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Abstract

Objective

This study aimed to investigate inflammatory cytokine expression profiles in the aqueous humor (AH) of diabetic cataract (DC) patients.

Methods

A quantitative multiplexed antibody assay was performed to measure the expression levels of 40 inflammatory cytokines in AH samples from DC and age-related cataract (ARC) patients. Bioinformatics analysis was used to examine the functions of the cytokines. Enzyme-linked immunosorbent assays (ELISAs) and western blots were performed to verify the data.

Results

The multiplexed antibody assay revealed that the expression levels of IL-6, sIL-6R, IL-17A, IL-8, MCP-1, TNF-β, RANTES, TIMP-1, and TIMP-2 were higher in the AH of DC patients compared with ARC patients. However, IL-1ra and IL-1a expression levels were lower in the DC patient AH samples. Pathway analysis indicated that IL-6 and sIL-6R belong to the class I helical cytokine family, which is associated with many biological functions. ELISA and western blot results confirmed that IL-6R and IL-6 expression levels were significantly higher in DC patients compared with ARC patients.

Conclusions

Our results revealed the status of 40 inflammatory cytokines in the AH by quantitative multiplexed assays. Additionally, IL-6 and sIL-6R were expressed markedly higher in DC compared with ARC, which may play critical roles in DC pathophysiology.

Keywords

Diabetic cataract aqueous humor IL-6 sIL-6R quantitative multiplexed assay cytokine

Introduction

Cataracts are the leading cause of blindness worldwide.¹ Diabetes duration is significantly associated with the risk of being diagnosed with a cataract.² However, the etiology of diabetic cataracts (DCs) has not been clearly revealed. According to the location of opacification, cataracts can be classified as cortical, nuclear, or posterior subcapsular. Posterior subcapsular cataract (PSC) is the most common opacification position of DCs, which may be caused by inflammatory cytokines.^3,4 However, the expression profiles of inflammatory cytokines in DC have not been fully investigated.

The aqueous humor (AH) contains numerous cytokines, and their expression levels vary significantly among different eye conditions, including diabetic retinopathy, myopic, and inflammation.^5–9 Analyzing the AH may provide a useful tool for understanding the pathophysiology and treatment responses of DC. In recent years, the safety and effectiveness of high-throughput quantitative chip technology for detecting cytokines in the AH have been recognized.⁶^,⁸^,⁹

One study reported 27 AH cytokine profiles in patients with type 2 diabetes with or without retinopathy using this quantitative chip technology.⁹ To identify additional AH inflammatory cytokine profiles and analyze the cytokine status of DC patients, we collected AH from patients with or without DR using 40 high-throughput quantitative multiplexed assays. We then further analyzed correlations of the differentially expressed cytokines (DECs) using bioinformatics approaches to uncover possible therapeutic targets for DC.

Materials and Methods

Type of study

This was designed as a cross-sectional study. The reporting of this study conforms to Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines.¹⁰

Subjects

We recruited age-related cataract (ARC) patients (without a diabetes history) and DC patients (with a diabetes history of over 10 years) who underwent uneventful cataract surgery at our center between January and February 2021. This case-control study was approved by Shanghai Jiao Tong University Affiliated Sixth People’s Hospital (Approval No. 2021-KY-019). Written informed consent was obtained from each patient before participation. All procedures adhered to the Declaration of Helsinki and were conducted in accordance with the approved research protocol.

