Brief Report Ophthalmic Res 2022;65:111–120 Proinflammatory Cytokine Profile Differences between Primary Open-Angle and Pseudoexfoliative Glaucoma Beatriz Vidal-Villegas a Bárbara Burgos-Blasco a Jose Luis Santiago Alvarez b Laura Espino-Paisán c Jose Fernández-Vigo a Vanessa Andrés-Guerrero a Julián García-Feijoo a Jose María Martínez-de-la-Casa a aServicio de Oftalmología, Hospital Clínico San Carlos and Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain; bServicio de Inmunologia, Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain; cLaboratorio de Investigación en Genética de Enfermedades Complejas, Hospital Clínico San Carlos and Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain Received: June 2, 2021 Accepted: September 13, 2021 Published online: October 18, 2021 Correspondence to: Beatriz Vidal-Villegas, beatrizvidalvillegas @ gmail.com © 2021 The Author(s). Published by S. Karger AG, Basel karger@karger.com www.karger.com/ore DOI: 10.1159/000519816 Keywords Glaucoma biomarkers · Proinflammatory cytokines · Primary open-angle glaucoma · Pseudoexfoliative glaucoma · Aqueous humor · Tear · Ocular hypotensive medication Abstract Introduction: Few studies have investigated glaucoma bio- markers in aqueous humor and tear and have found eleva- tions of proinflammatory cytokines in patients with primary open-angle glaucoma (POAG) and pseudoexfoliative glau- coma (PXG). In this study, we investigate differences in in- flammatory cytokines between POAG and PXG patients to find specific disease biomarkers. Methods: For this purpose, tear and aqueous humor samples of 14 eyes with POAG and 15 eyes with PXG undergoing cataract surgery were immu- noassayed for 27 proinflammatory cytokines. The concen- trations of cytokines in tear and aqueous humor and their association with clinical variables were analyzed, correlated, and compared between the groups. Results: We found that the levels of three cytokines differed significantly in the aque- ous humor of POAG and PXG patients: IL-12 and IL-13 were higher in the POAG group, while monocyte chemoattractant protein-1 (monocyte chemotactic and activating factor) was higher in the PXG group. The number of topical hypotensive medications was correlated with diminished levels of two cytokines (IL-7 and basic fibroblast growth factor) in aque- ous humor in the POAG group and with diminished levels of IL-12 in tear in the PXG group. Conclusion: We conclude that both POAG and PXG show elevated concentrations of proin- flammatory cytokines in tear and aqueous humor that could be used as biomarkers for these types of glaucoma and that the concentrations in aqueous humor of three cytokines, IL-12, IL-13, and monocyte chemoattractant protein-1 (mono- cyte chemotactic and activating factor), could be used to dif- ferentiate POAG and PXG. © 2021 The Author(s). Published by S. Karger AG, Basel Introduction There are various types of glaucoma that affect predom- inantly certain age groups, but the most common form in adults is the open-angle glaucoma, which may be pri- mary or secondary. Primary open-angle glaucoma (POAG) represents 75% of all glaucoma and, although various risk This is an Open Access article licensed under the Creative Commons Attribution-NonCommercial-4.0 International License (CC BY-NC) (http://www.karger.com/Services/OpenAccessLicense), applicable to the online version of the article only. Usage and distribution for com- mercial purposes requires written permission. Vidal-Villegas et al.Ophthalmic Res 2022;65:111–120112 DOI: 10.1159/000519816 factors for this type of glaucoma have been identified, its exact cause remains unknown [1, 2]. The most common form of secondary open-angle glaucoma is the pseudoex- foliative glaucoma (PXG) [1]. This occurs in the context of pseudoexfoliation syndrome (PXS), which is characterized by the deposition of fine fibrillary material in the anterior segment of the eye, along with other organs [1, 3]. Because POAG and PXG often course without symp- toms, its