Abstract
Purpose :
Primary Sjögren's syndrome (pSS) is an autoimmune disease of the lacrimal and salivary glands. Recently, multiple clinical studies revealed an overactivated DNA sensing mechanism in pSS patients. Cytosolic double-stranded DNA activates both AIM2 and cyclic GMP–AMP synthase (cGAS). The activation of cGAS produces Cyclic-GMP-AMP (cGAMP), which then, directly interacts with the stimulator of interferon genes (STING) leading to its activation and production of type I-IFN. We reported the activation of the AIM2 inflammasome stimulated by self-genomic DNA (gDNA) in myoepithelial cells (MECs). Herein, we identified the components of the gDNA sensing signaling pathway via STING in MECs.
Methods :
Primary MECs were cultured from lacrimal glands collected from 8-week-old female C57BL6J mice. Self-genomic DNA was extracted from the spleen and introduced to the MECs 18h prior to the analysis. Immunofluorescence microscopy (IFM) and Western Blotting (WB) were used to detect the activation of signaling molecules. ELISA was used to analyze the secretion of type-I IFN. Cell death was determined by propidium iodine. For in vivo experiments, 12-week-old male NOD.B10.H2B mice were used with age/sex-matched Balb/C as the controls.
Results :
Treatment with gDNA translocated STING from the nucleus to the cytoplasm and induced its dimerization. gDNA also stimulated the secretion of IFN-β but not IFN-α. The secretion of IFN-β was completely blocked by a STING-specific inhibitor H151, indicating that IFN-β secretion was dependent on STING activation. Both downstream signaling molecules TBK1 and NFκB were activated by gDNA, and the activation of NFκB was independent of the TBK1 signal. IFN-β further enhanced the secretion of IL-1β stimulated by gDNA and induced cell death. A strong positive fluorescent signal of STING was observed in the lacrimal glands from the NOD.B10.H2B mice, but not in the Balb/C controls. Increased interferon regulator factor (IRF)-9 expression was also observed in NOD.B10.H2B mice, indicating the transcription of interferon-stimulated genes in the lacrimal gland was activated, potentially by IFN-β produced from STING activation.
Conclusions :
We conclude that Self-gDNA is proinflammatory to lacrimal gland MECs by activating STING pathways, leading to the secretion of IFN-β, which promotes the AIM2 inflammasome activity and cell death.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.