*3.1. Vitamin C Sufficient Mice Demonstrate Reduced Lung NETs and Lower cf-DNA Following Peritonitis-Induced Sepsis*

We have previously shown that fecal peritonitis promotes PMN infiltration of the lungs in VitC deficient mice [19]. Here we used immunofluorescence staining and DIC microscopy to examine the extent of NETs in lungs of mice following FIP induced sepsis. Immuno-positive staining for platelet CD-41 (green), nuclear histones (red), and myeloperoxidase (grey) are visible in the lungs of saline exposed mice (Figure 1A). No appreciable immuno-positive staining differences were seen in the lungs of saline exposed VitC deficient mice (Figure 1C). FIP induced a mild increase in CD-41 immuno-positivity as well as some cytosolic histone staining (Figure 1B). However, no significant histological changes were evident in the VitC sufficient septic mice. In contrast, FIP induced significant NETs in VitC deficient mice as evidenced by dramatically increased co-staining for platelet CD-41 (green), histones (red), and myeloperoxidase (grey) in the vascular and alveolar spaces of septic mice (arrowheads, Figure 1D). Moreover, extensive extra-nuclear staining of histones (arrows) is also evident in this representative section along with thickened alveolar walls. Importantly, FIP exposed vitamin C deficient mice treated with ascorbic acid exhibited significant attenuation of NETs (Figure 1E).

In order to quantify NETs we determined levels of cf-DNA in the serum of VitC sufficient and deficient mice 16 h after sham treatment or FIP. Levels of serum cf-DNA were significantly elevated in the FIP exposed VitC deficient mice (Figure 1F, 5-fold, *p* < 0.05). Treatment of septic VitC deficient mice with ascorbic acid significantly lowered the cf-DNA values to control levels (*p* < 0.05). In addition peritoneal neutrophils from vitamin C deficient mice were more susceptible to NETosis than those from vitamin C deficient mice (Supplementary Material, Figure S1).
