*3.6. Vitamin C Deficient Neutrophils Exhibit Increased NFκB Activation*

The transcription factor NFκB modulates the expression of many immuno-regulatory mediators in the acute inflammatory response in sepsis. Yang *et al.* found that diminished nuclear translocation of NFκB in peripheral PMNs was associated with less time on the ventilator and improved survival in critically ill patients [32]. NFκB activation is also associated with increased ROS production and endoplasmic reticulum stress signaling [33]. Therefore, we examined nuclear translocation of NFκB in peritoneal PMNs isolated from VitC sufficient and deficient mice. As seen in Figure 6A, significantly increased NFκB translocation was observed in nuclei of VitC *deficient* PMNs (*p* < 0.05). Increased nuclear NFκB translocation was associated with induction of the NFκB dependent pro-inflammatory genes for TNFα and IL-1β (Figure 6B, *p* < 0.05).

**Figure 6.** Vitamin C deficient neutrophils exhibit increased NFκB activation. (**A**) Representative Western blot for nuclear expression of NFκBp65 and Lamin B from peritoneal PMNs of VitC sufficient and deficient Gulo−/− mice. Densitometry of NFκBp65/Lamin B from peritoneal PMNs of VitC sufficient and deficient Gulo−/− mice (*N* = 4 for each group, \* *p* < 0.05). (**B**) Real time QPCR for TNFα and IL-1β mRNA from peritoneal PMNs of VitC sufficient and deficient Gulo−/− mice, (*N* = 6 for each group, \* *p* < 0.05).

*3.7. Vitamin C Attenuates NET Formation in Activated Human Neutrophils*

Freshly isolated human PMNs formed NETs following activation by PMA (50 nM) for three hours as seen by immunofluorescence staining (Figure 7B,E). Loading the cells with VitC (3 mM) prior to PMA stimulation greatly reduced NET formation by human PMN (Figure 7C,F). Further, quantification of cf-DNA from the supernatants showed VitC (3 mM) loading significantly reduced NETs release from activated PMN (Figure 7G, *p* < 0.05).