diagnosis is often made when there is already extensive irreversible damage to the optic nerve. There- fore, it is important to find biomarkers of the disease that could allow an early diagnosis and deepen our knowledge of glaucoma pathogenesis. Recent studies have suggested that ischemia, oxidative stress, and, especially, inflamma- tion may be involved in PXS and POAG [4, 5]. In this regard, recent studies have documented an eleva- tion of inflammatory cytokines in POAG and PXG, with increased cytokine levels in tear of patients with POAG [6]. In addition, two earlier studies of our group revealed high- er cytokine levels in tear and aqueous humor of POAG patients and specifically in the tears of patients treated with topical drugs containing preservatives [7, 8]. Other authors have also found increased cytokine levels in the aqueous humor of patients with POAG [9] or in both PXG and POAG [10, 11]. Finally, various studies have found differ- ences in aqueous humor growth factors or cytokine levels, but only in patients with PXG [12–17]. Thus, although pre- vious studies investigating cytokine levels have reported variable results in POAG and PXG, they all suggest that inflammation involved in their pathophysiology could thus serve as a biomarker for these types of glaucoma. The main objective of the present study was to investi- gate differences in inflammation between POAG and PXG (the most common forms of the disease) that could give insight into their pathogenesis. For this purpose, we deter- mined the levels of 27 inflammatory cytokines in tear and aqueous humor of patients with POAG and PXG and ana- lyzed the differences between these types of glaucoma. Methods Patients This cross-sectional observational study was carried out in pa- tients with POAG or PXG of the Glaucoma Unit of the Hospital Clinico San Carlos in Madrid (Spain) who were controlled with top- ical hypotensive treatment and underwent cataract surgery. The study was approved by the Clinical Research Ethics Committee of the hospital and followed the Helsinki Declaration. All the patients were informed about the procedure and signed the consent form. Eighteen patients with POAG and 17 patients with PXG who were scheduled for cataract surgery between January and April of 2019 were included. The inclusion criteria were: age older than 40 years, diagnosis of POAG or PXG at least 2 years before, and topi- cal hypotensive treatment maintained for the last 6 months. The exclusion criteria were previous ocular surgery, YAG iridotomy, intraocular or intravitreal injections, or other ocular pathology. From the medical records of the patients, the following data were obtained: age, sex, previous diseases, surgeries, and treat- ments, including the number and type of ocular hypotensive drugs. From the data of the visit prior to surgery, we also anno- tated the best corrected visual acuity (BCVA), the intraocular pres- sure, the mean deviation, and corrected loss of variance of the 24-2 visual field (Octopus, Haag Streit), and the peripapillary nerve fi- ber layer thickness (Optic Coherence Tomography, Spectralis, Heidelberg). In the 3-month follow-up, the BCVA and number of topical medications were included. The patients were informed of the study on their arrival to the hospital for the cataract operation (between 8:30 and 12:30 a.m.). Before initiating the procedure for anesthesia or dilation of the pupil, a tear sample (3–5 µL) was obtained without anesthesia from the inferior fornix with a glass capillary tube (5 µL; Drummond Microcaps; Broomall, PA, USA). The aqueous humor (40–50 µL) sample was obtained through paracentesis with a 30G needle on a syringe as the first step of the surgery. Both samples were immedi- ately transported to the Immunology Department, where tear samples were diluted with buffered saline to give a final volume of 50 μL and then immediately frozen at −80°C until processing. Cytokine Determination Cytokine levels in tear and aqueous humor were