Cataract type and classification were determined using a standardized lens examination by slit lamp according to Lens Opacities Classification System III (LOCS III). The LOCS III system is based on standard photographs and provides separate, nearly continuous grades for posterior subcapsular opacities (P) (ranging from 0 to 5.9, using five standards), nuclear opalescence (NO) (ranging from 0 to 6.9, using six standards), nuclear color (NC) (ranging from 0 to 6.9), and cortical opacities (C) (ranging from 0 to 5.9, using five standards). Here, we present the associations of posterior subcapsular (P), cortical (C), nuclear (N), and mixed cataracts (M) with the major location of lens opacification by LOCS III.³ All cataract surgeries were performed by one surgeon (Prof. QW). No anti-inflammatory therapies, including topical non-steroidal anti-inflammatory drugs (NSAIDs), were used in these patients before cataract surgery. Eyes with uveitis, glaucoma, previous trauma, zonular weakness, myopic choroidal neovascularization, or diabetic retinopathy were excluded from this study.

AH collection

After swabbing the eyelids and the surrounding skin with disinfectant, we created a lateral corneal incision and a main corneal incision. We then gently inserted a 26 G needle through the lateral corneal incision to aspirate the AH (100 μL to 200 μL) before commencing cataract surgery. The samples were immediately stored at −80°C until further analysis.

Total protein quantification

Before the array assays, the total protein in each AH sample was estimated using a BCA Protein Assay Kit (Solarbio Life Sciences; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Briefly, 10 μL of bovine serum albumin (BSA) standard was diluted with 90 μL of PBS to obtain a final concentration of 0.5 mg/mL. Then, volumes of 0, 2, 4, 6, 8, 12, 16, and 20 μL of the 0.5 mg/mL BSA standard were added to each well of a 96-well plate along with PBS to a total volume of 20 μL. At the same time, 2, 4, and 8 μL of the AH samples were added to the plate. PBS was added to bring the total volume of each well to 20 μL. The samples were mixed with 200 μL of BCA Dye Reagent (the ratio of BCA reagent to Cu was 50:1). After incubation for 30 minutes at 37°C, the absorbance values of the standards and AH samples were measured at 562 nm (UV-Vis Spectrophotometer, ThermoFisher Scientific, Waltham, MA, USA).

Cytokine antibody array analysis

Cytokine antibody arrays were used to detect inflammatory cytokines in the AH samples obtained from four ARC patients and four DC patients. The Human Inflammation Array 3 (QAH-INF-3, RayBio; RayBiotech; Norcross, GA, USA) was used for the simultaneous analysis of 40 selected cytokines. The assay was performed according to the manufacturer’s instructions. The assay is based on the sandwich immunoassay principle. Briefly, 70 μL of sample was applied to each block. Antibodies targeting the selected cytokines were immobilized in specific locations on the surface of the array membrane. Cytokines present in the samples were captured by the corresponding antibodies, and a cocktail of biotinylated antibodies was added to detect the bound cytokines. The signals were visualized using a fluorescent dye conjugated with streptavidin (Cy3 equivalent) and were detected with a GenePix 4000B system (Axon Instruments, Foster City, CA, USA). GenePix Pro 6.0 software (Axon Instruments) was used for densitometric analysis of the spots.

Bioinformatics analysis of differential cytokine expression

After obtaining the expression data for 40 cytokines, we used Student’s t-test (two-sided) to calculate the P-value. DECs were determined using cutoffs of P-value <0.05 and fold change >1.5. The pheatmap package (v1.0.12) was used to generate a heatmap showing the levels of cytokine expression. From low to high, the expression levels were scaled from 0 to 1 across samples. The pathview package (Version 1.30.0) in R (www.r-project.org) was used to intuitively exhibit the DECs within the cytokine-cytokine receptor interaction Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. This data analysis was performed with R-scripts.

Enzyme-linked immunosorbent assay (ELISA)

The protein concentration of each AH sample was estimated using a BCA Protein Assay Kit (Solarbio Life Sciences; Beijing Solarbio Science & Technology Co., Ltd.). The Human IL-6 ELISA Kit (EL10023, Anogen, Mississauga, Ontario, Canada) and Human sIL-6R ELISA Kit (BMS214 and BMS214TEN, Invitrogen, Vienna, Austria) were used for ELISAs. Data were analyzed using a Bio-Rad Microplate Reader 550 (Bio-Rad, Hercules, CA, USA). The results are expressed in pg/mL.

