Background/Purpose: The type I IFNs (IFN-I), are strongly associated with Systemic Lupus Erythematosus (SLE).  It is generally considered that IFN-I is induced by immune complexes (IC) containing (ribo)nucleoprotein antigens. However, this conclusion is based on in vitro studies and doesn’t address how IFN-I may be induced prior to the formation of IC. Cytosolic DNA induces IFN-I and other cytokines which are important for antimicrobial defense but can also trigger autoimmunity. Cytoplasmic DNA frequently transduces signals via the adaptor protein, STING, and the transcription factor IRF3, however how cytosolic DNA is sensed in eukaryotic cells remains under intense investigation. Recently it was shown that a newly discovered enzyme called cyclic-di-GMP–AMP (cGAMP) synthase (cGAS) detects cytosolic DNA, synthesizes cGAMP which then acts as a second messenger to trigger a signaling pathway through STING and IRF3, resulting in production of IFN-β in mammalian cells. Our research aim is to determine whether the cGAS pathway could contribute to IFN I production in SLE.

Methods: cGAS and Interferon Stimulated Genes (ISGs) mRNA expression was quantified by quantitative PCR (qPCR). IRF3 phosphorylation was determined by anti-phospho-IRF3 antibody and western blot. cGAMP levels were monitored by Selective Reaction Monitoring (SRM) with High Performance Liquid Chromatography-tandem Mass Spectrometry (HPLC-MS/MS).

Results: When compared to normal controls (n=20), SLE patients (n=51) had a significant increase of the expression of cGAS (P=0.0045) in peripheral blood mononuclear cells (PBMC). cGAS expression positively correlated with ISGs expression and IFN score in SLE patients consistent with it being an IFN signature gene. We further quantified IRF3 activation by phosphorylation in PBMC and found that IRF3 was activated in a higher proportion of SLE patients compared to controls. To quantify c-GAMP in patient PBMC samples we used a reference cyclic GMP-AMP dinucleotide that is comprised of the unusual 2’-5’ and 3’-5’ phosphodiester linkage and demonstrated the ability to distinguish the cyclic dinucleotide from other species by HPLC-MS/MS. Of 23 SLE patients tested, we could detect cGAMP in approximately one fourth of SLE patients but not in any of the normal controls and RA patients.

Conclusion:

Our findings implicate the activation of cGAS pathway in at least some patients with SLE. While increased cGAS gene expression may be induced in response to IFN-I, DNA binding to cGAS is required for synthesis of cGAMP. Defining the molecular mechanisms responsible for activation of the cGAS enzyme, will contribute to our understanding of IFN-I stimulation in SLE patients. Furthermore, since IFN-β primes other cells such as pDC for heightened expression of IFN-α, targeted approaches to reduce DNA/cGAS interaction may be a useful therapeutic strategy in SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-second-messenger-cyclic-gmp-amp-dinucleotide-cgamp-and-the-enzyme-cyclic-gmp-amp-synthase-cgas-are-expressed-in-systemic-lupus-erythematosus/