Ultrasound Neuromodulation of an Anti-Inflammatory Pathway at the Spleen Improves Experimental Pulmonary Hypertension

Abstract

BACKGROUND: Inflammation is pathogenically implicated in pulmonary arterial hypertension; 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. METHODS: Pulmonary hypertension was induced in rats either by Sugen 5416 (20 mg/kg SQ) injection, followed by 21 (or 35) days of hypoxia (sugen/hypoxia model), or by monocrotaline (60 mg/kg IP) injection (monocrotaline model). Animals were randomized to receive either 12-minute-long sessions of sFUS daily 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. RESULTS: Splenic denervation right before induction of pulmonary hypertension results in a more severe disease phenotype. In both sugen/hypoxia and monocrotaline models, sFUS treatment reduces right ventricular systolic pressure by 25% to 30% compared with sham treatment, without affecting systemic pressure, and improves right ventricular function and autonomic indices. sFUS reduces wall thickness, apoptosis, and proliferation in small pulmonary arterioles, suppresses CD3 + and CD68 + cell infiltration in lungs and right ventricular fibrosis and hypertrophy and lowers BNP (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-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 pulmonary arterial hypertension–associated genes. CONCLUSIONS: sFUS causes dose-dependent, sustained improvement of hemodynamic, autonomic, laboratory, and pathological manifestations in 2 models of experimental pulmonary hypertension. 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.

EnFUSiasm for Healing: Ultrasound Neuromodulation in PAH

“…Spleen-Focused Ultrasound Neuromodulation in PAH the study by Zafeiropoulos et al 11 did not replicate the published effects of sFUS on cytokine production, 10 observing no significant changes in IL-1β, TNF, or other inflammatory cytokines in the spleen, circulation, or lungs of PAH rodents. The precise analysis of circulating and pulmonary/splenic residential Th17, regulatory T cells, and macrophages could address the remaining question about the impact of sFUS on the function of immune cells.…”

“…In this issue of the Journal Circulation Research, Zafeiropoulos et al 11 implemented this innovative technology to investigate the therapeutic effect of focused ultrasound stimulation of the spleen (sFUS) in experimental PH (Figure). Using rats with SuHx-and monocrotalineinduced PH, the authors demonstrated that sFUS treatment led to a significant reduction in right ventricular systolic pressure, improved right ventricular hypertrophy, and decreased remodeling of small PAs with no effect on mean arterial pressure, heart rate, and right ventricular end-diastolic pressure.…”