2.1 × 10−5; DRYlip, p = 1.2 × 10−4), and the tear break-up time at T3 vs. T1 (DRY, p = 3.8 × 10−2; DRYlip, p = 1.4 × 10−2). Thealoz® Duo significantly ameliorated expression of inflammatory response proteins (p < 0.05) at T3, which was observed at T1 (DRY, p = 3.4 × 10−4; DRYlip, p = 7.1 × 10−3; DRYaqlip, p = 2.7 × 10−8). Protein S100-A8 (S100A8), Alpha-1-antitrypsin (SERPINA1), Annexin A1 (ANXA1), and Apolipoprotein A-I (APOA1) were found to be significantly reduced in all the DED subgroups. The application of Thealoz® Duo showed the therapeutic characteristic of the anti-inflammatory mechanism by promoting the expression of (Metalloproteinase inhibitor 1) TIMP1 in all the DED subgroups. Conclusions: Thealoz® Duo substantially improved the DED symptoms and restored the functionality of the tear lipid layer to near normal in DRYlip and DRY patients by ameliorating inflammation. Notably, this study unravels the novel mechanistic alterations underpinning the healing effects of Thealoz® Duo in DED subgroups in a time-dependent manner, which supports the improvement in corresponding clinical attributes.

The disruption of microglial homeostasis and cytokine release are critical for neuroinflammation post-injury and strongly implicated in retinal neurodegenerative diseases like glaucoma. This study examines microglial responses to chemical hypoxia induced by cobalt chloride (CoCl2) in BV-2 murine microglial cells, focusing on signaling pathways and proteomic alterations. We assessed the protective effects of monoclonal antibodies against TNFα and IL-1β. CoCl2 exposure led to decreased cell viability, reduced mitochondrial membrane potential, increased lactate dehydrogenase release, elevated reactive oxygen species generation, and activation of inflammatory pathways, including nitric oxide synthase (iNOS), STAT1, and NF-κB/NLRP3. These responses were significantly mitigated by treatment with anti-TNFα and anti-IL-1β, suggesting their dual role in reducing microglial damage and inhibiting inflammatory reactivity. Additionally, these treatments reduced apoptosis by modulating ATF4 and the p38 MAPK/caspase-3 pathways. Label-free quantitative mass spectrometry-based proteomics and Gene Ontology revealed that CoCl2 exposure led to the upregulation of proteins primarily involved in endoplasmic reticulum and catabolic processes, while downregulated proteins are associated with biosynthesis. Anti-TNFα and anti-IL-1β treatments partially restored the proteomic profile toward normalcy, with network analysis identifying heat shock protein family A member 8 (HSPA8) as a central mediator in recovery. These findings offer insights into the pathogenesis of hypoxic microglial impairment and suggest potential therapeutic targets.

Aims/Purpose: The detrimental effects of tear hyperosmolarity on human corneal epithelial (HCE) cells is a primary cause of dry eye syndrome (DES). However, the molecular mechanisms underlying inflammatory processes and cellular dysfunction in HCE are largely unknown. This study characterized the proteome changes in an optimized hyperosmolarity‐induced DES model in vitro. Methods: An in vitro hyperosmolar (400–550 mOsm) DES model was established in a simian virus 40 (SV40)‐immortalized HCE cell line. Cells incubated in medium with physiological osmolarity (312 mOsm) were used as control. Cell viability, cell death and intracellular reactive oxygen species (ROS) levels were analysed with MTS, Annexin‐V‐FLUOS, crystal violet and DCFDA assays. Mass spectrometry‐based proteomics analyses characterized the proteome and elucidated the underlying molecular changes. Results: The HCE cell viability was significantly decreased in a concentration‐dependent manner with increasing hyperosmolarity compared to control at 48 hr (450 mOsm, −47%, p = 2.7 × 10 −8 ; 500 mOsm, −71%, p = 1.1 × 10 −4 ; 550 mOsm, −84%, p = 2.6 × 10 −5 ). Notably, cell adherence was significantly decreased ( p = 5.8 × 10 −9 ), while levels of ROS ( p = 3.6 × 10 −2 ) and apoptosis ( p = 3.6 × 10 −2 ) were significantly increased at 450 mOsm compared to control. Proteomics analysis identified ~2000 proteins (FDR <1%) and, as many as 215 proteins were found to be significantly ( p < 0.05) differentially abundant in the 450 vs. 312 mOsm. The effects of high hyperosmolarity (450 mOsm)‐induced damage resembling DES on the HCE cell were further demonstrated by the expression of proteins involved in the activation of inflammatory response ( p = 5.5 × 10 −3 ), apoptosis ( p = 5.3 × 10 −4 ) and generation of ROS (e.g., glutathione S‐transferase P) ( p = 7.1 × 10 −3 ). Moreover, many differentially expressed proteins were also implicated in the regulation of cell viability ( p = 1.1 × 10 −6 ) and organization of cytoplasm (e.g., cyclase associated actin cytoskeleton regulatory protein 1) ( p = 2.4 × 10 −2 ). Conclusions: In summary, an optimized hyperosmolarity‐induced DES cell culture model for proteomics analysis was established, and specific alterations in the proteome were characterized in this model. This in vitro model would be instrumental for testing therapeutic targets for DES in the future.

