Anitra C. Carr, and Silvia Maggini
Vitamin C is an essential micronutrient for humans, with pleiotropic functions related to its ability to donate electrons. It is a potent antioxidant and a cofactor for a family of biosynthetic and gene regulatory enzymes. Vitamin C contributes to immune defense by supporting various cellular functions of both the innate and adaptive immune system. Vitamin C supports epithelial barrier function against pathogens and promotes the oxidant scavenging activity of the skin, thereby potentially protecting against environmental oxidative stress. Vitamin C accumulates in phagocytic cells, such as neutrophils, and can enhance chemotaxis, phagocytosis, generation of reactive oxygen species, and ultimately microbial killing. It is also needed for apoptosis and clearance of the spent neutrophils from sites of infection by macrophages, thereby decreasing necrosis/NETosis and potential tissue damage. The role of vitamin C in lymphocytes is less clear, but it has been shown to enhance differentiation and proliferation of B- and T-cells, likely due to its gene regulating effects. Vitamin C deficiency results in impaired immunity and higher susceptibility to infections.
In turn, infections significantly impact on vitamin C levels due to enhanced inflammation and metabolic requirements. Furthermore, supplementation with vitamin C appears to be able to both prevent and treat respiratory and systemic infections. In contrast, treatment of established infections requires significantly higher (gram) doses of the vitamin to compensate for the increased inflammatory response and metabolic demand.
Introduction
The immune system is a multifaceted and sophisticated network of specialized organs, tissues, cells, proteins, and chemicals, which has evolved in order to protect the host from a range of pathogens, such as bacteria, viruses, fungi, and parasites, as well as cancer cells.
Vitamin C is an essential nutrient which cannot be synthesized by humans due to loss of a key enzyme in the biosynthetic pathway. Severe vitamin C deficiency results in the potentially fatal disease scurvy. Scurvy is characterized by weakening of collagenous structures, resulting in poor wound healing, and impaired immunity. Individuals with scurvy are highly susceptible to potentially fatal infections such as pneumonia.
In turn, infections can significantly impact on vitamin C levels due to enhanced inflammation and metabolic requirements. Early on, it was noted that scurvy often followed infectious epidemics in populations, and cases of scurvy have been reported following respiratory infection.
The recommended dietary intakes for vitamin C are up to one hundred-fold higher than that for many other vitamins. There are several reasons why vitamin C dietary recommendations are not met, even in countries where food availability and supply would be expected to be sufficient. These include poor dietary habits, life-stages and/or lifestyles either limiting intakes or increasing micronutrient requirements (e.g., smoking and alcohol or drug abuse), various diseases, exposure to pollutants and smoke (both active and passive), and economic reasons (poor socioeconomic status and limited access to nutritious food). Even otherwise ‘healthy’ individuals in industrialized countries can be at risk due to lifestyle-related factors, such as those on a diet or eating an unbalanced diet, and people facing periods of excessive physical or psychological stress.
Vitamin C has a number of activities that could conceivably contribute to its immune-modulating effects. It is a highly effective antioxidant, due to its ability to readily donate electrons, thus protecting important biomolecules (proteins, lipids, carbohydrates, and nucleic acids) from damage by oxidants generated during normal cell metabolism and through exposure to toxins and pollutants (e.g., cigarette smoke).
Barrier Integrity and Wound Healing
Vitamin C is actively accumulated into the epidermal and dermal cells via the two sodium-dependent vitamin C transporter (SVCT) isoforms 1 and 2, suggesting that the vitamin has crucial functions within the skin. Clues to the role of vitamin C in the skin come from the symptoms of the vitamin C deficiency disease scurvy, which is characterized by bleeding gums, bruising, and impaired wound healing. These symptoms are thought to be a result of the role of vitamin C as a cofactor for the prolyl and lysyl hydroxylase enzymes that stabilize the tertiary structure of collagen. Further research has shown that vitamin C can also increase collagen gene expression in fibroblasts of the skin. The elevated antioxidant status of the skin following vitamin C supplementation could potentially protect against oxidative stress induced by environmental pollutants. The antioxidant effects of vitamin C are likely to be enhanced in combination with vitamin E. Cell culture and preclinical studies have indicated that vitamin C can enhance epithelial barrier functions via a number of different mechanisms.
