M. L. Joachims, B. Khatri, K. L. Tessneer, A. M. Stolarczyk, G. B. Wiley, A. Rasmussen, J. Guthridge, J. A. James, R. H. Scofield, K. L. Sivils, I. Adrianto, C. Lessard
2020 Annals of the Rheumatic Diseases  
Background:Sjögren's syndrome (SS) is a chronic, heterogenous autoimmune disease characterized by inflammatory destruction of the exocrine glands. Long non-coding RNAs (lncRNAs) have emerged as a functionally diverse class of non-protein coding RNA (ncRNA) with increasing implications in interferon signaling, immune cell regulation, and autoimmune disease pathology. The potential role of lncRNAs in SS pathogenesis is unknown.Objectives:To identify and characterize candidate lncRNAs with
more » ... l relevance to SS pathology.Methods:RNA-seq was used on whole blood from SS patients (n=30 antibody negative (Ro-); n=27 antibody positive (Ro+)) and healthy controls (HC, n=27) to identify differentially expressed (DE) lncRNAs (log2 fold change (FC) ≥ 2 or ≤ 0.5; padj<0.05). Bioinformatic and pathway analyses were used to predict lncRNA function.In vitrotime course experiments in HSB2 T cell lymphoblasts stimulated with PMA/Ionomycin (PMA/I) or type I interferon (IFN) were used to assess biological relevance.LINC01871function was further investigated by RNA-seq on a single cell clone of HSB2 with confirmed CRISPR-targetedLINC01871deletion (LINC01871-/-).Results:We identified a total of 1054 unique DE ncRNAs between Ro+, Ro-and/or a combined analysis relative to HC; of these, 45 (1 long intergenic ncRNA (lincRNA), 1 antisense, 43 pseudogenes) were overexpressed in all 3 SS subsets. To begin investigating the function of the previously undescribed lincRNA,LINC01871(SSRo-: FC=2.85; padj=1.1x10-4), we performed a correlation analysis of the SSRo-transcriptome, which found several co-expressed protein coding RNAs involved in immune regulation (THEMIS,TBX21,IL10RA,IL2RB,among many others). Similarly, Ingenuity Pathway Analysis of the SS transcriptome compared to HC, as well as several gene ontology enrichment analyses of publicly available RNA expression correlation databases, identified shared immune-related pathways including cytotoxic T cell, natural killer cell, and T cell regulation. To further study the role ofLINC01871in cytotoxic T cells, we used qRT-PCR to resolve the effects of PMA/I or type I IFN stimulation onLINC01871expression in the T lymphoblastoid HSB2 cells.LINC01871expression was downregulated after PMA/I stimulation, but unchanged with type I IFN stimulation. To explore the regulatory function ofLINC01871in T cells, we targetedLINC01871in HSB2 cells using CRISPR. To this end, we generated a single cellLINC01871-/-clone with no RNA expression by qPCR and confirmed homozygous deletion using DNA sequencing. RNA-seq analysis ofLINC01871-/-compared to unmodified HSB2 cells identified 1166 DE transcripts. Pathway analyses clustered the DE transcripts into similar immune regulatory, cytotoxic and T cell pathways identified in SSRo-whole blood RNA-seq and publicly available RNA-seq databases. Further, several prominent T cell regulatory transcripts that exhibited correlated upregulation withLINC01871in SSRo-whole blood RNA-seq also demonstrated downregulation afterLINC01871deletion:CD109(FC=-9.7; padj=5.3x10-16),IL22(FC=-8.1; padj=7.6x10-11),PDCD1(FC=-6.2; padj=1.1x10-6),THEMIS(FC=-3.8; padj=2.7x10-165) andTBX21(FC=-2.1; padj=3.3x10-25).Conclusion:LncRNAs are emerging as important regulators of immune function with increasing evidence of autoimmune disease relevance. Here, we leveraged RNA-seq, extensive bioinformatic data, and CRISPR technology to identify and functionally characterizeLINC01871as a potential mediator of the dysregulated T cell inflammatory response pathways implicated in SS pathogenesis.Disclosure of Interests:Michelle L Joachims: None declared, Bhuwan Khatri: None declared, Kandice L Tessneer: None declared, Anna M Stolarczyk: None declared, Graham B Wiley: None declared, Astrid Rasmussen Speakers bureau: Novartis, ThermoFischer, Joel Guthridge Grant/research support from: Xencor, Bristol Myers Squibb, DXterity, Judith A. James Grant/research support from: Progentec Diagnostics, Inc, Consultant of: Abbvie, Novartis, Jannsen, R Hal Scofield Grant/research support from: Pfizer, Kathy L Sivils: None declared, Indra Adrianto: None declared, Christopher Lessard: None declared
doi:10.1136/annrheumdis-2020-eular.3950 fatcat:sjgbersfkbc47hmajb3i74ovlq