Western blot analysis

After determining the protein concentration, each AH sample (containing 4 mg of total AH protein) was mixed with 5× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and denatured at 100°C for 5 minutes. Then, the samples were separated by 15% gradient acrylamide SDS-PAGE. The separated protein bands were transferred onto a polyvinylidene difluoride (PVDF) blotting membrane (Millipore, Burlington, MA, USA) and incubated with primary rabbit anti-human antibodies for IL-16R (1:1000; ab2573; Abcam, Cambridge, MA, USA), IL-6 (1:1000; ab151538; Abcam), and β-actin (1:1000; ab8226; Abcam) at 4°C overnight. The membranes were then incubated with a goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000; ab6721; Abcam) for 30 minutes at room temperature. The immunoblotted bands were visualized using an enhanced chemiluminescence reagent (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific). The gray band values of the blots were analyzed using Scion Image (Version: Beta 4.0.2 of Scion Image, Scion Corporation, Frederick, MD, USA).

Statistical analysis

All data are expressed as the mean ± standard deviation. Student’s t tests were used after testing the homogeneity of variance to compare quantitative data differences between the two groups of patients. The relative expression level for western blot data was obtained by dividing the gray value of the band of interest by the β-actin band value. The χ² test was used to examine differences between categorical variables, as well as gender and which eye was operated on. P-values less than 0.05 were considered statistically significant. All analyses were performed using SPSS software (version 19.0; IBM Corp., Armonk, NY, USA).

Results

Patient characteristics

Table 1 shows the characteristics of the participants. The average age of the 29 ARC patients was significantly higher than that of the 29 DC patients (P < 0.001). There were no significant differences between the two patient groups for gender or the eye that was operated on. Table 2 shows the proportions of different types of cataracts. The proportion of PSCs in DC patients (41.4%) was significantly higher than that in ARC patients (3.4%) (P < 0.001), and the proportion of nuclear cataracts in DC patients (20.7%) was relatively lower than that in ARC patients (41.4%) (P = 0.005).

Table 1.

Patient details.

	Cytokine antibody array		ELISA		Western blot
	ARC	DC	ARC	DC	ARC	DC	P-value
N	4	4	22	22	3	3
Age, years	75.8 ± 3.1	63.8 ± 3.3	75.8 ± 8.8	67.9 ± 6.6	79.3 ± 1.5	63.7 ± 6.4	<0.001
Gender (M/F)	2/2	2/2	11/11	12/10	1/2	2/1	0.812
History of diabetes, years	0	12.8 ± 1.7	0	14.8 ± 3.2	0	13.0 ± 2.0	<0.001
Operated eye (right/left)	2/2	2/2	11/11	11/11	2/1	1/2	0.793

ELISA, enzyme-linked immunosorbent assay; ARC, age-related cataract; DC, diabetic cataract.

Table 2.

Proportion (%) of different cataract types in all patients.

	ARC	DC	P-value
PSC	1 (3.4%)	12 (41.4%)	<0.001
Cortical	8 (27.6%)	9 (31.1%)	0.625
Nuclear	12 (41.4%)	6 (20.7%)	0.005
Mixed	8 (27.6%)	2 (6.8%)	0.002
Total	29	29

ARC, age-related cataract; DC, diabetic cataract; PSC, posterior subcapsular cataract.

Cytokine profiles in AH by antibody array

Table 3 shows the results of the inflammatory cytokines tested with this array. Of the 40 inflammatory cytokines examined, the expression levels of IL-6, IL-6R, IL-17A, IL-8, MCP-1, TNF-β, RANTES, TIMP-1, and TIMP-2 were significantly higher in DC patients compared with ARC patients (all P < 0.01). However, the expression levels of IL-1ra and IL-1a were significantly lower in DC patients than in ARC patients (all P < 0.001) (Figure 1).