Thus, vitamin C appears to be particularly essential during wound healing, also decreasing the expression of pro-inflammatory mediators and enhancing the expression of various wound healing mediators. Vitamin C is thought to influence several important aspects of neutrophil function: migration in response to inflammatory mediators (chemotaxis), phagocytosis and killing of microbes, and apoptosis and clearance by macrophages.
Vitamin C and Leukocyte Function
Leukocytes, such as neutrophils and monocytes, actively accumulate vitamin C against a concentration gradient, resulting in values that are 50- to 100-fold higher than plasma concentrations. These cells accumulate maximal vitamin C concentrations at dietary intakes of ~100 mg/day, although other body tissues likely require higher intakes for saturation.
Accumulation of millimolar concentrations of vitamin C into neutrophils, particularly following activation of their oxidative burst, is thought to protect these cells from oxidative damage.
Vitamin C is a potent water-soluble antioxidant that can scavenge numerous reactive oxidants and can also regenerate the important cellular and membrane antioxidants glutathione and vitamin E. Upon phagocytosis or activation with soluble stimulants, vitamin C is depleted from neutrophils in an oxidant-dependent manner. An alteration in the balance between oxidant generation and antioxidant defenses can lead to alterations in multiple signaling pathways.
3.1. Neutrophil Chemotaxis
Neutrophil infiltration into infected tissues is an early step in innate immunity. In response to pathogen- or host-derived inflammatory signals (e.g., N-formyl methionyl-leucyl-phenylalanine (fMLP), interleukin (IL)-8, leukotriene B4, and complement component C5a), marginated neutrophils literally swarm to the site of infection. Migration of neutrophils in response to chemical stimuli is termed chemotaxis, while random migration is termed chemokinesis. Neutrophils express more than 30 different chemokine and chemoattractant receptors in order to sense and rapidly respond to tissue damage signals. These findings suggest that vitamin C deficiency may impact on the ability of phagocytes to migrate to sites of infection. Patients with severe infection exhibit compromised neutrophil chemotactic ability. This neutrophil ‘paralysis’ is believed to be partly due to enhanced levels of anti-inflammatory and immune-suppressive mediators (e.g., IL-4 and IL-10) during the compensatory anti-inflammatory response observed following initial hyper-stimulation of the immune system.
Furthermore, supplementation of neonates with suspected sepsis with 400 mg/day vitamin C dramatically improved neutrophil chemotaxis. Recurrent infections can also result from genetic disorders of neutrophil function, such as chronic granulomatous disease (CGD), an immunodeficiency disease resulting in defective leukocyte generation of ROS, and Chediak-Higashi syndrome (CHS), a rare autosomal recessive disorder affecting vesicle trafficking.
3.2. Phagocytosis and Microbial Killing
Various intracellular granules are mobilized and fuse with the phagosome, emptying their arsenal of antimicrobial peptides and proteins into the phagosome. Components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase assemble in the phagosomal membrane and generate superoxide, the first in a long line of ROS generated by neutrophils to kill pathogens. The enzyme superoxide dismutase converts superoxide to hydrogen peroxide, which can then be utilized to form the oxidant hypochlorous acid via the azurophilic granule enzyme myeloperoxidase. Hypochlorous acid can further react with amines to form secondary oxidants known as chloramines. These various neutrophil-derived oxidants have different reactivities and specificities for biological targets, with protein thiol groups being particularly susceptible. Neutrophils isolated from scorbutic guinea pigs exhibit a severely impaired ability to kill microbes, and studies have indicated impaired phagocytosis and/or ROS generation in neutrophils from scorbutic compared with ascorbate replete animals.
