B. Pan1, B. Liu1, E. Chen1, Y. Wang2, Y. Liang1, Y. Li1, H. B. Alam1 1University Of Michigan Hospital,General Surgery/Surgery/Medical School,Ann Arbor, MI, USA 2Penn State University,Department Of Biochemistry And Molecular Biology,University Park, PA, USA
Introduction: Usefulness of many biomarkers is limited by their short half-life in the circulation. We previously showed that citrullinated histone H3 (CitH3) is a potential diagnostic biomarker and mediator in sepsis, but it is unclear if it persists in blood long enough to be clinically useful. The present study was designed to test the hypothesis that circulating CitH3 is a long-lasting biomarker, and that treatment with YW3-56, a peptidylarginine deiminase-4 (PAD4) inhibitor, can improve survival in a lethal lipopolysaccharide (LPS) induced shock model while attenuating blood levels of CitH3.
Methods: Three experiments were carried out. In experiment I, CitH3 specific enzyme-linked immunosorbent assay (ELISA) was established by our laboratory. A 96-well plate was coated with CitH3 monoclonal antibody overnight, incubated with mouse serum for 2 hours (h) followed by incubation with CitH3 polyclonal antibody and HRP-linked anti-rabbit antibody. Synthesized CitH3 was used to generate a standard curve (0–20 ng/ml). In experiment II, C57BL/6J mice were randomized to the following 3 groups with intraperitoneal injection of different reagents (n=7/group): (1) Dimethyl sulfoxide (DMSO), (2) LPS (35 mg/kg) +DMSO, and (3) LPS+YW3-56 (5 mg/kg) dissolved in DMSO (post LPS). Survival was monitored for 10 days. In experiment III, mice were subjected to the same treatment as the experiment II, and blood samples were collected at 0, 0.5, 3, 12, and 24 h after treatment. Plasma CitH3 were measured by both Western blot and ELISA. ANOVA was used for multiple comparisons and Kaplan-Meier curves for survival.
Results: All mice in the DMSO group survived. The LPS injection was universally lethal, with the majority of deaths within 24 h. YW3-56 treatment significantly improved survival compared to the LPS alone group (p<0.0001). No circulating CitH3 was found in DMSO group. LPS injection was associated with elevated plasma CitH3 at 0.5, 3, 12, and 24 h (87pg/ml, 245 pg/ml, 1180 pg/ml, and 318 pg/ml, respectively) with peak level at the 12-h time point. YW3-56 treatment significantly attenuated LPS-induced CitH3 levels in blood (13pg/ml, 73pg/ml, 183pg/ml, and 3pg/ml, respectively), compared to the untreated LPS group. The elevated CitH3 was detectible by Western blot and ELISA, but only ELISA could accurately quantify the concentration of blood CitH3 (Figure 1).
Conclusion: Our study demonstrated for the first time that (1) the CitH3 specific ELISA developed by us is a more reliable and accurate quantitative method than Western blot; (2) CitH3 protein in the peripheral blood is an ideal biomarker for sepsis because it responds to an insult early (0.5 h), lasts long, and is responsive to therapeutic interventions.
