Multiple Cytokine Analysis of Aqueous Humor in Uveitis With or Without Secondary Glaucoma

doi:10.21203/rs.3.rs-4302564/v1

To investigate the concentration of pro- and anti-inflammatory cytokines of aqueous humor (AH) in uveitis with or without glaucoma, and compare them with patients with primary open-angle glaucoma (POAG) and age-related cataract (ARC). By using Luminex xMAP® multiplex assays analyses, we assessed levels of 11 cytokines and chemokines, and compared them across groups, including uveitis-secondary glaucoma (USG) (n=16), uveitis without glaucoma (UwoG), (n=16), POAG (n=16), and ARC (n=16) to analyze the correlation between these cytokines and uveitis and intraocular hypertension (IOP). Pro-inflammatory factors MCP-1, MIP-1β, IL-6, IL-8, and transforming growth factors TGF-β1 and TGF-β2 were significantly elevated in the AH of USG eyes. In the case of enhanced anti-inflammatory in the perioperative period, the pro-inflammatory factors in the USG group were still significantly higher than those in the UwoG group (P<0.01). The concentrations of IL-6, IL-8, and MCP-1 in the AH of the USG group and POAG group had the same trend, which was significantly higher than those of the ARC group (P<0.01). Significantly increased levels of MCP-1, MIP-1β, IL-6, IL-8, TGF-β1, and TGF-β2 were found in the AH of USG patients, suggesting that these molecules may contribute to the clinical development of glaucoma in patients with uveitis. Besides there was no statistically significant difference in the levels of cytokines in the AH of USG eyes whether the preoperative baseline IOP was greater than 30 mmHg or not, indicating that the safety of antiglaucoma surgery in USG patients even with baseline high IOP.

aqueous humor

pro-inflammatory cytokines

TGF-β1

uveitic glaucoma

timing of surgery

Disruption of the blood-retinal barrier can trigger retinal or uveal disease and lead to changes in the biochemical or protein composition of the aqueous humor. Uveitis is an autoimmune disease in which T lymphocytes, especially Th cells and the cytokines they secrete play an important role in the pathogenesis[1]. Uveitis-secondary glaucoma (USG) is a common and serious complication of uveitis that can occur in many types of ocular inflammation. It has been reported that multiple bioactive molecules are elevated in the aqueous humor of patients with uveitis using complex bead immunoassays[2-8], such as interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and interferon-γ (IFN-γ) were increased in idiopathic uveitis aqueous humor; IFN-γ was detected elevating in both anterior uveitis group and panuveitis group; higher levels of IL-6, IL-8 and interferon-inducible protein-10 (IP-10) in uveitis patients; active intermediate uveitis is characterized by higher levels of IL-6, IL-8, activator of normal T cell secretion (RANTES), and MCP-1. The sample sizes detected in these previous studies were small, and there are no reports on multiple cytokines and their related background factors in the aqueous humor of USG patients.

Some patients with uveitis develop angle-closure glaucoma, but open-angle glaucoma is the most common type in USG. About one-third of adult patients with USG need surgery [9]. Many researchers believe that secondary open-angle glaucoma is caused by chronic changes to the outflow pathway of the trabecular meshwork (TM) The changes in the TM include deposition of extracellular matrix (ECM), different composition of ECM protein, and loss of trabecular endothelial cells, ultimately leading to reduced phagocytosis and altered protein cleavage [10,11]. Another explanation for elevated intraocular pressure (IOP) is the mechanical obstruction of the outflow pathway by debris and cells [10-12] In addition, studies have shown that excessively IOP elevated may also lead to damage to the blood-aqueous humor barrier, resulting in the presence of more inflammatory cells and inflammation-related mediators in the aqueous humor. And the concentration of pro-inflammatory factors is positively correlated with IOP, suggesting that IOP can potentially stimulate cytokine production, while increased cytokine concentrations may in turn affect aqueous humor dynamics and lead to increased IOP[13,14]. Therefore, local immune responses may differ in USG or uveitis without glaucoma (UwoG) patients, thereby mediating different outflow rates of the TM.

Whether the pathogenesis of POAG is immune-related has also been controversial. Such local immune responses may be controlled by proinflammatory and anti-inflammatory cytokines, which can be secreted by immune cells and certain other cell types to regulate inflammation. In recent years, it has been found that immune factors are one of the non-stress-dependent risk factors for optic nerve damage, and cytokines can participate in IOP elevation and apoptosis through multiple pathways, accelerating the progression of glaucoma. Proinflammatory cytokines are predominantly produced by helper T cell and macrophages to upregulate inflammatory reactions [15]. Therapies against inflammatory diseases often involve monoclonal antibodies targeting pro-inflammatory cytokines or their receptors. Proinflammatory cytokines [e.g., tumor necrosis factor-alpha (TNF-α) or IL-1β] may participate in the degradation of ECM and the regulation of IOP[16]. Anti-inflammatory cytokines like trans-forming growth factor beta (TGF-β) often have both pro- and anti-inflammatory effects. TGF-β may convert an active site of inflammation into one dominated by resolution and repair[16]. TGF-β1 and TGF-β2 may increase ECM production in the anterior chamber and also inhibit the degradation of existing ECM [17,18]. The effect of surgery on USG patients is unsatisfactory and controversial[19], they are relatively more resistant to filtering surgery modalities compared with POAG[20,21]. However, there is currently no literature reporting the difference in inflammation between POAG and USG and it is not clear which cytokines can significantly affect the outcome of filtration surgery.

Aqueous humor is easier to collect than the vitreous and can better reflect the microenvironment in the eye than blood. We hypothesized that altered outflow rates might be reflected by alterations in the IL-8, MCP-1 and TGF-β pathways. Therefore, evaluating cytokine concentrations in the aqueous humor of uveitis with and without glaucoma could be helpful to clarify the pathogenetic processes of the outflow pathway and may help to provide a reference for clinical treatment and prognosis of glaucoma development in uveitis patients. The Luminex xMAP® multiplex assays are highly specific and allows simultaneous capture of monoclonal antibodies coupled to different fluorescently labeled magnetic beads, and the different types of magnetic beads are mixed and suspended in a 96-well microplate. This study aimed to detect cytokine profiles in uveitis patients with or without glaucoma and compare with POAG and ARC groups.