Aims/Purpose: Reflex tears triggered by various external and internal stimuli play vital functions in the protection of the ocular surface and visual acuity. The impairment of the molecular processes involved in reflex tearing is conjectured to be associated with the progression of dry eye syndrome (DES). Therefore, this study was undertaken to unravel the intricate proteome changes in the reflex and DES tears. Methods: Thirty six subjects underwent basic secretory test (BST) via Schirmer I (without anaesthetics) to determine the secretory rate of the lacrimal gland due to the irritating nature of the strip and were divided into reflex (RF, n = 12, BST = 35.0 ± 0.0), normal (NR, n = 12, BST = 15.3 ± 3.0) and severe aqueous‐deficient DES (DRY, n = 12, BST = 3.4 ± 1.4). The individual tear samples were analysed employing the state‐of‐the‐art mass spectrometry‐based proteomics strategy, followed by bioinformatics analyses to elucidate the underlying molecular processes. Results: A total of 293 and 237 proteins were found to be significantly ( p < 0.05) differentially regulated in the RFL vs. NR (up‐regulated, UR = 12; down‐regulated, DR = 281) and DRY vs. NR (UR = 197; DR = 40), respectively. The hallmark of this study was the identification of 10 lacrimal gland‐specific proteins (e.g., LYZ, LCN1, LTF, PRR4, ZG16B) secreted mainly via the exocytotic pathway, which were significantly up‐regulated in the RFL vs. NR but down‐regulated in the DRY vs. NR. Antibacterial response was also significantly activated in RFL ( p = 1.9 × 10 −2 ) compared to DRY ( p > 0.05). The activated inflammatory response (e.g. S100A8/A9, ANXA1) associated with DRY ( p = 2.7 × 10 −8 , z = 4.0) was found to be inhibited in the RFL ( p = 2.7 × 10 −9 , z = −3.5). Likewise, proteins involved in glycolysis ( z = −2.8), acute phase response ( z = −2.7) and actin cytoskeleton signalling ( z = −2.1) were also inhibited in the RFL compared to DRY. Conclusions: In summary, this study has demonstrated that protein markers involved in the exocytotic pathway play major roles in the production of reflex tears and their regulation is impaired in the DES. Importantly, the abrogation of the vicious cycle of pathways involved in the progression of DES, especially inflammation, was observed in the reflex tears, which may be the key for the protection and maintenance of dynamic balance of the ocular surface.

