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.

Gut-derived butyrate suppresses ocular surface inflammation

“…Finally, we show that in the conjunctiva of mice subjected to desiccating stress, there is upregulation of genes involved in Type I IFN signaling, and tributyrin treatment reduces expression of these genes. This result agrees with our previously published NanoString results comparing gene expression in human conjunctival cells from Sjögren Syndrome (SS) patients with dry eye and healthy control subjects, where we observed upregulation of Type 1 IFN signaling in SS 35 . SS is an autoimmune inflammatory disorder characterized by secretory dysfunction in the eye and mouth; in the eye, this results in tear film instability, reduced tear production, and corneal barrier disruption.…”

“…nCounts of mRNA transcripts were normalized using the geometric means of 6 reference genes ( Cltc , Gapdh , Gusb , Hprt , Pgk1 , Tubb5 ). Data was analyzed by ROSALIND® ( https://rosalind.onramp.bio/ ), with a HyperScale architecture developed by ROSALIND, Inc. (San Diego, CA) as described previously 35 . Heatmaps were constructed using GraphPad Prism 9.0 software (GraphPad Software Inc., San Diego, CA, USA).…”

Autoimmune Epithelitis and Chronic Inflammation in Sjögren’s Syndrome-Related Dry Eye Disease

“…Genetic predisposition and/or hormonal factors influence the integrity of glandular epithelia [22]. Recent studies showed that innate and adaptive immune-related genes are upregulated in SS conjunctiva [23] and systemic SS [24]. Hormone imbalance, especially androgen deficiency has been reported to be linked to meibomian gland dysfunction [25] and lacrimal gland dysfunction in SS [25,26].…”

Ocular Surface Inflammatory Disorders (OSID): A Collective of Systemic Etiologies Which Cause or Amplify Dry Eye Syndrome

“…The OSID collective pathological sign is cellular inflammation that chronically infiltrates the subconjunctival ocular surface tissues, MGs, and LG, further perpetuating the autoinflammatory insult to the ocular surface, causing loss of homeostasis that results in signs and symptoms of DED ( Figure 1 ). The findings include: (1) Persistent conjunctival leukocyte infiltration, leading to conjunctival vasodilation and trasudation ( 3 ); (2) Cellular infiltration around meibomian glands with release of myeloperoxidase ( 4 ), leading to meibomian gland disorders ( 5 ); (3) Cellular infiltration into the LG acini, which leads to gland atrophy and decreased tear production ( 6 ); (4) Infiltration into the conjunctiva subepithelial layer leading to direct inflammatory injury of the conjunctival epithelium ( 7 ); (5) Low-grade inflammatory infiltration into the cornea leading to corneal nerve damage and/or injury to the corneal epithelium ( 8 – 10 ). Nonetheless, the confirmatory diagnosis of these disorders includes a conjunctival biopsy which is seldomly performed, since these illnesses are diagnosed clinically.…”

“…Can the persistent conjunctival inflammation play a role in modifying gene expression of epithelial cells? A recent study comparing the conjunctival gene expression of SS patients with controls resulted in the discovery of 53 differentially expressed genes between both groups, indicating that SS patients, and potentially the OSIDs, exhibit a phenotype of immune activation which contributes to dysregulation of the conjunctiva and ocular surface ( 7 ). Interestingly, SS patients have also been reported to exhibit greater number of lymphocytes in the tarsal conjunctiva than controls ( 49 ), which supports that OSIDs have an impact not only the classically defined targets (as the LG in in SS), but likewise in the conjunctival epithelium ( 48 ) and MG ( 49 ) ( Figure 1 ).…”