Ischemia-Reperfusion Injury in Liver Transplantation

Types and Stages of Liver Ischemia-Reperfusion Injury

Two types of liver injury elicited by ischemia-reperfusion (IR) can be distinguished. The “warm” IRI, initiated by hepatocellular damage, develops in situ during liver surgery, shock, or trauma and may lead to liver or multiorgan failure. The “cold” IRI, initiated by damage to hepatic sinusoidal endothelial cells (SECs) and disruption of the microcirculation, occurs during ex vivo preservation and is usually coupled with warm IRI during liver transplant surgery. Although initial cellular targets of the two IRI types may be different, they do share a common disease cause (i.e., local inflammatory innate immune activation). Mechanistic appreciation of different IRI types is of current interest because, as later discussed, cold static hepatic preservation and warm ex vivo liver perfusion are being compared in large animal models for future clinical use.

The activation of liver Kupffer cells (KCs), neutrophils, cytokine/chemokine production, generation of ROS, increased expression of adhesion molecules, and infiltration by circulating lymphocytes/monocytes constitute the immunological cascades in both types of IR. Distinctive from alloreactive responses against liver grafts, IR-triggered tissue inflammation occurs immediately after reperfusion. It constitutes predominantly an innate immune-dominated response, mediated by sentinel pattern recognition receptor (PRR) system. Endogenous ligands generated from liver damage, damage-associated molecular patterns (DAMPs), and exogenous pathogen-associated molecular patterns play the key role in tissue inflammation. However, IR elicits an equally robust adaptive immune response in the liver that is CD4 T-cell dependent. Our own studies imply that although innate activation may induce IR-damage both in situ and in liver transplants, the cold preservation injury favors an early and massive T-cell influx into ischemic liver grafts.

Two distinct stages of liver IRI, with unique mechanisms of hepatic damage, have been identified ( Fig. 105-1 ). The ischemic injury, a local process of cellular metabolic disturbances, results from glycogen consumption, lack of oxygen supply, and adenosine triphosphate (ATP) depletion, leading to the parenchymal cell death. The reperfusion injury, which follows, results from continuous metabolic disturbances and inflammatory responses. Indeed, liver inflammation is critical, because prevention of immune activation uniformly ameliorates IRI. Hence dissection of innate immune activation is a key for identifying novel therapeutic targets to alleviate proinflammatory while sparing or augmenting antiinflammatory mechanisms. Furthermore, IR-triggered innate adaptive crosstalk readily converts an immunologically quiescent liver to an inflammatory organ, even in the sterile environment. Most of the discussed studies were performed in a mouse in situ model of segmental hepatic warm IR. Although only partially reflecting the real-life transplant setting, the model takes advantage of the genetically targeted mouse strains. The clinically more relevant mouse model, combining cold and warm IRI components followed by orthotopic liver transplantation, has only recently been established.

Toll-Like Receptors in Liver Ischemia-Reperfusion Innate Immune Activation

Based on observations from renal transplant patients in the mid 1990s, it has been suggested that IRI activates a cascade of innate-dominated proinflammatory immune responses, which trigger the adaptive immune response that culminates in graft rejection. Evidence indicates that vertebrates use the same sentinel innate immune receptor systems, PRRs, in response to tissue damage in the absence of infections. There are four different classes of PPRs: Toll-like receptors (TLRs) and C-type lectin receptors are transmembrane proteins; retinoic acid–inducible gene-I-like receptors and NOD-like receptors (NLRs) are cytoplasmic proteins. These PRRs are expressed primarily in macrophages and dendritic cells (DCs), and their activation triggers upregulation of inflammatory gene programs. The TLR system consists of at least 13 members that function as homo dimers or heterodimers of type I transmembrane glycoproteins. Most TLRs are expressed on the cell surface, except TLR3, 7, 8, 9, which reside in intracellular compartments. Coreceptors are required for some TLRs to recognize their ligands (e.g., MD2 and CD14 for TLR-4 to bind lipopolysaccharide [LPS]). TLR ligation triggers multiple intracellular signaling pathways and results in activation of transcription factors NF-κB and AP-1 and interferon regulatory factors (IRFs), to initiate expression of genes encoding cytokines, chemokines, and costimulatory molecules. TLRs are the most thoroughly studied PRRs in liver IRI immune cascade ( Fig. 105-2 ), with at least three TLRs (3, 4, and 9) identified to be relevant.