Aims/Purpose: Hypoxia is a vital cause of neurodegenerative diseases such as glaucoma and retinal ischemia. We investigated the potential neuroprotective effects of anti‐cytokine antibodies against hypoxic damage. Methods: An in vitro hypoxia model was established using cobalt chloride (CoCl 2 ) stimulation in microglial cells (BV‐2). Immunofluorescence staining was performed to detect the expression markers of different microglial phenotypes in BV‐2. Different concentrations of CoCl 2 (0–150 μM) were tested to generate dose–response curves. Following exposure to CoCl 2 at an approximate IC 50 of 0.1 mM, BV‐2 were treated with anti‐tumour necrosis factor‐alpha (anti‐TNF‐α) or anti‐ interleukin‐1 beta (anti‐IL‐1β) antibodies for 24 h. Cell viability was determined by MTS assay. Proteome changes in BV‐2 were analysed with the mass spectrometry‐based proteomics approach. Results: Immunocytochemistry revealed the expression of M1 marker consisting CD68 and M2 marker CD206 in the BV‐2 cells. Hypoxia induced by mimetic CoCl 2 led to a concentration‐dependent viability impairment in BV‐2 and cells treated with 0.1 mM CoCl 2 for 24 h showed decreased viability of ~46%‐ 50%. Interestingly, the application of anti‐TNF‐α (2ug/ml) and anti‐IL‐1β (4ug/ml) antibodies significantly improved the BV‐2 cell viability to 80% ( p < 0.0001) and 76% ( p < 0.0001), respectively. Proteomics and bioinformatics analyses of the differentially expressed proteins revealed that CoCl 2 induced proteome alterations involved in mitochondrial dysfunction signalling pathway ( p = 1.10 × 10 −3 ), which were eliminated by the treatment with both anti‐TNF‐α and anti‐IL‐1β. Besides, CoCl 2 ‐induced activation of endoplasmic reticulum (ER) stress response ( p = 3.33 × 10 −9 ) was alleviated by anti‐IL‐1β and anti‐TNF‐α treatment ( p = 1.7 × 10 −7 and p = 8.82 × 10 −7 , respectively). Particularly, anti‐TNF‐α antibody significantly downregulated proteins involved in apoptosis ( p = 1.58 × 10 −10 ). Anti‐IL‐1β induced Nrf2‐mediated antioxidant response and exerts protection against oxidative stress ( p = 1.00 × 10 −6 ). Conclusions: Antibodies targeting cytokines provide neuroprotective effects on microglial cells from hypoxic stress. These findings demonstrated the potential use of anti‐cytokines in the treatment of neurodegenerative diseases.

Age-related macular degeneration (AMD) is a severe retinal disease that causes irreversible visual loss and blindness in elderly populations worldwide. The pathological mechanism of AMD is complex, involving the interactions of multiple environmental and genetic factors. A poor understanding of the disease leads to limited treatment options and few effective prevention methods. The discovery of autoantibodies in AMD patients provides an opportunity to explore the pathogenesis and treatment direction of the disease. This review focuses on the mitochondria-associated autoantibodies and summarizes the functional roles of mitochondria under physiological conditions and their alterations during the pathological states. Additionally, it discusses the crosstalk between mitochondria and other organelles, as well as the mitochondria-related therapeutic strategies in AMD.

Aims/Purpose: Neurovascular dysfunction in glaucoma is exacerbated by aging. We recently deciphered the crucial role of the cytochrome P450 (CYP) metabolic pathway in the retina (R) and ophthalmic artery (OA). Vasoactive CYP metabolites are hydrolysed by soluble epoxide hydrolase (sEH), thereby diminishing their protective effects. Hence, sEH has emerged as a novel inhibitory target. This study defined the neuro‐vasculoprotective molecular signatures of Ephx2 gene deletion (KO) that impinge senescence in the neural R and OA. Methods: Retinae and OA were isolated from young (3–5 months) and old (12–24 months) male wild type (WT) and KO mice. Samples were pooled ( n = 5 mice/group/replicate) to yield 4 biological replicates per group and subjected to label‐free mass spectrometry‐based proteomics and in‐silico bioinformatics analyses. Results: A total of 3706 retinal and 644 OA proteins were identified. Aging profoundly affected the R and OA proteome of WT mice, as demonstrated by the differential expressions of 183 and 111 proteins, respectively. The key changes attributed to age‐related R cellular dysfunction and DNA damage were reflected in the inactivation of NAD + ( p = 9.1 × 10 −4 ) and sirtuin ( p = 6.2 × 10 −3 ) signalling pathways. Remarkably, KO had a major influence on the regulation of R proteins involved in bioenergetics reprogramming via mitochondrial homeostasis in young mice, which was shown by the activation of oxidative phosphorylation ( p = 1.3 × 10 −6 ) and inhibition of mitochondrial dysfunction ( p = 3.4 × 10 −3 ), both of which corresponded to upstream involvement of UQCC3 ( p = 7.3 × 10 −7 ). A translation regulator, LARP1, which regulates cell proliferation and protein synthesis, was highly activated in the aged KO retina ( p = 10 × 10 −16 ) compared to aged WT ( p = 3.1 × 10 −5 ). In the OA, vascular remodelling processes observed in the aged WT mice, primarily compromised microtubule dynamics (3.6 × 10 −8 ) and cell–cell contact (3.7 × 10 −6 ), were significantly attenuated in the KO. Noteworthy, KO significantly mitigated proteome changes attributed to inflammatory response in the OA of both young and aged mice. Conclusions: In gist, the Ephx2 gene deletion significantly ameliorated detrimental senescesome in ocular neurovascular tissues. The potential use of sEH inhibitors for glaucoma needs further investigation.