Subjects

The Institutional Review Board of the Peking Union Medical College Hospital in Beijing, China, approved this study, which followed the tenets of the Declaration of Helsinki. Written informed consent was obtained from all patients.

In the present study, the samples consisted of 64 eyes from 64 patients, which included 16 USG eyes and 16 POAG eyes with anti-glaucoma surgeries, and 16 UowG eyes and 16 ARC eyes with cataract surgeries. Uveitis was classified according to the recommendations put forth by the International Uveitis Study Group (IUSG), under consideration of the recent modifications[22]. All patients were treated at the Peking Union Medical College Hospital between June 2021 and January 2022.

Inclusion and exclusion criteria were used to improve diagnostic accuracy and minimize potential bias caused by confounding factors. The inclusion criteria of USG and POAG patients were as follows: (1) conform to the diagnosis of USG or POAG; (2) patients over 18 years old underwent modified CLASS or dual-access surgery in our hospital; (3) no history of ocular trauma, and the history of intraocular surgery should be more than 1 year away from the current anti-glaucoma surgery; The exclusion criteria were as follows: (1) suspicion of steroid-induced ocular hypertension; (2) shallow anterior chamber (the depth of the peripheral anterior chamber does not exceed 1/4 of the corneal thickness); (3) corneal opacity or opaque refractive media that may interfere with optic nerve evaluations；(4) patients with blindness in the other eye.

The inclusion criteria of UowG and ARC patients were as follows: (1) undergone cataract surgery at our hospital and have no active inflammation (anterior chamber cells ≥0) during the 3 months prior to AH collection; (2) no changes at the optic nerve and had not received any topical or systemic IOP-lowering medication at any time during the previous clinical course; (3) IOP was never measured above 21 mmHg; (4) no history of other eye diseases, eye trauma, or eye surgery. The exclusion criteria were as follows: patients with blindness in the other eye. If patients underwent surgery in both eyes, the first eye to be treated was included. All patients were relatively healthy with no systemic diseases, such as diabetes, infectious diseases, history of malignant tumors, serious mental and psychological diseases, etc., and the follow-up data were complete.

Aqueous humor collection

For patients with USG or POAG, the collection of aqueous humor should be performed after anti-glaucoma surgery to make a scleral flap (before breaking the blood-aqueous humor barrier). In patients with UwoG or ARC, the collection of aqueous humor is performed after a clear corneal incision during cataract surgery. The aqueous humor sample (70–100 µL) for each patient was obtained by performing paracentesis through the anterior chamber with a 30-gauge needle and a 1.0 mL syringe. The aqueous samples were immediately frozen at -80 °C for further analysis.

Luminex xMAP® multiplex assays

The selection of cytokines in this study was based on their potential role in intraocular inflammatory diseases and the availability of detection kits. In this experiment, the Human XL Cytokine Premixed Kit (FSCSTM18-08, R&D Systems, USA) and TGF-β Premixed Kit (FSCSTM17-03, R&D Systems, USA) were used according to the manufacturer’s instructions for the determination of cytokines in four groups of aqueous humor samples. Its detection concentration range is large, which can be accurately quantified to pg/ml, thus reducing the error[23]. Human XL Cytokine Premixed Kit contains IL-6, IL-8, interleukin-10 (IL-10), TNF-α, IFN-γ and normal T cell secretion activator (RANTES), MCP-1, macrophage inflammatory protein-1β (MIP-1β) factors; TGF-β Premixed Kit contains TGF-β, including TGF-β1, TGF-β2, TGF-β3. In the TGF-β assay, samples were acidified and activated using the Sample Activation Kit1 activation kit from R&D systems.

Cytokine standards were serially diluted in a 1:3 ratio, and the aqueous samples were diluted in a 1:2 ratio with the supplied diluents. In total, 50 µL of diluted aqueous samples were used. Fluorescence intensity readings for all the standards and samples were converted into the corresponding cytokine concentrations using Luminex200 (Luminex Corp., Austin, TX, USA) following the manufacturer’s instructions, and xPONENT3.1 software for further analysis. Each sample was measured only once because of the limited quantity of the collected aqueous humor.

Statistical Analysis

The software used was SPSS 23.0. Visual acuity data were collected from medical records and converted to a logarithm of the minimal angle of resolution (logMAR). Differences between each pair of groups including USG, POAG, UowG group and ARC group were assessed by the ANOVA test or Mann-Whitney U test for continuous variables and by the Chi-square test for categorical variables. ANOVA test was used for data measurement when data met normal distribution, Mann-Whitney test was used when data were abnormal distribution. The correlations in this study were assessed by Spearman’s rank correlation coefficient. P<0.05 was deemed to be statistically significant.

Demographic Data

The patient characteristics are shown in Table 1. The age distribution of the patients in the POAG group and the senile cataract group was significantly higher than that in the USG group (P=0.039; P<0.001), and the difference was related to the natural onset time of uveitis. The preoperative mean baseline IOP in the USG group was significantly higher than that in the POAG group (P=0.001), but no statistical difference was found in the number of glaucoma medications. The clinical characteristics of patients with uveitis and the treatment of baseline uveitis are shown in Table 2. The etiologies of uveitis in patients with USG are Posner-Schlossman Syndrome (n=4), BD (n=3), VKH and Fuchs Syndrome were both 1 case, and the other 7 cases of unknown etiology, respectively. Nine of 16 UwoG subjects had received steroids systemically, while only 3 of 16 cases were in USG patients. All patients in the USG group were in a quiescent phase or only had mild inflammation (indicated by anterior chamber flare and inflammatory cells <0.5+) within two weeks before surgery, and the average duration of inflammation control was 3.94±3.47 months.