3.3. Neutrophil Apoptosis and Clearance
Following microbial phagocytosis and killing, neutrophils undergo a process of programmed cell death called apoptosis. This process facilitates subsequent phagocytosis and clearance of the spent neutrophils from sites of inflammation by macrophages, thus supporting resolution of inflammation and preventing excessive tissue damage. Caspases are key effector enzymes in the apoptotic process, culminating in phosphatidylserine exposure, thus marking the cells for uptake and clearance by macrophages.
Thus, vitamin C may be expected to protect the oxidant-sensitive caspase-dependent apoptotic process following activation of neutrophils. In support of this premise, in vitro studies have shown that loading human neutrophils with vitamin C can enhance Escherichia coli-mediated apoptosis of the neutrophils. Plasma from septic patients has been found to suppress apoptosis in healthy neutrophils, suggesting that proinflammatory cytokines were responsible for the increased in vivo survival of neutrophils during inflammatory conditions. Interestingly, high-dose vitamin C administration has been shown to modulate cytokine levels in patients with cancer and, although this has not yet been assessed in patients with severe infection, could conceivably be another mechanism by which vitamin C may modulate neutrophil function in these patients.
3.4. Neutrophil Necrosis and NETosis
Neutrophils that fail to undergo apoptosis instead undergo necrotic cell death . The subsequent release of toxic intracellular components, such as proteases, can cause extensive tissue damage.
Patients with sepsis, or who go on to develop sepsis, have significantly elevated levels of circulating cell-free DNA, which is thought to indicate NET formation. Pre-clinical studies in vitamin C-deficient Gulo knockout mice indicated enhanced NETosis in the lungs of septic animals and increased circulating cell-free DNA. The levels of these markers were attenuated in vitamin C sufficient animals or in deficient animals that were administered vitamin C. Administration of gram doses of vitamin C to septic patients over four days, however, did not appear to decrease circulating cell-free DNA levels, although the duration of treatment may have been too short to see a sustained effect. It should be noted that cell-free DNA is not specific for neutrophil-derived DNA, as it may also derive from necrotic tissue; however, the association of neutrophil-specific proteins or enzymes, such as myeloperoxidase, with the DNA can potentially provide an indication of its source.
3.5. Lymphocyte Function
Like phagocytes, B- and T-lymphocytes accumulate vitamin C to high levels via SVCT. The role of vitamin C within these cells is less clear, although antioxidant protection has been suggested. In vitro studies have indicated that incubation of vitamin C with lymphocytes promotes proliferation, resulting in enhanced antibody generation, and also provides resistance to various cell death stimuli.
Furthermore, vitamin C appears to have an important role in developmental differentiation and maturation of immature T-cells. Similar proliferative and differentiation/maturation effects have been observed with mature and immature natural killer cells, respectively.
Administration of vitamin C to elderly people was also shown to enhance ex vivo lymphocyte proliferation, a finding confirmed using combinations of vitamin C with vitamins A and/or E. Exposure to toxic chemicals can affect lymphocyte function, and both natural killer cell activity and lymphocyte blastogenic responses to T- and B-cell mitogens were restored to normal levels following vitamin C supplementation. Although the human studies mentioned above are encouraging, it is apparent that more human intervention studies are needed to confirm these findings.
3.6. Inflammatory Mediators
Cytokines are important cell signaling molecules secreted by a variety of immune cells, both innate and adaptive, in response to infection and inflammation. They comprise a broad range of molecules, including chemokines, interferons (IFNs), ILs, lymphokines, and TNFs, which modulate both humoral and cell-based immune responses, and regulate the maturation, growth, and responsiveness of specific cell populations. Cytokines can elicit pro-inflammatory or anti-inflammatory responses, and vitamin C appears to modulate systemic and leukocyte-derived cytokines in a complex manner. Incubation of vitamin C with peripheral blood lymphocytes decreased lipopolysaccharide (LPS)-induced generation of the proinflammatory cytokines TNF-α and IFN-γ, and increased anti-inflammatory IL-10 production, while having no effect on IL-1β levels. Furthermore, in vitro addition of vitamin C to peripheral blood monocytes isolated from pneumonia patients decreased the generation of the proinflammatory cytokines TNF-α and IL-6.
