Biomarkers in endotoxemia with a special interest in citrullinated histone H3

Background: Sepsis with multi-organ failure has an unacceptably high mortality rate of 25-30% despite the use of antibiotics and modern intensive care. Attenuating the multi-organ failure and finding new biomarkers for early detection of septic shock would be of high clinical relevance in order to improve outcome.

Aim: The aim of this thesis was to study if inhaled nitric oxide (iNO) in combination with steroids could attenuate multi-organ failure in a porcine model of endotoxemia. We further aimed to investigate the dynamics of circulating citrullinated histone H3 (H3Cit), a recently proposed biomarker in sepsis, in a human model of endotoxemia. Since microvesicles (MVs) have been shown to be elevated in sepsis, a further objective of this thesis was to investigate whether H3Cit could be detected bound to MVs.

Methods: A randomized controlled trial (RCT) with 30 domestic piglets exposed to lipopolysaccharide (LPS)-alone, LPS + iNO, LPS + IV steroid, LPS + iNO + IV steroid or anesthesia only (Control) was conducted in paper I. Various biomarkers were measured at endpoint in order to evaluate organ function after 30 hrs of endotoxic shock. In paper II, an ELISA-based assay quantifying plasma H3Cit was developed and methodologically validated in accordance to recommended ELISA validation requirements. This ELISA, as well as a flow cytometric assay quantifying MV-bound H3Cit, was used in plasma samples at baseline and then 2, 4 and 7 hrs after LPS-injection in a placebo controlled RCT including 22 healthy volunteers in Paper III.

Results: LPS + iNO+ IV steroid tended to require less norepinephrine and were significantly less acidotic (p < 0.05) compared to LPS-only in the porcine model of endotoxemia. No significant differences could, however, be detected in other clinical variables. Circulating H3Cit, quantified by the ELISA assay, which was validated with high specificity, precision and stability, rose significantly after LPS injection in the human model of endotoxemia. Similar elevations were seen when quantifying H3Cit-bearing neutrophil- and platelet derived MVs.

Conclusions: Our data suggest that combined therapy with iNO and IV steroid is at least partially, protective after experimental LPS infusion. We furthermore show that circulating H3Cit, quantified by two distinct methods, is elevated after experimental LPS injection, suggesting that H3Cit may be a novel biomarker even in sepsis. Our data also show that H3Cit can be detected bound to MVs, proposing a novel mechanism by which H3Cit can be transported throughout the vasculature.

List of scientific papers

I. Paues Goranson S, Gozdzik W, Harbut P, Ryniak S, Zielinski S, Haegerstrand C. G, Kubler A, Hedenstierna G, Frostell C, Albert J. Organ dysfunction among piglets treated with inhaled Nitric Oxide and intravenous hydrocortisone during prolonged endotoxin infusion. PLoS One. 2014 May 14; 9(5):e96594.
https://doi.org/10.1371/journal.pone.0096594

II. Thålin C, Daleskog M, Paues Göranson S, Schatzberg D, Lasselin J, Laska AC, Kallner A, Helleday T, Wallén H, Demers M. Validation of an enzymelinked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma. Immunol Res. 2017 Jun; 65(3):706-712.
https://doi.org/10.1007/s12026-017-8905-3

III. Paues Göranson S, Thålin C, Lundström A, Hållström L, Lasselin J, Wallén H, Soop A, Mobarrez F. Circulating H3Cit is elevated in a human model of endotoxemia and can be detected bound to microvesicles. [Submitted]