Table 3.

Results of the cytokine antibody array.

Cytokine (pg/mL)	ARC (n = 4)		DC (n = 4)		P-value
Cytokine (pg/mL)	Mean	SD	Mean	SD	P-value
BLC	2.1	0.1	2.1	0.3	1.000
Eotaxin-1	7.1	0.1	7.9	2.0	0.451
Eotaxin-2	7.7	0.2	7.4	1.5	0.702
G-CSF	17.6	2.3	16.6	3.0	0.619
GM-CSF	5.8	1.9	5.9	1.6	0.937
I-309	4.8	0.1	4.9	1.1	0.897
ICAM-1	455.5	52.2	48.8	89.1	0.591
IFN-γ	11.3	5.0	17.6	5.5	0.139
IL-1a	22.1	4.2	4.2	2.0	<0.001*
IL-1b	5.7	0.7	5.9	0.9	0.749
IL-1ra	3.0	0.3	1.5	0.3	<0.001*
IL-2	4.0	1.3	2.9	1.2	0.259
IL-4	13.8	5.3	13.2	2.2	0.829
IL-5	6.5	1.3	7.9	0.4	0.071
IL-6	6.3	1.5	16.4	3.4	0.002*
sIL-6R	14.0	1.8	33.9	1.4	<0.001*
IL-7	23.2	9.3	24.9	3.2	0.745
IL-8	0.7	0.4	2.8	0.5	0.001*
IL-10	2.7	1.2	2.2	0.7	0.517
IL-11	15.3	4.8	17.3	5.5	0.603
IL-12p40	46.4	3.6	44.6	8.2	0.707
IL-12p70	1.0	0.1	1.0	0.2	1.000
IL-13	1.0	2.8	10.0	2.9	0.972
IL-15	18.3	6.5	14.9	5.8	0.467
IL-16	7.9	2.4	7.0	0.8	0.50
IL-17A	1.8	0.7	3.4	3.3	0.005*
MCP-1	87.1	39.6	231.8	11.0	<0.001*
M-CSF	11.9	1.2	12.6	1.8	0.519
MIG	31.5	0.8	32.3	5.8	0.787
MIP-1a	8.4	0.7	8.9	0.9	0.468
MIP-1b	2.1	1.7	1.5	1.2	0.570
MIP-1d	40.4	2.21	44.9	7.3	0.281
PDGF-BB	1.9	1.73	1.7	2.1	0.241
RANTES	10.7	1.64	17.9	3.5	0.01*
TIMP-1	737.5	238.9	1528.8	272.4	0.005*
TIMP-2	1345.0	275.0	3103.3	704.6	0.001*
TNF-α	7.6	2.52	5.8	1.4	0.246
TNF-β	2.5	0.82	64.6	4.6	<0.001*
TNF-RI	146.5	16.6	159.2	22.7	0.399
TNF-RII	632.4	85.0	658.0	117.5	0.737

ARC, age-related cataract; DC, diabetic cataract; BLC, B lymphocyte chemoattractant; G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte and macrophage colony stimulating factor; I-309, recombinant human C-C motif chemokine 1; ICAM-1, intercellular adhesion molecule-1; IFN-γ, interferon-gamma; IL, interleukin; sIL, secretory interleukin; MCP, monocyte chemotactic protein; MIG, monokine induced by IFN-γ; MIP-1, mixed protein-1; PDGF-BB, platelet-derived growth factor BB; RANTES, activation regulatory factor; TIMP, tissue inhibitor of metalloproteinase; TNF, tumor necrosis factor.

Cytokines in bold have a statistically significant difference between ARC and DC patient samples (*P < 0.05, Student’s t test was used after the homogeneity test of variance.)

Figure 1.

