Aqueous humor cytokines are associated with baseline optical coherence tomography imaging features in eyes with diabetic macular edema

• Introduction
• Methods
• Results
• Discussion

Abstract

Purpose: This study aimed to assess the association between cytokine expression and optical coherence tomography (OCT) features of diabetic macular edema (DME).

Methods: This is a post-hoc analysis of the baseline data of a prospective study designed to evaluate the effect of intravitreal ranibizumab injections on aqueous humor cytokine levels in patients with center-involved DME. OCT images were evaluated for baseline imaging features of DME, including central macular thickness (CMT), macular volume (MV), subretinal fluid (SRF) height, morphological pattern of DME, intraretinal cyst (IRC) size, status of the ellipsoid zone/external limiting membrane (EZ/ELM), presence of disorganization of retinal inner layers (DRIL), number of hyperreflective foci (HRF), and the presence of epiretinal membrane (ERM) or vitreomacular traction (VMT). Hierarchical cluster analysis (HCA) was used to identify two groups (clusters) of subjects that shared similar cytokine characteristics (low and high expression profiles), and differences in OCT imaging parameters across clusters were compared using multivariable logistic regression models.

Results: Aqueous cytokine concentrations and OCT images were obtained from 30 eyes of 25 patients recruited. The HCA demonstrated that eyes in the low-concentration cytokine profile group were associated with better BCVA (OR = 0.90, [95% CI 0.81–0.99], p = 0.05), thinner CMT (OR = 1.08, [95% CI 1.01–1.17], p = 0.03), and lower MV (OR = 2.08, [95% CI 1.10–3.90], p = 0.02). Reduced BCVA, greater CMT, and larger MV were individually correlated with multiple cytokines, including IL-7, IL-9, MIP-1α, and TNF-α. Increased SRF was significantly associated with IL-15 (r = 0.40, p = 0.03) and eotaxin (r = 0.47, p = 0.008). Increased levels of IL-6 (H = 39, p = 0.04), IL-13 (H = 44, p = 0.02), and sVEGFR-1 (H = 25.5, p = 0.05) were associated with the presence of DRIL, whereas GM-CSF was protective (H = 131, p = 0.01).

Conclusions: These findings associate higher cytokine expression with worse BCVA, CMT, and MV. Although it is likely that multiple cytokines are associated with the functional and anatomic features of DME, we identified specific cytokines that may drive the process, such as TNF-α, IL-6, IL-7, and IL-9.

Diabetic retinopathy is the leading cause of blindness among working-aged adults with diabetic macular edema (DME), the most common vision-impairing complication. DME is caused by changes in the retinal microvasculature encompassing endothelial cell dysfunction, capillary basement membrane thickening, and a reduced number of pericytes. The resulting vascular permeability and leakage cause fluid accumulation manifested as DME, culminating in retinal dysfunction and vision impairment [1].

There is growing emphasis on the role of inflammation and immune dysregulation in the pathophysiology of DME. Metabolic and oxidative stress from chronic hyperglycemia induces leukostasis and inflammatory breakdown of the blood–retina barrier driven by the coordination of cytokines and growth factors further promoting inflammation [2]. Elevated inflammatory cytokines, such as TNF-α, IL-1β, IL-6 and MCP-1, have been regularly associated with the severity of DME [3-6]. In the DISCERN study [7], we demonstrated changes in the concentrations of multiple cytokines after treatment with intravitreal ranibizumab (IL-1β, IL-7, IL-8, IL-10, IL-12, IL-17, MCP-1, and TNF-α). More recent studies have associated various aqueous cytokines with visual or anatomic responses to treatment with anti-VEGF agents [8-10].

Anti-VEGF-resistant cases of DME classified by worsening or lack of improvement in central macular thickness (CMT) or best-corrected visual acuity (BCVA) can be seen in up to 32%–66% of patients, despite intensive treatment regimes [11-13]. The underlying mechanisms by which these cases differ in their response to anti-VEGF agents is still unclear; however, alternative treatments, such as intravitreal corticosteroids, have proven effective in providing anatomic and functional improvement in such cases [14,15], thereby implicating a significant role of inflammation in DME. Furthermore, intravitreal dexamethasone and triamcinolone have been shown to improve DME by not only reducing VEGF levels but also by targeting the synthesis of proinflammatory mediators involved in DME, such as IL-6, IL-8, MCP-1, ICAM-1, TNF-α, VEGF, and Ang 2 [16-18].

