Erythropoietin‐producing hepatocellular receptor B2 receptor tyrosine kinase: A novel regulator of infection‐ and inflammation‐induced liver fibrosis
Authors: Noah S. Butler, Nathan W. Schmidt
Journal: Hepatology (2015)

Potential conflict of interest: Nothing to report. This work was supported by grants from the National Institute of General Medical Sciences (8P20GM103447; to N.S.B.), the American Heart Association (13BGIA17140002; to N.S.B.), the National Institute of Allergy and Infectious Disease (1R21AI113386; to N.W.S.), and the American Cancer Society (Research Scholar Grant, RSG‐14‐057‐01‐MPC; to N.W.S.). See Article on Page 900 Erythropoietin‐producing hepatocellular (Eph) receptors are the largest family of receptor tyrosine kinases. Engagement by their ligands, known as ephrins (Eph receptor‐interacting proteins), stimulates an array of cell‐signaling pathways that regulate key aspects of cell motility, adhesion, proliferation, survival, and tissue patterning during health and disease.1 Sixteen Eph receptors have been identified in vertebrates and are broadly classified into two groups that include 10 EphA receptors and six EphB family members, which bind preferentially to ephrinA and ephrinB ligands, respectively. Beyond their well‐described roles in regulating tissue patterning and development, Eph‐ephrin interactions also play important roles in immune cell adhesion during inflammatory innate and adaptive immune reactions.2 Eph receptors have been functionally linked to leukocyte adhesion to endothelial cells (ECs), alteration of vascular permeability, disruption of vascular integrity, regulation of hematopoiesis, platelet aggregation, and modulation of T‐cell activity.2 One receptor family member, EphB2, which preferentially binds the ligands, ephrinB1 or ephrinB2, has been implicated in many of the immunological processes described above. EphB2 is highly expressed on monocytes and macrophages and regulates trafficking and adhesion of these cells to endothelial tissue.5 Strikingly, despite having been first identified in a hepatocellular cell line,6 the role and contribution of the Eph‐ephrin axis in regulating hepatic mononuclear cell infiltration, liver inflammation, tissue remodeling, and fibrogenesis had not been previously examined. Fibrosis is a common consequence of excessive or persistent inflammation. Chronic inflammation in the liver drives cascades of cellular and molecular wound‐healing processes that can eventually result in deposition of scar tissue and replacement of normal parenchymal tissue, thus resulting in severe architectural changes, dysfunction of liver vasculature, and, ultimately, loss of liver function. Fibrotic changes are a consequence of several forms of liver damage, including microbial infection, autoimmune reactions, and alcoholic and nonalcoholic steatohepatitis. Malaria, caused by infection with protozoan parasites of the genus Plasmodium, is intimately linked to liver health and disease. Sporozoites, the mosquito‐transmitted form of the parasite, undergo an obligatory developmental transition in the liver during the first week of infection. Parasites eventually erupt from infected hepatocytes as newly differentiated merozoites. Merozoities subsequently target and infect red blood cells (RBCs), initiating the clinically relevant "erythrocytic" stage of Plasmodium infection. The clinical signs and symptoms of malaria are the result of cyclic rupture and release of merozoites from infected RBCs, which drives inflammation, fever, chills, and malaise. Life‐threatening, severe malaria is further linked to adhesion of parasite‐infected RBCs to the endothelium of the central nervous system or placental tissues, triggering recruitment of leukocytes and immunopathology.7 Additional and significant immunopathological changes also occur within liver sinuses, resulting in severe malaria‐associated hepatomegaly, remodeling of liver tissue, and fibrosis.9 Though the adhesion of infected RBCs to liver ECs, sequestration of parasites in liver sinuses, and induction of liver fibrosis during malaria is well documented, the host cellular and molecular factors responsible for these hepatic alterations had not been determined. In this issue of Hepatology, Mimche et al.11 demonstrate that EphB receptor expression is functionally linked to the progression of malaria‐ and inflammation‐induced liver fibrosis in mice. Using multiple rodent models of Plasmodium infection that recapitulate key aspects of hepatic inflammation and fibrosis observed in humans infected with Plasmodium falciparum, the parasite responsible for the most severe forms of clinical malaria, the investigators initially identified that experimental malaria was linked to marked up‐regulation of EphB receptor expression in livers of infected mice, including the EphB2 family member. Notably, malaria‐induced up‐regulation of EphB receptors was largely restricted to the liver, given that receptor expression was relatively unchanged in other tissues in which infected RBCs sequester and drive inflammation during experimental malaria. To explore the biological relevance of these initial observations, the investigators performed a series of comparative experiments in Plasmodium‐infected wild‐type (WT) and EphB2‐deficient littermate mice. Strikingly, EphB2‐deficient mice were resistant to malaria‐induced liver fibrosis, despite harboring equivalent numbers of parasite‐infected RBCs. Marked reductions in collagen deposition and expression of alpha‐smooth muscle actin in hepatic stellate cells (HSCs) were observed throughout the course of infection in EphB2‐deficient mice, which also correlated with reduced