Background/Purpose: Chronic anterior uveitis (CAU) is a potentially sight-limiting complication of pediatric rheumatic diseases, including juvenile idiopathic arthritis. However, the underlying inflammatory biology of CAU in the eye is poorly understood, and there is a critical need for improved biomarkers for CAU development, progression, and damage. Here, we developed methods for RNA collection and transcriptional profiling from tear fluid of children with CAU.

Methods: Tear fluid was collected using Schirmer strips. CAU was graded as active (anterior chamber [AC] cell ≥0.5+) or inactive (AC=0) by Standardization of Uveitis Nomenclature (SUN) criteria. RNA was extracted from fresh or frozen absorbed Schirmer strips using three methods. MagMAX-96 Total RNA Isolation Kit was used after incubating with either 140uL of Lysis/Binding, or with 500uL Phosphate Buffered Saline (PBS). Alternatively, 500uL of TRIzol Reagent (ThermoFisher) was added to each tear strip, followed by RNA extraction. Samples with RNA Integrity Number (RIN) > 4 were selected for transcriptional profiling using the Ampliseq Transcriptome Panel on the Ion Torrent sequencing system. Sequencing data was processed and aligned using Ion Torrent, and differential gene expression for genes with mean reads per million > 5 was determined using AltAnalyze (p < 0.05, fold change >2). Pathway analysis was performed using GO-Elite.

Results: For both RNA extraction methods using the MagMAX-96 Total RNA Isolation kit, the concentration of RNA was low and of poor quality. In contrast, for 32 samples that were extracted via TRIzol, RNA concentrations ranged from 6.9ng/uL – 69.8ng/uL with RIN numbers 1.0 – 7.8. We then performed Ampliseq Transcriptome profiling on 6 tear RNA samples from 4 patients with active CAU and 7 samples from 5 patients with inactive CAU (Table 1). One patient with active CAU was excluded after sequencing for low read quality. The remaining samples were sequenced to a mean depth of 6.4 million reads (range 4.0-9.9 million) and we identified 11,595 genes with mean expression >5 RPM. Comparing active to inactive CAU, we found 113 significantly upregulated and 35 significantly downregulated genes (Figure 1). Among the most enriched gene pathways of upregulated genes in active CAU were laminin complex (z-score 13.3, adjusted p=0.026), extracellular matrix (z-score 7.6, adjusted p=0.003), and regulation of cell migration (z-score 5.1, adjusted p=0.09). The most significantly enriched gene pathway of downregulated genes in active CAU was immune system processes (z-score 6.2, adjusted p=0.0001), including IL7R, IRF5, NAIP, and NCF1. Among the marker genes for patients with inactive CAU was MAPILC3A, which encodes LCA3, expressed in the retinal pigment epithelium, which has a key role in blood-retinal barrier and retinal immune regulation.

Conclusion: This pilot work demonstrates our ability to obtain high-quality RNA suitable for transcriptomics from tear fluid of children with CAU. We also find that active CAU is associated with transcriptional activation in pathways associated with eye injury and blood-retinal barrier, including laminin complex, extracellular matrix, and cellular migration, as well as downregulation of inflammatory response genes.

Figure 1: Transcriptomic profiling of tear RNA from chronic anterior uveitis. Top: principal component analysis of differentially expressed genes. Red= active uveitis, blue= inactive uveitis. Bottom: hierarchical clustering of differentially expressed genes between active and inactive uveitis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/transcriptiomics-of-tear-rna-from-children-with-active-and-inactive-chronic-anterior-uveitis/