**4.2 Phagocytosis and microbial elimination**

The phagocyte function of ascorbic acid has been associated with: (a) chemotaxis' role that augments neutrophils migration after chemoattractants induction, (b) phagocytosis' action that builds up engulfment of pathogens, and (c) encourages free radical production and eliminates pathogens. Ascorbic acid helps caspase-dependent

apoptosis increasing macrophages uptake and elimination, and suppresses necrosis, including NETosis, thus helping resolution of the inflammatory response and regenerating cellular destruction [14, 16].

Neutrophils proceed to engulf the microbes after they have come to the area of inflammation. Antimicrobial peptides and proteins are emptied into the phagosome by intracellular granules that move and combine with the phagosome [14]. Superoxide, the initial product of ROS produced by neutrophils to eliminate microbes, is created when components of the Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase assemble in the phagosomal membrane. Superoxide is changed into hydrogen peroxide by the enzyme superoxide dismutase, which can subsequently be utilized by the azurophilic granule enzyme myeloperoxidase to generate the oxidant hypochlorous acid. In a subsequent reaction, hypochlorous acid can form chloramines, which are secondary oxidants. The sensitivities and specificities of these distinct neutrophil-derived oxidants for biological targets vary with protein thiol groups being particularly vulnerable [14–16].

Neutrophils isolated from scorbutic guinea pigs showed a capability damage to eliminate pathogens, and researches have showed that damaged phagocytosis and/ or free radical production in neutrophils from scorbutic compared with subjects with ascorbic acid supplementation [14]. The production of ROS by neutrophils from insufficient ascorbic acid level subjects can be elevated by 20% after administration with ascorbic acid, and elevates in phagocytosis and oxidant production were observed following administration of elderly volunteers with a combination of vitamins E. Patients with recurrent infections, or the genetic disorders such as CGD or CHS have damaged neutrophil bacterial elimination and/or phagocytosis, which can be significantly increased after administration with gram doses of ascorbic acid, which leads to sustained clinical improvement. Some of researches showed no development of ex vivo anti-microbial activity in neutrophils isolated from CGD or CHS patients with ascorbic acid. The cause for these variances is not yet understood, although it may relate on the baseline ascorbic acid concentration of the subjects, which is not evaluated in most studies. Additionally, different pathogens have different vulnerability to the oxidative and non-oxidative anti-microbial mechanisms of neutrophils. For instance, although other microbes are more sensitive to non-oxidative mechanisms, *Staphylococcus aureus* is sensitive to oxidative mechanisms. Therefore, the species of pathogens used to evaluate the ex vivo neutrophil ability could affect the studies [14, 16].

Patients with critical infection (sepsis) showed a reduced function to engulf pathogens and a decreased function to produce ROS, and furthermore increased patient death [14]. Interestingly, Stephan et al. (2002) postulated disturbance of neutrophil elimination function in critically ill individuals to get nosocomial infections, suggesting that critical diseases alone, without underlying infection, can also lower neutrophil capacity. As a result, the patients are susceptible to hospital-acquired infections. The impaired phagocytic and the capacity to produce ROS of leukocytes in sepsis subjects has been associated to the compensatory anti-inflammatory response, resulting in increased levels of immunosuppressive mediators such as IL-10, as well as to the hypoxic states of inflammatory areas, which reduce substrate for free radical production. The higher quantities of immature neutrophils that are produced from the bone marrow as a result of increased demands during sepsis are another factor. These immature neutrophils have reduced ability than differentiated neutrophils. Conflicting conclusions in sepsis could be due to the variation in the overall numbers of underactive immature neutrophils compared with activated fully-differentiated neutrophils. Despite having an activated basal state, the mature neutrophils from

subjects with sepsis do not produce ROS to the same amount as healthy neutrophils after ex vivo induction. The impact of ascorbic acid administration on phagocytosis, oxidant production, and pathogen elimination by leukocytes from critical infection subjects has not yet been studied [14–16].