Background: Age-related macular degeneration (AMD) is a multifactorial disorder, and there is growing evidence of immunological involvement in its pathogenesis. To address this, we aimed to identify biomarker candidates related to retinal antigens in patients with neovascular AMD treated with ranibizumab and healthy subjects. Materials and Methods: This study was designed as a prospective, open, parallel-group, interventional, single-center phase IV trial. Fifty subjects with neovascular AMD and twenty healthy volunteers were enrolled. The primary objective was to assess the efficacy of intravitreally (IVT) administered ranibizumab in terms of the change in best-corrected visual acuity in subjects with all subtypes of neovascular AMD and in a subgroup of pretreated AMD subjects. A secondary objective was to assess the efficacy of the same in terms of the change in central retinal thickness (CRT) in the same subjects. Another secondary objective was to identify antibodies against retinal antigens in patients with neovascular AMD treated with ranibizumab and healthy subjects. The last secondary objective was to correlate functional and structural parameters with the identified biomarker candidates to differentiate between initial and deferred responders to IVT administered ranibizumab. Serum was analyzed using customized antigen microarrays containing 58 antigens. Results: After 12 weeks of ranibizumab treatment, treated patients gained 4.02 letters on average. The central retinal thickness (CRT) measured in the complete AMD study population was significantly (p < 0.001) decreased at Week 24 compared to the baseline measurement, and the mean CRT dropped from 393.4 to 296.8 µm. A significant increase in the following autoantibodies was detected between the control group and AMD group at Week 24, as well as in the AMD group between baseline and Week 24: antibodies targeting the proteins serotransferrin, opioid growth factor receptor, 60 kDa chaperonin 2, neurotrophin-4, dermcidin, clusterin and vascular endothelial growth factor. Conclusions: The present trial was able to confirm the efficacy of ranibizumab treatment in neovascular AMD, and treatment-naïve patients benefitted the most. Up- and downregulations of antibodies were observed over the course of treatment with ranibizumab. Some antibodies seemed to have a fair correlation with the classification of initial and deferred responders.

Aims/Purpose: Ferroptosis is known to be involved in the pathogenesis of age‐related macular degeneration (AMD). We elucidated the potential roles and related pathways of ferroptosis in an in vitro AMD cell culture model and the mass spectrometry (MS)‐based proteomics analysis. Methods: To mimic ferroptosis in AMD, ARPE‐19 cells were treated with erastin (ERS; 1, 2, 5 and 10 μM) or RSL3 (1, 2, 4, and 6 μM) for 24 h. Cells without treatment were used as control. Cell viability and glutathione peroxidase 4 (Gpx4) expression levels were used to determine the optimal AMD model. The MS‐based proteomics approach was employed to profile the protein expressions in ferroptotic RPE cells compared to controls. Next, bioinformatics analysis identified the significantly affected functions and pathways of the differentially expressed proteins. Results: Cell viability and Gpx4 expression levels of ARPE‐19 cells were decreased in a dose‐dependent manner after induction with ERS (1, 2, 5 and 10 μM) and RSL3 (1, 2, 4, and 6 μM). Doses corresponding to ~60% cell viability (5 μM ERS and 2 μM RSL3) were selected for the proteomics analysis. A total of 1356 proteins were identified in all groups, with 300 and 209 significantly ( p < 0.05) differentially expressed proteins in the ERS and RSL3 group, respectively. The induction of ferroptosis in this optimized model with ERS ( p = 6.3 × 10 −3 ) and RSL3 ( p = 1.5 × 10 −4 ), and disruption of iron homeostasis signalling pathway ( p = 1.9 × 10 −4 in RSL3) were further confirmed by proteomics analysis. Iron metabolism‐related proteins ferritin light chain and heme oxygenase 1 were significantly up‐regulated in the RSL3 group. Notably, a large cluster of proteins were highly significantly implicated in the eukaryotic initiation factor 2 signalling pathway ( p = 2.0 × 10 −30 in ERS and 1.0 × 10 −5 in RSL3), which was correlated with ferroptosis. Furthermore, the upregulation of autophagy‐related Ras‐related protein Ral‐A and involvement of mTOR signalling pathway were found in both groups ( p = 3.2 × 10 −6 in ERS and p = 1.9 × 10 −3 in RSL3). Conclusions: In summary, this study established an optimum model for ferroptosis and demonstrated that it is involved in RPE cell death and is autophagy‐dependent. Our results also suggested that the disruption of iron uptake and intracellular storage in RPE cells are involved in AMD progression.

