*3.3. Autophagy Signaling Is Induced in Vitamin C Deficient Neutrophils*

Autophagy is a vital process for the catabolism of cytosolic proteins and organelles, but has also been shown to be required for NETosis [17,28]. To examine whether VitC regulates autophagy in PMNs we assessed the expression of several autophagy genes in thioglycollate elicited PMNs from VitC sufficient and deficient mice. As seen in Figure 3A, the expression of autophagy related signaling molecules (except for ATG6) were significantly elevated in the VitC *deficient* PMNs (*p* < 0.05).

**Figure 2.** Vitamin C deficient neutrophils show increased PAD4 mRNA. Real time QPCR for PAD4 shows two-fold increase in mRNA expression from peritoneal PMNs of VitC deficient Gulo−/− mice when compared to PMNs from VitC sufficient Gulo−/− mice (*N* = 6 for each group, \* *p* < 0.05).

Activation of the autophagic process causes lipidation of ATG8/LC3B (LC3B-I to LC3B-II conversion) and the lipid-modified LC3B-II translocates to autophagosomes. This LC3B-I to LC3B-II conversion is considered a critical marker of autophagy activation [29]. We observed significantly enhanced LC3B-I to LC3B-II conversion in cell lysates of VitC *deficient* PMNs by immunoblotting (Figure 3B, *p* < 0.05).

**Figure 3.** Autophagy signaling is induced in Vitamin C deficient neutrophils. (**A**) Real time QPCR for ATG3, ATG5, ATG6, ATG7, and ATG8 mRNA from peritoneal PMNs of VitC sufficient and deficient Gulo−/− mice, (*N* = 6 for each group, \* *p* < 0.05). (**B**) Representative Western blot for expression of LC3B-I and LC3B-II from peritoneal PMNs of VitC sufficient and deficient Gulo−/− mice. Densitometry of LC3B-II/actin from peritoneal PMNs of VitC sufficient and deficient Gulo−/− mice (*N* = 6 for each group, \* *p* < 0.05). (**C**) Representative Western blot for expression of p62 and actin from peritoneal PMNs of VitC sufficient and deficient Gulo−/− mice. Densitometry of normalized p62 expression from peritoneal PMNs of VitC sufficient and deficient Gulo−/− mice (*N* = 6 for each group, ns *p* = 0.3).

To further investigate the regulation of autophagy signaling by VitC in PMNs we examined the accumulation of p62/sequestosome I in these cell lysates. The loss of p62 in cells is typically indicative of increased autophagic activity [30]. Detection of p62 by immunoblotting showed a trend towards decreases p62 levels in the VitC *deficient* PMNs (Figure 3C). However this decline did not reach statistical significance (*p* = 0.3).

**Figure 4.** Endoplasmic reticulum stress associated gene expression in up-regulated in vitamin C deficient neutrophils. Real time QPCR for activating transcription factor 4 (ATF4), glucose-regulated protein 78 (Grp78, BiP), C/EBP homologous protein (CHOP), ER degradation-enhancing α-mannosidase-like protein (EDEM), X-box binding protein-1 spliced (XBP-1s), and unspliced (XBP-1un) mRNA from peritoneal PMNs of VitC sufficient and deficient Gulo−/− mice, (*N* = 6 for each group, \* *p* < 0.05).
