**5. Neutrophil necrosis and NETosis**

Neutrophil necrosis will be experienced by neutrophils that fail to face apoptosis, and the toxic parts (such as proteases) may cause tissue destruction. NETosis

is the form of neutrophil necrosis, that resulted from the release of "Neutrophil Extracellular Traps" (NETs) containing neutrophil DNA, histones, and enzymes [14, 21]. Even though, NETs have been advanced to involve a special way of pathogen elimination, they have also been involved in tissue destruction and organ failure. NET-associated histones can function as damage-associated molecular pattern proteins, inducing the immune system and accelerating destruction. Subjects in severe infection, or who go on to develop severe infection, have remarkably increased concentrations of circulating cell-free DNA, which is assumed to show NET production [14, 20].

Pre-clinical researches in ascorbic acid-deficient Gulo knockout mice exhibited increased NETosis in the lungs of severe infection subjects and enhanced circulating cell-free DNA [14]. The concentrations of these markers were influenced in subjects with normal level of ascorbic acid or in insufficient subjects that were supplemented with ascorbic acid. The same studies exhibited that in vitro administration of human neutrophils with ascorbic acid influenced phorbol ester-induced NETosis. Supplementation of gram doses of ascorbic acid to severe infection subjects over 4 days did not seem to reduce circulating cell-free DNA concentrations, although the length of supplementation may have been too early to look a remarkable impact. It should be highlighted that cell-free DNA is not specific for neutrophil-derived DNA, as it may also origin from necrotic tissue; however, the relationship of neutrophilspecific proteins or enzymes, such as myeloperoxidase, with the DNA can potentially give a sign of its origin [14, 15, 20].

Delaying apoptosis is the pathway of the transcription factor HIF-1α assists neutrophil survival at hypoxic regions. It's interesting to note that ascorbic acid is a cofactor for the iron-containing dioxygenase enzymes that modulate HIF-1α concentration and function. These hydroxylase enzymes inhibit HIF-1α function by facilitating the breakdown of constitutively expressed HIF-1α and preventing transcription coactivators from binding. In ascorbic acid-deficient Gulo knockout mice, HIF-1α up-regulation was reported in normal states, associated with the change of neutrophil apoptosis and elimination by macrophages [14]. Ascorbic acid may be able to inhibit the formation of NET by neutrophils by downregulating HIF-1α, which has also been proposed as a modulator of NET production by these cells [14, 20].

## **5.1 Lymphocyte function**

B- and T-lymphocytes accumulate ascorbic acid to high concentrations through SVCT, such as phagocytes [14]. The effect of ascorbic acid within lymphocyte is rarely understanding, although antioxidant protection has been recommended. Ascorbic acid will induce differentiation and maturation of lymphocytes and natural killer cells; and also increase Ig production [14, 15, 22].

Intraperitoneal ascorbic acid increased proliferation of lymphocytes and immunoglobulin concentrations in research using guinea pigs. Human experimental research reported correlation of immunoglobulin concentrations (IgM, IgG, and IgA) and ascorbic acid administration [14]. Other research by Anderson et al. (1980) reported that oral and intravenous administration of ascorbic acid to pediatric patients with asthma and control subjects increased lymphocyte differentiation, an ex vivo measure of mitogen-induced proliferation and enlargement of T-lymphocytes. Supplementation of ascorbic acid to aged subject was also exhibited to increase ex vivo lymphocyte proliferation, a result supported using combinations of ascorbic acid with vitamins A and/or E. Exposure to hazard materials can influence lymphocyte
