Intertwined roles for GDF-15, HMGB1, and MIG/CXCL9 in Pediatric Acute Liver Failure

Abstract

Introduction: Pediatric Acute Liver Failure (PALF) presents as a rapidly evolving, multifaceted, and devastating clinical syndrome whose precise etiology remains incompletely understood. Consequently, predicting outcomes-whether survival or mortality-and informing liver transplantation decisions in PALF remain challenging. We have previously implicated High-Mobility Group Box 1 (HMGB1) as a central mediator in PALF-associated dynamic inflammation networks that could be recapitulated in acetaminophen (APAP)-treated mouse hepatocytes (HC) in vitro. Here, we hypothesized that Growth/Differentiation Factor-15 (GDF-15) is involved along with HMGB1 in PALF.

Methods: 28 and 23 inflammatory mediators including HMGB1 and GDF15 were measured in serum samples from PALF patients and cell supernatants from wild-type (C57BL/6) mouse hepatocytes (HC) and from cells from HC-specific HMGB1-null mice (HC-HMGB1^-/-) exposed to APAP, respectively. Results were analyzed computationally to define statistically significant and potential causal relationships.

Results: Circulating GDF-15 was elevated significantly (P < 0.05) in PALF non-survivors as compared to survivors, and together with HMGB1 was identified as a central node in dynamic inflammatory networks in both PALF patients and mouse HC. This analysis also pointed to MIG/CXCL9 as a differential node linking HMGB1 and GDF-15 in survivors but not in non-survivors, and, when combined with in vitro studies, suggested that MIG suppresses GDF-15-induced inflammation.

Discussion: This study suggests GDF-15 as a novel PALF outcome biomarker, posits GDF-15 alongside HMGB1 as a central node within the intricate web of systemic inflammation dynamics in PALF, and infers a novel, negative regulatory role for MIG.

Keywords: biomarker; inflammation; network analysis; serum; systems biology.

FIGURE 1

Time-dependent release of GDF-15 in PALF patients. (A) Serum samples from PALF patients [spontaneous survivors (n = 14) and non-survivors (n = 7)] were assessed for GDF-15 using Luminex technology as described in Materials and Methods. Results represent the mean ± SEM, analyzed by Mann-Whitney Rank Sum test (*P < 0.001) as described. (B) Analysis of AUC ROC for HMGB1 and GDF-15 in PALF suggests that GDF-15 levels might be more helpful in prognosticating survival. Fig. shows the ROC curves and serum levels (survivors vs. non-survivors) of HMGB1 (left) and GDF-15 (right) calculated using MetaboAnalyst as described in Materials and Methods. The black dots represent the concentrations of the selected feature (e.g., HMGB1 protein) from all samples (all time points) in each patient group. The notch indicates the 95% confidence interval around the median of each group, defined as ± 1.58*IQR/sqrt(n). The mean concentration of each group is indicated with a yellow diamond. The horizontal red line represents an optimal cutoff calculated automatically by the algorithm. (C) Volcano Plot Analysis of statistical significance vs. magnitude of change in the inflammatory mediator data obtained by Luminex™ shows GDF-15 as one of the top genes that surpassed the fold change threshold (set at 2.0) and exhibited the smallest P-values (significance set at P < 0.05).

FIGURE 2

Dynamic Bayesian Network (DyBN) analysis of circulating inflammatory mediators in PALF patients. Circulating inflammatory mediators in serum samples from PALF spontaneous survivors (S, n = 14 patients) and non-survivors (NS, n = 7 patients) were measured and DyBN analysis was performed as described in Materials and Methods. Inflammatory mediators are shown as nodes, and the arrows connecting them suggest an influence of one mediator on the one(s) to which it is connected. The arrows do not distinguish positive from negative influences of one mediator on another. Semi-circular arrows suggest either positive or negative feedback of a given mediator on itself.

FIGURE 3

Dynamic Network Analysis (DyNA) of inflammatory mediators in PALF patients. (A) An overview of all the dynamic networks and mediator connections over seven time-intervals (d0-d1, d1-d2, d2-d3, d3-d4, d4-d5, d5-d6, d6-d7) of two PALF patient subgroups determined by DyNA (stringency level 0.85) as described in Materials and Methods. Closed red circles represent mediators directly connected to GDF-15. Black and red lines connecting two mediators represent positive and negative correlations, respectively. Panel (B) shows the complexity of networks shown in Panel (A) and Panel (C) highlights the detailed HMGB1, GDF-15 and MIG connectivity resulting from DyNA in PALF non-survivors and survivors calculated as described in Materials and Methods.

FIGURE 4

Circulating levels of GDF-15 in PALF patients and primary mouse hepatocytes in the context of APAP toxicity. (A) Time‐dependent release of GDF-15 in PALF survivors diagnosed with APAPo (n = 3) or non-APAP (n = 11) as described in Figure 1 and analyzed by Mann-Whitney Rank Sum test (*P < 0.05) as described. (B) Supernatant levels of HMGB1, GDF-15 and MIG (measured using Luminex technology as described in Materials and Methods) in cultures of mouse hepatocytes treated with 10 mM APAP for the times indicated. Cells were from n independent experiments/animas as follows: C57BL/6 [Control (n = 3), APAP (n = 3); HMGB1^−/−: Control [n = 4], APAP (n = 5)]. Results represent the mean ± SEM, analyzed by Two‐Way ANOVA (*P < 0.05).

FIGURE 5

Dynamic Network Analysis (DyNA) of inflammatory mediators in mouse hepatocytes. Cell supernatants from freshly isolated mouse hepatocytes from C57BL/6 mice or HC-HMGB1^−/− with or without 10 mM APAP treatment for 1–48 h were assayed for 23 inflammatory mediators as described in Materials and Methods. Panels (A, B) show an overview of all the dynamic networks (stringency level 0.85) and mediator connections over four time-intervals (1–3, 3–6, 6–24, and 24–48 h) for control and APAP-treated C57BL/6 HCs and HC-HMGB1^−/−, respectively. Red and yellow circles represent mediators connected to other mediators and mediators without specific connections, respectively. Panels (C, D) highlight the detailed GDF-15 connectivity resulting from DyNA in C57BL/6 HCs and HC-HMGB1−/−, respectively. Panel (E) displays the total number of connections for HMGB1, GDF-15 and MIG across all experimental groups. These connections were determined and calculated according to the methods outlined in the Materials and Methods section.