Scleral Proteome in Noninfectious Scleritis Unravels Upregulation of Filaggrin-2 and Signs of Neovascularization

Abstract

Purpose: Scleritis is a severe inflammatory ocular disorder with unknown pathogenesis. We investigated healthy sclera as well as sclera affected by noninfectious scleritis for differentially expressed proteins using a mass spectrometry approach.

Methods: We collected scleral samples of enucleated eyes due to severe noninfectious scleritis (n = 3), and control scleral tissues (n = 5), all exenterated eyes for eyelid carcinomas (n = 4), or choroidal melanoma (n = 1) without scleral invasion. Samples were prepared for the nano liquid-chromatography mass spectrometer (LC-MS), data were analyzed using proteomics software (Scaffold), and is available via ProteomeXchange (identifier PXD038727). Samples were also stained for immuno-histopathological evaluation.

Results: Mass spectrometry identified 629 proteins within the healthy and diseased scleral tissues, whereof collagen type XII, VI, and I were the most abundantly expressed protein. Collagen type II-XII was also present. Filaggrin-2, a protein that plays a crucial role in epidermal barrier function, was found upregulated in all scleritis cases. In addition, other epithelial associated proteins were upregulated (such as keratin 33b, 34, and 85, epiplakin, transglutaminase-3, galectin 7, and caspase-14) in scleritis. Further, upregulated proteins involved in regulation of the cytoskeleton (vinculin and myosin 9), and housekeeping proteins were found (elongation factor-2 and cytoplasmic dynein 1) in our study. Upregulation of filaggrin-2 and myosin-9 was confirmed with immunohistochemistry, the latter protein showing co-localization with the endothelial cell marker ETC-related gene (ERG), indicating neovascularization in scleral tissue affected by scleritis.

Conclusions: We found upregulation of filaggrin-2 and signs of neovascularization in scleral tissue of patients with noninfectious scleritis. Further research, ideally including more scleritis cases, is needed to validate our findings.

Figure 1.

Processing of scleral tissue samples for histopathological and mass spectrometry analysis. Fresh enucleated eyes from human donors (3 affected by scleritis, and 5 due to eyelid carcinoma without any scleral involvement) were fixated in neutral buffered-formalin 10%. The formalin fixed paraffin embedded (FFPE) block was cut at 4 µm sections using a microtome, and mounted on slides. Slides were used for immunohistochemistry or for mass spectrometry, scleral tissue was carefully scraped off slides using a scalpel and binocular preparation microscope, without encountering episcleral, conjunctival, or choroidal tissue. Scleral tissue was placed in an Eppendorf tube for enzymatic digestion, where after mass spectrometry was performed.

Figure 2.

Histopathological findings of scleral tissues affected by scleritis (N = 3) versus healthy scleral tissue (N = 1). HE staining shows scleral edema within the collagen fibrils in all tissues affected by scleritis, whereas necrosis was seen only in the third case. CD4 staining for helper-T-cells was positive mainly in cases 1 and 2. CD8 staining for cytotoxic T-cells showed a similar staining pattern (figures not shown). Macrophage staining with CD68 and CD163 (figures not shown), and HLA-DR, a marker for major histocompatibility complex (MHC) class II expression, were positive in all cases. CD20 staining for B-cells showed minimal positivity in cases 1 and 2, and was negative in case 3 (figures not shown). All healthy controls show comparable staining patters (only one is shown), with low amounts of CD4, and CD20 positive T-cells and B-cells. A number of resting macrophages and antigen-presenting cells in healthy scleral tissue is seen with moderate CD68, CD163 (figure not shown), and HLA-DR staining. HE, hematoxylin and eosin. Magnification = 20 times.

Figure 3.

Comparative proteomics of scleral tissue affected by scleritis (N = 3) versus control scleral tissue (N = 5). (A) Venn diagram of the presence of individual proteins in control scleral tissues (N = 5) versus scleral tissues affected by scleritis (N = 3) analyzed by mass-spectrometry. One hundred forty-six (146) proteins were exclusively found in scleral tissues affected by scleritis. (B) Volcano plot of protein differences in scleral tissues affected by scleritis versus control scleral tissues. Only filaggrin-2 was found to be significantly different taking into account the Benjamini-Hochberg method for multiple testing. (C) Heatmap of differentially expressed proteins (N = 30) in sclera affected by scleritis (N = 3) versus control sclera (N = 5).

Figure 4.

Immunohistochemical validation of filaggrin-2 and myosin-9 upregulation in scleritis, and characterization of myosin-9 positive scleral cells using CD163 and ETS-related gene (ERG) transcription factor. (A) Filaggrin-2 (FLG2) staining (brown) shows extracellular matrix coloring in diseased scleral tissue versus no staining in control scleral tissue. (B) Myosin-9 (MYH9) staining (purple) is upregulated in the diseased sclera (vascular wall and stromal cells). (C) Double staining of diseased sclera with myosin-9 (purple) and CD163 (yellow, cytoplasmic stain), a macrophage marker, shows little overlap. Double staining with myosin-9 (purple) and ETC-related gene (ERG) transcription factor, expressed in endothelial cells (yellow, nuclear stain), shows extensive overlap. FLG2, Filaggrin-2; MYH9, Myosin-9. Magnification = 40 times.