analyzed using the Bio-Plex ProTM Human Cytokine 27-Plex Immunoassay kit (Lab- oratorios Bio-Rad SA, Madrid, Spain) according to the manufac- turer’s instructions. This kit uses fluorescent magnetic surfaces and antibodies and allows the quantitation of 27 proinflammatory cyto- kines: Interleukin (IL)-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL9, IL-10, IL-12, IL-13, IL-15, IL-17A, eotaxin, basic fibroblast growth factor, granulocyte colony-stimulating factor, granulocyte- macrophage colony-stimulating factor, interferon gamma (IFN-γ), interferon gamma-induced protein 10, monocyte chemoattractant protein-1 (monocyte chemotactic and activating factor) (MCP-1 [MCAF]), macrophage inflammatory proteins (MIP) forms 1α and 1β (MIP-1α and MIP-1β), platelet-derived growth factor BB dimer, Regulated on Activation, Normal T Cell Expressed and Secreted fac- tor, tumor necrosis factor-α, and vascular endothelial growth factor (VEGF). For this assay, 50 µL of each sample was placed in a well and the platform was read using Luminex MAGPIXTM (Luminex Corpo- ration, Austin, TX, USA). Cytokine concentrations were determined by interpolation of the fluorescence measurements of each sample and adjusting it to their standard curves. Also, for each sample a cor- rection factor according to the dilution was used. Finally, the soft- ware Bio-Plex ManagerTM (Bio-Rad, Hercules, CA, USA) calculated the concentration of the 27 cytokines. Data Analysis The data from the medical records and cytokine concentra- tions were introduced in Microsoft Excel sheets and analyzed with the software SPSS (IBM SPSS Statistics for Mac 22.0 (IBM Corp., Armonk, NY, USA). Data are expressed as means ± stan- dard deviation (SD). Quantitative variables were compared using the t test or the Mann-Whitney test depending on the normality of the data. The correlation between cytokine levels in tear and Proinflammatory Cytokines as Glaucoma Biomarkers 113Ophthalmic Res 2022;65:111–120 DOI: 10.1159/000519816 aqueous humor and number of treatments was analyzed using the Spearman’s Rho correlation coefficient. A p value <0.05 was considered statistically significant. Results Patients Although 18 eyes of patients with POAG and 17 eyes of patients with PXG were initially included in the study, some samples had to be discarded because of insufficient quan- tity and finally only 14 eyes with POAG and 15 eyes with PXG could be included. Table 1 depicts the characteristics of both study groups. Mean age of the patients was similar in both groups. BCVA increased after cataract surgery in both groups, and was significantly higher in the POAG than in the PXG group both at baseline and postoperative- ly. The number of topical hypotensive drugs used preop- eratively and postoperatively was similar in both groups and all patients had been on that treatment for at least 6 months. Preoperative and postoperative intraocular pres- sure was also similar in both groups. The mean deviation of the visual field was −6.49 ± 6,28 and −10.45 ± 6.35 dB in the POAG and PXG group, respectively, with significant differences between them (Table 1). The global and sectorial pRNFL thicknesses were similar between groups, except for the superotem- poral sector that was significantly thinner in the PXG group (Table 1). Cytokine Concentrations in Tear and Aqueous Humor Cytokine concentrations (pg/mL) can be observed in Figures 1 and 2 and Table 2. Cytokine concentrations var- ied greatly in aqueous humor and tear in both experimen- tal groups. However, when cytokine concentrations in tear or aqueous humor were compared between groups, three cytokines showed significant differences: IL-12 and IL-13 were significantly higher in the POAG group and MCP-1 (MCAF) was significantly increased in PXG pa- tients (Fig. 1, 2; Table 2). Correlation between Cytokine Concentrations in Tear and Aqueous Humor There were no significant correlations between the cytokine concentrations in tear and aqueous humor in the POAG group (Table 2), but