Identification of intraocular inflammatory mediators in patients with endophthalmitis

Abstract

Purpose: Endophthalmitis is mediated by inflammatory cytokines. We employed a quantitative antibody array, which profiles protein expression and function in a high-throughput manner, to identify inflammatory mediators in the infectious aqueous and vitreous humor from patients with endophthalmitis.

Methods: In this prospective study, aqueous humor (AH) and vitreous humor (VH) samples were obtained from 30 patients with endophthalmitis and were collected during anterior chamber paracentesis and vitrectomy. Control samples were obtained from 32 healthy donors. We examined the expression of 20 inflammatory mediators in AH and VH using a quantitative antibody protein array. Hierarchical cluster analysis based on the expression of the quantified cytokines was applied to identify the specificity of endophthalmitis disease. Validation analysis using enzyme-linked immunosorbent assay (ELISA) was performed to confirm the expression of the cytokines identified in the AH and VH samples.

Results: Our results demonstrated elevated expression of interleukin (IL)-1β, IL-6, and macrophage inflammatory protein (MIP)-3α in AH or VH from patients with endophthalmitis. The concentration of IL-17 was upregulated in AH from the patients. The expression of IL-2, IL-5, IL-21, and transforming growth factor (TGF)-β1 was downregulated in AH from the patients. The cluster analysis demonstrated that the cytokine profile expression in AH or VH significantly differed between the patients with endophthalmitis and the healthy controls. Confirmation with ELISA validated the increase in IL-1β, IL-6, and MIP-3α in the AH and VH samples from the patients with endophthalmitis.

Conclusions: Increased expression of IL-1β, IL-6, IL-17, and MIP-3α and decreased expression of IL-2, IL-5, IL-21, and TGF-β in the AH and VH suggests an abnormal cytokine profile in patients with endophthalmitis. Knowledge of this will aid in the diagnosis of infectious endophthalmitis.

Figure 1

Anterior segment ocular manifestations and Gram staining of patients with endophthalmitis. A: Identification of bacteria strain by Gram staining. Gram-positive coccus indicated by arrow. Magnification, ×100. B: Anterior chamber inflammation and hypopyon in patients with infectious endophthalmitis following post-traumatic globe injury.

Figure 2

The cytokine profile in the aqueous humor from patients with endophthalmitis (n=15) and controls (n=16). Abbreviations: IFN-γ represents interferon gammar; IL represents interleukin; MIP represents Macrophage inflammatory protein; GM-CSF represents granulocyte-macrophage colony-stimulating factor; TGF represents transforming growth factor; TNF represents tumor necrosis factor. A: The levels of GM-CSF, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-28, IFN-γ, MIP-3α, TNF-α, TGF-β1, and TNF-β were detected using an antibody cytokine array. Data are represented as mean±SD *p<0.05. B: Heatmap of cluster analysis. Cluster analysis was performed with data from 20 cytokines and represented in a dendrogram. The horizontal bar on the left of the heatmap indicates cytokine expression. Higher expression is displayed in red, and lower expression is displayed in blue. Upregulation or downregulation of cytokines is indicated on a scale from red to green, respectively. The vertical lines indicate patients and controls. C: A heatmap of the cluster analysis was performed with eight of the chosen 20 cytokines. The differentially expressed cytokines were clearly associated with endophthalmitis with bacterial infection. D: Representative fluorescent mean intensities images for antibody array of AH. Additional cytokines in patients with endophthalmitis is marked in red.

Figure 3

The cytokine profile in the VH from patients with endophthalmitis (n=15) and controls (n=16). A: The expression of 20 cytokines was detected using antibody cytokine array. The interleukin (IL)-1β, IL-6, and macrophage inflammatory protein (MIP)-3α levels were significantly upregulated in the patients with endophthalmitis compared to the healthy control samples. Data are represented as mean±SD *p<0.05. B: Heatmap of cluster analysis. Cluster analysis was performed with the 20 chosen cytokines. The horizontal bar on the left of the heatmap indicates the cytokine expression. C: Heatmap of cluster analysis of three of the 20 cytokines. The endophthalmitis group and the control group completely segregated into two principle clusters. D: Representative fluorescent signal images for the antibody array in the vitreous humor (VH). The additional cytokines are marked in red.

Figure 4

Validation analysis with ELISA of the AH and VH from patients with endophthalmitis (n=15) and healthy controls (n=16). A: The expression of interleukin (IL)-1β, IL-6, and macrophage inflammatory protein (MIP)-3α was significantly upregulated in the aqueous humor (AH) from patients with endophthalmitis. B: The production of IL-1β, IL-6, and MIP-3α was also elevated in vitreous humor (VH) from the patients compared to the health control samples. Data are represented as mean±SD *p<0.05, **p<0.001.