EphB2 Receptor Tyrosine Kinase: A Novel Regulator of Infection- and Inflammation-induced Liver Fibrosis

Erythropoietin 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 ten 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, alteration of vascular permeability, disruption of vascular integrity, regulation of hematopoiesis, platelet aggregation, and modulation of T cell activity.^2–4 One receptor family member, EphB2, which preferentially binds the ligands ephrinB1 or ephrinB2, has been implicated in many of the immunologic processes described above. EphB2 is highly expressed on monocytes and macrophages and regulates trafficking and adhesion of these cells to endothelial tissue⁵.

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 the deposition of scar tissue and replacement of normal parenchymal tissue. Thus results in severe architectural changes, dysfunction of liver vasculature and ultimately the 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 the 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 (RBC), initiating the clinically relevant ‘erythrocytic’ stage of Plasmodium infection. The clinical signs and symptoms of malaria are due to cyclic rupture and release of merozoites from infected RBC, which drives inflammation, fever, chills, and malaise. Life-threatening, severe malaria is further linked to the adhesion of parasite-infected RBC to the endothelium of the central nervous system or placental tissues, triggering recruitment of leukocytes and immunopathology.⁷,⁸ Additional and significant immunopathologic changes also occur within liver sinuses resulting in severe malaria-associated hepatomegaly, remodeling of liver tissue, and fibrosis.⁹,¹⁰ While the adhesion of infected RBC to liver endothelial cells, 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 authors initially identified that experimental malaria was linked to marked upregulation of EphB receptor expression in the livers of infected mice, including the EphB2 family member. Notably, malaria-induced upregulation of EphB receptors was largely restricted to the liver, as receptor expression was relatively unchanged in other tissues in which infected RBC sequester and drive inflammation during experimental malaria.

To explore the biological relevance of these initial observations, the authors performed a series of comparative experiments in Plasmodium-infected wild-type and EphB2-deficient littermate mice. Strikingly, EphB2-deficient mice were resistant to malaria-induced liver fibrosis, despite harboring equivalent numbers of parasite-infected RBC. Marked reductions in collagen deposition and expression of α-smooth muscle actin (α-SMA) in hepatic stellate cells (HSC) were observed throughout the course of infection in EphB2-deficient mice, which also correlated with reduced expression of inflammatory mediators in the liver, including TNF, IL-6 and inducible nitric oxide synthase (iNOS). Additional experiments revealed that primary hepatocytes derived from EphB2-deficient mice were less responsive to inflammatory insults, compared to wild-type hepatocytes, as evidenced by reduced NFκB activation following stimulation with cytokines or lysates from parasite-infected RBC.

The hepatocyte-intrinsic role for EphB2 in modulating reactivity to inflammatory and parasite-derived stimuli was unexpected and suggests 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 author’s identification that multiple resident and infiltrating immune cells in the Plasmodium-infected liver also express EphB receptors, including HSC, monocytes, neutrophils, and macrophages. Thus, it is likely that EphB receptors have the capacity to regulate other pro-inflammatory 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 NFκB activation. 2. Activated hepatocytes express chemokines and cytokines that activate sinusoidal endothelial cells facilitating the recruitment of circulating monocytes (Mono) to the liver parenchyma. 3. EphB2-expressing resident Kupffer cells (KC) and infiltrating macrophages (Mϕ) accumulate in inflammatory foci in the liver leading to marked increases in levels of EphB2 expression. Activated Kupffer cells and macrophages express profibrotic chemokines and cytokines that further potentiate mononuclear cell infiltration. 4. Quiescent hepatic stellate cells (HSC) subsequently undergo transdifferentiation to myofibroblasts (MF). 5. Myofibroblasts drive liver fibrosis via deposition of extracellular matrix proteins such as collagen. Notably, depletion of Kupffer cells and macrophages reveals their essential roles in the initiation of HSC transdifferentiation and deposition of collagen by myofibroblasts following malaria or CCL₄-induced liver inflammation.

To further explore cellular and molecular pathways that regulate fibrogenesis during malaria, Mimche et al.¹¹ depleted infiltrating phagocytes (monocytes, macrophages, neutrophils) and tissue resident phagocytes (Kupffer cells) in Plasmodium-infected wild-type mice. Strikingly, in vivo depletion of Kupffer cells 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 Kupffer cells/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 the EphB2-mediated regulation of liver fibrosis is not restricted to the malaria models. Using a carbon tetrachloride (CCL₄) model of inflammation-induced liver fibrosis, the authors 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 the 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 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 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 healthcare 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 identify 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 the progression liver fibrosis.

Abbreviations

CCL₄

carbon tetrachloride

DC

dendritic cell

Eph

erythropoietin producing hepatocellular receptor

Ephrin

Eph receptor interacting protein

HSC

hepatic stellate cell

IL

interleukin

RBC

red blood cell