there were significant correlations in the concentrations of three cytokines in the PXG group: IFN-γ, MIP-1β, and VEGF (Table 2). Table 1. Demographic and clinical variables and comparisons between groups Variable Diagnosis Statistical comparison (p = test) POAG PXG Age, years 76.14±6.89 77.40±9.45 0.652 Mann-Whitney Baseline BCVA 0.43±0.13 0.29±0.15 0.019 Mann-Whitney Postoperative BCVA 0.85±0.12 0.61±0.32 0.019 Mann-Whitney Topical hypotensive drugs (baseline), n 1.69±0.63 1.64±0.84 0.864 t test Topical hypotensive drugs (postoperative), n 0.69±0.75 0.79±0.80 0.757 t test IOP (baseline) 19.08±3.45 19.79±2.75 0.488 Mann-Whitney IOP (postoperative) 16.54±1.81 16.71±4.08 0.756 Mann-Whitney MD of the VF 6.49±6.28 10.45±6.35 0.020 Mann-Whitney CLV of the VF 3.98±1.41 4.93±1.41 0.102 Mann-Whitney pRNFL thickness GLOBAL 83.43±20.88 70.93±18.56 0.085 Mann-Whitney Nasal 59.07±19.29 57.73±18.51 0.813 Mann-Whitney Nasal superior 93.64±25.32 78.20±29.96 0.158 Mann-Whitney Temporal superior 116.50±32.38 86.53±32.52 0.023 Mann-Whitney Temporal 66±17.66 58.07±13.35 0.158 Mann-Whitney Temporal inferior 119.29±45.80 91.87±32.08 0.057 Mann-Whitney Nasal inferior 87.36±27.99 79.80±22.89 0.146 Mann-Whitney Values are the mean ± SD. Postoperative values were obtained 3 months after cataract surgery. The value in bold indicates a significant difference between the groups. n = number IOP, intraocular pressure; MD, mean deviation; CLV, corrected loss of variance; VF, visual field; BCVA, best corrected visual acuity; POAG, primary open-angle glaucoma; PXG, pseudoexfoliative glaucoma. Vidal-Villegas et al.Ophthalmic Res 2022;65:111–120114 DOI: 10.1159/000519816 Correlation between Cytokine Concentration and the Number of Topical Treatments There was a significant negative correlation between the number of topical treatments used at baseline and the concentrations of two cytokines in POAG patients: IL-7 and basic fibroblast growth factor in aqueous hu- mor (Table  2). There was also a significant negative correlation between the number of topical treatments used at baseline and the concentration of IL-12 (Table 2) in tear in the PXG group. 0.01 0.1 1 10 100 1,000 10,000 100,0000.001 Tear concentrations, pg/mL ■ POAG ■ PXG IL-1β IL-1ra IL-2 IL-4 IL-5 IL-6 IL-7 IL-8 IL-9 IL-10 IL-12 (p70) IL-13 IL-15 IL-17 Eotaxina FGF basic G-CSF GM-CSF IFN-γ IP-10 MCP-1 (MCAF) MIP-1α PDGF-BB MIP-1β RANTES TNFα- VEGF Fig. 1. Cytokine concentrations (pg/mL ± SD) in tear in POAG and PXG. x-axis: logarithmic scale. POAG, primary open-angle glaucoma; PXG, pseudoexfoliative glaucoma. Proinflammatory Cytokines as Glaucoma Biomarkers 115Ophthalmic Res 2022;65:111–120 DOI: 10.1159/000519816 Discussion In the present study, we have used a sensitive immu- noassay to analyze the concentrations of 27 inflammatory cytokines in tear and aqueous humor of patients with POAG or PXG who were undergoing cataract surgery to discern whether these cytokines were involved differently in these types of glaucoma and could, therefore, be used as biomarkers. We documented great variations of cytokine concen- trations in both groups of patients, both in tear and aque- ous humor. This indicates a great variability of cytokine 0.01 0.1 1 10 100 1,000 10,000 100,000 Concentrations in aqueous humor, pg/mL ■ POAG ■ PXG IL-1β IL-1ra IL-2 IL-4 IL-5 IL-6 IL-7 IL-8 IL-9 IL-10 IL-12 (p70) IL-13 IL-15 IL-17 Eotaxina FGF basic G-CSF GM-CSF IFN-γ IP-10 MCP-1 (MCAF) MIP-1α PDGF-BB MIP-1β RANTES TNFα- VEGF Fig. 2. Cytokine concentrations (pg/mL ± SD) in aqueous humor in POAG and PXG. x-axis: logarithmic scale. POAG, primary open-angle glaucoma; PXG, pseudoexfoliative glaucoma. Vidal-Villegas et al.Ophthalmic Res 2022;65:111–120116 DOI: 10.1159/000519816 Ta b le 2 . C yt ok in e co nc en tr at io ns (p g/ m L) in te ar a nd a qu eo us h um or , c om p ar is on a nd c or re la tio ns b et w ee n th em a nd w ith n um b er o f t op ic al tr ea tm en ts C yt ok in e D ia gn os is Te ar C om p ar is on of P O A G /P X G (t ea r) A qu eo us h um or C om p ar is on o f P O A G / PX G (a qu eo us h um or ) C or re la tio n w ith te ar /a qu eo us C or re la tio n w ith n um b er o f tr ea tm en ts (S p ea rm an , p ) m ea n SD M an n- W hi tn ey p m ea n SD M an n- W hi tn ey p Pe ar so n te ar aq ue ou s hu m or IL -1 β PO A G 2. 