Eleven differentially expressed cytokines (DECs) in the aqueous humor (AH) of age-related cataract (ARC) and diabetic cataract (DC) patients analyzed by antibody array. (a) The values of these upregulated DECs were less than 70 pg/mL. (b) The values of these upregulated cytokines were greater than 0.8 ng/mL and. (c) The downregulated DECs. ***P < 0.001, **P < 0.01, *P < 0.05.

DECs in a pathway context

We next further investigated the DECs between ARC and DC patients. Among the 40 analyzed cytokines, we identified 11 DECs using a cutoff P-value of less than 0.05 and fold change of greater than 1.5. We generated a heatmap of these 11 DECs, of which IL1A (IL-1a) and IL1RN (IL-1ra) were downregulated and IL6 (IL-6), IL6R (IL-6R), IL17A (IL-17A), CXCL8 (IL-8), CCL2 (MCP-1), LTA (TNF-β), CCL5 (RANTES), TIMP1 (TIMP-1), and TIMP2 (TIMP-2) were upregulated in DC compared with ARC (Figure 2). To analyze these DECs in a pathway context, we presented them in the cytokine-cytokine receptor interaction KEGG pathway. Strikingly, we found that IL-6 and IL-6R were commonly upregulated, suggesting that they possibly play critical roles in the pathophysiology of DC (Figure 3).

Figure 2.

Heatmap generated using the pheatmap package. Among the 40 analyzed cytokines, 11 differentially expressed cytokines (DECs) had a P-value <0.05 and fold change >1.5. Of these DECs, IL1A (IL-1a) and IL1RN (IL-1ra) were downregulated and IL6 (IL-6), IL6R (IL-6R), IL17A (IL-17A), CXCL8 (IL-8), CCL2 (MCP-1), LTA (TNF-β), CCL5 (RANTES), TIMP1 (TIMP-1), and TIMP2 (TIMP-2) were upregulated in diabetic cataract (DC) patients versus age-related cataract (ARC) patients.

Figure 3.

Differentially expressed cytokines between age-related cataract (ARC) and diabetic cataract (DC) patients in the cytokine-cytokine receptor interaction Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. IL1A (IL-1a) and IL1RN (IL-1ra), which belong to the IL1-like cytokine family, were downregulated in DC versus ARC. IL6 (IL-6: class I helical cytokine family IL6/12 subfamily), IL6R (IL-6R: class I helical cytokine family IL6/12 subfamily), IL17A (IL-17A: IL17-like cytokine family), CXCL8 (IL-8: chemokine family CXC subfamily), CCL2 (MCP-1: chemokine family CC subfamily), CCL5 (RANTES: chemokine family CC subfamily), and LTA (TNF-β: TNF family) were upregulated in DC versus ARC. Red: upregulated; Green: downregulated.

IL-6 and sIL-6R concentrations measured by ELISA

To verify the results of the cytokine antibody array, we examined IL-6 and soluble IL-6 receptor (sIL-6R) levels using ELISAs. We found that the expression levels of IL-6 in DC patients were significantly higher than those in ARC patients (ARC: 10.4 ± 5.0 pg/mL, DC: 44.3 ± 18.8 pg/mL; P < 0.001). The sIL-6R expression levels were also significantly higher in DC patients compared with ARC patients (ARC: 36.5 ± 19.4 pg/mL, DC: 116.9 ± 43.6; P < 0.001). For ARC patients, sIL-6R expression was almost 4.3 times that of IL-6. For DC patients, sIL-6R expression was almost 3.2 times that of IL-6 (Figure 4).

Figure 4.

Protein expression levels of IL-6 and sIL-6R in the aqueous humor (AH) of age-related cataract (ARC) and diabetic cataract (DC) patients measured by enzyme-linked immunosorbent assays (ELISAs). IL-6 and sIL-6R expression levels in DC patients were significantly higher than those in ARC patients. ***P < 0.001.

