Prognostic value of plasma exosomal levels of histone H3 protein in patients with heat stroke

Abstract

Heat stroke (HS) is a condition that can lead to multiple organ dysfunction syndrome and death; however, there is no reliable method for stratifying mortality risk in HS. The abundance of exosomes in the circulation and their contents may be used as potential biomarkers of HS. The present study aimed to examine whether histone H3 levels in plasma exosomes could be used to determine HS prognosis. Blood samples were collected from patients with HS (36 survivors and 8 non-survivors) at admission to the intensive care unit and 4 days after admission. Blood samples were additionally collected from 15 healthy volunteers. Plasma exosomes were isolated using high-speed differential centrifugation. Correlation between histone H3 level and organ function and disease severity was examined. The results suggested differential expression and enrichment of histone H3 in the plasma exosomes of patients with HS (survivors, 249.3±04.6; non-survivors, 500.4±216.8; healthy controls, 161.1±52.49 pg/100 µg; P<0.05). The increased expression of histone H3 was associated with increased disease severity and duration. Plasma exosomal levels of histone H3 were significantly correlated with both organ dysfunction and disease severity (P<0.0001) and were significantly different between non-survivors and survivors (area under the receiver operating characteristic curve, 0.9668). A cutoff value of 307 pg/100 µg demonstrated optimized sensitivity (95%) and specificity (91.67%) for predicting mortality risk, suggesting that histone H3 levels in plasma exosomes may be a reliable biomarker for HS prognosis.

Keywords: biomarker; exosome; heat stroke; histone; multiple organ failure.

Figure 1

Characterization of plasma exosomes in healthy controls and surviving and non-surviving patients with HS. (A) Morphology of plasma exosomes visualized under transmission electron microscopy. White arrow indicates the bilayer round vesicles of ~100 nm in diameter (scale bar=100 nm). (B) Size distribution of exosomes detected by nanoparticle tracking analysis. (C) Expression of the characteristic exosomal surface markers CD9, CD63 and CD81 following western blot analysis. All experiments were repeated three times. HS, heat stroke.

Figure 2

Histone H3 levels in plasma exosomes and free plasma of healthy controls and patients with HS (survivors and non-survivors) on days 1 and 4. Plasma exosomal levels of histone H3 were significantly higher in non-survivors than in healthy controls and survivors on both days 1 and 4. Plasma exosomal levels of histone H3 in non-survivors were significantly higher on day 4 than on day 1. Plasma histone H3 levels on day 1 and 4 were significantly higher in the HS non-survivors than healthy controls. There were no significant changes in plasma exosome histone H3 levels between survivors and healthy controls both on day 1 and 4. On day 4, plasma free histone H3 levels were elevated in non-survivors and remain unchanged in survivors compared to day 1. HS, heat stroke. ^*P<0.05, ^**P<0.01, ^****P<0.0001.

Figure 3

ROC curves for the discrimination of survivors and non-survivors among patients with HS. (A) ROC curve of plasma exosomal levels of histone H3 for discriminating between survivors and non-survivors among patients with HS. (B) ROC curve of plasma AST for the discrimination of survivors and non-survivors among patients with HS. (C) ROC curve of plasma ALT for the discrimination of survivors and non-survivors among the patients with HS. ROC, receiver operator characteristic; HS, heat stroke; AST, aspartate aminotransferase; ALT, alanine aminotransferase; AUC, area under the curve; CI, 95% confidence interval.