Histamine is an immune mediator produced by basophils, eosinophils, and mast cells during the immune response to pathogens and stress. Histamine stimulates vasodilation and increased capillary permeability, resulting in the classic allergic symptoms of runny nose and eyes.
Vitamin C Insufficiency Conditions
Numerous environmental and health conditions can have an impact on vitamin C status. In this section we discuss examples which also have a link with impaired immunity and increased susceptibility to infection. For example, exposure to air pollution containing oxidants, such as ozone and nitrogen dioxide, can upset the oxidant-antioxidant balance within the body and cause oxidative stress. Oxidative stress can also occur if antioxidant defenses are impaired, which may be the case when vitamin C levels are insufficient. Air pollution can damage respiratory tract lining fluid and increase the risk of respiratory disease, particularly in children and the elderly who are at risk of both impaired immunity and vitamin C insufficiency. Vitamin C is a free-radical scavenger that can scavenge superoxide and peroxyl radicals, hydrogen peroxide, hypochlorous acid, and oxidant air pollutants.
Several immune-related changes are observed in obesity that contribute towards the development of type 2 diabetes. A major factor is persistent low-grade inflflammation of adipose tissue in obese subjects, which plays a role in the progression to insulin resistance and type 2 diabetes, and which is not present in the adipose tissue of lean subjects.
Acute and chronic diseases that are prevalent in this age group may also play an important part in the reduction of vitamin C reserves. Institutionalization in particular is an aggravating factor in this age group, resulting in even lower plasma vitamin C levels than in non-institutionalized elderly people. It is noteworthy that elderly hospitalized patients with acute respiratory infections have been shown to fare significantly better with vitamin C supplementation than those not receiving the vitamin.
Vitamin C and Infection
A major symptom of the vitamin C deficiency disease scurvy is the marked susceptibility to infections, particularly of the respiratory tract, with pneumonia being one of the most frequent complications of scurvy and a major cause of death. Patients with acute respiratory infections, such as pulmonary tuberculosis and pneumonia, have decreased plasma vitamin C concentrations relative to control subjects. Administration of vitamin C to patients with acute respiratory infections returns their plasma vitamin C levels to normal and ameliorates the severity of the respiratory symptoms.
This vitamin C-dependent clearance of neutrophils from infected lungs could conceivably be due to enhanced apoptosis and subsequent phagocytosis and clearance of the spent neutrophils by macrophages. Meta-analysis has indicated that vitamin C supplementation with doses of 200 mg or more daily is effective in ameliorating the severity and duration of the common cold, and the incidence of the common cold if also exposed to physical stress.
Beneficial effects of vitamin C on recovery have been noted in pneumonia. In elderly people hospitalized because of pneumonia, who were determined to have very low vitamin C levels, administration of vitamin C reduced the respiratory symptom score in the more severe patients. It is likely that the low vitamin C levels observed during respiratory infections are both a cause and a consequence of the disease.
Conclusions
Overall, vitamin C appears to exert a multitude of beneficial effects on cellular functions of both the innate and adaptive immune system. Although vitamin C is a potent antioxidant protecting the body against endogenous and exogenous oxidative challenges, it is likely that its action as a cofactor for numerous biosynthetic and gene regulatory enzymes plays a key role in its immune-modulating effects. Vitamin C stimulates neutrophil migration to the site of infection, enhances phagocytosis and oxidant generation, and microbial killing.
Thus, it is apparent that vitamin C is necessary for the immune system to mount and sustain an adequate response against pathogens, whilst avoiding excessive damage to the host. Vitamin C appears to be able to both prevent and treat respiratory and systemic infections by enhancing various immune cell functions. Increased needs occur due to pollution and smoking, fighting infections, and diseases with oxidative and inflammatory components, e.g., type 2 diabetes, etc. Ensuring adequate intake of vitamin C through the diet or via supplementation, especially in groups such as the elderly or in individuals exposed to risk factors for vitamin C insufficiency, is required for proper immune function and resistance to infections.
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