Aqueous humor cytokine levels in patients with diabetic macular edema refractory to anti-VEGF treatment
Aqueous humor cytokine levels in patients with diabetic macular edema refractory to anti-VEGF treatment
Jin-woo Kwon 0 1
Donghyun Jee 0 1
0 Department of Ophthalmology and Visual Science, St. Vincent's Hospital, College of Medicine, Catholic University of Korea , Suwon, Kyunggi-do , Korea
1 Editor: Alfred S Lewin, University of Florida , UNITED STATES
To determine aqueous cytokines association with response to intravitreal bevacizumab (IVB) injection in diabetic macular edema (DME). We compared the concentrations of IL (interleukin)-1?, IL-2, IL-8, IL-10, IL-17, placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) in the aqueous humor of 64 na?ve DME patients with those of 13 cataract patients. Factors associated with central subfield thickness (CST) in DME patients were identified. DME patients were then subgrouped in terms of responsiveness to three IVB injections; cytokine concentrations were compared, and factors associated with responsiveness were identified.
Levels of IL-2, IL-8, PlGF, and VEGF were significantly elevated in DME patients (p = 0.007,
p < 0.001, p < 0.001, and p = 0.004 respectively). Regression analysis showed that the
preoperative CST was associated with the preoperative best-corrected visual acuity and
the aqueous IL-10 level (p < 0.001, p = 0.006, respectively). Of the 64 DME patients, 28
(43.75%) exhibited either CST < 300 ?m or reduction in CST 50 ?m after three
consecutive IVB injections. On sub-group analysis, the mean IL-8 concentration was higher in
the refractory group than in the responsive group, and multivariate logistic regression
analysis showed that the IL-8 was the only factor associated with responsiveness (OR = 0.95,
p = 0.017).
The IL-8 concentration in the aqueous humor was associated with responsiveness to IVB in
Funding: This work was supported by the National
Research Foundation of Korea Grant funded by the
Korean government (MSIP),
Donghyun Jee. The funder had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Diabetic retinopathy (DR) is one of the most significant causes of visual impairment
worldwide, despite advances in laser and surgical treatments.[1?5] The visual impairment associated
with DR is principally attributable to neovascularization or diabetic macular edema (DME).
[5?7] The early pathogenesis of DME features damage to the blood?retina barrier,
characterized by loss of pericytes and endothelial tight junctions induced by metabolic alterations and
] DME is affected by not only inflammatory and glial cells but also the
expression levels of inflammatory molecules, including interleukins (ILs), vascular endothelial
growth factor (VEGF), tumor necrosis factor-?, transforming growth factor-?, and matrix
metalloproteinases (MMPs).[8?10] Based on fundamental studies of the effects of VEGF and
anti-VEGF antibodies, several types of anti-VEGF agents have been shown to effectively treat
] Recently, intravitreal steroid implants have also been proven to reduce central
subfield thickness (CST) and improve visual acuity in DME patients.[
Many studies have shown that the aqueous humor of DME patients contains elevated levels
of inflammatory cytokines, growth factors, and MMPs, associated with retinal status.[15?17]
The individual roles played by these factors in pathogenesis remain unclear; many studies have
sought to determine the detailed mechanisms of action. We thus compared the levels of IL-1?,
IL-2, IL-8, IL-10, IL-17, placental growth factor (PlGF), and VEGF in the aqueous humor of 64
na?ve DME patients with those of a control group. We also identified factors associated with
responsiveness to intravitreal bevacizumab (IVB).
We compared IL-1?, IL-2, IL-8, IL-10, IL-17, PlGF, and VEGF levels in the aqueous humor of
na?ve DME patients with type II diabetes mellitus (DM). We followed all relevant tenets of the
Declaration of Helsinki. The protocol was approved by the Institutional Review/Ethics Board
of the Catholic University of Korea. All methods were performed in accordance with the
relevant guidelines and regulations by the protocol. All participants gave written informed consent
to the use of their clinical records.