Table 1. Demographics and ophthalmologic history of patients at baseline.

Characteristic	USG	POAG	UwoG	ARC
Patients	16	16	16	16
Male/Female	9/7	8/8	5/11	7/9
Age（years）
Mean±SD	43.0±13.3*	54.3±12.8	44.5±11.7	68.3±11.7†
Range	20-65	29-71	26-66	43-86
IOP（mmHg）
Mean±SD	34.4±12.8*	22.6±9.7	15.1±3.1	15.1±1.5
Range	20.6-59	14-49	10-21.0	12.7-17.5
Anti-glaucoma medications
Numbers, Mean±SD	3.2±0.8	2.7±0.8	0	0
Range	2.0-4.0	1.0-4.0	0	0
β-blockers	14（87.5）	8（50.0）	0	0
Prostaglandin analogs	7（43.8）	14（87.5）	0	0
Carbonic anhydrase inhibitor	15（93.4）	13（81.3）	0	0
α₂_-receptor agonists	13（81.3）	8（50.0）	0	0
Others	2（12.5）	0	0	0
Glaucoma treatment time（months）
Mean±SD	37.2±31.7	68.9±50.7	0	0
Range	1-120	4-180	0	0
Previous phacoemulsification	6（37.5）	3（18.6）	0	0
Pupil seclusion with peripheral iridectomy	7（43.8）	3（18.6）	0	0

UwoG: Uveitis without glaucoma.

*P<0.05, USG vs POAG.

†P<0.05, USG vs ARC.

Table 2. Clinical characteristics of patients with uveitis and treatment of baseline uveitis.

Characteristic	USG（n=16）	UwoG（n=16）
Diagnosis
Idiopathic uveitis	7（43.8）	7（43.8）
Bechet’s disease	3（18.8）	1（6.3）
Vogt-Koyanagi-Harada syndrome	1（6.3）	5（31.3）
Posner-Schlossman syndrome	4（25.0）	0
Fuchs’ heterochromic iridocyclitis	1（6.3）	3（18.8）
Anterior segment complications
Peripheral anterior synechia（<180 degree）	7（43.8）	1（6.3）
Extensive posterior synechia	1（6.3）	4（25.0）
Pupil seclusion with peripheral iridectomy	1（6.3）	1（6.3）
Duration of uveitis (years)	7.9±5.2	6.2±4.3
Inflammation control duration (months)	3.94±3.47	4.56±1.28
Medication
Topical steroid eye drops, n (%)	13（81.3）	9（56.3）
Oral steroid，n (%)	3（18.8）	9（56.3）
Interferon，n (%)	2（12.5）	0

UwoG: Uveitis without glaucoma.

Aqueous cytokines analysis

Table 3 shows the mean values and ranges of all cytokines detected in the aqueous humor of the four groups of patients. The cytokines in the aqueous humor of the USG group and the three control groups were significantly different, including MCP-1, MIP-1β, IL-6, IL-8, TNF-α, TGF-β1, and TGF-β2. All patients had RANTES concentrations below the lower limit of the detection range and were excluded from further analysis.

Figure 1 shows the comparison of six pro-inflammatory cytokines between USG and UwoG patients. The aqueous humor IL-6 (P=0.001), IL-8 (P<0.001), MCP-1 (P=0.008), and MIP-1β (P=0.007) of the USG patients were significantly higher than those of UwoG patients. There was no statistical difference in IFN-γ and TNF-α between the two groups.

Figure 2 shows that the six pro-inflammatory cytokines were not statistically different between USG and POAG groups. However, the levels of IL-6 (P=0.001; P<0.001), IL-8 (P<0.001; P<0.001), and MCP-1 (P<0.001; P=0.003) in the USG group and POAG group were significantly higher than those in ARC group.

The levels of TGF-β1, TGF-β2 and TGF-β3 were measured in all four groups of patients. Figure 3 shows that the USG patients had significantly higher concentrations of TGF-β1 and TGF-β2 than the other three groups (TGF-β1: all P<0.001; TGF-β2: P=0.022; P=0.045; P=0.004). TGF-β3 showed a similar trend but was not statistically different because most of the TGF-β3 were below the lowest detectable value (63.21 pg/mL). The anti-inflammatory cytokine IL-10 had no statistical difference among the four groups.

Table 3. Concentrations of aqueous cytokines in four groups of patients（pg/ml）.

Cytokines	USG		POAG		UwoG		ARC
	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range
MCP-1	2425.15±2044.13	73.38-6466.54	1585.90±1401.70	1.91-4608.98	1004.81±782.51	404.79-3373.85	521.65±118.17	347.81-741.36
MIP-1β	95.93±15.72	50.71-120.72	98.26±21.21	50.71-137.43	82.11±17.51	50.71-120.72	81.98±29.75	23.53-120.72
IL-6	1197.21±1287.199	1.17-3532.78	1142.82±2077.84	2.81-8070.88	47.81±72.20	6.02-268.48	19.61±44.97	1.74-161.7
IL-8	143.94±103.58	3.37-394.51	161.60±185.46	0.4-660.42	26.81±31.05	4.73-119.81	4.68±2.11	1.87-10.13
IL-10	27.64±29.23	7.69-98.63	14.25±8.49	2.59-27.99	34.20±60.46	5.14-204.2	12.13±7.86	5.14-22.92
IFN-γ	12.92±7.02	2.73-23.39	12.48±4.28	4.85-20.68	14.89±10.10	5.56-43.5	10.71±3.91	5.56-17.97
TNF-α	4.87±1.41	2.6-7.68	3.83±0.86	2.6-5.12	4.28±2.17	2.29-10.94	3.31±1.00	1.37-5.12
TGF-β1	523.69±474.78	111.89-1951.95	148.66±83.93	25.96-261.53	153.64±168.37	12.98-626.84	46.71±23.93	12.98-92.67
TGF-β2	1484.65±827.57	387.33-3470.67	867.96±642.77	299.61-2726.22	922.57±415.91	363.63-1852.44	731.56±223.13	377.43-1123.5
TGF-β3	38.31±43.82	14.92-131.72	14.92±0	14.92-14.92	20.24±7.52	14.92-25.55	14.92±0	14.92-14.92

UwoG: Uveitis without glaucoma.

