Citrullinated histone H3: A novel target for the treatment of sepsis

Abstract

We have recently demonstrated that in a rodent model of lipopolysaccharide (LPS)-induced shock, an increase in circulating citrullinated histone H3 (Cit H3) is associated with lethality of sepsis, and treatment with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor (HDACI), significantly improves survival. However, the role of Cit H3 in pathogenesis and therapeutics of sepsis are largely unknown. The present study was designed to test whether treatment with HDACI could inhibit cellular Cit H3 production, and inhibition of peptidylarginine deiminase (PAD, an enzyme producing Cit H3) with Cl-amidine (PAD inhibitor) or neutralization of blood Cit H3 with anti-Cit H3 antibody could improve survival in a clinically relevant mouse model of cecal ligation and puncture (CLP)-induced septic shock. Three experiments were carried out. In experiment I, HL-60 neutrophilic cells grown on a coverslip were treated with LPS (100 ng/mL) in the presence or absence of SAHA (5 μmol) for 3 hours, and subjected to immunostaining with anti-Cit H3 antibody to assess effect of SAHA on Cit H3 production under a fluorescence microscope. The ratio of Cit H3 positive cells was calculated as mean values ± SD (n = 3). In experiment II, male C57BL/6J mice were subjected to CLP, and 1 hour later randomly divided into 2 groups for intraperitoneal injection as follows: (1) Dimethyl sulfoxide (DMSO), (2) SAHA (50 mg/kg) in DMSO, and (3) Cl-amidine (80 mg/kg) in DMSO (n = 10/group). In experiment III, male C57BL/6J mice were divided into control and treatment groups, and subjected to CLP. Two hours later, immunoglobulin (Ig)G and Cit H3 antibody (20 mg/kg IV; n = 5/group) were injected into the control and treatment groups, respectively. Survival was monitored for ≤10 days. In experiment I, LPS induced Cit H3 production in the HL-60 cells, and SAHA treatment inhibited H3 citrullination significantly (P < .05). In experiment II, all vehicle-injected mice died within 3 days with increased circulating Cit H3 levels, whereas treatment with HDACI or Cl-amidine notably improved long-term survival (P < .01). In experiment III, administration of IgG did not improve survival, but a single treatment with Cit H3 specific antibody significantly improved survival (P < .014). Inhibition of HDAC or PAD significantly suppresses Cit H3 production in vitro and improves survival in vivo. Neutralization of Cit H3 significantly improves survival in septic mice. Collectively, our findings indicate for the first time that Cit H3 could not only serve as a potential biomarker, but also a novel therapeutic target in sepsis.