Because the liver receives portal vein blood draining the gastrointestinal system, where commensal bacteria reside, gut-derived endotoxin may be translocated into the liver circulation. This occurs during liver IR when the portal vein occlusion results in the congestion of the intestinal wall, leading to its increased permeability during reperfusion. TLR-4 was the first innate immune receptor tested in liver IRI. Indeed, using murine warm ischemia models, livers in TLR-4–deficient mice were protected from IRI, and local hepatic inflammation was suppressed in the absence of TLR-4. In contrast, TLR-2 was dispensable in the development of liver IRI, which is distinctive from its role in the kidney or heart. The critical role of TLR-4 specific activation in triggering liver IRI was confirmed in orthotopic liver transplantation, which comprises warm and cold IRI components, and in a steatotic liver IRI model. Indeed, donor TLR-4 deficiency was sufficient to confer liver protection in the transplant model, and TLR-4 signaling on liver nonparenchymal cells rather than parenchymal cells seems more relevant for liver IRI, although a recent study showed a unique role of TLR-4 in liver parenchymal cells at the late stage of the disease process.

The function of TLR-4 in liver IRI is dependent on downstream signaling mediated by IRF-3 but not MyD88. MyD88-deficient animals not only developed hepatocellular damage, but their IRI “signature” proinflammatory cytokine (tumor necrosis factor-α [TNF-α], interleukin [IL]-1, IL-6) and chemokine (CXCL10) programs were unaffected. Because the MyD88-independent, TRIF-dependent pathway triggers a delayed NF-κB activation, it seems that MyD88-mediated early phase NF-κB activation is not necessary for liver proinflammatory response against IR. This is different from renal and heart IRI, in which either MyD88 and TRIF or MyD88 only are required. The fact that IRI peaks at 6 hours of reperfusion in the liver, whereas it lasts for days in the kidney and heart may partially explain this discrepancy. The other potential reason is that liver TLR-4 may have developed partial tolerance as a result of gut-derived endotoxin, which targets more toward the MyD88-dependent pathway. TLR-4 activation triggers both proinflammatory and antiinflammatory programs in macrophages in vitro and livers in vivo. Gsk3β, a serine/threonine kinase, was found to differentially regulate these two programs. Moreover, a Gsk3β inhibitor was shown to be an effective therapeutic agent by inhibiting proinflammatory cytokines while sparing immune-regulatory IL-10 in liver IRI.

Although the role of endotoxin has been implicated, the question of whether it provides the triggering signal for liver inflammatory immune response against IR remains controversial. Increased LPS levels were detected in portal and systemic circulation after IR in both animal models and liver transplant patients. A protective effect of antiendotoxin monoclonal antibodies was detected in a steatotic hepatic IRI model. However, endotoxin-neutralizing peptides failed to show any organ protection in the early phase of liver IR. One key point that might differentiate the latter result from the former is the time post reperfusion (6 hours versus 24 hours). Perhaps endotoxin is not responsible for triggering liver innate activation during IR, but instead it may participate in sustaining the inflammation response.

