Neurologic cues modulate immune‐mediated liver injury and regeneration
Authors: Benjamin L. Woolbright, Hartmut Jaeschke
Journal: Hepatology (2016)

Potential conflict of interest: Nothing to report. This work was supported by National Institutes of Health grant See Article on Page 1689 Hepatic ischemia/reperfusion (I/R) injury occurs during a number of different surgical procedures, including liver transplantation (LT) and hepatic resection, and during hemorrhagic shock/resuscitation and other hypotensive events. Whereas limited cell death may occur during the ischemic phase, especially when ischemia is kept to shorter periods, tissue reperfusion activates an inflammatory cascade that dramatically exacerbates liver injury.1 This is largely mediated by resident Kupffer cells (KCs) and recruited neutrophils, which produce cytotoxic reactive oxygen species (ROS) that postischemic hepatocytes cannot effectively detoxify, resulting in considerable cell necrosis.1 As such, immune‐modulatory agents and antioxidants have been proposed as a way to limit hepatic reperfusion injury and improve outcomes during LT, liver surgery, or during hypoxic hepatitis. However, despite numerous reports of successful therapeutic interventions in animal models, there are, as of yet, no specific and effective therapeutics in clinical use that can inhibit abnormal immune cell activation during hepatic I/R injury without increasing patient risk for infection. Netrin‐1 (Ntn1) is a neuroimmune guidance protein that, during development, provides chemoattractive and ‐repulsive signals for neuronal cells in the central nervous system. However, Ntn1 is also expressed in vascular endothelial cells and can regulate inflammatory cell recruitment.3 The importance of Ntn1 has been recognized in a number of inflammatory conditions, including I/R injury in the heart and kidney.4 In this issue of Hepatology, Schlegel et al. studied the potential role for Ntn1 as a mediator of inflammation in a murine hepatic I/R model.5 The investigators first provided evidence for Ntn1 messenger RNA and protein expression in the liver. Hepatic I/R (30 minutes/3 hours) reduced Ntn1 expression. By using a mouse with partial deficiency of the Ntn1 gene (Ntn1+/−), the investigators were able to further demonstrate that reduced expression of Ntn1 in the liver exacerbated I/R injury by enhanced recruitment of neutrophils to the liver. This increased injury correlated with elevated serum levels of proinflammatory cytokines (e.g., tumor necrosis factor alpha [TNF‐α]) and neutrophil‐chemoattractant chemokines, such as mouse keratinocyte‐derived chemokine in Ntn1+/− mice.5 In a reciprocal experiment, intravenous administration of recombinant Ntn1 protein had the opposite effects; netrin reduced liver injury as well as chemokine production and leukocyte adhesion. Importantly, these experiments demonstrated that Ntn1 only affected the reperfusion injury phase and that it can be used as a therapeutic agent. Interestingly, these effects appear not to be mediated by the Ntn1 receptor, unc‐5 netrin receptor B, which is expressed on leukocytes,3 but by the A2B adenosine receptor given that Ntn1 administration was ineffective in A2BAR−/− mice exposed to hepatic I/R.5 However, it remained unclear why the investigators' A2B receptor knockout KO mice had similar reperfusion injury as the wild‐type controls, in contrast with previous studies suggesting that A2BAR−/− mice had more‐severe liver pathology after undergoing hepatic I/R.6 The role of UNC5B and the A2B adenosine receptor may need to be investigated in more detail. Perhaps most important, administration of Ntn1 at the time of reperfusion resulted in faster injury resolution and enhanced liver regeneration, likely attributed to enhanced production of injury‐resolving lipid mediators and increased expression of regenerative markers, such as hepatocyte growth factor and vascular endothelial growth factor.5 The improved recovery correlated with reduced neutrophil infiltration and improved phagocytosis of dead cells. In support of these findings, Ntn1+/− mice showed the opposite effects, that is, decreased growth factor expression and reduced proliferating cell nuclear antigen‐positive cells. This inhibited proregenerative response in Ntn1+/− mice could be reversed by recombinant Ntn1 administration.5 Although these findings provided strong evidence that Ntn1 supports tissue recovery after extensive injury, a number of issues need to be investigated. In particular, the cellular sources of endogenous Ntn1 and the actual target cells of both therapeutically administered and endogenously generated Ntn1 remain unclear. Furthermore, the neutrophil‐mediated injury phase includes 12‐24 hours of reperfusion based on both reactive oxygen formation by neutrophils accumulated in the liver7 and by release of necrotic biomarkers into the blood.8 Treatment of animals with Ntn1 at the end of ischemia and even at 3 hours of reperfusion falls well within the neutrophil‐induced injury. Thus, it cannot be excluded, based on the experimental design, that the improved regeneration is caused, at least in part, by the effect on proinflammatory injury mechanisms rather than by directly stimulating resolution of inflammation and recovery. Addressing this issue could considerably broaden the therapeutic potential of Ntn1 in hepatic I/R injury and other liver disease models. KCs, activated by complement factors, were identified as the primary source of a vascular oxidant stress during the initial phase of hepatic I/R injury in rodent models.1 Necrotic cell death and release of damage‐associated molecular patterns triggers a second injury phase dominated by neutrophil recruitment into the liver, extravasation, and a neutrophil‐induced oxidant stress.1 The importance of neutrophil‐derived ROS, such as hypochlorous acid, was shown by the presence of intracellular protein modifications specific for these oxidants.7 Using antibodies against chlorotyrosine, Schlegel et al. provided evidence that, in the murine model of hepatic I/R injury, Ntn1 treatment did not only reduce the number of neutrophils, but also attenuated neutrophil cytotoxicity (oxidant stress) after 3 hours of reperfusion.5 In addition, the investigators delineated the subpopulations of macrophages during the early reperfusion phase. Ntn1 administration resulted in an increase in total F4/80+ resident macrophages (KCs), which was caused by a reduction in CD11b+ cells and a concomitant increase in CD68+ KCs. Whereas the CD11b+ subset of KCs has a high cytokine‐producing capacity in response to endotoxin, the CD68+ KCs produce large amounts of reactive oxygen and effectively phagocytose debris.10 These characteristics of the resident macrophage population correlated with the reduction in cytokine levels and increased phagocytic capacity in Ntn1‐treated mice. Overall, this resulted in an attenuated proinflammatory milieu and increased neutrophil efferocytosis.5 During the late reperfusion phase, the investigators also characterized Ly6C expression on newly recruited hepatic monocytes. Classical Ly6Chigh monocytes (M1) are considered proinflammatory because of their high capacity to produce reactive oxygen and generate cytokines, such as TNF‐α. In contrast, the nonclassical Ly6Clow monocytes (M2) are considered anti‐inflammatory because of their ability to generate cytokines, such as interleukin (IL)‐10, which down‐regulates TNF‐α formation, and because of the high phagocytic capacity of M2 macrophages. Schlegel et al. found that Ntn1 treatment decreased the number of classical Ly6Chigh monocytes and increased the levels of Ly6Clow nonclassical monocytes, which created an anti‐inflammatory and proregenerative environment that limited additional injury and supported recovery.5 Though IL‐10 was not measured in this study, it is likely that the higher proportion of Ly6Clow, compared to Ly6Chigh, monocytes led to increased IL‐10 formation, which contributed to the injury‐resolving nature of Ntn1 administration. Together, these results suggest that neuroimmune guidance proteins such as Ntn1 have a previously unrecognized role in modulating immune responses in clinically relevant models of liver injury. The observations reported by Schlegel et al. advance both our understanding of the basic science behind the regulation of the sterile inflammatory response in hepatic I/R injury and identified a promising novel therapeutic approach.