A total of 77 eyes from 77 patients were included in this study, comprising 31 eyes diagnosed with Posner-Schlossman syndrome (PSS), 26 with primary open-angle glaucoma (POAG), and 20 with age-related cataract (ARC, serving as controls). All participants were recruited from the Eye and ENT Hospital, Fudan University, between August 2022 and November 2024. Patients in the control group exhibited no signs of ocular pathology upon comprehensive ophthalmic examination and had intraocular pressure (IOP) values below 21 mmHg.

All participants enrolled in this study were carefully screened and confirmed to have no underlying systemic diseases (e.g., diabetes, autoimmune disorders, cardiovascular diseases, etc.). Furthermore, all samples were collected prior to the initiation of any study-related or relevant medications, ensuring that the sample status reflected the natural history of the condition under investigation without pharmacological confounding. In patients with PSS, aqueous humor sampling was performed during an active episode characterized by elevated intraocular pressure and mild anterior chamber inflammation, but before the initiation of treatment, to ensure that cytokine profiles reflected the intrinsic inflammatory status of the disease.

The diagnosis of PSS was based on the following criteria: (1) recurrent unilateral episodes of elevated IOP (>21 mmHg) with spontaneous return to normal between episodes; (2) mild anterior chamber inflammation, characterized by fine keratic precipitates on slit-lamp examination; (3) open iridocorneal angle confirmed by gonioscopy; and (4) a healthy fellow eye with normal IOP, visual field, and optic disc morphology (11). POAG diagnosis was established according to standard criteria, including an open iridocorneal angle, documented history of elevated IOP prior to anti-glaucoma treatment, and exclusion of secondary glaucoma.

The study protocol adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Ethics Committee of the Eye and ENT Hospital, Fudan University. Written informed consent was obtained from all participants.

A 12-plex human cytokine detection kit (Catalog No. 191217) was purchased from Hangzhou R&D Biological Technology Co., Ltd. Inflammatory cytokines in aqueous humor—including IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, TNF-α, IFN-α, and IFN-γ—were measured using a bead-based multiplex flow cytometry immunoassay. The procedure employed a microsphere-based sandwich immunofluorescence technique, integrated with enzyme-linked immunosorbent assay (ELISA) principles.

Qualitative polymerase chain reaction (PCR) for CMV DNA detection was performed using a TaqMan PCR kit (Shanghai ZJ BioTech Co., Ltd., China) on an ABI 7500 real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA).

Aqueous humor samples (100 μL each) were obtained via anterior chamber paracentesis prior to any intraocular surgery or pharmacological treatment. All samples were collected under aseptic conditions and immediately stored at –80 °C until analysis.

Visual Acuity: Visual acuity was measured using an international logMAR visual acuity chart. Visual Field Examination: Visual fields were measured using an Octopus 900 automated perimeter. Corneal Endothelial Cell Density: Corneal endothelial cell density was measured using a corneal endothelial microscope (NSP-9900II).

Data analysis was conducted using IBM SPSS Statistics 21.0. The Shapiro–Wilk test was used to assess normality. For normally distributed variables, results are expressed as mean ± standard deviation (SD), and comparisons between groups were made using independent-sample t-tests. Non-normally distributed variables are presented as median with interquartile range [IQR], and analyzed using the Mann–Whitney U test. Categorical variables were compared using Fisher’s exact test.

Spearman’s rank correlation analysis was performed to evaluate associations between cytokine concentrations and clinical parameters (e.g., IOP, CECD, visual field). Correlation strength was defined as: strong (|r| ≥ 0.8), moderate (|r| = 0.5–0.8), weak (|r| = 0.3–0.5), and negligible (|r| < 0.3). A P-value <0.05 was considered statistically significant. Given the exploratory nature of this study and the biological correlations among cytokines, formal correction for multiple comparisons was not applied. The findings should therefore be interpreted with caution and viewed as hypothesis-generating.

