University of Washington Seattle, WA, United States
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Jie An1, Stephen Wilson2, Jill Henault3 and Keith Elkon1, 1University of Washington, Seattle, WA, 2Bristol Myers Squibb, Cambridge, MA, 3Bristol Myers Squibb, Acton, MA
Background/Purpose: The majority of patients with SLE show a striking Type I Interferon (IFN-I) signature in their peripheral blood. Although this signature can be generated by immune complex activation of Toll Like Receptors (TLRs) in vitro, the in vivo mechanisms are not known. Using ex vivo derived peripheral blood mononuclear cell (PBMC) samples from patients, we previously showed that another IFN-I pathway, called cGAS-STING, is activated in ~15% of SLE patients. In the present study, our aims were i) to determine the frequency of cGAS-STING activation in additional SLE cohorts as well as patients with Sjogren's syndrome; ii) to perform longitudinal studies of cGAS-STING positive (cGAMP+) SLE patients; and iii) to evaluate whether activation of downstream pathways such as phospho-STING (P-STING) could be detected.
Methods: The following groups were studied: Healthy Controls (HC) (n=25), SLE (n=52), primary Sjögren's syndrome (SS) (n=20), and secondary SS (SLE/SS) (n=8). SLE disease activity was determined by the SLEDAI. PBMCs were purified from blood by Ficoll density gradient centrifugation and the cells were lysed with sonication. cGAMP concentrations were quantified with 2'-3' cGAMP ELISA kit (Cayman Chemical). Phosphorylated STING and total STING protein were measured with Homogeneous Time Resolved Fluorescence (HTRF) Phospho-STING Ser366 and Total STING Cellular detection Kit. Results were expressed as the ratio of P-STING to STING.
Results: When compared to HC, the new cohorts of SLE patients had a significant increase of cGAMP concentration in PBMCs (p< 0.05). There was no statistically significant increase in cGAMP concentrations in SS and SLE/SS PBMC compared to HC. The concentrations of cGAMP in PBMC from SLE patients were also higher compared to SS (p< 0.01) but not SLE/SS.The overall frequency of detection of cGAMP by ELISA was 17% in SLE, 0% in SS, and 25% in SLE/SS patients. When longitudinal studies were performed in 6 SLE patients who had tested positive for PBMC cGAMP, 3 of the 6 SLE patients (50%) showed cGAMP positivity on at least two different occasions whereas the other 3 SLE patients only showed cGAMP positivity in their initial clinic visit. Of note, the SLEDAI score and cGAMP level from one patient showed same trend of continued decrease over the course of three different clinic visits. Although no statistically significant difference in the P-STING/STING ratio determined by the HTRF assay was detected when SLE, SS and SLE/SS were compared to HC, it is of interest that SLE patients with higher cGAMP expression also had higher P-STING/STING ratios.
Conclusion: Detection of both cGAMP and phospho-STING in new cohorts of SLE patients confirm activation of the cGAS-STING pathway in a proportion of SLE patients and indicate that the cGAS-STING pathway likely contributes to IFN-I production in SLE. Longitudinal detection of cGAMP in 50% of cGAMP+ patients suggest that activation of this pathway may be persistent. Further defining the molecular mechanisms responsible for activation of the cGAS-STING pathway will contribute to our understanding of IFN-I stimulation and targeted approaches to inhibit cGAS-STING pathway activation may be a useful therapeutic strategy in a subset of SLE patients.
J. An: None; S. Wilson: Bristol-Myers Squibb(BMS), 3; J. Henault: Bristol-Myers Squibb(BMS), 3; K. Elkon: None.