expression of inflammatory mediators in the liver, including tumor necrosis factor, interleukin‐6, and inducible nitric oxide synthase. Additional experiments revealed that primary hepatocytes derived from EphB2‐deficient mice were less responsive to inflammatory insults, compared to WT hepatocytes, as evidenced by reduced nuclear factor kappa B (NF‐κB) activation after stimulation with cytokines or lysates from parasite‐infected RBCs. The hepatocyte‐intrinsic role for EphB2 in modulating reactivity to inflammatory and parasite‐derived stimuli was unexpected and suggests that functional crosstalk occurs between EphB receptor and pattern recognition or cytokine receptor signaling (Fig. 1). The potential significance of these surprising aspects of EphB receptor biology are further underscored by the investigators' identification that multiple resident and infiltrating immune cells in the Plasmodium‐infected liver also express EphB receptors, including HSCs, monocytes, neutrophils, and macrophages. Thus, it is likely that EphB receptors have the capacity to regulate other proinflammatory signaling cascades in multiple cell types during malaria, although further studies are required to confirm this hypothesis. Indeed, the extent to which EphB receptor signaling impacts other signaling pathways in various immune cell subsets warrants further investigation.Figure 1: EphB2 impacts multiple aspects of malaria‐ and inflammation‐induced liver fibrosis. (1) Malaria or other inflammatory insults to liver tissue activate hepatic EphB2 signaling, which likely acts in concert with cytokine or TLR signaling to trigger hepatocyte NF‐κB activation. (2) Activated hepatocytes express chemokines and cytokines that activate sinusoidal ECs, facilitating recruitment of circulating monocytes (Mono) to liver parenchyma. (3) EphB2‐expressing resident KCs and infiltrating macrophages (Mϕ) accumulate in inflammatory foci in the liver, leading to marked increases in levels of EphB2 expression. Activated KCs and macrophages express profibrotic chemokines and cytokines that further potentiate mononuclear cell infiltration. (4) Quiescent HSCs subsequently undergo transdifferentiation to myofibroblasts (MF). (5) Myofibroblasts drive liver fibrosis by deposition of ECM proteins, such as collagen. Notably, depletion of KCs and macrophages reveals their essential roles in the initiation of HSC transdifferentiation and deposition of collagen by myofibroblasts after malaria‐ or CCL4‐induced liver inflammation. Abbreviation: TLR, Toll‐like receptor.To further explore cellular and molecular pathways that regulate fibrogenesis during malaria, Mimche et al.11 depleted infiltrating phagocytes (monocytes, macrophages, and neutrophils) and tissue resident phagocytes (Kupffer cells; KCs) in Plasmodium‐infected WT mice. Strikingly, in vivo depletion of KCs and macrophages, but not neutrophils or monocytes, markedly reduced both liver expression of EphB2 and malaria‐associated fibrosis (Fig. 1). These data identify the essential role that EphB2‐expressing KCs/macrophages play in orchestrating and propagating malaria infection‐induced fibrosis. Another key finding in the study by Mimche et al.11 is the demonstration that EphB2‐mediated regulation of liver fibrosis is not restricted to malaria models. Using a CCL4 model of inflammation‐induced liver fibrosis, the investigators illustrate the generalizable role and contribution of EphB2 during liver fibrogenesis. Therefore, the cellular and molecular pathways mapped by Mimche et al.11 are broadly applicable to inflammation‐induced liver fibrosis. Despite this being the first report to establish functional links between Eph receptor expression and liver fibrosis induced by infection or inflammation, several important questions remain to be addressed. For example, it will be of interest to determine patterns of ephrin ligand expression during liver fibrosis. Moreover, it will be of interest to determine whether the pathways regulated by Eph receptor expression and signaling are conserved in other tissues where fibrosis is associated with important clinical disease, including skin and lung. Indeed, whether EphB or ephrin family members can serve as therapeutic targets to limit or prevent the cascade of inflammatory cell infiltration and deposition of extracellular matrix (ECM) in multiple tissues during multiple diseases remains to be determined. Finally, determining the relative contribution of EphB2 expression on hepatocytes versus liver‐infiltrating and resident immune cell subsets (Fig. 1) will be important for identifying strategies and developing interventions to limit EphB2‐mediated fibrogenesis. Indeed, new reagents targeting Eph receptors and ephrin ligands are being developed and evaluated for their ability to modulate the biology of this signaling axis12 and may have the potential for treating inflammatory liver fibrotic disease. Mimche et al.11 have, for the first time, functionally linked Eph receptor expression to liver fibrosis as well as identified key immune cell subsets that potentiate both inflammation‐ and malaria‐induced liver pathology (Fig. 1). The health care burden of hospitalizations and comorbidities associated with chronic liver fibrosis remain major health problems. As such, identifying targets and opportunities to reduce or reverse liver fibrosis or cirrhosis through therapeutic intervention remains a priority. The recent work by Michime et al.11 identifies a new player in the mechanistic cascade of events that drive liver fibrosis, raising the possibility for new interventions or therapies that target the Eph‐ephrin axis to limit progression of liver fibrosis.