66 3. 87 1 1. 23 1. 90 0. 42 5 0. 52 2 0. 15 9 − 0. 15 1 PX G 14 .9 5 39 .4 3 0. 86 2. 22 − 0. 03 − 0. 17 4 − 0. 08 3 IL -1 ra PO A G 8, 00 5. 29 10 ,5 35 .0 4 0. 84 7 2, 42 5. 90 3, 08 5. 20 0. 78 0. 48 1 0. 02 1 − 0. 20 6 PX G 3, 14 8. 25 4, 99 3. 16 7, 50 6. 10 19 ,6 93 .0 8 − 0. 16 9 − 0. 34 7 − 0. 16 6 IL -2 PO A G 8. 73 9. 52 0. 42 5 8. 96 12 .8 6 0. 71 5 0. 32 5 − 0. 24 3 − 0. 47 5 PX G 2. 75 2. 01 7. 16 13 .9 3 0. 48 7 − 0. 10 7 − 0. 34 7 IL -4 PO A G 0. 15 0. 15 0. 12 3 0. 14 0. 12 0. 71 5 0. 13 4 − 0. 18 8 0. 09 2 PX G 0. 22 0. 15 0. 12 0. 09 0. 14 8 0. 20 9 − 0. 01 0 IL -5 PO A G 43 .4 1 68 .3 9 0. 68 3 45 .0 0 56 .2 9 0. 50 5 0. 52 2 0. 21 6 − 0. 23 5 PX G 32 .4 4 68 .1 4 42 .3 3 62 .5 2 0. 39 9 − 0. 40 6 0. 02 3 IL -6 PO A G 8. 52 10 .3 1 0. 37 7 6. 54 6. 91 1 0. 48 4 − 0. 00 9 − 0. 14 1 PX G 10 .6 8 12 .1 0 13 .1 3 23 .2 8 0. 14 8 0. 02 1 0. 02 2 IL -7 PO A G 25 .6 7 41 .0 8 0. 88 14 .3 0 16 .6 3 0. 78 0. 31 7 0. 10 1 − 0. 65 9 p = 0 .0 14 PX G 17 .5 2 19 .7 6 24 .5 0 54 .8 7 0. 28 9 0. 00 9 − 0. 12 3 IL -8 PO A G 53 .9 7 91 .3 4 0. 74 7 40 .7 3 66 .6 6 1 0. 16 3 − 0. 34 3 − 0. 44 6 PX G 36 .2 5 46 .1 8 22 8. 86 80 1. 84 0. 15 8 − 0. 28 5 − 0. 20 2 IL -9 PO A G 81 .2 9 86 .3 2 0. 56 1 58 .5 3 76 .2 3 0. 81 3 0. 08 9 − 0. 04 0 0. 02 8 PX G 10 6. 63 15 7. 65 75 .4 2 10 1. 62 0. 25 1 − 0. 30 8 − 0. 19 8 IL -1 0 PO A G 39 .8 6 45 .9 5 0. 45 1 38 .1 8 38 .3 3 0. 18 6 0. 35 3 − 0. 11 7 − 0. 22 3 PX G 27 .4 3 42 .4 8 24 .2 6 37 .0 5 0. 42 9 − 0. 44 4 − 0. 05 9 IL -1 2 (p 70 ) PO A G 7. 03 10 .6 4 0. 17 2 12 .5 2 10 .0 7 0. 00 0 0. 29 3 − 0. 16 7 − 0. 24 9 PX G 1. 75 3. 62 0. 63 0 0. 47 3 − 0. 61 9 p = 0 .0 18 − 0. 21 4 IL -1 3 PO A G 4. 10 4. 74 0. 62 1 5. 37 4. 73 0. 03 7 − 0. 44 0. 00 8 − 0. 45 2 PX G 6. 29 6. 83 2. 89 3. 99 0. 23 6 − 0. 35 5 0. 09 IL -1 5 PO A G 97 0. 05 96 6. 32 0. 84 7 1, 11 5. 45 1, 04 1. 91 0. 25 2 − 0. 27 9 − 0. 12 5 − 0. 15 9 PX G 1, 05 8. 39 1, 19 9. 51 69 7. 87 1, 04 1. 52 0. 31 4 − 0. 37 0. 05 4 IL -1 7 PO A G 53 .6 3 77 .0 2 0. 13 4 31 .4 8 62 .0 3 0. 42 5 − 0. 26 7 0. 19 4 − 0. 48 3 PX G 17 .7 6 40 .2 6 29 .4 2 29 .6 0 0. 35 5 − 0. 36 2 0. 20 5 Eo ta xi n PO A G 9. 80 8. 30 0. 4 7. 20 6. 37 0. 20 1 − 0. 07 7 − 0. 01 7 − 0. 15 6 PX G 7. 19 3. 71 14 .2 8 19 .6 8 0. 19 2 0. 04 − 0. 16 6 Proinflammatory Cytokines as Glaucoma Biomarkers 117Ophthalmic Res 2022;65:111–120 DOI: 10.1159/000519816 C yt ok in e D ia gn os is Te ar C om p ar is on of P O A G /P X G (t ea r) A qu eo us h um or C om p ar is on o f P O A G / PX G (a qu eo us h um or ) C or re la tio n w ith te ar /a qu eo us C or re la tio n w ith n um b er o f tr ea tm en ts (S p ea rm an , p ) m ea n SD M an n- W hi tn ey p m ea n SD M an n- W hi tn ey p Pe ar so n te ar aq ue ou s hu m or FG F b as ic PO A G 71 .3 8 10 3. 32 0. 47 7 85 .9 1 12 3. 26 0. 71 5 − 0. 14 9 0. 20 9 − 0. 66 1 p = 0 .0 14 PX G 66 .5 6 53 .5 5 59 .6 5 83 .9 8 0. 25 − 0. 32 2 − 0. 14 6 G -C SF PO A G 21 5. 21 30 0. 05 0. 47 7 38 8. 29 47 1. 57 0. 29 − 0. 44 4 0. 04 1 − 0. 02 3 PX G 18 4. 83 43 5. 76 20 7. 27 34 1. 44 0. 44 9 − 0. 28 6 − 0. 20 1 G M -C SF PO A G 16 .4 1 18 .1 5 0. 37 7 6. 57 7. 28 0. 25 2 − 0. 48 7 − 0. 02 8 − 0. 07 5 PX G 7. 63 8. 98 5. 46 12 .8 8 − 0. 07 9 − 0. 43 2 0. 20 7 IF N -γ PO A G 11 9. 38 15 9. 26 0. 27 0 37 .8 7 49 .2 3 0. 62 1 − 0. 19 5 − 0. 05 2 − 0. 29 5 PX G 39 .5 0 51 .0 2 31 .5 8 52 .6 3 0. 79 2 p < 0 .0 01 − 0. 35 5 − 0. 18 2 IP -1 0 PO A G 12 ,2 35 .0 7 19 ,1 01 .5 9 0. 45 1 10 ,0 45 .6 8 13 ,8 87 .7 8 0. 91 4 − 0. 26 8 − 0. 14 1 − 0. 49 0 PX G 8, 42 9. 80 13 ,3 99 .6 4 10 ,9 81 .9 4 24 ,9 34 .3 1 − 0. 29 6 − 0. 28 4 − 0. 14 3 M C P- 1 (M C A F) PO A G 32 4. 43 27 4. 71 0. 56 1 14 4. 12 12 2. 24 0. 01 4 − 0. 13 7 − 0. 25 3 0. 05 8 PX G 25 1. 