We enrolled 64 na?ve DME eyes (of 64 patients) of CST > 300 ?m; 13 diabetes-free patients
with cataracts served as controls. The exclusion criteria included glaucoma, retinal
degeneration, and macular edema attributable to other causes including an epiretinal membrane or
vitreo-macular traction. We excluded eyes with concurrent diseases such as retinal vascular
occlusion, and eyes with histories of prior ocular conditions, uveitis, or intraocular surgery
that could influence enzyme levels in the aqueous humor.
Every patient underwent a full ophthalmological examination including measurement of
visual acuity, refraction, and intraocular pressure (IOP), in addition to a dilated fundus
examination. All eyes were classified using the Early Treatment of Diabetic Retinopathy criteria as
having mild non-proliferative diabetic retinopathy (NPDR), moderate or severe NPDR, or
proliferative diabetic retinopathy (PDR). Macular thickness was measured using optical
coherence tomography (OCT) (Cirrus High- Definition OCT; Carl Zeiss Meditec, Dublin, CA,
USA) and axial length employing an IOL Master instrument (Carl Zeiss Meditec).
We classified DME patients as either IVB-responsive or -refractory. Responsiveness was
defined as either CST < 300 ?m or a CST reduction 50 ?m at 1 month after 3 consecutive
monthly injections of IVB.[
Assay of cytokines and growth factors
Concentrations of IL-1?, IL-2, IL-8, IL-10, IL-17, PlGF, and VEGF in 75 ?L of aqueous humor
from the anterior chamber (collected via anterior paracentesis during the first IVB injection or
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cataract surgery, and were immediately stored at -80?C until analysis) were measured. The
assays used human antibodies against IL-1?, IL-2, IL-8, IL-10, IL-17, PlGF, and VEGF. The
antibodies were immobilized on beads; 75-?L humor samples with 75 ?L Calibrator Diluent
RD6-52 were added to the bead preparations. The samples were incubated for 2 h at room
temperature (20?25?C) after bead addition, for a further 1 h at room temperature after the
addition of detection antibodies, and for 30 min at room temperature after the addition of
streptavidin-phycoerythrin reagent. A Luminex-x-MAP suspension array system (Luminex,
Austin, TX, USA) was used for detection; this is a multiplexed, microsphere suspension
immunoassay that detects and quantitates spectrally unique microspheres attached to specific
antibodies. The technique enables many samples to be analyzed in a single reaction.
The detection limits and dynamic ranges are as follows: 0.8 pg/mL with a dynamic range to
3,950 pg/mL for IL-1?, 1.8 pg/mL with a dynamic range to 8,510 pg/mL for IL-2, 1.8 pg/mL
with a dynamic range to 1,140 pg/mL for IL-8, 1.6 pg/mL with a dynamic range to 890 pg/mL
for IL-10, 1.8 pg/mL with a dynamic range to 2,090 pg/mL for IL-17, 1.9 pg/mL with a
dynamic range to 470 pg/mL for PlGF, and 2.1 pg/mL with a dynamic range to 2,170 pg/mL
Statistical analyses were performed using SPSS for Windows software (ver. 20.0; SPSS,
Chicago, IL, USA) and R (ver. 3.2.3, 2015-12-10, Platform: x86_64-redhat-linux-gnu, R Core
Team (2015) [R: A language and environment for statistical computing. R Foundation for
Statistical Computing, Vienna, Austria.URL https://www.R-project.org/.])
We used the t-test, Mann?Whitney U-test, and the chi-squared test to compare the values
and the ratio of the participants groups. The Wilcoxon signed-rank test was used to compare
changes in IOP, CST, and BCVA, after placement of intravitreal dexamethasone implants.
Linear regression analysis was employed to identify CST-associated factors. Logistic regression
was employed to identify factors associated with responsiveness to IVB injection. The
statistical significance level was set at p<0.05.
