Ultrasound neuromodulation of an anti-inflammatory pathway at the spleen produces sustained improvement of experimental pulmonary hypertension

Rationale: Chronic inflammation is pathogenically implicated in pulmonary arterial hypertension (PAH), however, it has not been adequately targeted therapeutically. Objectives: We investigated whether neuromodulation of an anti-inflammatory neuroimmune pathway using noninvasive, focused ultrasound stimulation of the spleen (sFUS) can improve experimental pulmonary hypertension (PH). Methods: PH was induced in rats by SU5416 (20 mg/kg SQ) injection, followed by 21 (or 35) days of hypoxia (10% FiO2). Animals were randomized to receive either daily, 12-min-long sessions of sFUS or sham-stimulation, for 14 days. Invasive hemodynamics, echocardiography, autonomic function parameters, lung and heart histology/immunohistochemistry, and lung single-cell-RNA sequencing were performed after treatment to assess effects of sFUS. Results: Compared to sham, sFUS treatment reduces right ventricular (RV) systolic pressure by 25-30%; it improves RV function and indices of autonomic function. sFUS treatment reduces wall thickness in small pulmonary arterioles, suppresses inflammatory cell infiltration in lungs and RV fibrosis and hypertrophy, and lowers serum levels of brain natriuretic peptide. Beneficial effects persist for weeks after sFUS treatment discontinuation and are more robust when treatment is initiated earlier and delivered for longer. Selective ablation of the splenic nerve abolishes the therapeutic benefits of sFUS. sFUS treatment downregulates several inflammatory genes and pathways in nonclassical and classical monocytes, and macrophages in the lung; differentially expressed genes in those cell types are significantly enriched for genes associated with human PAH. Conclusions: Noninvasive, sFUS treatment causes sustained improvement of hemodynamic, autonomic, laboratory and pathological manifestations of experimental PH, and downregulates inflammatory genes and pathways in the lung, many of which are relevant in human disease. ### Competing Interest Statement The authors have declared no competing interest.