D19-01 Enhanced Hydrogen Sulfide Signaling Inhibits Fibroblast Ferroptosis in Pulmonary Fibrosis

Rationale Pulmonary Fibrosis (PF) is a fibroproliferative disease characterized by increased proliferation, constant extracellular matrix (ECM) deposition, and dysfunctional antioxidant systems. In PF, an emerging initiator is abnormal iron regulation and lipid peroxidation: consequences of iron catalyzed cell death, i.e. ferroptosis (FP). Abnormal iron regulation and lipid peroxidation lead to diminished glutathione (GSH)/glutathione peroxidase 4 (GPX4) antioxidant systems which promote ECM signaling in fibroblasts. Accordingly, inhibiting ferroptosis and/or correcting iron dysregulation via increasing GSH can help with early PF intervention. We hypothesize that enhanced hydrogen sulfide (H2S) signaling through exogenous H2S donors GYY4137 (slow-releasing) and AP39 (mitochondrial-specific), can blunt FP/lipid peroxidation leading to reduced ECM deposition in the context of PF. Methodsand Results Human fibroblasts were isolated from lung tissue of patients with PF and healthy patients (Mayo IRB approved) and were treated with exogenous H2S donors. Prussian blue staining of FFPE lung sections from patients with PF revealed increased ferric iron in alveolar regions. Immunostaining of PF human lung tissue showed lower expression of H2S generating enzyme, CBS, and higher expression of the lipid peroxidation enzyme, ACSL4, compared to non-PF patients. At the cellular level, Western blot was performed in fibroblasts to understand the effect of PF on Fe regulation. PF fibroblasts showed higher expression of transferrin receptor (TfR1) and ferritin, as well as higher expression of ACSL4. The impact of exogenous H2S donors on PF fibroblasts was tested. While H2S donors did not impact TfR1 or ferritin, they did blunt lipid peroxidation as determined using live cell BDP 581/591 C11- fluorescent imaging and ACSL4 protein expression. Furthermore, GYY4137 increased the cysteine transport protein xCT(SLC7A11; part of xC), the H2S synthesis enzyme CBS and GPX4, overall showing an antioxidant effect. This was further substantiated though measuring GSH/GSSG ratios via luminescence assay. In PF fibroblasts, GSH/GSSG levels were low comparatively to normal fibroblasts. However, with exogenous H2S, PF fibroblasts showed increased GSH/GSSG levels indicating a restoration of antioxidant function. Deposition of collagen-1 was higher in PF fibroblasts, and was blunted by GYY4137, which was more potent than the FP inhibitor ferrostatain-1. Conclusion Our data suggests ferroptosis/impaired antioxidant systems are significant factors in PF and that exogeneous H2S can be leveraged to replenish antioxidant systems and blunt FP, thus potentially reducing fibrotic activity.