Targeting IL-6-STAT3/STAT4 Signaling Restores FOXP3 Expression in Pulmonary Arterial Endothelium and Reveals Novel Biomarkers for PAH

Background Interleukin-6 (IL-6) is a central driver of pulmonary vascular remodeling in idiopathic, heritable, and connective tissue disease-associated pulmonary arterial hypertension (PAH). Elevated IL-6 correlates with right ventricular (RV) dysfunction and poor survival. However, the specific downstream mechanisms by which IL-6 drives pathogenesis remain poorly defined. We investigated the therapeutic impact of direct IL-6 neutralization and its regulation of a novel epigenetic signaling axis in PAH.

We evaluated species-specific IL-6-neutralizing antibodies in murine Sugen/hypoxia and rat monocrotaline models of PAH. RV structure and function were assessed using cardiac MRI and invasive hemodynamics. Lung transcriptomic profiling was performed by RNA sequencing mouse lung tissue. Key findings were validated in explanted human PAH lungs, serum, and peripheral blood mononuclear cells (PBMCs), and further interrogated through mechanistic in vitro studies in human pulmonary artery endothelial cells (PAECs).

IL-6 neutralization significantly improved RV function, reduced pulmonary arterial pressures, and attenuated pulmonary vascular remodeling in both experimental models. Transcriptomic analysis identified a dysregulated FOXP3 signaling axis. Mechanistically, IL-6 induced cooperative binding of phosphorylated STAT3 and STAT4 to the FOXP3 promoter, facilitating DNMT1-mediated DNA methylation and stable gene silencing. IL-6 blockade restored downstream FOXP3 expression, rescued downstream BMPR2 signaling, and re-established endothelial homeostasis. In clinical PAH cohorts, FOXP3 expression was markedly reduced and inversely correlated with circulating IL-6 levels and indices of disease severity.

IL-6 drives pulmonary hypertension through STAT3/STAT4- and DNMT1-dependent epigenetic repression of FOXP3, linking chronic inflammation to BMPR2 dysfunction and pulmonary vascular remodeling. IL-6 neutralization reverses this pathogenic program in experimental PAH. FOXP3 emerges as a mechanistic biomarker of disease severity and a potential tool for precision stratification of patients likely to benefit from IL-6-targeted therapies. What Is New? Synergistic Gene Repression: The study demonstrates that phosphorylated STAT3 functional transcriptional complex that binds directly to the FOXP3 promoter. Acting as a molecular scaffold, this complex actively represses FOXP3 expression within the pulmonary endothelium. Epigenetic Silencing Mechanism: IL-6 does not merely induce a transient suppression of FOXP3. Instead, IL-6/STAT3 signaling recruits DNMT1 to the FOXP3 promoter, leading to site-specific DNA methylation and long-term epigenetic silencing. This mechanism provides a direct molecular link between chronic systemic inflammation and sustained pulmonary vascular injury. The FOXP3-BMPR2 Connection: The findings identify FOXP3 as a critical positive regulator of BMPR2 expression. Neutralization of IL-6 disrupts this pathological inhibitory loop, permitting restoration of BMPR2 signaling, the central gatekeeper of pulmonary vascular integrity and homeostasis. Translational Biomarkers: Importantly, the molecular changes within the lung are reflected systemically. Suppressed FOXP3 expression is readily detectable in PBMCs and serum, offering a non-invasive biomarker that correlates with pulmonary hemodynamic status and vascular health. Clinical Implications Precision Patient Stratification: FOXP3 expression in PBMCs and serum functions as a surrogate marker of pulmonary vascular epigenetic health. This could identify a specific inflammatory endotype of PAH patients most likely to respond to IL-6-targeted therapies. Prognostic Biomarker: Because FOXP3 levels integrate both systemic inflammatory burden and dysfunction of the RV-pulmonary vascular axis, they may provide a more sensitive tool for monitoring therapeutic response and predicting clinical worsening than current standard-of-care markers. Competing Interest Statement The authors have declared no competing interest. List of Abbreviations - Abbreviation - Full Term - α-SMA - Alpha-Smooth Muscle Actin - ANP - Atrial Natriuretic Peptide - BAL - Bronchoalveolar Lavage - BNP - Brain Natriuretic Peptide - βMHC - Beta-Myosin Heavy Chain - cDNA - Complementary DNA - cMRI - Cardiac Magnetic Resonance Imaging - Col1a1 - Collagen Type I Alpha 1 - Col1a2 - Collagen Type I Alpha 2 - Col3a1 - Collagen Type III Alpha 1 - DAPI - 4′,6-Diamidino-2-Phenylindole - ECM - Extracellular Matrix - EF - Ejection Fraction - FOXP3 - Forkhead Box P3 - H&E - Hematoxylin and Eosin - IL-6 - Interleukin-6 - IL-6R - Interleukin-6 Receptor - LGALS3 - Galectin-3 - LV - Left Ventricle - MAPSE - Mitral Annular Plane Systolic Excursion - MCT - Monocrotaline - mPAP - Mean Pulmonary Arterial Pressure - nAb - Neutralizing Antibody - PAEC - Pulmonary Artery Endothelial Cell - PAH - Pulmonary Arterial Hypertension - PASMC - Pulmonary Artery Smooth Muscle Cell - PCA - Principal Component Analysis - RV - Right Ventricle - RVEDV - Right Ventricular End-Diastolic Volume - RVEF - Right Ventricular Ejection Fraction - RVESV - Right Ventricular End-Systolic Volume - RVF - Right Ventricular Failure - RVLS - Right Ventricular Longitudinal Strain - RVSP - Right Ventricular Systolic Pressure - RVSV - Right Ventricular Stroke Volume - STAT3 - Signal Transducer and Activator of Transcription 3 - SV - Stroke Volume - SuHx - Sugen/Hypoxia - TAPSE - Tricuspid Annular Plane Systolic Excursion - TGF-β1 - Transforming Growth Factor Beta 1 - Th17 - T-helper 17 - TNF-α - Tumor Necrosis Factor Alpha - Treg - Regulatory T Cell