Aims/Purpose: Microglia‐mediated neuroinflammation is involved in many neurodegenerative diseases, including glaucoma and diabetic retinopathy. This study examined the effects of anti‐cytokine antibodies on LPS‐induced inflammatory response of microglial cells, and explored the underlying molecular mechanisms by proteomic analysis. Methods: A lipopolysaccharide (LPS)‐induced neuroinflammation model was established in murine BV‐2 microglial cells in vitro. The expression of microglial cell markers (CD68 and CD206) were detected by immunocytochemistry. BV‐2 was stimulated with LPS (1 μg/mL) with or without anti‐tumour necrosis factor‐alpha (anti‐TNF‐α, 2 μg /ml) or anti‐interleukin‐1 beta (anti‐IL‐1β, 4 μg /ml) for 24 h. LPS‐induced inflammation level was analysed using the Griess assay to determine nitric oxide (NO) concentration. Proteome alterations were characterized employing the mass spectrometry‐based proteomics and bioinformatics analyses. Results: Immunocytochemistry confirmed the expression of microglial markers in BV‐2 cells. LPS stimulation induced excessive activation of BV‐2, with significant increase in NO generation ( p < 0.0001). Anti‐IL‐1β significantly attenuated 34% of the LPS‐induced NO generation ( p < 0.0001). Interestingly, anti‐TNF‐α showed a stronger anti‐inflammatory effect by reducing 57% of LPS‐induced NO generation ( p < 0.0001). Bioinformatics analyses revealed that differentially expressed proteins involved in inflammation ( p = 3.8 × 10 −5 ) and apoptotic ( p = 3.2 × 10 −11 ) signalling pathways were significantly activated by LPS. Particularly, LPS‐mediated upregulation of STAT1 ( p = 8.6 × 10 −6 ) was restored by anti‐IL‐1β and anti‐TNF‐α ( p = 3.1 × 10 −4 and p = 2.7 × 10 −3 , respectively). Anti‐IL‐1β induced proteome alterations involved in anti‐inflammatory signalling pathways and promote glial cell polarization towards neuroprotective M2 phenotype ( p = 5.3 × 10 −6 ), while anti‐TNF‐α treatment significantly regulated proteins involved in immune response of macrophages ( p = 2.7 × 10 −8 ). Conclusions: In summary, anti‐TNF‐α and anti‐IL‐1β antibodies alleviated LPS‐induced neuroinflammation and altered proteins involved in neuroprotective pathways. Hence, anti‐cytokine antibodies have therapeutic potential as neuroprotective agents.

Background: Observational studies have noted that patients with certain retinal degenerative diseases exhibit iron disturbances in the retina or vitreous compared to healthy controls. However, the connection between serum iron status and these diseases remains unclear. This study aims to explore the potential causal relationship between serum iron status biomarkers and the development of age-related macular degeneration (AMD), retinitis pigmentosa (RP), and diabetic retinopathy (DR). Methods: A two-sample Mendelian randomization (MR) analysis was conducted to investigate the causal relationship between serum iron status and several retinal degenerative diseases. Genome-wide association study (GWAS) summary-level data were extracted from public GWAS databases. Inverse-variance weighting (IVW), MR-Egger regressions, Simple model, Weighted median, and Weight mode were used as MR methods. Sensitivity analysis was conducted to confirm the robustness of the results by examining horizontal pleiotropy and heterogeneity through MR-Egger intercept and leave-one-out analysis. Results: The MR analysis revealed causal relationships between genetically predicted serum iron status biomarkers and various retinal diseases. Transferrin was positively associated with the odds of AMD (whether dry or wet) (OR = 1.167, 95% CI = 1.045-1.304, p = 0.006) and wet AMD (OR = 1.194, 95% CI = 1.018-1.402, p = 0.030). Ferritin was negatively associated with the odds of wet AMD (OR = 0.555, 95% CI = 0.333-0.927, p = 0.024). Serum iron (OR = 0.508, 95% CI = 0.260-0.993, p = 0.048) and transferrin saturation (OR = 0.508, 95% CI = 0.260-0.993, p = 0.048) were negatively associated with the odds of RP. Conclusions: These findings provide evidence supporting a potential causal relationship between serum iron status and various retinal degenerative diseases, highlighting a direction for future research into the underlying mechanisms of these diseases.