Receiver operating characteristic (ROC) curves were generated using MedCalc 19.6 (MedCalc Software Ltd., Ostend, Belgium) to assess the diagnostic performance of candidate cytokines. For ROC curve analyses, optimal cutoff values were determined using Youden’s index, defined as sensitivity + specificity − 1.

Table 1 summarizes the demographic and preoperative clinical characteristics of the study population. There were no statistically significant differences in age or gender distribution among the PSS, POAG, and control groups (P > 0.05). Both the PSS and POAG groups exhibited significantly elevated intraocular pressure (IOP) compared with controls (P < 0.0001), while the IOP difference between PSS and POAG groups was not statistically significant (P = 0.0534).

Among the 12 measured cytokines, IL-2, IL-4, IL-12, and IFN-α were below the lower limit of detection in the majority of samples across all three groups and were therefore excluded from subsequent quantitative analyses.

As shown in Table 2, the levels of IL-1β, IL-5, IL-6, IL-8, IL-10, IFN-γ, and TNF-α in the aqueous humor of PSS patients were significantly higher than those in the cataract control group (P < 0.05). Furthermore, IL-1β, IL-6, IL-10, and IFN-γ levels were also significantly elevated in PSS compared to POAG eyes (P < 0.05), while IL-5 and TNF-α showed no significant differences between the two.

Receiver operating characteristic (ROC) curve analysis was conducted to assess the potential discriminatory ability of IL-1β, IL-6, IL-10, and IFN-γ in differentiating PSS from POAG. The results are presented in Table 3 and Figure 1.

Among the cytokines analyzed, IL-1β demonstrated the highest diagnostic performance, with an area under the curve (AUC) of 0.867 (95% CI: 0.750–0.942), sensitivity of 83.87%, and specificity of 76.92% at a cutoff of 3.14 pg/mL. IL-6 also showed moderate discriminative value (AUC = 0.739), followed by IFN-γ and IL-10.

Stratified analyses based on sex, age, presence of coexisting cataract, and CMV DNA status revealed no significant differences in aqueous humor cytokine levels among PSS subgroups (Table 4, all P > 0.05).

Among the 31 PSS patients, 17 (54.8%) tested positive for CMV DNA in aqueous humor by PCR. However, no statistically significant differences were observed in cytokine concentrations between CMV-positive and CMV-negative individuals (Table 4).

Correlation analysis between cytokine levels and ophthalmic measurements in the PSS group revealed that IL-6 was significantly and positively correlated with IOP (r = 0.395, P = 0.007) and relative endothelial cell loss (RECL; r = 0.453, P = 0.039) (Table 5). No other cytokines showed statistically significant correlations with CECD, visual field indices, or axial length. In multivariable regression analyses adjusted for age, sex, and intraocular pressure, IL-6 remained significantly associated with intraocular pressure and relative endothelial cell loss.

Spearman’s rank correlation analysis revealed a complex cytokine regulatory network within the aqueous humor of patients with Posner-Schlossman syndrome (PSS) (Table 6). IL-6 exhibited strong positive correlations with both IL-8 (r = 0.688, P < 0.0001) and IL-10 (r = 0.487, P = 0.006), suggesting coordinated upregulation among these inflammatory mediators. Similarly, IL-8 was positively correlated with IL-10 (r = 0.506, P = 0.004) and IFN-γ (r = 0.404, P = 0.024), showing a significant positive correlation, which is consistent with (or may reflect) coordinated expression within a shared inflammatory pathway. IL-1β showed a positive correlation with IL-17 (r = 0.457, P = 0.010), but was negatively correlated with TNF-α (r = –0.491, P = 0.005), IL-1β levels showed a significant positive correlation with IL-17 and a significant negative correlation with TNF-α. This pattern of correlations suggests a complex, non-linear regulatory network among these cytokines in PSS, rather than a simple linear association. Additionally, IL-5 was negatively correlated with IFN-γ (r = –0.440, P = 0.013), reflecting a potential counterbalance between Th2- and Th1-type immune responses. Collectively, these findings highlight the intricate and dynamic cytokine interactions that may underlie the immunopathogenesis of PSS.