Construction of a lentiviral vector containing shRNA targeting ADAM17 and its role in attenuating endotoxemia in mice

Abstract

Systemic inflammatory response syndrome is a pathophysiological inflammatory response mediated largely by tumor necrosis factor-α (TNF-α), in response to infectious or non-infectious stimuli. TNF-α secretion in response to bacterial lipopolysaccharide (LPS) is regulated in part by disintegrin and metalloproteinase domain-containing protein 17 (ADAM17). Therefore, the present study aimed to identify an effective inhibitor of ADAM 17, in order to control inflammation and associated processes. In the present study, a lentiviral vector expressing short hairpin (sh)RNA targeting the ADAM17 gene was constructed. U937 cells were infected with the lentivirus and stimulated with LPS. ADAM17 expression was assessed by western blotting and TNF-α secretion was assessed by ELISA analysis. The lentivirus was additionally tested in vivo in a mouse model of endotoxemia and sTNF-α expression was assessed by flow cytometry in peritoneal macrophages. In vitro, the ADAM17 shRNA lentivirus reduced ADAM 17 expression, and prevented TNF-α maturation in U937 cells. In vivo, mice exposed to the ADAM17 shRNA lentivirus prior to LPS-induced endotoxemia exhibited fewer signs of inflammation and less tissue damage compared with the control mice. In conclusion, the present study successfully constructed a shRNA lentiviral vector targeting the ADAM 17 gene that exhibited apparent in vitro and in vivo effects on TNF-α processing in response to an LPS challenge. The results of the present study may aid the design and improvement of drugs designed to inhibit the function of ADAM17, and suggested a novel means of controlling inflammation and associated processes.