Differentially Expressed Gene Pathways in the Conjunctiva of Sjögren Syndrome Keratoconjunctivitis Sicca

Abstract

Sjögren syndrome (SS) is an autoimmune condition that targets the salivary and lacrimal glands, with cardinal clinical signs of dry eye (keratoconjunctivitis sicca, KCS) and dry mouth. The conjunctiva of SS patients is often infiltrated by immune cells that participate in the induction and maintenance of local inflammation. The purpose of this study was to investigate immune-related molecular pathways activated in the conjunctiva of SS patients. Female SS patients (n=7) and controls (n=19) completed a series of oral, ocular surface exams. Symptom severity scores were evaluated using validated questionnaires (OSDI and SANDE). All patients fulfilled the ACR/EULAR criteria for SS and the criteria for KCS. Fluorescein and lissamine green dye staining evaluated tear-break-up time (TBUT), corneal and conjunctival disease, respectively. Impression cytology of the temporal bulbar conjunctiva was performed to collect cells lysed and subjected to gene expression analysis using the NanoString Immunology Panel. 53/594 differentially expressed genes (DEGs) were observed between SS and healthy controls; 49 DEGs were upregulated, and 4 were downregulated (TRAF5, TGFBI, KLRAP1, and CMKLRI). The top 10 DEGs in descending order were BST2, IFITM1, LAMP3, CXCL1, IL19, CFB, LY96, MX1, IL4R, CDKN1A. Twenty pathways had a global significance score greater or equal to 2. Spearman correlations showed that 29/49 upregulated DEGs correlated with either TBUT (inverse) or OSDI or conjunctival staining score (positive correlations). Venn diagrams identified that 26/29 DEGs correlated with TBUT, 5/26 DEGs correlated with OSDI, and 16/26 correlated with conjunctival staining scores. Five upregulated DEGs (CFB, CFI, IL1R1, IL2RG, IL4R) were uniquely negatively correlated with TBUT. These data indicate that the conjunctiva of SS patients exhibits a phenotype of immune activation, although some genes could be inhibitory. Some of the DEGs and pathways overlap with previous DEGs in salivary gland biopsies, but new DEGs were identified, and some of these correlated with symptoms and signs of dry eye. Our results indicate that gene analysis of conjunctiva imprints is a powerful tool to understand the pathogenesis of SS and develop new therapeutic targets.

Keywords: Sjögren syndrome; conjunctiva; dry eye; gene expression; immune pathways.

Figure 1

Differential expression of immune genes in the conjunctiva of SS patients. (A) A volcano plot shows −log10(p-value) and Log 2-fold change in gene expression in SS-KCS patients compared to control subjects (fold increase >1.5 or fold decrease <1.5 and p adjusted value of 0.05). P-value thresholds were adjusted using the Benjamini–Hochberg method of estimating false discovery rates. The horizontal line indicates p=0.05, and the two vertical lines indicate greater or lower than 1.5-fold. (B) Pathway descriptors are included in the panel. Dark bars indicate modulated genes (up or down), and white bars show unaffected genes. SS-KCS, Sjögren Syndrome keratoconjunctivitis sicca. Numbers above the bars indicate the number of unaffected genes (left) followed by the affected genes (right).

Figure 2

A heatmap of expression of immune pathways that were upregulated in SS KCS conjunctiva. Heatmaps show Type I and II Interferon, MHC class I and II antigen presentation, phagocytosis and degradation, and B cell receptor signaling pathways.

Figure 3

Heatmaps of expression of immune pathways with significant genes upregulated or downregulated gene (TRAF5) in SS KCS conjunctiva. Heatmaps show Cytokine signaling, Host-pathogen interaction, Innate immune system, Nod-like-receptor (NLR) signaling, and Complement pathways.

Figure 4

Heatmaps of Adaptive immune system, Lymphocyte activation, NFkB, TLR signaling, Transcriptional regulation, Immunometabolism, Cell adhesion, and apoptosis. The downregulated genes TRAF5, CMKLR1, and TGFBI, are found in the NFkB signaling, Immunometabolism, and Cell adhesion pathways.

Figure 5

A subset of upregulated genes correlates with ocular symptoms and signs. Spearman correlations of clinical symptoms (OSDI), Tear-break-up time (TBUT), and conjunctival (CJ) staining score with up or downregulated DEGs were calculated using GraphPad Prism software using clinical data from all subjects (19 healthy controls and 7 SS KCS subjects). (A) Representative graph showing Spearman correlations of CD74 and LAMP3 vs. OSDI, TBUT, corneal staining score, and conjunctival staining score. Additional genes are shown in Table 3 . (B) Venn diagram showing common and unique correlation of genes and OSDI, TBUT, and conjunctival staining score. R = coefficient of correlation. Please note that GraphPad Prism does not plot all dots if they overlap.