IL-6 and sIL-6R concentrations measured by western blots

To further confirm the results of the cytokine arrays and ELISAs, we performed western blot analysis for IL-6 and sIL-6R. Consistent with the ELISA data, the expression levels of sIL-6R were markedly higher than those of IL-6 in both the ARC and DC groups. A more prominent band was noted in the DC group than in the ARC group for sIL-6R. For IL-6, a relatively strong band was detected in the DC group, while a very faint band was detected in the ARC group. The relative levels of IL-6 in the DC group were 5.7 times higher than those in the ARC group, while the relative levels of sIL-6R in the DC group were 3.4 times higher than those in the ARC group (P = 0.009 and <0.001, respectively) (Figure 5).

Figure 5.

Protein expression levels of IL-6 and sIL-6R in the aqueous humor (AH) of age-related cataract (ARC) and diabetic cataract (DC) patients measured by western blot assays. (a) Western blot assay results. (b) Relative IL-6R and IL-6 protein expression levels. The relative expression of IL-6 in the DC group was 1.7 ± 0.1 times higher than that in the ARC group. The relative expression of IL-6R in the DC group was 1.5 ± 0.1 times higher than that in the ARC group. ***P < 0.001, **P < 0.01.

Discussion

Inflammatory cytokines may play important roles in DC pathogenesis.^3,4 However, the cytokine status in the AH of DC patients and the mechanisms involved need to be further explored. The development of high-throughput quantitative multiplexed assay technology for the detection of inflammatory cytokines at low protein concentrations has allowed more studies to focus on changes in inflammatory cytokine levels in the AH of various types of cataracts.⁶^,⁸^,⁹^,¹¹

In this study, we included patients with a diabetic history of at least 10 years because DC can be largely affected by the duration of diabetes.³ While obtaining the AH samples, we created both a main corneal incision and lateral incision. We then inserted a needle through the lateral incision rather than creating one incision to directly draw the AH. Using this improved method with two incisions, the negative pressure of the anterior chamber could be reduced and we were able to successfully extract 100 μL to 200 μL of AH each time.

Of the 40 inflammatory cytokines screened by quantitative multiplexed assays in this study, we identified 11 DECs between DC and ARC samples. The concentrations of IL-6, sIL-6R, IL-8, IL-17A, MCP-1, TNF-β, RANTES, TIMP-1, and TIMP-2 were significantly higher in the AH of DC patients compared with those of ARC patients. However, the concentrations of IL-1a and IL-1ra were significantly lower in the AH of DC patients compared with those of ARC patients. To the best of our knowledge, most of the previous studies aimed to explore cytokine changes in AH samples of diabetic retinopathy (DR) and diabetic macular edema, rather than that of diabetic cataracts.^5–7^,⁹^,¹¹^,¹² Our results, such as those for IL-6, IL-8, and MCP-1, were mostly consistent with these previous studies.⁵^,⁹^,¹¹^,¹² Dong et al. measured 27 AH cytokines with a multiplex assay.⁹ They found that the IL-1β, IL-6, IL-8, MCP-1, and interferon gamma-induced protein-10 levels in the AH increased as the severity of DR increased. Cheung et al. measured the concentrations of 22 human cytokines.¹¹ They found that compared with diabetic patients without DR, the IL-6 and VEGF levels were significantly higher in diabetic patients with DR. In our study, we screened 40 inflammatory cytokines by quantitative multiplexed assays.

To analyze these DECs in a pathway context, we used the cytokine-cytokine receptor interaction KEGG pathway. These results indicated that the higher expressed factors in the DC group mainly belong to chemokine family, class I helical cytokine family, IL-17-like cytokine family, and TNF family. These cytokine families play key roles in host defense, immune regulation, reproduction, and the regulation of neural growth.¹³^,¹⁴ The lower expressed factors in the DC group, IL-1a and IL-1ra, belong to IL-1-like cytokine family, which plays major roles in a wide range of inflammatory, infectious, and autoimmune diseases.¹⁵ Among these inflammatory cytokines, we became interested in IL-6 and sIL-6R and further verified their expression levels in our samples using ELISAs and western blot analysis. Collectively, our experiments confirmed that IL-6 and sIL-6R were significantly upregulated in DC patients compared with ARC patients, which was consistent with work performed by Chen et al.¹⁶