Correlation of cytokine levels with baseline IOP and age

According to whether the preoperative baseline IOP of the USG group was greater than 30mmHg, they were divided into the baseline IOP greater than or equal to 30mmHg (≥30mmHg) group and the baseline IOP less than 30mmHg (<30mmHg) group. The expression levels of the six pro-inflammatory factors were not different between the two groups of samples, as shown in Figure 4. The correlation of cytokines in the USG and UwoG groups showed no significant differences concerning age.

Correlation between cytokines

The correlations among chemokines, growth factors and cytokines levels in all subjects are shown in Table 4. TGF-β1 and MCP-1 were positively correlated with IL-6, IL-8, and TNF-α. A negative correlation was not calculated between any of the various cytokines.

Table 4. Correlations between chemokines, growth factors, and pro-inflammatory factors.

	MCP-1		IL-6		IL-8		IL-10		TNF-α		TGF-β1
	r	P	r	P	r	P	r	P	r	P	r	P
MCP-1	——	——	0.729	<0.001*	0.819	<0.001*	0.443	0.005*	0.486	<0.001*	0.662	<0.001*
IL-6	0.729	<0.001*	——	——	0.833	<0.001*	0.281	0.083	0.220	0.097	0.642	<0.001*
IL-8	0.819	<0.001*	0.833	<0.001*	——	——	0.199	0.225	0.316	0.016*	0.694	<0.001*
IL-10	0.443	0.005*	0.281	0.083	0.199	0.225	——	——	0.369	0.021*	0.222	0.200
TNF-α	0.486	<0.001*	0.220	0.097	0.316	0.016*	0.369	0.021*	——	——	0.429	0.002*
TGF-β1	0.659	<0.001*	0.662	<0.001*	0.694	<0.001*	0.222	0.200	0.429	0.002*	——	——

*P<0.05.

This study reveals the significant alterations in the cytokine levels from the aqueous humor of USG patients in comparison with the other three groups, and it greatly enriches our understanding of the aqueous humor in patients with USG. In the current study, even in the preoperative use of topical combined with systemic corticosteroids for anti-inflammatory, the proinflammatory factors in the aqueous humor of the USG group were still significantly higher than those of the UwoG group. Secondly, the pro-inflammatory factors such as IL-6, IL-8 and MCP-1 in the aqueous humor have the same trend in the USG group and POAG group, and they are all significantly higher than those in the ARC group, and they could play a role in the pathogenesis of ocular hypertension. Additionally, there is currently no guideline on the timing of anti-glaucoma surgery in eyes with USG. Our study is one of the first pilot studies reporting that there is no difference in the expression of cytokines in the aqueous humor between groups of USG with or without baseline high IOP before surgery.

Many studies have shown that inflammatory factors and chemokines play a certain role in the occurrence and development of uveitis. It has been reported that uveitis in the clinical quiescent stage has higher levels of chemokines and soluble adhesion molecules in aqueous humor, and the levels of MCP-1, MIP-1β, and IL-8 are significantly increased, among which MCP-1 and cataract Inflammation recurrence after surgery[24]. Ohira et al.[25] reported that multiple cytokines, including IL-6, IL-8, MCP-1, TNF-α, and platelet-derived growth factor-AA, were elevated in the aqueous humor of patients with neovascular glaucoma. However, since the volume of aqueous humor that can usually be sampled from USG patients is only 50-150 μl, there was a lack of reports on USG in the past. In this study, we found a variety of cytokines closely related to USG, such as MCP-1, MIP-1β, IL-6, IL-8, TNF-α, TGF-β1, TGF-β2.

MCP-1 is a typical pro-inflammatory chemokine in the C-C chemokine family and also has fibrogenic effects, and is a potent chemotactic activator of human monocytes and macrophages. High levels of MCP-1 promote the recruitment and activation of mononuclear cells in uveitis eyes. Several studies have shown that macrophage infiltration plays a key role in uveitis tissue damage. The increase of MCP-1 in the aqueous humor can recruit macrophages to accumulate in the anterior chamber and trabecular meshwork, and macrophages are more likely to phagocytose inflammatory factors. The trabecular meshwork is blocked, resulting in poor filtration of aqueous humor and increased IOP. The more severe the local inflammatory response, the higher the expression of MCP-1, and the more obvious cell migration and inflammatory infiltration, which is consistent with the preoperative IOP and MCP-1 levels in the USG group were significantly higher than those in the other three groups in this study.

MIP-1β, as the main factor produced by macrophages after stimulation, has the function of regulating other peripheral immune cells[26]. IL-8 belongs to the CXC chemotaxis superfamily, which can be produced by macrophages and various ocular resident cells and is an inflammatory factor with chemotaxis. The chemotactic activity of IL-8 on neutrophils was observed in vitreous samples from uveitis and other vitreoretinal diseases[27]. In addition, the inner retina and optic nerve and trabecular meshwork tissue produce endogenous IL-8[28,29]. Studies have reported that IL-8 can inhibit the activity of RGC-5 cells by inducing apoptosis[30]. In addition, ischemia hypoxia and elevated IOP can both induce increased secretion of IL-8[31], and the concentration of IL-8 is positively correlated with IOP, suggesting that elevated IL-8 can positively enhance the outflow resistance of aqueous humor[32]. Both MIP-1β and IL-8 can recruit immune cells to participate in pro-inflammatory chemokines in the inflammatory response. In the present study, their expression was increased in both the USG and POAG groups at almost the same level, which was significantly higher than in the ARC group. This is consistent with the theory that the immune system has a significant effect on retinal nerve cells.[33,34]. Moreover, the content of IL-8 and TGF-β was positively correlated, suggesting that high concentrations of IL-8 and TGF-β may reflect the severe decrease in the outflow capacity of aqueous humor at the trabecular meshwork, and both inflammatory reactions are involved in the occurrence of these two types of glaucoma.

