Laser microdissection coupled with RNA-seq reveal cell-type and disease-specific markers in the salivary gland of Sjögren's syndrome patients.

OBJECTIVES:Little is known about the molecular details regarding the contribution of different cell types of the salivary gland to the altered gene expression profile seen in Sjögren's syndrome (SS). METHODS:Using laser microdissection, tissue samples enriched in acini, ducts and inflammatory foci in subjects with and without SS were isolated for RNA-seq analysis. Gene expression profiles were analysed and selected enriched genes were further examined using real time PCR and by immunofluorescence. RESULTS:RNA-seq analysis of salivary biopsies from subjects with and without SS revealed marked differences in gene expression occurring in the ductal and infiltrating cells compared to acinar cells. Up-regulated genes in the SS ductal cells included C4A complement and the SLC26A9 ion channel. The inflammatory infiltrate showed the most dramatic differences in gene expression and contained up-regulated genes associated with T-cells, natural killer, dendritic and basophils/mast cells. qPCR with total salivary gland mRNA confirmed the differential mRNA expression of several genes (MMP9, FOL1HB, CCL21, CCR7), thereby validating the approach. Additional immunofluorescence studies demonstrated high expression and co-localisation of CCL21 chemokine and CCR7 chemokine receptor within the SS infiltrates. CONCLUSIONS:Major gene expression changes in the salivary gland of SS were detected in the ductal and inflammatory cells and not in the acinar cells. Two chemokines involved in immune cell trafficking to secondary lymphoid tissue, CCR7 and CCL21, showed markedly increased expression and may contribute to the recruitment of diverse immune cells to the salivary glands, causing inflammation and loss of secretory function.