16 18 4. 59 34 5. 76 40 7. 48 0. 25 5 0. 06 1 − 0. 23 9 M IP -1 α PO A G 3. 55 3. 24 0. 59 1 2. 96 4. 12 0. 88 0. 42 3 − 0. 16 0 − 0. 43 5 PX G 2. 79 2. 63 2. 50 3. 00 0. 00 5 − 0. 43 4 0. 01 6 PD G F- BB PO A G 12 7. 72 14 8. 31 0. 56 1 12 9. 87 11 8. 31 0. 29 0 − 0. 50 2 − 0. 12 5 − 0. 16 3 PX G 75 .5 0 12 2. 13 12 1. 57 19 5. 56 0. 26 9 − 0. 27 4 − 0. 04 0 M IP -1 β PO A G 22 .5 3 19 .4 5 0. 4 28 .3 9 37 .0 7 0. 94 9 0. 06 1 − 0. 06 4 − 0. 29 1 PX G 18 .4 8 25 .4 7 19 .9 9 21 .3 8 0. 75 0 p < 0 .0 01 − 0. 28 2 − 0. 31 1 RA N TE S PO A G 58 .3 1 67 .4 0 0. 88 42 .7 4 52 .2 8 0. 33 1 − 0. 48 3 − 0. 06 7 − 0. 24 3 PX G 51 .0 3 59 .5 6 38 .7 0 64 .2 8 0. 35 6 − 0. 21 9 − 0. 01 8 TN F- α PO A G 76 .9 3 93 .5 0 0. 81 3 59 .3 3 76 .9 3 0. 50 5 − 0. 46 3 − 0. 18 9 − 0. 44 1 PX G 49 .6 6 52 .2 9 42 .5 3 71 .6 3 0. 16 8 − 0. 09 7 − 0. 12 9 VE G F PO A G 1, 68 1. 76 3, 44 8. 88 1 2, 71 3. 41 4, 55 1. 83 0. 81 3 − 0. 46 2 0. 15 1 − 0. 16 0 PX G 2, 39 0. 89 6, 38 1. 50 1, 90 0. 25 3, 33 8. 63 0. 62 1 p = 0 .0 14 − 0. 15 2 − 0. 08 3 Va lu es in b ol d in di ca te a s ig ni fic an t d iff er en ce o r a s ig ni fic an t c or re la tio n b et w ee n th e gr ou p s. PO A G , p rim ar y op en -a ng le g la uc om a; P X G , p se ud oe xf ol ia tiv e gl au co m a; V EG F, v as cu la r e nd ot he lia l g ro w th fa ct or ; T N F- α, tu m or n ec ro si s fa ct or -α ; R A N TE S, R eg ul at ed o n A ct iv at io n, N or m al T C el l E xp re ss ed a nd S ec re te d; M IP -1 β, m ac ro p ha ge in fla m m at or y p ro te in fo rm 1 β; M IP -1 α, m ac ro p ha ge in fla m m at or y p ro te in fo rm 1 α; P D G F- BB , p la te le t- de riv ed g ro w th fa ct or B B di m er ; M C P- 1 (M C A F) , m on oc yt e ch em oa tt ra ct an t p ro te in -1 (m on oc yt e ch em ot ac tic a nd a ct iv at in g fa ct or ); IP -1 0, in te rf er on g am m a- in du ce d p ro te in 1 0; G M -C SF , g ra nu lo cy te -m ac ro p ha ge c ol on y- st im ul at - in g fa ct or ; G -C SF , g ra nu lo cy te c ol on y- st im ul at in g fa ct or ; F G F, fi b ro b la st g ro w th fa ct or ; I L, in te rle uk in . Ta b le 2 (c on tin ue d) Vidal-Villegas et al.Ophthalmic Res 2022;65:111–120118 DOI: 10.1159/000519816 expression between patients even within a diagnostic group, and it has been documented previously in normal patients and in patients with different pathologies such as Sjögren syndrome or glaucoma [6–8, 18–20]. Our results document a tendency for the cytokines to show a high or low concentration both in tear and in aqueous humor. However, we could not document a sig- nificant correlation between the cytokine concentrations in tear and aqueous humor in the POAG group and we could only document a significant correlation in the con- centrations of three cytokines in the PXG group: IFN Gamma, MIP-1β, and VEGF. When comparing cytokine concentrations in tear and aqueous humor, other authors have found higher levels of cytokines in tears than in the aqueous humor [20], but in this study and in a previous study of our group we have failed to do so [8]. Our main objective was to add insight into the patho- genesis of POAG and PXG and for this purpose we have analyzed the levels of inflammatory cytokines in these two types of glaucoma and the differences between them. We have found high levels of four cytokines, IL-1RA, IL-15, interferon gamma-induced protein 10, and VEGF, both in tear and aqueous humor of POAG and PXG patients. Elevated levels of some of these cytokines in open-angle glaucoma patients when compared to the control group have also been observed in previous works of our group [7, 8], and increased levels of VEGF, tumor necrosis factor-α, and IL-6 have also been previously documented in tear of POAG patients [6]. It has been proposed that elevated levels of VEGF and TGF-β in the aqueous humor could be responsible for trabeculectomy failure in POAG patients [21], but other authors have argued that only di- minished levels of IFN-γ, GM-CSF, and IL-5 in tear are associated with