IL-1β contribute to NGF-induced alterations in pulmonary hypertension

Abstract

Introduction

Pulmonary Hypertension (PH) is a severe disease leading to right heart failure and death. We have previously demonstrated a pathological role of the nerve growth factor NGF in PH, particularly in pulmonary arterial inflammation. We have here studied the link between NGF, monocytes/macrophages and interleukin-1β (IL-1β), a pro-inflammatory cytokine secreted in response to NGF and overexpressed in PH.

Methods

In vivo, PH was induced in rats by monocrotaline (60 mg/kg, intraperitoneal (ip) injection) in the absence or presence of a preventive treatment with anti-NGF blocking antibodies (10 μg/kg, ip injection). IL-1β pulmonary levels were then determined (ELISA). Expression of inflammasome components (NLRP3/pro-caspase-1/ASC/caspase-1/pro-IL-1β), of NGF and of monocytes/macrophages markers (CD₁₁b/CD₆₈) was evaluated by Western blotting (WB) in lung homogenates. Expression of CD₆₈was also evaluated by immunohistochemistry in pulmonary arteries.

In vitro, both on the monocytic cell line THP-1 and on freshly isolated human monocytes, cell migration (Transwell assay) and/or proliferation (counting) were studied in response to NGF. Control human pulmonary arterial smooth muscle (hPASMC) or endothelial cells (hPAEC) were treated with IL-1β, and cell proliferation (counting), migration (Transwell assay), and interleukin-6 (IL-6) secretion (ELISA) were then assessed. In hPAEC, expression of endothelial nitric oxide synthase (eNOS), intercellular adhesion molecule-1 (ICAM-1), CD₃₁, VE-cadherin, Snail and Twist 1 was also assessed (WB).

Results

In vivo, IL-1β pulmonary levels were increased in PH rats, as observed in PH patients. Expression of inflammasome components such as procaspase-1, ASC, caspase-1 and pro-IL-1β was significantly increased in lung homogenates from PH rats compared to controls. CD₆₈expression was also significantly increased in both the lung and pulmonary arteries (PA) from PH rats compared to controls. Treatment with anti-NGF antibodies prevented PH development and reduced expression of inflammasome proteins and of CD₆₈in both the lung and PA. In vitro, NGF induced migration and/or proliferation of both THP-1 cells and freshly isolated human monocytes. In hPASMC and hPAEC, IL-1β significantly increased cell proliferation, migration and IL-6 secretion in a NF-kB and/or AP-1-dependent manner, with IL-6 secretion induced by IL-1β playing a role in IL-1β-dependent cell proliferation and migration. In hPAEC, IL-1β also significantly increased ICAM-1 and Snail expression and significantly decreased eNOS, CD₃₁and VE-cadherin expression, mechanisms known to contribute to endothelial dysfunction and loss of endothelial phenotype in PH.

Conclusion

Our results suggest that NGF can trigger monocytes/macrophages migration into the lung and PA, thus contributing to an inflammatory environment in particular by release of IL-1β. These mechanisms may then participate in PA dysfunction in PH by stimulating both hPASMC and hPAEC to induce remodelling, inflammation pathways and altered endothelial function. Since NGF contributes to IL-1β increased levels in PH, IL-1β may therefore contribute to NGF-dependent PA alterations in this disease.