High Mobility Group Box 1 Release by Cholangiocytes Governs Biliary Atresia Pathogenesis and Correlates With Increases in Afflicted Infants

Abstract

Background and aims: Biliary atresia (BA) is a devastating cholangiopathy of infancy. Upon diagnosis, surgical reconstruction by Kasai hepatoportoenterostomy (HPE) restores biliary drainage in a subset of patients, but most patients develop fibrosis and progress to end-stage liver disease requiring liver transplantation for survival. In the murine model of BA, rhesus rotavirus (RRV) infection of newborn pups results in a cholangiopathy paralleling that of human BA. High-mobility group box 1 (HMGB1) is an important member of the danger-associated molecular patterns capable of mediating inflammation during infection-associated responses. In this study, we investigated the role of HMGB1 in BA pathogenesis.

Approach and results: In cholangiocytes, RRV induced the expression and release of HMGB1 through the p38 mitogen-activated protein kinase signaling pathway, and inhibition of p38 blocked HMGB1 release. Treatment of cholangiocytes with ethyl pyruvate suppressed the release of HMGB1. Administration of glycyrrhizin in vivo decreased symptoms and increased survival in the murine model of BA. HMGB1 levels were measured in serum obtained from infants with BA enrolled in the PROBE and START studies conducted by the Childhood Liver Disease Research Network. High HMGB1 levels were found in a subset of patients at the time of HPE. These patients had higher bilirubin levels 3 months post-HPE and a lower survival of their native liver at 2 years.

Conclusions: These results suggest that HMGB1 plays a role in virus induced BA pathogenesis and could be a target for therapeutic interventions in a subset of patients with BA and high HMGB1.

Fig. 1

Quantification of serum levels of HMGB1 in vivo and temporal release from cholangiocytes in vitro. Serum harvested from RRV‐injected, Ro1845‐injected, and saline‐injected mice at 3, 5, 7, and 12 days postinfection demonstrates significantly higher levels of HMGB1 in RRV‐injected mice starting at day 7 when compared to both saline‐injected and Ro1845‐injected pups (A). Cholangiocytes infected with RRV at an MOI of 10 resulted in a significant increase in HMGB1 release into the supernatant at 24 hours postinfection; conversely, H2.35 infection demonstrated no increase of HMGB1 even at a 10‐fold higher MOI (B). Following RRV infection of cholangiocytes, HMGB1 release was detected in the supernatant at 16 hours and continued until 24 hours (C) (*P < 0.05).

Fig. 2

HMGB1 release from cholangiocytes is replication‐dependent, strain‐dependent, and dose‐dependent. Infection with inactivated RRV resulted in no HMGB1 release from cholangiocytes at 16 hours postinfection when compared to infectious RRV (A). Cholangiocytes infected with multiple strains of rotavirus demonstrated HMGB1 release from RRV and GRV, while no release was observed from Ro1845. Additionally, HMGB1 release is facilitated by the rotavirus gene VP4 as Ro1845^RRV(VP4) is capable of inducing HMGB1 secretion, while the incorporation of Ro1845’s VP4 to RRV (RRV^Ro1845(VP4)) results in loss of HMGB1 release (B). Cholangiocytes infected with increasing MOIs of RRV lead to a dose‐dependent increase in HMGB1 release; conversely, no increase was witnessed with increasing MOIs of Ro1845 (C) (*P < 0.05).

Fig. 3

Effect of EP on HMGB1 release. Cholangiocytes treated with EP showed significantly reduced HMGB1 levels following infection (A). EP had no effect on viral titers (B) (*P < 0.05). Abbreviation: FFU, focus‐forming unit.

Fig. 4

HMGB1 release is p38‐dependent and STAT1‐dependent. Protein isolated from cholangiocytes 8 hours postinfection revealed a significant increase in the phosphorylation of both p38 and STAT1 in a similar pattern by those virus strains which were capable of inducing HMGB1 release (A). Cholangiocytes treated with SB203580, a p38 inhibitor, resulted in reduced levels of p‐MAPKAPK2 (control) and p‐STAT1 along with reduced HMGB1 release (B). A significant decrease in HMGB1 release was witnessed following cholangiocyte treatment with fludarabine, a STAT1 inhibitor (C) (*P < 0.05).

Fig. 5

HMGB1 release is ROS‐dependent. At 2 hours postinfection of cholangiocytes with RRV, a significant increase in ROS was measured by MFI. This increase was not observed by infection with Ro1845. Treatment with a ROS inhibitor, EUK‐8, significantly reduced the levels of ROS within the cells (A), without affecting the virus titer (B). Additionally, treatment with EUK‐8 was able to reduce the phosphorylation levels of p38 at 8 hours and HMGB1 release from the cells at 16 hours (C) (*P < 0.05). Abbreviation: FFU, focus‐forming unit.

Fig. 6

Glycyrrhizin treatment attenuates a murine model of BA. Newborn mice were infected with RRV (7.5 × 10⁵ focus‐forming units/pup) followed by injections of glycyrrhizin or saline on days of life 1, 3, 5, and 7. Symptoms were monitored for 21 days postinjection. There was a significant reduction in symptoms following glycyrrhizin treatment (A). A significant decrease in mortality was also observed in glycyrrhizin‐treated pups versus saline (36.4% versus 87.5% respectively; P < 0.05) (B). Glycyrrhizin treatment had no effect on viral titer (C) but did decrease both AST and ALT levels (D and E, respectively). Histological evaluation of the extrahepatic bile duct performed by hematoxylin and eosin 10 days postinfection with RRV demonstrated a complete obstruction, while, in contrast, the bile ducts from pups infected with RRV and treated with glycyrrhizin remained patent (×10 magnification) (F) (*P < 0.05). Abbreviation: FFU, focus‐forming unit.

Fig. 7

Liver immune cell profile following glycyrrhizin treatment. Histological evaluation performed by hematoxylin and eosin illustrated a reduced inflammatory response in the liver after glycyrrhizin treatment (A). Immunohistochemistry was used to quantify specific immune cells, which demonstrated a significant reduction in macrophages stained with CD68 (B) and NK cells stained with CD49b (C) in the glycyrrhizin‐treated pups; conversely, there was no difference in neutrophils stained with Ly6G (D) (*P < 0.05). Abbreviation: Ly6G, lymphocyte antigen 6 complex locus G.

Fig. 8

HMGB1 in human cholangiocytes and patients with BA. Infection with RRV and Ro1845^RRV(VP4) triggered the release of HMGB1 from human cholangiocytes (H69); conversely, Ro1845 failed to induce HMGB1 secretions, a pattern similar to that seen in the mCLs (A). A mean ± 1 standard deviation based on the logN of reference fluorescence units in healthy controls was used to identify a subpopulation of subjects with BA expressing higher levels of HMGB1 (BAhiH) versus the remaining patients (BAloH) (B). Comparison analysis of total bilirubin at 3 months post‐HPE showed higher levels in the BAhiH group versus the BAloH group, which approached significance (C). At 2 years post‐HPE a significantly lower percentage of patients initially in the BAhiH group retained their native liver compared to those in the BAloH group (D) (*P < 0.05). Abbreviation: RFU, relative fluorescence units.