IL-6 levels are reportedly increased after cataract surgery and may modulate the activities of immune cells to promote PSCs.³^,⁴ Additionally, a recent study revealed that miR-29a could mitigate high glucose-induced oxidative injury and exert protective effects via modulation of IL-6/STAT3 signaling in DC.¹⁷ These studies indicated that IL-6 may play an important role in DC through the trans signal transduction pathway. The roles of IL-6 can be divided into three types: the classical signal transduction pathway, the trans signal transduction pathway, and the trans presentation pathway, which are induced by IL-6R, sIL-6R, and IL-6R, respectively. Therefore, IL-6 acts through its soluble receptor (sIL-6R) or its cellular receptor (IL-6R).¹⁸ While both molecules are IL-6 cognate receptors, sIL-6R does not have the cytoplasmic domain present in IL-6R.¹⁹ IL-6R is present on the surface of cells that express it, while sIL-6R exists in serum or even in cells that lack IL-6R.¹⁸ The main role of IL-6 is to amplify inflammatory responses by activating the trans-signaling pathway.^20,21

In our study, sIL-6R expression levels were nearly 2 to 4 times higher than those of IL-6 in the AH samples of both ARC and DC patients. A possible reason for this markedly higher expression pattern is that there could be more sIL-6R molecules ready to bind IL-6 to improve the efficiency of the inflammatory response when inflammation is induced.

IL-6 is produced by a variety of cells, including inflammatory cells, like lymphocytes and macrophages, and non-inflammatory cells, such as fibroblasts, myocytes, and endothelial cells.²² A large number of dendritic cells are found in the corneal stroma of individuals with diabetes mellitus. Corneal epithelial cells and ocular uveal cells, such as fibroblasts, macrophages, and T lymphocytes, have also been reported to secrete IL-6.²³ Therefore, IL-6 in the AH may be produced by cells of the cornea or ocular uvea.

Quantitative inflammatory multiplexed antibody arrays have some limitations because their design does not avoid potential non-specific reactions between different antibodies and cytokines. Therefore, the data generated using such antibody arrays need to be carefully analyzed and further verified. In our study, we performed ELISAs and western blots to verify the quantitative multiplexed assay results for IL-6 and sIL-6R.

In conclusion, our study identified the differentially expressed inflammatory factors in the AH of DC samples using a quantitative multiplexed assay. The significant upregulation of IL-6 and sIL-6R in DC samples was validated by ELISAs and western blots. Our findings may provide a valuable therapeutic target for the non-surgical treatment of DC.

Footnotes

Acknowledgements

The authors thank AJE for providing helpful writing revision.

Declaration of conflicting interest

The authors declare that there is no conflict of interest.

Funding

This work was supported by the Clinical Medical Department of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital (X113 to YC).

ORCID iD

Yan Chen

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Cytokine status and significant increase of IL-6 and sIL-6R in the aqueous humor of diabetic cataract patients revealed by quantitative multiplexed assays

Abstract

Objective

Methods

Results

Conclusions

Keywords

Introduction

Materials and Methods

Type of study

Subjects

AH collection

Total protein quantification

Cytokine antibody array analysis

Bioinformatics analysis of differential cytokine expression

Enzyme-linked immunosorbent assay (ELISA)

Western blot analysis

Statistical analysis

Results

Patient characteristics

Cytokine profiles in AH by antibody array

DECs in a pathway context

IL-6 and sIL-6R concentrations measured by ELISA

IL-6 and sIL-6R concentrations measured by western blots

Discussion

Footnotes

Acknowledgements

Declaration of conflicting interest

Funding

ORCID iD

References