Hypothermic oxygenated perfusion inhibits HECTD3-mediated TRAF3 polyubiquitination to alleviate DCD liver ischemia-reperfusion injury

Ischemia-reperfusion injury (IRI) is an inevitable and serious clinical problem in donations after heart death (DCD) liver transplantation. Excessive sterile inflammation plays a fateful

role in liver IRI. Hypothermic oxygenated perfusion (HOPE), as an emerging organ preservation technology, has a better preservation effect than cold storage (CS) for reducing liver IRI, in

which regulating inflammation is one of the main mechanisms. HECTD3, a new E3 ubiquitin ligase, and TRAF3 have an essential role in inflammation. However, little is known about HECTD3 and

TRAF3 in HOPE-regulated liver IRI. Here, we aimed to investigate the effects of HOPE on liver IRI in a DCD rat model and explore the roles of HECTD3 and TRAF3 in its pathogenesis. We found

that HOPE significantly improved liver damage, including hepatocyte and liver sinusoidal endothelial cell injury, and reduced DCD liver inflammation. Mechanistically, both the DOC and HECT

domains of HECTD3 directly interacted with TRAF3, and the catalytic Cys (C832) in the HECT domain promoted the K63-linked polyubiquitination of TRAF3 at Lys138. Further, the ubiquitinated

TRAF3 at Lys138 increased oxidative stress and activated the NF-κB inflammation pathway to induce liver IRI in BRL-3A cells under hypoxia/reoxygenation conditions. Finally, we confirmed that

the expression of HECTD3 and TRAF3 was obviously increased in human DCD liver transplantation specimens. Overall, these findings demonstrated that HOPE can protect against DCD liver

transplantation-induced-liver IRI by reducing inflammation via HECTD3-mediated TRAF3 K63-linked polyubiquitination. Therefore, HOPE regulating the HECTD3/TRAF3 pathway is a novel target for

improving IRI in DCD liver transplantation.

Owing to the shortage of donor organs, marginal donor allografts are increasing becoming the main source of grafts1. Particularly, liver allografts from donations after cardiac death (DCD)

may increase the pool of organs by as much as 20%2. Unfortunately, warm ischemia, cold ischemia, and subsequent reperfusion phases during DCD liver transplantation can lead to inevitable

ischemia-reperfusion injury (IRI), which results in delayed graft dysfunction and ultimately decreases long-term graft survival3,4,5. The ischemic phase can activate innate immune cells to

initiate the tissue repair process required to restore homeostasis and provide defense against microbial invasion. However, in the subsequent reperfusion phase, the excessive activation of

immune responses causes oxidative stress and, local and systemic inflammation, thereby aggravating liver damage6,7. To alleviate IRI, dynamic preservation technology for grafts has been

developed. Specifically, hypothermic oxygenated perfusion (HOPE) has been demonstrated to decrease oxidative stress and cellular inflammation in DCD liver transplantation1,8,9,10. However,

little is known about its mechanism of action.

Ubiquitin modification plays an indispensable role in regulating inflammation and immune responses11. Ubiquitin-protein ligases (E3s) are commonly divided into two types: HECT and RING12.

HECT ligases comprise 28 members grouped into three subfamilies12. The homologous to the E6-associated protein carboxyl terminus domain containing 3 (HECTD3), a member of the third subfamily

of HECT ligases, is strongly expressed in the human liver13. HECTD3 consists of 861 amino acid residues, with a DOC domain (219–397) at the N-terminus and a HECT domain (512–857) at the

C-terminus14. After a specific substrate combines with the DOC domain, it is usually ubiquitinated and modified via a ubiquitin-HECT thioester complex11,14, thereby exerting its biological

activities. Itch, a HECT E3 ubiquitin ligase, promotes the degradation of RORγt via K48-linked polyubiquitination to suppress colonic inflammation15. In addition, HECTD3 promotes

nondegradative K27-linked and K29-linked polyubiquitination of Malt1 at K648 and K27-linked polyubiquitination of Stat3 at K180 to activate the pathogenic Th17 lineage, thereby aggravating

the severity of experimental autoimmune encephalomyelitis (EAE) in mice16. These results show that HECTD3 plays a vital role in coordinating inflammation and immune responses via

ubiquitination, and that it regulates ubiquitination through different sites depending on substrates. However, whether HECTD3 regulates inflammation in liver IRI is unclear.

Tumor necrosis factor receptor-associated factor 3 (TRAF3), the other type of E3s, is a representative member of the TRAF family. As an adaptor molecule, TRAF3 associates the TNF receptor

family with numerous signaling pathways related to cell survival and stress responses17. However, owing to the postnatal lethality of global TRAF3-deficiency, the functions of TRAF3 are

identified until the application of gene conditional knockout18,19. Mainly, TRAF3 is involved in inflammation and immune responses by regulating NF-κB, MAPK, and type I interferon (IFN-I)

pathways20,21. Upon bacterial infection, HECTD3 promotes the polyubiquitination of TRAF3, leading to the promotion of type I IFN production and inflammatory response11. However, it is

unclear whether HETCD3 and TRAF3 interact directly during bacterial inflammation. In addition, whether their interaction in sterile inflammation is unknown. Although a previous study has

revealed that TRAF3 may be involved in the regulation of liver damage and inflammation by activating the NF-κB pathway during liver IRI18, the specific action site of TRAF3 and its regulator

are still unclear.

Given the effect of HOPE on inflammation in DCD liver transplantation and the interaction between HECTD3 and TRAF3 during bacterial inflammation, we speculated that HOPE may regulate the

inflammatory injury induced by ischemia-reperfusion (IR) via HECTD3/TRAF3 pathway in DCD liver transplantation. Herein, in this study, we sought to investigate that the effect of HOPE on

liver IRI in a DCD rat model and explore the mechanisms of action of HECTD3 and TRAF3 in its pathogenesis, thereby seeking a new therapeutic target for improving IRI in DCD liver

transplantation.

To verify the effect of HOPE on liver function in a DCD rat model, we measured alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in perfusate. Their activities

were significantly increased in the CS group compared with the sham group, whereas these increases were suppressed in the HOPE group (Fig. 1A, B). Moreover, haematoxylin-eosin (H&E) staining

of the liver tissues and their histological scores confirmed that HOPE can reduce liver IRI in a DCD rat model (Fig. 1Ea, F).

A, B ALT and AST activities in the perfusate. Data are mean ± SD, **P