The average age of the cataract patients was 67.92 ? 13.03 years and that of the DME patients
was 56.81 ? 7.96 years (p = 0.004). There were 30 males and 34 females in the study group and
8 males and 5 females in the control group. There was no significant difference in either axial
length or initial IOP between the study and control groups. Levels of IL-2, IL-8, PlGF, and
VEGF were significantly elevated in the DME group (p = 0.007, p < 0.001, p < 0.001, and
p = 0.004 respectively) (Table 1).
In the subgroup analysis, there was no significant difference in levels of aqueous cytokines
and responsiveness of IVB between PDR and NPDR patients (Table 2).
Preoperative CST was associated with preoperative BCVA and aqueous humor IL-10 level
on multivariate regression analysis (p < 0.001, p = 0.006, respectively) (Table 3).
Of the 64 DME patients, 28 (43.75%) exhibited either CST < 300 ?m or a reduction in
CST 50 ?m 1 month after the last IVB injection. On sub-group analysis, the mean IL-8
concentration of the refractory group was higher than that of the responsive group, and
multivariate logistic regression analysis showed that the IL-8 was the only factor associated with
responsiveness (OR = 0.95, p = 0.017) (Table 4).
Of the 36 refractory patients, 23 received intravitreal dexamethasone implants. Of these, 17
(73.91%) exhibited CST < 300 ?m and 20(86.96%) exhibited a CST reduction 50 ?m at the
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1-month follow-up. The CST and BCVA of 23 patients improved significantly compared with
the values prior to implantation (Fig 1).
Many studies have found that DME is mediated by inflammatory cytokines and VEGFs, the
actions of which are interconnected.[
] Analysis of vitreous samples yields valuable
information on retinal status, but obtaining such samples is invasive and data quality may be
compromised if the samples are contaminated with blood. Other studies have also shown that
the aqueous humor reflects retinal status; levels of many cytokines are elevated during retinal
Values are expressed as mean ? SD or median and interquartile range, as appropriate
Abbreviations: DME, diabetic macular edema; IOP, intraocular pressure; IL, interleukin; PlGF, placental growth factor; VEGF, vascular endothelial growth factor,
BCVA best-corrected visual acuity; CST, central subfield thickness
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? ? SE
CST, central subfield thickness; DME, diabetic macular edema; IL, interleukin; NPDR, non-proliferative diabetic retinopathy; PDR, proliferative diabetic retinopathy;
PlGF, placental growth factor; VEGF, vascular endothelial growth factor.
hypoxia or inflammation.[
17, 22, 23
] Most studies on aqueous humor have simply shown that
the concentrations of various materials differed between control and DME patients.[
Few studies have explored the associations between prognosis and individual cytokine levels.
] We thus sought associations between cytokine and VEGF levels, and responsiveness
to anti-VEGF therapy. We first determined that IL-8 concentrations may be associated with
IL-8 is a well-known pro-inflammatory cytokine that acts as a neutrophil chemoattractant
and a T-cell activator.[
] In the eye, hypoxia induces IL-8 synthesis by endothelial and glial
cells, associated with neovascularization in patients with ischemic retinal disease.[
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Fig 1. Box-and-whisker plots for central subfield thickness (CST), best-corrected visual acuity (BCVA), and intraocular pressure (IOP)
changes in patients receiving dexamethasone implants due to poor responsiveness to 3 months of intravitreal bevacizumab (IVB)
injections. The circles represent outliers. (A) CST was significantly reduced both after IVB injection and placement of dexamethasone implants.
(B) BCVA did not improve significantly after IVB injection, but did upon dexamethasone treatment. (C) The baseline IOP was 15.52 ? 3.23
mmHg, and increased significantly to 16.61 ? 2.21 mmHg after placement of dexamethasone implants.
Many studies have shown that elevated IL-8 levels in the aqueous humor of DME patients
may be associated with inflammation-induced damage to the blood-retina barrier.[
article has suggested that IL-8 may play a role in DME development that is not adequately
controlled by anti-VEGF antibodies or steroids. However, another study found that
intravitreal triamcinolone was effective in patients unresponsive to IVB, and that the efficacy was
associated with the IL-8 level in the aqueous humor.[
] This result matches our study, and we
suggest that the DME more related with inflammation could manifest higher aqueous IL-8.