Compared with primary angle-closure glaucoma, POAG has lower IOP, less intraocular ischemia and hypoxia, and less tissue damage. Therefore, the changes in the concentration of cytokines in the aqueous humor are more likely to be caused by damage to the optic nerve and trabecular meshwork. At present, the source of IL-6 in the aqueous humor is not clear, but both clinical and animal experiments have shown that IL-6 plays an important role in the pathogenesis of various autoimmune diseases including rheumatoid arthritis[35]. IL-6 is also associated with a variety of central nervous system diseases, including nerve damage, neurodegenerative diseases, etc[36]. The relationship between IL-6 and nerve cells is still debated, some scholars believe that IL-6 accelerates nerve cell death and causes neuroinflammation[37]; and apoptosis of nerve cells[38]. In addition, different studies have reported the changes in IL-6 concentration in the aqueous humor of POAG patients. For example, Borkenstein et al.[39] and Takai et al.[40] found that the concentration of IL-6 in the aqueous humor of POAG patients was significantly lower than that of the cataract control group; Kuchtey et al.[28] found that the IL-6 concentration in the aqueous humor of POAG patients was not significantly different from that of the cataract control group. The above differences may be related to the inclusion criteria and the size of the sample. This study found that the concentration of IL-6 in the USG group and POAG group was significantly higher than that in the ARC group, and had a significant positive correlation with IOP. And some studies have found that IL-6 can activate the STAT3 signaling pathway, affect the proliferation of fibroblasts, and mediate the production of extracellular matrix[41], indicating that IL-6 is likely to mediate the increase in IOP by a certain mechanism and participate in the production of IOP. Pathogenesis of USG and POAG. We, therefore, recommend perioperative hormonal therapy for patients planning antiglaucoma surgery to achieve both anti-inflammatory and optic nerve protection goals[42,43].

Uveitis is thought to be mainly mediated by T cells[44,45]. RANTES is a chemoattractant that recruits CD4+ monocytes and T cells to antigenically challenging sites[46,47]. In the present study, RANTES was below the lowest detectable value in all subjects, and the intraocular chemotactic activity of CD4+ T cells was not elevated, suggesting that patients in either the USG group or the UwoG group were in a period of relative quiescent stage in the context of strong perioperative anti-inflammatory efforts.

TGF-β is a cytokine with an immunomodulatory activity that can be secreted and expressed in many tissues, but its role in the pathophysiology of USG is not fully understood. Previous literature reported that TGF-β1 played a profibrotic role in fibroblasts, and TGF-β2 could promote the proliferation of extracellular matrix and fibroblasts[48]. Because TGF-β1 and TGF-β2 in the aqueous humor can promote the contraction of trabecular meshwork cells in the early stage, and promote the fibrosis of the trabecular meshwork tissue in the late stage, which leads to the increase of IOP[49], which can reflect the damage of the trabecular meshwork. In this study, the positive correlation between TGF-β1 and TGF-β2 and the baseline preoperative IOP also confirmed this. Therefore, we speculate that the increase in aqueous outflow resistance and IOP in USG patients is related to the concentration of TGF-β[49-51]. Interestingly, TGF-β can also inhibit IL-2-dependent T cell proliferation by inhibiting the induction of IL-2 and transferrin receptors[52]. In addition to neuropeptides such as α-stimulating melanocyte hormone, vasoactive intestinal peptide, etc., TGF-β is also believed to be involved in maintaining the state of intraocular immunosuppression[53], thus TGF-β is a multifunctional factor. It has been reported that inflammation in patients with idiopathic uveitis is reduced with increasing TGF-β2 concentrations[2]; Min et al.[54] found that POAG and neovascular glaucoma aqueous humor increased TGF-β2 levels, while open-angle glaucoma The level of TGF-β2 in the aqueous humor of type USG was in the normal range. In this study, the levels of TGF-β1 and TGF-β2 in the aqueous humor of the USG group were significantly higher than those of the other three groups. Secondly, TGF-β1 was positively correlated with MCP-1 and TNF-α. The up-regulation of MCP-1 expression induced by the acyl-inositol 3-kinase signaling pathway was consistent. There are also animal experiments that found that TGF-β will increase responsively and antagonize the early production of IL-6 during the development of inflammation, thereby inhibiting the development of inflammation[55]. Based on these findings, we believe that the marked increase of TGF-β in USG eyes is related to its responsive antagonism of other significantly increased pro-inflammatory factors and whether TGF-β plays a key role in USG and its exact mechanism remains to be further elucidated.

Anti-glaucoma surgery is an effective way to preserve vision in patients with USG, and there are no guidelines regarding the timing of surgery. As a preliminary study, we found that there was no difference in the concentration of cytokines in the USG group according to whether the preoperative baseline IOP was greater than 30 mmHg, suggesting that there was no significant correlation between the baseline IOP and the increased expression of pro-inflammatory factors in patient eyes. A high IOP in the affected eye does not necessarily imply surgery failure. Further prospective trials are needed to determine the specific timing and corresponding requirements for anti-glaucoma surgery in patients with uveitis.

This study has several limitations. First, the sample size of our study was small, and each sample was measured only once due to the limited volume of aqueous humor. However, the results between the four groups were statistically significant and convincing; second, due to the limited sensitivity of the corresponding factor of the kit, some cytokine levels could not be measured, but it cannot be ruled out that they are altered in the disease. Third, because the surgical patients selected in this study are in the quiescent stage of inflammation or only have mild inflammation for treatment, further experimental exploration is needed for the changes of cytokines in the active stage of inflammation and the effects on cytokine expression before and after drug treatment.

