Extracellular histones are clinically associated with primary graft dysfunction in human liver transplantation

Abstract

Extracellular histones have been involved in numerous inflammatory conditions such as ischemia/reperfusion (I/R) injury, trauma, and infection. There is growing evidence of I/R injury associated with primary graft dysfunction (PGD) following organ transplantation. Here we investigated whether extracellular histones are clinically involved with PGD in human liver transplantation. In total 58 patients undergoing liver transplantation were studied. We collected blood samples from the recipients before and serially after transplantation (24 h, 72 h). We measured extracellular histones, myeloperoxidase (MPO), S100A8/A9, and multiple inflammatory cytokines. Additionally, we exposed human L02 hepatocytes or U937 monocytic cells to the recipient's sera overnight, and assessed cellular viability and cytokine production respectively. Lastly, we assessed the effect of histone-targeted interventions by administration of heparin or an anti-histone antibody. It showed that extracellular histones increased immediately after transplantation, peaked within 24 hours and remained at high levels up to 72 hours (all p < 0.01). Notably, extracellular histone levels were significantly higher in recipients with PGD (n = 9) than recipients without PGD (n = 49, p = 0.004). Extracellular histones correlated positively with MPO, S100A8/A9 and most detected cytokines. Ex vivo analysis demonstrated that the patients' sera after graft markedly induced L02 cell death and caused profound cytokine production in cultured U937 cells, which could be abrogated by heparin or an anti-histone antibody. Collectively, extracellular histones were increased significantly after liver transplantation, which may contribute to the occurrence of PGD through direct cytotoxicity and enhancement of systemic inflammation. Targeting extracellular histones may provide a promising approach for preventing PGD or other complications in clinical practice.

Fig. 1. Extracellular histones were significantly elevated in the sera of patients undergoing liver transplantation. (A) Sequential extracellular histone levels in patients undergoing liver transplantation at T0 (before graft), T1 (24 hours after graft) and T2 (within 72 hours after graft). (B) Median extracellular histones (T2) were significantly higher in patients with PGD than those in patient without PGD (p = 0.004). Variables were expressed as median (interquartile range).

Fig. 2. MPO (A) and S100A8/A9 (B) were increased in the sera of patients undergoing liver transplantation. As compared with T0 (before graft), both MPO and S100A8/A9 levels were increased remarkably at T1 (24 hours after graft), and showed a decreasing trend at T2 (within 72 hours after graft) but were still significantly higher than T0. Variables were expressed as median (interquartile range).

Fig. 3. Detection of systemic inflammation in the sera of patients undergoing liver transplantation. Multiplex immunoassay for a panel of multiple cytokines was performed. Only 10 cytokines with significant differences (*p < 0.01 in contrast to T0) were shown. #p < 0.01 denotes a significant difference between T1 and T2. Variables were expressed as median (interquartile range).

Fig. 4. Liver transplantation patients' sera induced human liver cell damage. It showed that the patients' sera at T1 that contained high levels of extracellular histones induced significant human liver cell damage after overnight incubation as compared with the sera collected at T0, whereas addition of anti-histone H4 antibody or heparin remarkably improved cell integrity. Variables were expressed as mean d standard deviation (SD).

Fig. 5. Liver transplantation patients' sera stimulated human monocytic cells. It showed that 6 histone-related cytokines were all notably increased in the supernatant of human monocytic U937 cells treated by liver transplantation patients' sera collected at T1 (24 hours after graft), whereas addition of anti-histone H4 antibody or heparin could decrease these cytokine levels (all p < 0.05). Variables were expressed as mean .0standard deviation (SD).