**Vitamin C: A Novel Regulator of Neutrophil Extracellular Trap Formation**

**Bassem M. Mohammed 1,2, Bernard J. Fisher 3 , Donatas Kraskauskas <sup>3</sup> , Daniela Farkas <sup>3</sup> , Donald F. Brophy <sup>1</sup> , Alpha A. Fowler III <sup>3</sup> and Ramesh Natarajan 3,\*** 


*Received: 13 May 2013; in revised form: 30 July 2013 / Accepted: 5 August 2013 / Published: 9 August 2013* 

**Abstract: Introduction**: Neutrophil extracellular trap (NET) formation was recently identified as a novel mechanism to kill pathogens. However, excessive NET formation in sepsis can injure host tissues. We have recently shown that parenteral vitamin C (VitC) is protective in sepsis. Whether VitC alters NETosis is unknown. **Methods**: We used Gulo−/− mice as they lack the ability to synthesize VitC. Sepsis was induced by intraperitoneal infusion of a fecal stem solution (abdominal peritonitis, FIP). Some VitC deficient Gulo−/− mice received an infusion of ascorbic acid (AscA, 200 mg/kg) 30 min after induction of FIP. NETosis was assessed histologically and by quantification for circulating free DNA (cf-DNA) in serum. Autophagy, histone citrullination, endoplasmic reticulum (ER) stress, NFκB activation and apoptosis were investigated in peritoneal PMNs. **Results**: Sepsis produced significant NETs in the lungs of VitC deficient Gulo−/− mice and increased circulating cf-DNA. This was attenuated in the VitC sufficient Gulo−/− mice and in VitC deficient Gulo−/− mice infused with AscA. Polymorphonuclear neutrophils (PMNs) from VitC deficient Gulo−/− mice demonstrated increased activation of ER stress, autophagy, histone citrullination, and NFκB activation, while apoptosis was inhibited. VitC also significantly attenuated PMA induced NETosis in PMNs from healthy human volunteers. **Conclusions**: Our *in vitro* and *in vivo* findings identify VitC as a novel regulator of NET formation in sepsis. This study complements the notion that VitC is protective in sepsis settings.

**Keywords:** vitamin C; sepsis; neutrophils; NETosis; cell free DNA; nuclear factor κB
