Self‐DNA induced inflammation impairs the normal function of myoepithelial cells of the lacrimal gland

Abstract

Sjögren's Syndrome (SS) is an autoimmune disease that mainly affects the lacrimal glands and salivary glands. Patients with SS suffer from severe dry eye which leads to corneal ulceration. The pathogenic nuclear acid triggered autoimmune response is playing a key role in autoimmune disease like SS. Previously our group observed a significantly higher expression of a DNA sensing mechanism Absent in Melanoma (AIM)2 inflammasome in two mice models of primary SS compared to the wildtype. AIM2 is a member of innate immune sensors that detects the presence of DNA in the cytoplasm, and then forms the AIM2 inflammasome to activate caspase‐1, which cleaves immature pro‐IL‐1ß to mature IL‐1ß that contribute to inflammation. In this study we investigate the effect of the AIM2 inflammasome DNA sensing pathway on primary myoepithelial cell (MEC) function of the lacrimal gland.Primary MECs culture was acquired from female wildtype C57Bl/6 mice aged 4‐6 weeks. Genomic DNA (gDNA) extracted from MECs was used to resemble the self‐DNA. The MECs were treated with gDNA for 6 hours, with random double‐stranded DNA poly A:T as a positive control. Immunofluorescent (IF) staining result shows the accumulation of AIM2 signals and a speck of Apoptosis‐associated speck‐like protein containing a CARD (ASC), indicating the assembly of AIM2 inflammasome in both gDNA and poly AT treated group (Fig 1). The activation of caspase‐1 by gDNA treatment was confirmed using FAM‐FLICA assay.We next determined the primary function of MECs which is contraction, under the effect of DNA‐induced cell response. A time lapse microscope was used to record a 2‐hour video of the MECs upon ATP (10‐5M) stimulation, as ATP is an important neurotransmitter released by the parasympathetic nerve end which is responsible for tear production. The first and last images will be used as a quantification study, and the reduction (%) of the area after the 2hrs period is calculated to represent the contractility. The area reduction in the gDNA as well as the positive control poly A:T treated group was significantly decreased from the non‐treated (NT) group. We next investigated the mechanism of the reduced contractility caused by DNA. One of the most important signals for muscle cell contraction is the Ca2+. We measured the change of intracellular Ca2+ concentration ([Ca2+]i) in MECs upon ATP stimulation but observed no difference between gDNA and NT group. We then determined the actin and cytoskeleton of the MECs upon DNA‐induced inflammation. The structure of ß‐tubulin which supports the cell structure, and F‐actin which enables the mobility, were investigated by IF. The result shows that the poly A:T treated group presents with higher ß‐tubulin but lower F‐actin signals compared to the control.To conclude, self‐DNA induces inflammasome formation and caspase‐1 activation in MECs, which results in the impairment of contraction by altering the cytoskeletal proteins.