The current pilot study identifies ipsilateral changes in the tear fluid proteome of patients with unilateral AAU. Accordingly, Schirmer’s test sampling of tear fluid potentially represents an adjuvant non-invasive approach to investigate the pathophysiological pathways in AAU.
For all patients, IPA identified the LXR/RXR and FXR/RXR pathways among the top canonical pathways affected. LXRs have predominantly been linked to regulation of lipid metabolism, but there is also mounting evidence of an anti-inflammatory role . LXRs modulate the immune response in lipopolysaccharide-activated macrophages by inhibiting expression of inflammatory mediators, including IL6 . LXR activation has also been associated with inhibition of the nuclear transcription factor-κB (NF-κB) , which is a major transcription factor in the regulation of immune responses. In rats, inhibition of NF-κB ameliorated anterior uveitis in experimental autoimmune uveitis (EAU) . Furthermore, in a mouse model of EAU, a synthetic LXR agonist reduced ocular inflammation . The authors of the latter study suggested that LXR agonists had potential for becoming a therapeutic option for uveitis patients. LXRs were expressed in the native rat corneal epithelium and stroma in a rat model of inflammatory corneal angiogenesis . Following induction of inflammatory corneal angiogenesis, LXR expression temporarily decreased in the corneal epithelium, while removal of the inflammatory stimulus (corneal suture) was associated with activation of the LXR/RXR pathway and resolution of inflammation. Consequently, the authors suggested that LXR/RXR pathway activation could contribute to resolution of corneal inflammation. To our knowledge, the FXR/RXR pathway has not been directly linked to the pathogenesis of uveitis. In a study on endotoxin-induced uveitis in rats, however, the FXR antagonist guggulsterone suppressed ocular inflammation . The authors related this effect to the inhibition of NF-κB by guggulsterone. Ultimately, engagement of the LXR/RXR and FXR/RXR pathways in the current study indicates that signs of AAU-associated inflammation are detectable in ipsilateral tear fluid samples.
In the current study, IPA determined that the changes to the tear proteome in AAU were associated with an inflammatory process (i.e., inflammatory response and immune cell trafficking were among the top diseases and bio functions). The upstream analysis by IPA further underscored the inflammatory tear profile by identifying TNF and IL6 among the top upstream regulators in four of the five patients. The role of TNF in the pathogenesis of uveitis has been well-established in animal studies [16, 17], and the concentration of TNF was also raised in the aqueous humor of patients with uveitis . Most importantly, the essential role of TNF in uveitis has been confirmed by the recent success of employing the TNF-inhibitor adalimumab in the treatment of non-infectious uveitis [19, 20]. IL6 promotes the differentiation of CD4+ T-helper (Th) cells into Th17 cells, thereby contributing to the pathogenesis of autoimmune diseases . In uveitis, IL6 has been proposed to be especially important in HLA-B27 positive cases . The latter study also reported a higher concentration of IL6 in the aqueous humor of HLA-B27 positive uveitis patients compared to patients with Vogt-Koyanagi-Harada disease, sarcoidosis, or idiopathic granulomatous uveitis. This is in line with our study, in which IPA identified IL6 as an upstream regulator in the two HLA-B27 positive patients.
We found a significantly higher level of APOBEC3A, a DNA cytidine deaminase, in tear fluid of the diseased eye relative to the healthy control. Interferon, a regulating cytokine in autoimmune uveitis , can induce APOBEC3A in monocytes. This enables deamination of foreign double-stranded DNA and inhibits replication of retroviruses and retrotransposons . In this way, APOBEC3A partakes in the innate immune system, and its increase in tear fluid from AAU eyes indicates local disease activity.
The level of TGM2 was reduced in tear fluid from the diseased eye. This is contradictory to a study on endotoxin-induced uveitis, in which the level of TGM2 was increased in aqueous humor . In agreement with our results, however, studies on Crohn’s disease patients have reported an inverse relationship between TGM2 and disease activity .
In total, 242 unique proteins were identified in the tear fluid samples. Only 32 proteins, however, were identified in all patients. The highest number of matching tear fluid proteins was seen in the three youngest patients that also had the shortest disease duration. On the other hand, HLA-B27 status was not obviously related to the number of matching tear fluid proteins. Nevertheless, as the anterior chamber cytokine profile differs between HLA-B27 positive and negative patients, the heterogeneity in HLA-B27 status might have contributed to the relative lack of matching proteins between patients in the current study.
In our study, tear fluid proteins were identified using a single Schirmer’s test strip in each patient. Previous studies have reported a higher number of identified proteins when pooling tear fluid samples from multiple patients . Although proteomics is useful as a screening method for AAU biomarkers, the method has limited sensitivity for low-concentration proteins. Alternative methods, including enzyme-linked immunosorbent assay, are more suitable for detecting proteins at low concentrations. Accordingly, restricted sensitivity may have contributed to the rather poor correspondence of tear fluid proteins between the patients in the current study. Conclusively, we believe that failure to identify certain proteins across all patients in the present study should not lead to the exclusion of these as potential biomarkers.
Serpent family A member 3 (SERPINA3), also known as serine protease inhibitor 3 (SPI-3), was more than 14-fold elevated in tear fluid from the diseased eye in one patient, despite disease duration of only a single day. In addition, this protein was mildly elevated in two other patients. SERPINA3, also referred to as SPI-3, is normally found in the aqueous humor , but it is also part of the acute phase response. In endotoxin-induced uveitis in rats following lipopolysaccharide injection, SERPINA3 mRNA in epithelial cells of the iris and ciliary body was rapidly upregulated as part of the initial inflammatory response . Yet another potential biomarker protein was increased in tear fluid from the diseased eye in three patients: orosomucoid 2 (ORM2), also known as alpha-1-acid glycoprotein. Similar to SERPINA3, ORM2 is an acute phase response protein. In a previous study, serum levels of ORM2 were increased in moderate and severe idiopathic AAU . Furthermore, in the latter study, ORM2 levels correlated with disease severity and returned to normal following resolution.
In conclusion, unilateral AAU induces inflammation-associated changes in the ipsilateral tear fluid proteome; in a clinical setting, Schirmer’s strips can be used to collect tear samples for proteomics. The inflammation-associated LXR/RXR was among the top canonical pathways affected. Further studies are necessary to determine whether APOBEC3A, SERPINA3, and ORM2 are potential AAU biomarkers.