trabeculectomy complications [20]. When we compared cytokine concentrations in tear and aqueous humor of POAG and PXG patients, we found statistically significant differences only in the aque- ous humor. IL-12 and IL-13 were significantly higher in the POAG group, while MCP-1 (MCAF) was significant- ly increased in the PXG group. Thus, these cytokines could be used as aqueous humor biomarkers for these types of glaucoma and may be involved in the different pathogenesis of each type of glaucoma. IL-12 is secreted by macrophages and dendritic cells. It acts by activating NK cells and promoting Th1 differentiation, as well as stimulating IFN-γ synthesis. Therefore, despite not being a commonly tested cytokine, IL-12 could serve as a marker for diagnosis and be specifically involved in POAG patho- genesis. However, the pleiotropic characteristics of cyto- kines render an exact assignment difficult. In accordance with this, previous studies of our group have also found increased levels of IL-12 in tear and aqueous humor of POAG patients and increased levels of IL-12 and IL-13 in patients with glaucoma treated with preserved latano- prost [7, 8]. Cytokine elevations in the aqueous humor may depend on the type of glaucoma. In this regard, Du- vesh et al. [22] found that IL-8, eotaxin, and interferon gamma-induced protein 10 are significantly increased in closed-angle glaucoma, others revealing differences in IL- 6, IL-8, granulocyte colony-stimulating factor, MCP-1, MCP-3, and VEGF [23, 24]. Our results document increased MCP-1 (MCAF) con- centrations in aqueous humor of the PXG group when compared to the POAG group and may reflect an in- creased inflammation in the PXG group. MCP-1 (MCAF), also known as CCL2, is a potent chemotactic factor for monocytes and macrophages and plays a role in various inflammatory diseases and probably in conjunctival scar- ring [25, 26]. Notwithstanding, the role of this cytokine in PXG pathogenesis is still unclear. Given that the blood- aqueous barrier breakdown in PXG patients results in the entry of inflammatory cytokines and extracellular matrix material into the anterior chamber, the differences in cy- tokine levels could be due to this blood–aqueous break- down rather than inflammation [27]. Although in our work MCP-1 (MCAF) is differentially elevated in PXG, it has been reported to be elevated in the aqueous humor in both POAG and PXG [11]. PXG is characterized by rapid evolution and severe prognosis and other cytokines, in- cluding IL-6, IL-17 [14], tumor necrosis factor-α [17], TGF-β, PDGF, and IL-8 [15], have been described to be elevated in the aqueous humor of PXG. All the patients included in our POAG and PXG groups had cataracts and glaucoma and were receiving topical hy- potensive medication (with and without preservatives) and this may have influenced the cytokine profiles. It is not known how cataracts affect cytokine concentrations in the aqueous humor, but it has been documented that ocular hypotensive treatments with preservatives influence the tear cytokine profile [6, 7]. As most patients in both groups were using >1 drug, it was not possible to correlate cytokine levels and the type of drug. However, all patients using one drug were on prostaglandins, and patients with two or more drugs were also on β-blockers and/or carbonic anhy- drase inhibitors. We analyzed if cytokine concentrations were correlated to the number of ocular hypotensive drug medications that the patients were receiving. We found that the number of ocular hypotensive medications produced a decrease in the IL-7 and basic fibroblast growth factor concentrations in aqueous humor of the POAG group, and Proinflammatory Cytokines as Glaucoma Biomarkers 119Ophthalmic Res 2022;65:111–120 DOI: 10.1159/000519816 of the IL-12 concentrations in tear of the PXG group. Thus, the topical treatment may have decreased inflammation and influenced in part the results. Although more patients were initially included in the study, the samples of