Abstract
Purpose :
In dry eye, a variety of recirculating T effector memory cells are increased in the conjuctival. Although primary Sjogren syndrome (pSS) and Rheumatoid arthritis (RA) are both autoimmune diseases associated with exocrine gland destruction and dry eye, it is unclear if there are differences in the conjunctival immune infiltration in these conditions. To address this question, a global transcriptomics study was performed.
Methods :
Patients with pSS or RA were recruited from dry eye clinic at Singapore National Eye Center. There were no significant differences between groups in age, Schirmer test, tear break up times or conjunctival redness. CD45+ cells from conjunctival impression samples were sorted. Total RNA was extracted, converted to cDNA and amplified. Libraries were prepared for next generation sequencing. Multiplexed paired end sequencing was run on the Ilumina Hiseq High output system. Alignment was done using HISAT2 followed by HTSeq. R package EdgeR was used for differential expression analysis. Genewise empirical Bayes quasi-likelihood F-test was used to find out which genes are differentially expressed followed by a FDR correction. Pathway analysis was performed using String Interaction Network online analysis. Cibersort was used to determine the relative composition of immune cells using LM22 library as reference.
Results :
Transcripts of 57708 genes were detected in all samples and out of which, 18479 pseudo genes, ribosomal related genes were detected and removed. Ten of the genes were dysregulated at p<0.001, 126 were dysregulated at p<0.01 and 516 genes were dysregulated at p<0.05. Only TLL2 and CCL2 were expressed higher in RA, whereas all the others were higher in pSS. Transcripts COBLL1, POTEG, CCDC181 and COL11A1 were detected only in pSS but not in RA patients. Three clusters of genes were relatively upregulated in pSS compared to RA. One cluster consists of TNF, TNFSF13B, CD47, TRADD, MyD88, CCL3, CD2, IL10RA, FOXP1, IL7R, CERK, FCGR2A and AQP5. Cibersort analysis shows that of the cells analyzed, CD8 T cells, CD4 resting memory cells and activated NK cells together make up more than half of the total cells in each sample.
Conclusions :
There are differences in conjunctival immune cell gene expression between patients with pSS and RA, despite similar clinical severities of dry eye and treatment. These findings point to differences in the auto-antigens and etiologies of dry eye in these conditions.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.