More than 20 distinct endogenous TLR-2/TLR-4 ligands, representing intracellular proteins, extracellular matrix (ECM) proteins, oxidatively modified lipids, and other soluble mediators, have been identified. High-mobility group box 1 protein (HMGB1), originally discovered as a nuclear protein, has been identified as the key endogenous TLR-4 ligand responsible for liver immune activation during IR. HMGB1 can be released from damaged hepatocytes to subsequently stimulate liver nonparenchymal cells, including KC through TLR-4 signaling (see Fig. 105-2 ). Hypoxic hepatocytes release HMGB1 through an active process facilitated by TLR-4–dependent ROS production. ROS, in turn, induces HMGB1 release through a calcium-mediated kinase–dependent mechanism, and such a positive loop of HMGB1-TLR-4 signaling may encourage a sustained inflammatory response in IR liver. Of note, HMGB1 biology has recently become quite complex, and how this ubiquitously expressed protein may trigger inflammation responses is the matter of controversy. Questions concerning the molecular nature of its TLR-4 binding partners and putative roles of other receptors in its immune activation effects, such as receptor for advanced glycation end products (RAGE) and CXCR4 need to be further defined. Indeed RAGE has been shown to be essential in liver IRI by regulating MIP-2 via an early growth response-1 (Egr-1)–dependent mechanism, as well as influencing cell death and TNF-α production in an Egr-1–independent manner. TLR-4–mediated upregulation of hepatocyte IRF-1 has been also identified as a necessary step for HMGB1 release in response to hypoxia, consistent with the essential function of parenchymal rather than nonparenchymal IRF-1 in the mechanism of liver transplant IRI. HMGB1 was found to promote recruitment of inflammatory cells to damaged tissue by forming a complex with CXCL12 chemokine and signaling via CXCR4, independent of RAGE and TLRs.

In addition to the HMGB1, other DAMPs released from damaged/necrotic cells may stimulate innate immune cells via various receptors, such as heat shock proteins, S100 proteins via TLR-4, RNA via TLR-3, and DNA via TLR-9. TLR-9 was found to function in bone marrow–derived cells, particularly neutrophils, to boost production of proinflammatory cytokines and chemokines during liver IR. Its inhibition exerted additive protective effects with HMGB1 neutralization in livers subjected to IR. Nuclear histone proteins were identified as the potential endogenous ligand of TLR-9 in the liver. Indeed, liver IR insult resulted in increased levels of circulating histones, and their neutralization was cytoprotective. Extracellular histones enhanced DNA-mediated TLR-9 activation, whereas their infusion exacerbated liver IRI pathologic conditions via TLR-9 signaling. TLR-3, which recognizes necrotic cell-derived RNA products, has also been shown to sustain inflammation in a murine gastrointestinal ischemia model.

Polymorphonuclear neutrophil (PMN)-derived neutrophil elastase (NE) may also contribute to TLR-4 activation ( Fig. 105-3 ). Activated Kupffer cells and hepatocytes produce proinflammatory cytokines, including TNF-α. The latter drives hepatocyte apoptosis and triggers chemokine-mediated endothelial expression of adhesion molecules, with resultant transmigration of PMNs from the vascular lumen into liver parenchyma. NE stimulates proinflammatory CXCL1/CXCL2 by infiltrating PMNs. Inhibition of NE depresses CXC chemokine programs, prevents PMN/macrophage recruitment, and suppresses inflammation responses. However, NE not only accelerates IR-mediated damage in a feedback mechanism with recruited PMNs, but may also serve as an endogenous TLR ligand causing TLR-4 upregulation on Kupffer cells and hepatocytes.

The role of other PRRs in liver IRI has only recently become unraveled. The necrotic cells can be sensed by inflammasomes to release panels of proinflammatory mediators. One member of the NLR family, NLRP3 (NLR family, pyrin domain containing 3), was found to be involved in PMN recruitment to sites of focal hepatic necrosis in a model of sterile in vivo inflammation. Gene silencing of NALP3 attenuated liver damage, in association with reduced IL-1ββ, IL-18, TNF-α, and IL-6; diminished HMGB1; and decreased inflammatory cell infiltration. Interestingly, hemorrhagic shock–induced liver damage may develop independent of NLRP3, and caspase-1 was found cytoprotective during trauma by mitigating liver injury/inflammation.

Although an array of PRR-targeting studies have shown promise in different animal models, the caveat is most of these studies focus on “correlation” between genetic deletion and cytoprotection rather than establishing the actual cause of the reduced tissue damage. With limited mechanistic understanding of a successful anti-IRI therapy, exploring multiple PRR pathways with small molecules acting preferably in a synergistic manner may be required to effectively combat liver inflammation and local tissue damage.