Glaucoma is a heterogeneous group of optic neuropathies characterized by a progressive degeneration of the retinal ganglion cells (RGCs), leading to irreversible vision loss. Nowadays, the traditional therapeutic approach to glaucoma consists of lowering the intraocular pressure (IOP), which does not address the neurodegenerative features of the disease. Besides animal models of glaucoma, there is a considerable need for in vitro experimental models to propose new therapeutic strategies for this ocular disease. In this study, we elucidated the pathological mechanisms leading to neuroretinal R28 cell death after exposure to glutamate and hydrogen peroxide (H2O2) in order to develop new therapeutic approaches for oxidative stress-induced retinal diseases, including glaucoma. We were able to show that glutamate and H2O2 can induce a decrease in R28 cell viability in a concentration-dependent manner. A cell viability of about 42% was found after exposure to 3 mM of glutamate and about 56% after exposure to 100 µM of H2O2 (n = 4). Label-free quantitative mass spectrometry analysis revealed differential alterations of 193 and 311 proteins in R28 cells exposed to 3 mM of glutamate and 100 µM of H2O2, respectively (FDR < 1%; p < 0.05). Bioinformatics analysis indicated that the protein changes were associated with the dysregulation of signaling pathways, which was similar to those observed in glaucoma. Thus, the proteomic alteration induced by glutamate was associated with the inhibition of the PI3K/AKT signaling pathway. On the other hand, H2O2-induced toxicity in R28 cells was linked to the activation of apoptosis signaling and the inhibition of the mTOR and ERK/MAPK signaling pathways. Furthermore, the data show a similarity in the inhibition of the EIF2 and AMPK signaling pathways and the activation of the sumoylation and WNT/β-catenin signaling pathways in both groups. Our findings suggest that the exposure of R28 cells to glutamate and H2O2 could induce glaucoma-like neurodegenerative features and potentially provide a suitable tool for the development of new therapeutic strategies for retinal diseases.

Aims/Purpose: Dry eye syndrome (DES) is a common yet deplorable disease of the ocular surface with high global prevalence. The pathomechanisms underlying the impairment of the native tear protein complexes remain unexplored. This study established a quantitative proteomics approach to characterize the novel protein complexes and unravel their functional roles in tears of DES patients. Methods: Tear samples were collected using Schirmer's strips from aqueous‐deficient DES (DRY, n = 16) and healthy (CTRL, n = 16) subjects following comprehensive DES clinical examinations. The native tear protein complex profiles were elucidated employing a customized blue native gel electrophoretic (BN‐PAGE) combined with mass spectrometry (MS)‐based proteomics strategy. In‐silico bioinformatics tools were employed for gene annotation analysis of the complexes. Results: Five major native protein complexes designated as complex I (cI, 800 kDa), II (cII, 400 kDa), III (cIII, 240 kDa), IV (cIV, 140 kDa) and V (cV, 66 kDa) were characterized for the first time in human tears. Interestingly, a cluster of immune‐related proteins identified in cI (e.g., IGHA1) were annotated to complement pathways and was found to be significantly increased in abundance in DRY vs. CTRL ( p = 8.2 × 10 −3 ). Likewise, increased abundance of another cluster of proteins annotated to inflammatory response (e.g. S100A9) in DRY ( p = 1.7 × 10 −2 ) was associated to cIII. On the contrary, cII was largely composed of lacrimal‐specific proteins (e.g., LYZ), which was found to be decreased in abundance in DRY ( p = 2.4 × 10 −2 ). Similarly, decrement of antimicrobial proteins in cIV (e.g., LACRT) was observed in DRY ( p = 1.7 × 10 −2 ). The expression of a cluster of carrier proteins involved in protein–protein interactions (e.g., ALB) was annotated in cV, which was increased in DRY ( p = 4.4 × 10 −3 ). Noteworthy, 35 new DES markers were identified with this methodological approach. Conclusions: Collectively, a tear‐customized BN‐PAGE‐MS analysis was instrumental to decipher the multifaceted biological functions of novel native protein complexes in human tears. For the first time, the intricate molecular mechanistic changes underlying these protein complexes in DES were unravelled, which could be instrumental for potential development of therapeutic strategies in ameliorating DES.