Identification of citrullinated histone H3 as a potential serum protein biomarker in a lethal model of lipopolysaccharide-induced shock

Abstract

Background Circulating proteins may serve as biomarkers for the early diagnosis and treatment of shock. We have recently demonstrated that treatment with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, significantly improves survival in a rodent model of lipopolysaccharide (LPS)-induced septic shock. Preliminary proteomic data showed that LPS-induced shock altered a number of proteins in circulation, including histone H3 (H3) and citrullinated histone H3 (Cit H3). The present study was designed to confirm these findings and to test whether the pro-survival phenotype could be detected by an early alteration in serum biomarkers. Methods Three experiments were performed. Exp 1: Western blotting was performed on serum samples from male C57B1/6J mice (n=9, 3/group) that belonged to following groups (a) LPS (20 mg/kg)-induced septic shock, (b) suberoylanilide hydroxanic acid (SAHA)-treated septic shock, and (c) sham (no LPS, no SAHA). Exp II: HL-60 granulocytes were cultured and treated with LPS (100 ng/ml) in the absence or presence of SAHA (10 μM). Sham (no LPS, no SAHA) granulocytes served as the control. The medium and cells were harvested at 3 h, and proteins were measured with Western blots. Exp III: LPS large dose (LD, 35 mg/kg) or small dose (SD, 10 mg/kg) was injected intraperitoneally into the C57B1/6J mice (n=10/group). Blood was collected at 3 h, and serum proteins were determined by Western blots or enzyme-linked immunosorbent assay (ELISA). All of the Western blots were performed with antibodies against H3, Cit H3, and acetylated H3 (Ac H3). ELISA was performed with antibody against tumor necrosis factor (TNF-α). Survival rates were recorded over 7 days. Results In Experiment I, intraperitoneal (i.p.) injection of LPS (20 mg/kg) significantly increased serum levels of H3, which was prevented by SAHA treatment. In Experiment II, LPS (100 ng/ml) induced expression and secretion of Cit H3 and H3 proteins in neutrophilic HL-60 cells, which was decreased by SAHA treatment. In Experiment III, administration of LPS (LD) caused a rise in serum H3 and Cit H3 but not Ac H3 at 3 h, and all of these animals died within 23 h (100% mortality). Decreasing the dose of LPS (SD) significantly reduced the mortality rate (10% mortality) as well as the circulating levels of Cit H3 (non detectable) and H3. An increase in serum TNF-α was found in both LPS (LD) and (SD) groups, but in a non dose-dependent fashion. Conclusions Our results reveal for the first time that Cit H3 is released into circulation during the early stages of LPS-induced shock. Moreover, serum levels of Cit H3 are significantly associated with severity of LPS-induced shock. Therefore, Cit H3 could serve as a potential protein biomarker for early diagnosis of septic shock, and for predicting its lethality.

PAD4-deficiency does not affect bacteremia in polymicrobial sepsis and ameliorates endotoxemic shock

“…Furthermore, supplemental Figure 3C shows histone release and fragmentation over time, similarly to that reported by Xu et al 9 Although necrotic cells also release nucleosomes in endotoxemia, 45 we saw that with increasing DNA release over time, H3Cit, a NET biomarker, became detectable in the plasma of PAD4 1/1 mice (supplemental Figure 3D), as was previously shown using low and high doses of LPS, 43 indicating that NETs are formed in our model. To investigate the effect of NETs in the LPS model, we compared PAD4 2/2 to PAD4 1/1 mice.…”

“…9,35 In vivo, histones contribute substantially to mortality in sepsis 9 and can induce thrombocytopenia. 42 In order to study the effect of NETs and endotoxemia in mice without a live bacterial infection, we turned to established models of LPS-induced shock 9,43 to generate large amounts of circulating nucleosomes in vivo. While not an ideal model of human sepsis, LPS infusion allowed us to investigate the host response leading to endotoxemic shock, which has been extensively studied experimentally.…”

Neutrophil extracellular traps in bronchial aspirates: a quantitative analysis

“…This observation supports the idea that citrullination of histone H3 is mandatory for the process of NETosis before nuclear decondensation. A previous report suggested that Cit H3 is released into the circulation during the early stage of lipopolysaccharide-induced shock and it is associated with the severity of shock in a mouse model [20]. Our data suggests that the quantity of Cit H3 has potential as a clinical biomarker of the severity of infection, independent from other well-known inflammatory biomarkers such as C-reactive protein and PCT.…”

Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis

“…This phenotype was abolished after neutrophil depletion and exogenous administration of DNAse 1 and NEi, implicating NETs as the likely source of extracellular DNA. The finding of widespread NET deposition following CLP is not unexpected, and extensive data in the literature exists that describes NET formation in vivo in the context of sepsis (48,59,60).…”