**1. Introduction**

Polymorphonuclear neutrophils (PMN) play key roles in the host response to pathogens by regulating innate host defenses and modulating inflammation. PMN combat pathogens by multiple mechanisms including phagocytosis, followed by exposure to reactive oxygen intermediates (short-lived and long-lived) in phagolysosomes [1], degranulation, which involves release of anti-bacterial peptides and proteases to kill pathogens [2], as well as production of cytokines and other inflammatory mediators. Aside from these traditional mechanisms, another mechanism for pathogen killing, the formation of neutrophil extracellular traps (NETs) by NETosis, a novel cell death pathway different from apoptosis and necrosis, was recently identified [3,4]. Although NETosis plays a crucial role in host defense during local infection by trapping and killing pathogens, excessive NET formation during systemic infections becomes self-defeating by promoting tissue injury and organ damage [5].

Sepsis, a leading cause of death with high mortality rates, is characterized by excessive inflammation and exuberant immune responses that lead to increased circulating PMN levels and extensive PMN sequestration in the lung. This massive influx of PMNs to the lungs often leads to acute lung injury (ALI) [6]. One postulated mechanism by which PMNs cause ALI is NETosis [7]. In sepsis, NETs are formed in response to pro-inflammatory stimuli such as lipopolysaccharide (LPS) and interleukin-8 (IL-8) [8,9] by expulsion of genomic DNA into web-like extracellular structures that display antimicrobial proteins such as histones, neutrophil elastase, and myeloperoxidase [10]. During NETosis, various signaling pathways lead to dissolution of nuclear envelope, thus allowing the mixing of nuclear chromatin with granular antimicrobial proteins from cytoplasmic granules, and then, by releasing the DNA into lattice-like structures, NETs concentrate proteases and antimicrobial proteins in the vicinity of trapped pathogens. However, in sepsis, exposure to NETs also produces organ injury. Indeed, Dwivedi *et al.* recently showed that NETosis, as determined by the circulating cell free DNA (cf-DNA) content, could predict ICU mortality in severe sepsis better than existing severity of illness or organ dysfunction scoring systems and was also better than IL-6, thrombin, and protein C [11]. While effective targeting or inhibition of NET structures has been suggested as therapy to benefit sepsis [8], identification of agents with the potential to alter NET formation remains elusive.

Vitamin C (VitC) is an essential vitamin for humans. While its role as an endogenous antioxidant is well recognized, our recent research suggests that VitC beneficially impacts multiple pathways associated with sepsis [12]. Its pleiotropic mechanisms including attenuation of the pro-inflammatory response, enhancement of epithelial barrier function, increasing alveolar fluid clearance, and prevention of coagulation abnormalities constitute a primary line of defense that is protective in sepsis syndromes [13]. Intracellular levels of VitC in various tissues differ significantly from circulating plasma levels with high cellular concentrations considered to be indicative of essential metabolic function [14]. In particular, VitC accumulates in millimolar quantities in PMNs where it regulates neutrophil apoptosis [14,15]. We recently showed that VitC attenuated neutrophiliccapillaritis and improved survival in murine sepsis models [12,13]. However, whether VitC alters NETosis in sepsis settings remains unknown. Humans lack functional L-gulono-γ-lactone oxidase (Gulo), the final enzyme in the biosynthesis of VitC [16]. In contrast, mice express functional Gulo, resulting in tissues generally maintaining high levels of VitC. In order to translate data from VitC studies in mice to humans we have examined NETosis in septic mice lacking Gulo (Gulo−/−). Our studies show that VitC sufficiency attenuated NETosis in septic mice. Importantly, at a cellular level, we show that VitC deficient PMN were more susceptible to undergo NETosis through increased activation of endoplasmic reticulum (ER) stress and autophagy, processes considered vital for NETosis [17]. VitC deficient PMNs displayed increased expression of peptidylargininedeiminase 4 (PAD4), the key enzyme required for hypercitrullination of histones and chromatin decondensation [18]. Moreover, our studies show that the pro-survival transcription factor nuclear factor kappa B (NFκB) was augmented in the VitC deficient PMNs while apoptosis was suppressed. The inhibitory effect of VitC on NETosis was recapitulated in phorbolmyristate acetate (PMA) activated human PMN.