In summary, the present study provided a detailed profile of the various cytokines in the aqueous humor of USG patients, while revealing their differences from the intraocular microenvironment of POAG. Our study is the first to report significantly elevated levels of IL-6, IL-8, MCP-1, MIP-1β, TGF-β1, and TGF-β2 in USG, suggesting that USG-affected eyes have elevated pro-inflammatory cytokines, less the damage of the beam network and the disturbance of the extracellular matrix suggests that these cytokines may be involved in the development of USG.

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors' contributions: Junyan Xiao: Conceptualization, Investigation, Writing-Original draft preparation; Meifen Zhang, Chan Zhao and Gangwei Cheng: Supervision, Methodology; Hang Song and Yang Zhang: Resources, Data Curation. All authors reviewed the manuscript.

Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Peking Union Medical College Hospital approved this study (ZS-2945).

Informed Consent Statement: Informed consent was obtained from the patients included in the study.

Data Availability Statement: Some or all data, models, or codes generated or used during the study are available from the corresponding author by request.

Conflicts of interest/Competing interests: The authors declare no conflict of interest.

Consent for publication: All authors read and approved the final manuscript. The requirements for authorship as stated earlier in this document have been met, and each author believes that the manuscript represents honest work.

Horai R, Caspi RR (2011) Cytokines in autoimmune uveitis. Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research 31 (10):733-744. https://doi.org/10.1089/jir.2011.0042
Curnow SJ, Falciani F, Durrani OM, Cheung CM, Ross EJ, Wloka K, Rauz S, Wallace GR, Salmon M, Murray PI (2005) Multiplex bead immunoassay analysis of aqueous humor reveals distinct cytokine profiles in uveitis. Investigative ophthalmology & visual science 46 (11):4251-4259. https://doi.org/10.1167/iovs.05-0444
Ooi KG, Galatowicz G, Towler HM, Lightman SL, Calder VL (2006) Multiplex cytokine detection versus ELISA for aqueous humor: IL-5, IL-10, and IFNgamma profiles in uveitis. Investigative ophthalmology & visual science 47 (1):272-277. https://doi.org/10.1167/iovs.05-0790
Takase H, Futagami Y, Yoshida T, Kamoi K, Sugita S, Imai Y, Mochizuki M (2006) Cytokine profile in aqueous humor and sera of patients with infectious or noninfectious uveitis. Investigative ophthalmology & visual science 47 (4):1557-1561. https://doi.org/10.1167/iovs.05-0836
van Kooij B, Rothova A, Rijkers GT, de Groot-Mijnes JD (2006) Distinct cytokine and chemokine profiles in the aqueous of patients with uveitis and cystoid macular edema. American journal of ophthalmology 142 (1):192-194. https://doi.org/10.1016/j.ajo.2006.02.052
El-Asrar AM, Al-Obeidan SS, Kangave D, Geboes K, Opdenakker G, Van Damme J, Struyf S (2011) CXC chemokine expression profiles in aqueous humor of patients with different clinical entities of endogenous uveitis. Immunobiology 216 (9):1004-1009. https://doi.org/10.1016/j.imbio.2011.03.007
Kuiper JJ, Mutis T, de Jager W, de Groot-Mijnes JD, Rothova A (2011) Intraocular interleukin-17 and proinflammatory cytokines in HLA-A29-associated birdshot chorioretinopathy. American journal of ophthalmology 152 (2):177-182.e171. https://doi.org/10.1016/j.ajo.2011.01.031
Valentincic NV, de Groot-Mijnes JD, Kraut A, Korosec P, Hawlina M, Rothova A (2011) Intraocular and serum cytokine profiles in patients with intermediate uveitis. Molecular vision 17:2003-2010
Heinz C, Koch JM, Zurek-Imhoff B, Heiligenhaus A (2009) Prevalence of uveitic secondary glaucoma and success of nonsurgical treatment in adults and children in a tertiary referral center. Ocular immunology and inflammation 17 (4):243-248. https://doi.org/10.1080/09273940902913035
Siddique SS, Suelves AM, Baheti U, Foster CS (2013) Glaucoma and uveitis. Surv Ophthalmol 58 (1):1-10. https://doi.org/10.1016/j.survophthal.2012.04.006
Tektas OY, Heinz C, Heiligenhaus A, Hammer CM, Luetjen-Drecoll E (2011) Morphological changes of trabeculectomy specimens in different kinds of uveitic glaucoma. Current eye research 36 (5):442-448. https://doi.org/10.3109/02713683.2011.566409
Moorthy RS, Mermoud A, Baerveldt G, Minckler DS, Lee PP, Rao NA (1997) Glaucoma associated with uveitis. Surv Ophthalmol 41 (5):361-394. https://doi.org/10.1016/s0039-6257(97)00006-4
Chono I, Miyazaki D, Miyake H, Komatsu N, Ehara F, Nagase D, Kawamoto Y, Shimizu Y, Ideta R, Inoue Y (2018) High interleukin-8 level in aqueous humor is associated with poor prognosis in eyes with open angle glaucoma and neovascular glaucoma. Scientific reports 8 (1):14533. https://doi.org/10.1038/s41598-018-32725-3
Freedman J, Iserovich P (2013) Pro-inflammatory cytokines in glaucomatous aqueous and encysted Molteno implant blebs and their relationship to pressure. Investigative ophthalmology & visual science 54 (7):4851-4855. https://doi.org/10.1167/iovs.13-12274
Zhang JM, An J (2007) Cytokines, inflammation, and pain. International anesthesiology clinics 45 (2):27-37. https://doi.org/10.1097/AIA.0b013e318034194e
