Diphenyleneiodonium ameliorates acute liver rejection during transplantation by inhibiting neutrophil extracellular traps formation in vivo

Abstract

Neutrophil extracellular traps (NETs) play critical roles in hepatic ischemic reperfusion injury (IRI) induced immune responses to inflammation. Diphenyleneiodonium (DPI) is an NADPH oxidative inhibitor that has been implicated in the regulation of NETs formation. However, the effects of NETs and their underlying mechanisms during DPI treatment of acute rejection (AR) after liver transplantation have not been elucidated. This study tested the hypothesis that blocking NETs formation by DPI treatment could be a potential therapeutic target against AR after liver transplantation. NETs were found to be excessively formed within the livers and serum of transplantation models, which could be an independent risk factor for AR. DPI was shown to alleviate hepatic injury and maintain liver functions by inhibiting NETs formation through the nicotinamide adenine dinucleotide phosphate (NADPH)/ROS/peptidylarginine deiminase 4 (PAD4) signaling pathway. NETs are highly involved in AR after liver transplantation. By inhibiting NETs formation, DPI suppresses activation of the NADPH/ROS/PAD4 signaling pathway which acts against AR after liver transplantation. Therefore, DPI is a potential candidate for the therapeutic management of AR after liver transplantation. Combination treatment containing both DPI and tacrolimus revealed a better antidamage efficacy than adjusting either treatment alone, suggesting that the joint therapy might be a promising solution in AR after liver transplantation.

Introduction

Liver transplantation is a major therapeutic approach in the clinical management of end-stage liver disease [1,2]. However, graft failure and death after liver transplantation has been associated with acute rejection (AR) [3,4]. Immunosuppressive agents have been shown to reduce the acute rejection rates, but, the efficacies of several AR therapies are poor [5,6]. In particular, most allograft recipients suffer from serious complications, such as serious infections and fatal malignancies, which are associated with immunosuppressive agents [7,8]. Therefore, it is essential to evaluate the mechanisms involved in the occurrence and development of AR after liver transplantation, and to identify new therapeutic targets and treatment strategies.

Neutrophil extracellular traps (NETs) formation, also referred to as NETosis, is a novel neutrophil-specific cell death process that is characterized by the release of NETs to the extracellular space to protect against invading pathogens [[9], [10], [11]]. NETs are large, extracellular, web-like structures that are decorated by over 20 different kinds of granular antimicrobial proteins. During NETosis, NETs have an intrinsic ability to neutralize and kill bacteria, fungi and various pathogens [12]. However, when dysregulated, NETs can lead to disease conditions or some immune related diseases such as liver failure, non-alcoholic steatohepatitis (NASH), hepatocellular carcinoma (HCC) and hepatic ischemia reperfusion injury (IRI) [[13], [14], [15]]. It has been reported that reducing cell-free DNA and NETs formation improves orthotopic liver transplantation outcomes [16]. Inhibitors of NETs formation or agents that dissolve NETs can potentially reduce AR incidences post liver transplantation.

Previous studies founded that neutrophils can trigger NETosis in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner which is one of the classical activators NETs formation [17]. NADPH oxidase, which has been identified to play an important role in NETs formation and liver ischemia reperfusion injury [18,19]. NOX2 and NOX4 are two predominant NOX isoforms existing in hepatocytes in liver parenchyma [20]. Besides, researchers founded that liver transplantation leads to severe inflammation accompanied with NADPH oxidase NOX2 activation. Propofol postconditioning exerted prominently protective function reduce liver inflammation via inhibition NOX2 [21].

Diphenyleneiodonium (DPI) is a widely used NOX2 inhibitor that interacts with gp91^phox, the catalytic subunit of NOX2, leading to the formation of relatively stable covalent adducts [22]. There are plenty of researches proving that DPI have several different applications including anti-inflammation, anti-bacterial activity and improvement of acute lung injury [[23], [24], [25]]. Although DPI has been shown to exhibit anti-inflammatory effects, its underlying mechanisms in AR development after liver transplantation have not been established. In this study, we found that DPI inhibits the NADPH/ROS/PAD4 signaling pathway and NETs formation to alleviate AR after liver transplantation. Therefore, regulating neutrophil release of NETs may be a novel therapeutic target for AR after liver transplantation. Moreover, DPI is a potential therapeutic option for AR after liver transplantation.