only 14 eyes with POAG and 15 eyes with PXG could be analyzed. The composition of the groups was generally comparable: at the baseline visit, age, intraocular pressure, global thickness of the pRNFL, and the number of topical hypotensive medications was simi- lar in both groups. Nevertheless, the eyes included in the PXG group in this study could have more advanced cata- racts and increased functional and structural glaucoma damage, or different disease timeline and/or different pre- vious treatments. Indeed, the mean deviation of the vi- sual field was significantly more negative in the PXG group, the BCVA was lower in the PXG, both at baseline and postoperatively, and the thickness of the superotem- poral sector of the pRNFL was also significantly thinner in this group. However, we believe that the elevations in cytokine concentration in the tear aqueous humor both in POAG and PXG and the differences in cytokine concen- trations that we find between the groups cannot be solely explained on the basis of different severity of glaucoma damage. Finally, although further studies are needed to define the concentrations of cytokines in tear and aque- ous humor of POAG and PXG patients and their varia- tions with the clinical course and severity of the disease, it is possible that these could be used in the future as non- invasive (in the case of tears) and rapid methods to diag- nose and grade these diseases. Conclusions The analysis of 27 inflammatory cytokines in tear and aqueous humor of POAG and PXG patients has allowed us to document that the levels of three cytokines in the aqueous humor differed between the groups: IL-12 and IL-13 were significantly higher in the POAG group and MCP-1(MCAF) was significantly higher in the PXG group. Thus, although further studies are needed, these cytokines could be particularly involved in the pathogen- esis of these forms of glaucoma. Acknowledgments We would like to thank all the personnel of the Service of Oph- thalmology of our hospital for their help in obtaining the samples and the personnel in the Service of Immunology for their help in processing the samples. Statement of Ethics The study was conducted according to the guidelines of the World Medical Association Declaration of Helsinki, and approved by the Ethics Committee of Hospital Clinico San Carlos (protocol code 18/255-E in December 2018). Written informed consent was obtained from all subjects in- volved in the study. Conflict of Interest Statement The authors have no conflicts of interest to declare. Funding Sources Red Temática de Investigación Cooperativa Oftared (RETICS RD16/0008/0004) Enfermedades oculares: “Prevención, detec- ción precoz, tratamiento y rehabilitación de las patologías oculares” (ISCIII-FEDER “Una manera de hacer Europa”) and Research Group 920415-GR58/08 of the Universidad Complutense de Madrid funded the study. We would also like to acknowledge the support received from Thea Laboratories for the publication of this article. Author Contributions J.M.M.-C. and J.G.-F. contributed to conceptualization; J.M.M.- C., J.L.S.A., and L.E.-P. contributed to methodology; J.L.S. and L.E.-P. contributed to software; J.M.M.-C., J.G.-F., and J.F.-V. contributed to validation; B.V.-V. and B.B.-B. performed the formal analysis; B.V.- V., B.B.-B., and V.A.-G. carried out the investigation; V.A.-G., J.L.S.A., and L.E.-P. contributed to the resources; B.V.-V., B.B.-B., J.M.M.-C., J.L.S.A., and L.E.-P. involved in data curation; B.V.-V. and B.B.-B. in- volved in writing – original draft preparation; B.V.-V., B.B.-B., and J.M.M.-C. involved in writing – review and editing; J.F.-V. and J.G.-F. contributed to visualization; J.F.-V., J.M.M.-C., and J.G.-F. supervised the study; J.M.M.-C. and J.G.-F. contributed to project administra- tion; J.M.M.-C. and J.G.-F. contributed to funding acquisition. All authors have read and approved the final manuscript and agreed to publish the manuscript. 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