A wide range of phenotypic manifestations of DME are considered imaging biomarkers that may predict treatment response and outcomes. Such OCT features include central macular thickness (CMT), macular volume (MV), subretinal fluid (SRF), morphological patterns, size of intraretinal cysts (IRC), hyper-reflective foci (HRF), disruption of the ellipsoid zone or external limiting membrane, disorganization of retinal inner layers (DRIL), and presence of epiretinal membrane (ERM) [19]. Recent studies have suggested that certain imaging biomarkers may be associated with raised aqueous or vitreous cytokine levels, and thus be contributed by inflammatory components involved in the pathophysiology of DME [20,21]. Analyzing inflammatory cytokines and potential links to imaging biomarkers may improve our understanding of the mechanisms of DME and support individualized therapeutic decision making. The objective of this study was to investigate the association of intraocular cytokines with the imaging features of eyes with DME.

This study was approved by the Human Research and Ethics Committee of the Royal Victorian Eye and Ear Hospital, Melbourne, Australia as part of the DISCERN (Diabetic macular edema: aqueous and Serum Cytokine profiling to determine the Efficacy of RaNibizumab) study and registered with the Australian New Zealand Clinical Trial Registry (ANZCTR identification number 12615000830594). The research adhered to the tenets of the Declaration of Helsinki, and written informed consent was obtained from all participants before enrollment in the study.

This post hoc analysis is based on the cohort of patients enrolled in the DISCERN study. A detailed description of the study design, patient treatment schedule, and aqueous sampling and analysis has been published [7], but in brief, patients with center-involved DME were recruited from the medical retina clinics at the Royal Victorian Eye and Ear Hospital, Melbourne, Australia, between September 2015 and May 2016. All recruited patients had eyes with DME affecting the foveal center and best-corrected visual acuity (BCVA) of 17–72 logarithms of the minimum angle of resolution (LogMAR) letters (Snellen equivalent, 20/400 to 20/40). Although the DISCERN study’s treatment schedule involved treating patients with intravitreal ranibizumab from baseline up to 48 weeks, as per the RESTORE protocol [22], this study only assessed baseline cytokine measurements with baseline OCT characteristics.

This study demonstrated an association of several inflammatory cytokines with quantitative features of DME, such as BCVA, CMT, MV, and SRF, and qualitative features seen on OCT. From the hierarchical cluster analysis, we found that higher concentrations of inflammation-associated cytokines were associated with worse BCVA, greater CMT, and larger MV at baseline. Specifically, we found that higher concentrations of TNF-α, IL-7, and IL-9 were associated with worse BCVA, greater CMT, and a larger MV. Similarly, higher MIP-1α concentrations were associated with greater MV and worse BCVA but not CMT. Although CMT has evolved as the most commonly used OCT measurement of DME in both research and clinical practice, features greatly affecting CMT, such as large IRC, may not accurately reflect changes in MV, generalized macular thickening, disease severity, or BCVA [30,31]. Thus, MV has been suggested as the most appropriate parameter for assessing the anatomic severity of DME. Accordingly, multiple other cytokines, including IL-8, IL-10, IL-12, IL-13, IL-15, and VEGF, were also positively associated with greater MV. Interestingly, increased aqueous VEGF concentrations were only associated with increased MV, with no correlation with BCVA or other OCT features.

Our study also demonstrated the association of several cytokines with the qualitative OCT features of DME at baseline. Smaller but more numerous intraretinal cysts were associated with greater levels of TNF-α and IFN-γ, whereas increased levels of IL-6, IL-13, and sVEGFR-1 were associated with an increased likelihood of DRIL.

Previous reports have suggested a wide range of aqueous cytokines involved in DME, with a few cytokines such as TNF-α, IL-1β, IL-6 and MCP-1 frequently associated with DME or its features [3,4,20]. However, this has not been consistent across previous studies, which demonstrate conflicting and heterogeneous results with various other cytokines often identified [6,7,32,33].