Lee IT, Lin CC, Wu YC, Yang CM (2010) TNF-alpha induces matrix metalloproteinase-9 expression in A549 cells: role of TNFR1/TRAF2/PKCalpha-dependent signaling pathways. Journal of cellular physiology 224 (2):454-464. https://doi.org/10.1002/jcp.22142
Chakravarthy A, Khan L, Bensler NP, Bose P, De Carvalho DD (2018) TGF-β-associated extracellular matrix genes link cancer-associated fibroblasts to immune evasion and immunotherapy failure. Nature communications 9 (1):4692. https://doi.org/10.1038/s41467-018-06654-8
Wilson SE (2021) TGF beta -1, -2 and -3 in the modulation of fibrosis in the cornea and other organs. Experimental eye research 207:108594. https://doi.org/10.1016/j.exer.2021.108594
Kaburaki T, Koshino T, Kawashima H, Numaga J, Tomidokoro A, Shirato S, Araie M (2009) Initial trabeculectomy with mitomycin C in eyes with uveitic glaucoma with inactive uveitis. Eye (London, England) 23 (7):1509-1517. https://doi.org/10.1038/eye.2009.117-cme
Iwao K, Inatani M, Seto T, Takihara Y, Ogata-Iwao M, Okinami S, Tanihara H (2014) Long-term outcomes and prognostic factors for trabeculectomy with mitomycin C in eyes with uveitic glaucoma: a retrospective cohort study. J Glaucoma 23 (2):88-94. https://doi.org/10.1097/IJG.0b013e3182685167
Landers J, Martin K, Sarkies N, Bourne R, Watson P (2012) A twenty-year follow-up study of trabeculectomy: risk factors and outcomes. Ophthalmology 119 (4):694-702. https://doi.org/10.1016/j.ophtha.2011.09.043
Jabs DA, Nussenblatt RB, Rosenbaum JT (2005) Standardization of uveitis nomenclature for reporting clinical data. Results of the First International Workshop. American journal of ophthalmology 140 (3):509-516. https://doi.org/10.1016/j.ajo.2005.03.057
Vignali DA (2000) Multiplexed particle-based flow cytometric assays. Journal of immunological methods 243 (1-2):243-255. https://doi.org/10.1016/s0022-1759(00)00238-6
Pei M, Liu X, Zhao C, Gao F, Tao Y, Zhang M (2018) Chemokine and adhesion molecule profiles in aqueous humor of clinically quiescent uveitic cataracts. Current eye research. https://doi.org/10.1080/02713683.2018.1532012
Ohira S, Inoue T, Shobayashi K, Iwao K, Fukushima M, Tanihara H (2015) Simultaneous increase in multiple proinflammatory cytokines in the aqueous humor in neovascular glaucoma with and without intravitreal bevacizumab injection. Investigative ophthalmology & visual science 56 (6):3541-3548. https://doi.org/10.1167/iovs.14-15918
Ren M, Guo Q, Guo L, Lenz M, Qian F, Koenen RR, Xu H, Schilling AB, Weber C, Ye RD, Dinner AR, Tang WJ (2010) Polymerization of MIP-1 chemokine (CCL3 and CCL4) and clearance of MIP-1 by insulin-degrading enzyme. The EMBO journal 29 (23):3952-3966. https://doi.org/10.1038/emboj.2010.256
de Boer JH, Hack CE, Verhoeven AJ, Baarsma GS, de Jong PT, Rademakers AJ, de Vries-Knoppert WA, Rothova A, Kijlstra A (1993) Chemoattractant and neutrophil degranulation activities related to interleukin-8 in vitreous fluid in uveitis and vitreoretinal disorders. Investigative ophthalmology & visual science 34 (12):3376-3385
Kuchtey J, Rezaei KA, Jaru-Ampornpan P, Sternberg P, Jr., Kuchtey RW (2010) Multiplex cytokine analysis reveals elevated concentration of interleukin-8 in glaucomatous aqueous humor. Investigative ophthalmology & visual science 51 (12):6441-6447. https://doi.org/10.1167/iovs.10-5216
Shifera AS, Trivedi S, Chau P, Bonnemaison LH, Iguchi R, Alvarado JA (2010) Constitutive secretion of chemokines by cultured human trabecular meshwork cells. Experimental eye research 91 (1):42-47. https://doi.org/10.1016/j.exer.2010.04.001
Thirumangalakudi L, Yin L, Rao HV, Grammas P (2007) IL-8 induces expression of matrix metalloproteinases, cell cycle and pro-apoptotic proteins, and cell death in cultured neurons. Journal of Alzheimer's disease : JAD 11 (3):305-311. https://doi.org/10.3233/jad-2007-11307
Jo N, Wu GS, Rao NA (2003) Upregulation of chemokine expression in the retinal vasculature in ischemia-reperfusion injury. Investigative ophthalmology & visual science 44 (9):4054-4060. https://doi.org/10.1167/iovs.02-1308
Kokubun T, Tsuda S, Kunikata H, Yasuda M, Himori N, Kunimatsu-Sanuki S, Maruyama K, Nakazawa T (2018) Characteristic Profiles of Inflammatory Cytokines in the Aqueous Humor of Glaucomatous Eyes. Ocular immunology and inflammation 26 (8):1177-1188. https://doi.org/10.1080/09273948.2017.1327605
Chou JC, Cousins CC, Miller JB, Song BJ, Shen LQ, Kass MA, Wiggs JL, Pasquale LR (2018) Fundus Densitometry Findings Suggest Optic Disc Hemorrhages in Primary Open-Angle Glaucoma Have an Arterial Origin. American journal of ophthalmology 187:108-116. https://doi.org/10.1016/j.ajo.2017.12.024
Sihota R, Angmo D, Ramaswamy D, Dada T (2018) Simplifying "target" intraocular pressure for different stages of primary open-angle glaucoma and primary angle-closure glaucoma. Indian journal of ophthalmology 66 (4):495-505. https://doi.org/10.4103/ijo.IJO_1130_17
Scott LJ (2017) Tocilizumab: A Review in Rheumatoid Arthritis. Drugs 77 (17):1865-1879. https://doi.org/10.1007/s40265-017-0829-7
