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

Abstract

AbstractBackgroundInflammation is pathogenically implicated in pulmonary arterial hypertension (PAH); however, it has not been adequately targeted therapeutically. We investigated whether neuromodulation of an anti-inflammatory neuroimmune pathway involving the splenic nerve using noninvasive, focused ultrasound stimulation of the spleen (sFUS) can improve experimental pulmonary hypertension (PH).MethodsPH was induced in rats either by SU5416 (20 mg/kg SQ) injection, followed by 21 (or 35) days of hypoxia (SuHx model), or by monocrotaline (60 mg/kg IP) injection (MCT model). Animals were randomized to receive either daily, 12-min-long sessions of sFUS or sham stimulation, for 14 days. Catheterizations, echocardiography, indices of autonomic function, lung and heart histology and immunohistochemistry, spleen flow cytometry and lung single-cell-RNA sequencing were performed after treatment to assess the effects of sFUS.ResultsSplenic denervation right before induction of PH results in a more severe phenotype. In both SuHx and MCT models of PH, sFUS treatment reduces right ventricular (RV) systolic pressure by 25-30% compared to sham therapy, without affecting systemic pressure, and improves RV function and autonomic indices. sFUS reduces wall thickness, apoptosis, and proliferation in small pulmonary arterioles, suppresses CD3+ and CD68+ cell infiltration in lungs and RV fibrosis and hypertrophy and lowers brain natriuretic peptide. Beneficial effects persist for weeks after sFUS discontinuation and are more robust with early and longer treatment. Splenic denervation abolishes sFUS therapeutic benefits. sFUS partially normalizes CD68+ and CD8+ T-cells cell counts in the spleen and 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 human PAH-associated genes.ConclusionssFUS causes dose-dependent, sustained improvement of hemodynamic, autonomic, laboratory and pathological manifestations in two models of experimental PH. Mechanistically, sFUS normalizes immune cell populations in the spleen and downregulates inflammatory genes and pathways in the lung, many of which are relevant in human disease.