**4. Discussion**

In this study we show that VitC could play a critical role in regulating the ultimate fate of PMNs in sepsis. Activated PMNs undergo extensive NETosis in septic mice lungs, resulting in potential damage to lung alveolar and endothelial cells. This effect was predominant in PMNs from VitC deficient mice and could be rescued by VitC infusion after the onset of sepsis. In contradistinction, PMNs from VitC sufficient mice underwent attenuated NETosis. Importantly, at a molecular level, VitC deficient peritoneal PMNs were likely to be more pro-inflammatory, to resist apoptosis, and to preferentially undergo NETosis.

**Figure 7.** Vitamin C attenuates NET formation in activated human neutrophils. Representative image of immunofluorescent staining for NETs in human neutrophils: DNA (blue); myeloperoxidase (green). **Upper Panels**: Control PMNs (**A**, 20×); PMNs exposed to PMA (50 nM) for 3 h (**B**, 20×); PMNs loaded with VitC (3 mM) for 1 h and then exposed to PMA (50 nM) for 3 h (**C**, 20×). **Lower Panels**: Control PMNs (**D**, 60×); PMNs exposed to PMA (50 nM) for 3 h (**E**, 60×); PMNs loaded with VitC (3 mM) for 1 h and then exposed to PMA (50 nM) for 3 h (**F**, 60×). (*N* = 3 for each group, Magnification: upper panel 20×, lower panel 60×). (**G**) Quantification of cf-DNA in the supernatants above (*N* = 3 for each group, *p* < 0.05).

Although several signaling mechanisms responsible for NET formation have been reported, critical regulatory elements remain unidentified. This study advances our understanding of PMN function and NET biology by identifying a novel regulatory mechanism for NET formation in both murine and human PMNs. Using our previously well-characterized model of abdominal peritonitis induced sepsis we show that sepsis promotes NET formation in lungs of VitC deficient mice (Figure 1). NETosis in this model was accompanied by increased circulating cf-DNA (Figure 1F). VitC

sufficiency or infusion of VitC after initiation of sepsis significantly decreased NETosis in lungs and circulating cf-DNA content (Figure 1). NET formation in VitC deficient peritoneal PMNs required activation of well characterized signaling pathways including ROS generation (data not shown), activation of the peptidylargininedeiminase PAD4 (Figure 2), autophagy (Figure 3), endoplasmic reticulum stress (Figure 4), and inhibition of apoptosis (Figure 5). NFκB, a pro-inflammatory, pro-survival transcription factor was activated in the VitC deficient peritoneal PMNs (Figure 6). VitC sufficiency or treatment with VitC attenuated these signaling pathways in PMNs.

Intracellular chromatin decondensation is essential for NET formation. Chromatin decondensation is brought about by peptidylargininedeiminase 4 (PAD4), a nuclear enzyme that deiminates arginine residues on histone tails thereby converting positively charged arginines to uncharged citrullines [17,18]. The importance of PAD4 is that many NET forming stimuli including PMA, LPS, and IL-8 as well as various bacterial and fungal species converge to its activation. While PAD4 is expressed in PMNs and is localized to the nucleus [26,27], little is known about its mechanism of action or its transcriptional regulation. Ying *et al.* have shown that PAD1, which belongs to the same family of enzymes as PAD4, is transcriptionally regulated by NFκB [34]. We have previously shown that VitC blocks NFκB activation in septic mouse lungs [12]. Cárcamo *et al*. also demonstrated that VitC blocks IκB kinase activity and NFκB activation [35]. In this study we observed that nuclear NFκB levels were higher in the VitC deficient PMNs (Figure 6A). Further, PAD4 mRNA expression was also significantly higher in PMNs from VitC deficient mice (Figure 2). Therefore, we hypothesize that VitC decreases PAD4 expression by suppressing NFκB activation in PMNs. Further, by decreasing PAD4 expression VitC could decrease histone citrullination activity and therefore chromatin decondensation in VitC sufficient PMNs.

Autophagy has been identified as a well-conserved, homeostatic mechanism that clears damaged organelles or proteins and plays an essential role in cell survival during periods of nutrient depletion [36]. Despite the view that it might not occur in neutrophils, autophagy was recently shown to occur both in murine and human PMNs [37,38]. While Mitroulis *et al*. reported that autophagy occurs in human PMNs in response to PMA activation [39]. Remijsen *et al*. have shown that autophagy is necessary for the induction of intracellular chromatin decondensation during PMA-induced NETosis [17]. In our study, we found increased expression of autophagy genes (Figure 3A) as well as significantly enhanced LC3B-I to LC3B-II conversion in VitC deficient PMNs (Figure 3B) indicative of the presence of more autophagosomes in VitC deficient PMNs. However, LC3B-I to LC3B-II conversion is a static measure of autophagosome number, and does not measure the actual activity of the pathway. The increased LC3B-II could be interpreted as either high autophagic activity or a downstream block in the system that results in an accumulation of LC3B-II protein, even though autophagic degradation itself is diminished. To supplement our observations we examined levels of p62/sequestosome I, a cytosolic chaperone protein with an LC3B binding domain [40]. The normal function of p62 protein is to carry polyubiquitinated proteins to the autphagolysosome where it binds to LC3B before getting degraded. Thus, the loss of p62 protein is a measure of the flux of autophagy and indicative of increased autophagy [41]. In our studies we found a trend towards decreased p62 levels in the VitC deficient PMNs (Figure 3C). While this decline did not reach statistical significance, in combination with the increased autophagy gene expression and increased LC3B conversion, our data imply increased autophagy in VitC deficient PMNs.