This could be a good biomarker to predict responsiveness of therapeutic agents. The response
rate of DME patients with IL-8 levels <17.71 pg/mL (the median value of DME patients in this
study) was 19/31 (61.28%), but that of patients with IL-8 levels greater than the median value
was only 7/31(22.81%). These proportions differed significantly (p = 0.004; odds ratio (OR)
5.429). The role played by IL-8 in DME requires further investigation.
IL-10 produced by activated macrophages and T-cells exerts various effects that are
principally anti-inflammatory and immunosuppressive in nature.[
] In the eye, IL-10 may be
associated with an angiogenetic response to hypoxia, but the details remain unclear.[
levels in aqueous humor are elevated in patients with diabetes, retinal vascular occlusion, or
primary intraocular lymphoma.[36?38] However, few IL-10 data are available for DME
patients, and the results vary. One study reported that aqueous humor IL-10 levels correlated
negatively with VEGF level, and another study found that aqueous humor IL-10 levels
were elevated in patients in whom DR progressed. A recent study found that aqueous
humor baseline IL-10 levels were negatively associated with BCVA.[
] Our data suggest that
IL-10 may be associated with the CST of DME patients. Both BCVA and CST are markers of
DME severity and disease progression. Like other studies, we found that CST was significantly
associated with BCVA (p < 0.001, Table 3). Thus, IL-10 levels may also be associated with
DME severity; more studies are required.
In the protocol H study of the Diabetic Retinopathy Clinical Research Network, mean
reduction was 56 ?m after two 1.25-mg IVB injections.[
] Although the OCT platforms used
differed, we obtained a similar result after three consecutive IVB injections; the mean baseline
and postoperative CSTs were 416.03 ? 81.14 and 362.23 ? 95.64 ?m respectively; the mean
reduction was thus 53.80 ? 63.35 ?m. The protocol T study showed that, after 1-year IVB
treatment, CST decreased by 101 ? 121 ?m on average.[
] However we did not continue IVB until
1year and performed intravitreal dexamethasone implants in some of the refractory patients.
The mean CST reduction after implantation, compared with the baseline level, averaged
178.22 ? 107.41 ?m, and visual acuity improved significantly (Fig 1). The IOP increment was
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only 1.09 ? 2.41 mmHg; no patient exhibited IOP > 21 mmHg. However, in the first year of
the BEVORDEX study, IOP elevations
5 mmHg from baseline were evident in 46% of
patients, and a two-grade rise in cataract density was reported in 13% of patients receiving
] Thus, both the IOP and visual acuity require long-term
Our study had certain limitations. First, the aqueous levels of only IL-2, IL-8, PlGF, and
VEGF were significantly elevated in the DME group. This may be attributable to the small
number of control patients, who differed in mean age from the DME group. Also, differences
among the characteristics of patients enrolled in other studies may have affected our results.
Second, the relationships between biomarker levels and fluorescein angiographic or OCT
angiographic images should be studied in terms of DME pathogenesis. We plan a follow-up
study along these lines. Third, a 1-month follow-up of patients receiving dexamethasone
implants is too short to evaluate drug effects or side-effects. Changes in CST, BCVA, IOP, and
cataract progression must be evaluated long-term.
In summary, the aqueous humor concentrations of IL-10 were associated with CST and
those of IL-8 levels were associated with IVB responsiveness. Additional studies with more
patients are required to confirm our results and to elucidate DME pathogenesis. Such studies
may provide the basis for novel therapeutic approaches.
Conceptualization: Jin-woo Kwon.
Data curation: Jin-woo Kwon.
Funding acquisition: Donghyun Jee.
Supervision: Donghyun Jee.
Validation: Jin-woo Kwon.
Writing ? original draft: Jin-woo Kwon.
Writing ? review & editing: Donghyun Jee.
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occlusion. Acta ophthalmologica. 2017; 95(2):e119?e27. Epub 2016/07/16. https://doi.org/10.1111/
aos.13152 PMID: 27417275.
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