Erta M, Giralt M, Esposito FL, Fernandez-Gayol O, Hidalgo J (2015) Astrocytic IL-6 mediates locomotor activity, exploration, anxiety, learning and social behavior. Hormones and behavior 73:64-74. https://doi.org/10.1016/j.yhbeh.2015.06.016
Zahir-Jouzdani F, Atyabi F, Mojtabavi N (2017) Interleukin-6 participation in pathology of ocular diseases. Pathophysiology : the official journal of the International Society for Pathophysiology 24 (3):123-131. https://doi.org/10.1016/j.pathophys.2017.05.005
Fang XX, Jiang XL, Han XH, Peng YP, Qiu YH (2013) Neuroprotection of interleukin-6 against NMDA-induced neurotoxicity is mediated by JAK/STAT3, MAPK/ERK, and PI3K/AKT signaling pathways. Cellular and molecular neurobiology 33 (2):241-251. https://doi.org/10.1007/s10571-012-9891-6
Borkenstein A, Faschinger C, Maier R, Weger M, Theisl A, Demel U, Graninger W, Irene H, Mossböck G (2013) Measurement of tumor necrosis factor-alpha, interleukin-6, Fas ligand, interleukin-1α, and interleukin-1β in the aqueous humor of patients with open angle glaucoma using multiplex bead analysis. Molecular vision 19:2306-2311
Takai Y, Tanito M, Ohira A (2012) Multiplex cytokine analysis of aqueous humor in eyes with primary open-angle glaucoma, exfoliation glaucoma, and cataract. Investigative ophthalmology & visual science 53 (1):241-247. https://doi.org/10.1167/iovs.11-8434
王强刘A孟A刘A (2018) 白细胞介素-6在青光眼发病过程中的作用. 国际眼科杂志%J International Eye Science 18 (10):1803-1805. https://doi.org/10.3980/j.issn.1672-5123.2018.10.10
Pereiro X, Ruzafa N, Acera A, Fonollosa A, Rodriguez FD, Vecino E (2018) Dexamethasone protects retinal ganglion cells but not Müller glia against hyperglycemia in vitro. PloS one 13 (11):e0207913. https://doi.org/10.1371/journal.pone.0207913
Heiduschka P, Thanos S (2006) Cortisol promotes survival and regeneration of axotomised retinal ganglion cells and enhances effects of aurintricarboxylic acid. Graefes Arch Clin Exp Ophthalmol 244 (11):1512-1521. https://doi.org/10.1007/s00417-005-0164-7
Schwartzman S (2016) Advancements in the management of uveitis. Best practice & research Clinical rheumatology 30 (2):304-315. https://doi.org/10.1016/j.berh.2016.07.005
Degroote RL, Uhl PB, Amann B, Krackhardt AM, Ueffing M, Hauck SM, Deeg CA (2017) Formin like 1 expression is increased on CD4+ T lymphocytes in spontaneous autoimmune uveitis. Journal of proteomics 154:102-108. https://doi.org/10.1016/j.jprot.2016.12.015
Rezk AF, Kemp DM, El-Domyati M, El-Din WH, Lee JB, Uitto J, Igoucheva O, Alexeev V (2017) Misbalanced CXCL12 and CCL5 Chemotactic Signals in Vitiligo Onset and Progression. The Journal of investigative dermatology 137 (5):1126-1134. https://doi.org/10.1016/j.jid.2016.12.028
Connolly KA, Belt BA, Figueroa NM, Murthy A, Patel A, Kim M, Lord EM, Linehan DC, Gerber SA (2016) Increasing the efficacy of radiotherapy by modulating the CCR2/CCR5 chemokine axes. Oncotarget 7 (52):86522-86535. https://doi.org/10.18632/oncotarget.13287
Clouthier DE, Comerford SA, Hammer RE (1997) Hepatic fibrosis, glomerulosclerosis, and a lipodystrophy-like syndrome in PEPCK-TGF-beta1 transgenic mice. The Journal of clinical investigation 100 (11):2697-2713. https://doi.org/10.1172/jci119815
Hill LJ, Mead B, Thomas CN, Foale S, Feinstein E, Berry M, Blanch RJ, Ahmed Z, Logan A (2018) TGF-β-induced IOP elevations are mediated by RhoA in the early but not the late fibrotic phase of open angle glaucoma. Molecular vision 24:712-726
Tsukamoto T, Kajiwara K, Nada S, Okada M (2019) Src mediates TGF-β-induced intraocular pressure elevation in glaucoma. Journal of cellular physiology 234 (2):1730-1744. https://doi.org/10.1002/jcp.27044
Xu Y, Xing YQ (2018) Long non-coding RNA GAS5 contributed to the development of glaucoma via regulating the TGF-β signaling pathway. European review for medical and pharmacological sciences 22 (4):896-902. https://doi.org/10.26355/eurrev_201802_14367
Kehrl JH, Wakefield LM, Roberts AB, Jakowlew S, Alvarez-Mon M, Derynck R, Sporn MB, Fauci AS (1986) Production of transforming growth factor beta by human T lymphocytes and its potential role in the regulation of T cell growth. The Journal of experimental medicine 163 (5):1037-1050. https://doi.org/10.1084/jem.163.5.1037
Taylor A (2003) A review of the influence of aqueous humor on immunity. Ocular immunology and inflammation 11 (4):231-241. https://doi.org/10.1076/ocii.11.4.231.18269
Min SH, Lee TI, Chung YS, Kim HK (2006) Transforming growth factor-beta levels in human aqueous humor of glaucomatous, diabetic and uveitic eyes. Korean journal of ophthalmology : KJO 20 (3):162-165. https://doi.org/10.3341/kjo.2006.20.3.162
Ohta K, Wiggert B, Yamagami S, Taylor AW, Streilein JW (2000) Analysis of immunomodulatory activities of aqueous humor from eyes of mice with experimental autoimmune uveitis. Journal of immunology (Baltimore, Md : 1950) 164 (3):1185-1192. https://doi.org/10.4049/jimmunol.164.3.1185

No competing interests reported.

Multiple Cytokine Analysis of Aqueous Humor in Uveitis With or Without Secondary Glaucoma

Status:

Version 1

Abstract

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

DECLARATIONS

References

Additional Declarations

Status:

Version 1