The unfolded protein response (UPR) and autophagic machinery have been shown to be critically linked to each other. It is well established that activation of the UPR genes transcriptionally up-regulates several autophagy related genes required for induction and construction of the autophagy machinery [42]. However, it is not known whether activation of the UPR drives autophagy and eventually leads to NET formation in PMNs. Our study shows that most of the UPR genes examined except for CHOP were significantly up-regulated in PMNs from VitC deficient mice (Figure 4). This implies that VitC deficient PMNs could be actively undergoing ER stress, which in turn could drive autophagy genes and increase their susceptibility to undergo NETosis.

The transcription factor NFκB is central to pro-inflammatory/pro-survival responses in sepsis. It is normally sequestered to IκB in the cytosol. Upon appropriate stimulation, IκB is degraded allowing NFκB to migrate to the nucleus and drives transcription of numerous genes that regulate the immune response in sepsis. Moine *et al*. have demonstrated increased NFκB translocation in the lungs of patients with ALI [43]. Yang *et al*. found that increased nuclear levels of NFκB in unstimulated neutrophils were associated with a worse clinical outcome [32]. As discussed above, NFκB likely drives expression of PAD4 in PMNs. NFκB activation also drives expression of pro-survival genes [44]. In this study we found that nuclear NFκB translocation was higher in VitC deficient PMNs (Figure 6A). Further, NFκB translocation in these VitC deficient PMNs increased expression of the pro-inflammatory genes TNFα and IL-1β (Figure 6). NFκB activation also inhibited apoptosis as seen by the reduced activation of caspase 3 in VitC deficient PMNs (Figure 5). These results suggest that NFκB may play a critical role in modulating cell signaling pathways that eventually regulate the fate of PMNs. By activating PAD4 (chromatin decondensation), inducing ER stress and subsequent autophagy, and inhibiting apoptosis, NFκB may drive the cellular machinery of VitC deficient PMNs towards NET formation (Figure 8). VitC sufficiency or infusion of VitC allows PMNs to increase intracellular levels of VitC and attenuate NFκB activation. This could dampen the pathways required for NETosis and may allow PMNs to undergo apoptosis instead. While the decreased apoptosis rate in VitC deficient PMNs may benefit the host by giving more time for PMNs to perform their innate immune functions, studies show that it could also be detrimental in sepsis due to the PMN-dependent inflammation and tissue damage that could be heightened by a prolonged lifespan. Recent reports in the literature have implicated NETs in transfusion-related acute lung injury (TRALI), the leading cause of death after transfusion therapy [45,46]. NETs were shown to be present during TRALI both in mice and humans and so it was suggested that targeting NET formation may be a new approach for the treatment of acute lung injury. While we did not examine TRALI in our studies, it is conceivable that VitC infusion could be a useful adjunct for the prevention/treatment of TRALI or other disease states involving exuberant formation of NETs particularly in the lungs.

Our study has several limitations: (1) It is possible that the PMNs isolated within the peritoneal cavity by thioglycollate could be partially activated; (2) We examined PMN function *ex vivo*. Further *in vivo* studies are needed to characterize the fate of PMNs; (3) others have performed studies with PMNs isolated from bone marrow instead of thioglycollate elicitation. These PMNs would be less "activated" when compared to thioglycollate elicited PMNs, but would also have a large component of immature PMNs which have been shown to behave somewhat differently from mature PMNs [47].

**Figure 8.** Schematic hypothesis of regulation of signaling pathways that leads to NETosis by VitC. Septic stimuli activate NFκB in PMNs with increased activation observed in VitC deficient PMNs. NFκB nuclear translocation drives expression of PAD4, ER stress and autophagy signaling genes while inhibiting caspase 3 in activated PMNs. This drives the fate of activated PMN away from apoptosis and enhances NETosis. VitC likely blocks up-regulation of PAD4, ER stress and autophagy signaling genes by decreasing NFκB activation. Activated PMNs now undergo apoptosis while NETosis is attenuated.
