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Multiplex Bead Immunoassay Analysis of Aqueous Humor Reveals Distinct Cytokine Profiles In Uveitis

¹From the Institute of Biomedical Research, MRC Centre for Immune Regulation, Division of Immunity and Infection, Medical School, The University of Birmingham, Birmingham, United Kingdom; ²Academic Unit of Ophthalmology, Division of Immunity and Infection, University of Birmingham, Birmingham and Midland Eye Centre, City Hospital NHS Trust, Birmingham, United Kingdom; and ³School of Biosciences, The University of Birmingham, Birmingham, United Kingdom.

PURPOSE. To extensively characterize the complex network of cytokines present in uveitis aqueous humor (AqH), and the relationships between cytokines and the cellular infiltrate.

METHODS. AqH from noninflammatory control subjects and patients with idiopathic, Fuchs’ heterochromic cyclitis (FHC), and herpes-viral or Behçet’s uveitis were analyzed for IL-1ß, -2, -4, -5, -7, -8, -10, -12, -13, -15, TNF, IFN, CCL2 (MCP-1), CCL5 (RANTES), CCL11 (Eotaxin), TGFß2, and CXCL12 (SDF-1), using multiplex bead immunoassays. The cellular infiltrate was also determined for each sample.

RESULTS. Idiopathic uveitis AqH, compared with noninflammatory controls, was characterized by high levels of IL-6, IL-8, CCL2 and IFN, the levels of which correlated with each other. For IL-6 and IL-8 these levels were proportional to the number of neutrophils present. By contrast, the levels of both TGFß2 and CXCL12 decreased in idiopathic uveitis AqH with increasing inflammation. Cluster analysis showed a degree of segregation between noninflammatory and idiopathic uveitis AqH. Further examination using random forest analysis yielded a complete distinction between these two groups. The minimum cytokines required for this classification were IL-6, IL-8, CCL2, IL-13, TNF, and IL-2.

CONCLUSIONS. Application of multiplex bead immunoassays has allowed us to identify distinct patterns of cytokines that relate to both clinical disease and the cellular infiltrates present. Bioinformatics analysis allowed identification of cytokines that differentiate idiopathic uveitis from noninflammatory control AqH and are likely to be important for the pathogenesis of uveitis.

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