Objective: The lungs of patients with Systemic Sclerosis Associated Interstitial Lung Disease (SSc-ILD) contain inflammatory myofibroblasts arising in association with fibrotic stimuli and perturbed innate immunity. The innate immune DNA binding receptor Cyclic GMP-AMP synthase (cGAS) is implicated in inflammation and fibrosis, but its involvement in SSc-ILD remains unknown. We examined cGAS expression, activity, and therapeutic potential in SSc-ILD using cultured fibroblasts, precision cut lung slices (PCLS), and a well-accepted animal model. Methods: Expression and localization of cGAS, cytokines, and type 1 interferons were evaluated in SSc-ILD lung tissues, bronchoalveolar lavage (BAL), and isolated lung fibroblasts. CGAS activation was assessed in a publicly available SSc-ILD single cell RNA sequencing dataset. Production of cytokines, type 1 interferons, and alpha-SMA elicited by TGF-beta-1 or local substrate stiffness were measured in normal human lung fibroblasts (NHLFs) via qRT-PCR, ELISA, and immunofluorescence. Small molecule cGAS inhibition was tested in cultured fibroblasts, human PCLS, and the bleomycin pulmonary fibrosis model. Results: SSc-ILD lung tissue and BAL are enriched for cGAS, cytokines, and type 1 interferons. The cGAS pathway shows constitutive activation in SSc-ILD fibroblasts and is inducible in NHLFs by TGF-beta-1 or mechanical stimuli. In these settings, and in human PCLS, cGAS expression is paralleled by the production of cytokines, type 1 interferons, and alpha-SMA that are mitigated by a small molecule cGAS inhibitor. These findings are recapitulated in the bleomycin mouse model. Conclusion: cGAS signaling contributes to pathogenic inflammatory myofibroblast phenotypes in SSc-ILD. Inhibiting cGAS or its downstream effectors represents a novel therapeutic approach.

The authors have declared no competing interest.

This study was funded by the following. JZ was supported by T32HL007778. AG was supported by T32HL007778. GI was supported by a Scholar Award from the Pulmonary Fibrosis Foundation, Wit Family Distinguished Scholar in Inflammation Science, and Yale Physician Scientist Development Award. MH was supported by R01 AR073270. JG was supported by R01HL153604 and R03 HL154275. CR was supported by K08HL151970-01 and Boehringer-Ingelheim Discovery Award. ELH was supported by 5R01HL163984-02 and 5R01HL152677-04. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or other funding sources.

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Ethics committee/IRB at the University of Pittsburgh (IRB 970946) and Yale School of Medicine (HIC 2000024862) gave ethical approval for this work.

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