Most groups performed their analyses by examining a certain range of cytokines from ELISA or multiplex-based assays, which were then individually correlated to clinical features or outcomes [3,8-10,17,18,21,34-43]. However, with generally small numbers of subjects in these studies, it may be difficult to ascertain the involvement or contribution of individual cytokines to a wide range of OCT manifestations or treatment responses. This is because cytokines are involved in complex and unclear mechanisms in DME, where both pro- and anti-inflammatory cytokines are involved in multiple networks and cascades, with regulatory feedback mechanisms functioning in autocrine or paracrine loops [44]. Inflammatory cytokines also exhibit multiple and diverse functions, and their activity may fluctuate according to the pathological state of the disease, whether acute, chronic, worsening, or improving [45]. Thus, it would be helpful to first explore the association of DME with an overview of cytokines by clustering those concordantly expressed in higher concentrations.

The HCA analysis enabled this study to demonstrate that high cytokine expression is associated with DME severity. Performing HCA analysis enables a novel method of clustering inflammatory cytokines, BCVA, CMT, and MVT into relatively similar groups called clusters. The correlation of the high cytokine expression group with the main outcome measures of DME demonstrates the importance of the expression of multiple inflammatory cytokines in the pathophysiological process of DME. The analysis of a large range of cytokines has enabled the identification of multiple inflammatory cytokines associated with disease severity, including IL-7 and IL-9, which have not been previously assessed in DME but have been shown to induce the secretion of TNF-α, IL-1β and IL-6 in bone marrow-derived dendritic cells [46].

TNF-α appears to be a critical element in the pathogenesis of DR. We found that increased aqueous concentrations of TNF-α were not only associated with BCVA, CMT, and MV but were also specifically associated with the presence of microcystic edema. This may relate to the presence of a more acute inflammatory phenotype of DME, as opposed to the larger IRC typically seen in the chronic structural morphology of cystoid degeneration in DME [47,48].

TNF-α is a potent inducer of VEGF, which plays a central role in promoting retinal neovascularization and vascular leakage. TNF-α mediated retinal leukostasis and apoptosis may also contribute to DME by the progressive breakdown in the blood–retina barrier and the increase in retinal endothelial cell permeability via disruption of tight junction complexes [49,50]. Treatment with intravitreal anti-VEGF agents has also been shown to reduce intraocular TNF-α levels, suggesting that the mechanisms of VEGF and inflammatory pathways involving TNF-α are intimately tied in DR [7,51]. The correlation of TNF-α to important quantitative outcome measures of DME, including BCVA, CMT, MV, and IRC, supports its prominent and central role in DME. In the clinical setting, measuring intraocular cytokines may reveal a more inflammatory process or phenotype, thus allowing clinicians to adjust their treatment accordingly.

Our study has limitations that should be acknowledged. First, aqueous sampling was used as a measure of posterior segment pathology. Analysis of vitreous fluid may be more accurate in reflecting DME severity; however, sampling is more invasive and is less preferred by our group and other researchers [3,8,20,52]. Cytokine concentrations found below the lower limit of detection were recoded as zero values and included in the statistical analysis. Although there are various methods of handling these values, with none that are particularly agreed upon [53], they may be of some importance when measuring cytokines or biomarkers in the eye, where concentrations may be relatively low. Recoding these values as zero (instead of as missing values) and including them for statistical analysis may underestimate type II statistical errors, or reduce false positives, which we conceived to be more evaluative for our exploratory analyses.

In correlating cytokines with the OCT features of DME, our study had a small sample size, limiting statistical power. Despite this, the results were statistically significant. We are reassured by the results of the HCA that high expression levels of cytokines are influenced by the main clinical parameters of DME, as assessed based on BCVA, CMT, and MV. Furthermore, multivariate regression analysis found overlap of similar cytokines significantly associated with these parameters. Lastly, analyzing a large number of cytokines raises the possibility of statistical errors related to multiple testing. Although we applied the Benjami & Hochberg method to address this, there are no agreed criteria to apply multiple testing correction methods [54], and multiple comparisons in this study need to be interpreted with caution.

In conclusion, we note that correlating individual cytokines with features of DME is difficult, given the complexity of cytokine functions within inflammatory cascades and the considerable disease overlap of imaging features. The HCA in our study demonstrated that in addition to VEGF, there is a significant contribution of inflammation-associated cytokines to the severity of DME. Specifically, TNF-α, IL-7 and IL-9, appear to be associated with OCT features of worse DME severity. Correlating imaging biomarkers of DME with inflammatory cytokines may allow clinicians to easily assess cases on OCT that portray a greater inflammatory phenotype and thus influence individualized therapeutic decision making.