Inhalation of Ascorbic Acid Modulates Sinonasal Immune System

Abstract

Inhaled drugs, commonly used for the treatment of chronic lung disease, are intended to have their effect quickly reach the airways and are less absorbed into the bloodstream. As ascorbic acid can be dangerous at high dosages, the inhalation route can be a substitute for getting a rapid topical elevated level of ascorbic acid. Drug/vitamin with inhalation route has an advantage as a non-invasive route, minimal side-effect, fast-onset, no first-pass metabolism, and more potent pharmacodynamics drug. The relationship between the effect of ascorbic acid in the form of inhalation on the sinonasal mucosal immune system needs to be studied, considering the role of ascorbic acid in the prevention and plausible prognosis of pandemic era.

1. Introduction

Ascorbic acid is needed by the body as an antioxidant, protecting infection, and building the immune system. Oral or systemic routes of administration in high doses have the consequence of increasing blood plasma levels, so they must be excreted by the kidneys and produce darker yellow urine. Urine that is excess of ascorbic acid supplementation is excreted is termed “expensive urine.” Therefore, ascorbic acid supplementation by inhalation is an effective and efficient way in corona virus disease 2019 (COVID-19) pandemic. The hazard of transmission of the SARS CoV-2 virus through the air that hits the airway mucosa, can be anticipated to prevent it through increasing the mucosal immune system and anti-oxidative stress which has the potential to be contained in ascorbic acid.

Inhaled drugs, commonly used for the treatment of chronic lung disease, are intended to have their effect quickly reach the airways and are less absorbed into the bloodstream. Inhaled drugs, distributed via aerosol spray, mist, or powder that the patient inhales directly, which although the main effect of inhaled drugs is breathing, there may still be systemic effects as well.

Since time immemorial, there is a habit of squeezing the leaves, or fruit peels and inhaling the aroma for various diseases in the absence of sufficient information of pharmacokinetics and pharmacodynamics of this ingredients. Citrus fruit, in particular, has shown the effect for the inflammation and infections including the common cold, including the potential benefit of ascorbic acid administration against the replication inhibitory activity of the SARS CoV-2 virus. The impact of ascorbic acid in the form of inhalation to the sinonasal mucosal immune system needs to be studied, considering the impact of ascorbic acid in the avoidance and plausible prognosis of COVID-19 is becoming increasingly necessary.

2. Sinonasal immune system

The sinonasal mucosal immune system is the primary barrier in contrary to pathogens, consist of innate and adaptive immunological components, called Nasal-Associated Lymphoid Tissues (NALT) which is included in Mucosa-Associated Lymphoid Tissue (MALT) [1, 2, 3, 4, 5, 6, 7, 8, 9]. Human NALT consist of lymphoid tissue, formed by lymphocyte-containing follicles in junction with Follicle-Associated Epithelium (FAE) and High Endothelial Venules (HEVs), located in the nasal mucosa and pharyngeal tonsils (adenoids). NALT play a function as a barrier and as an induction site for immunological reactions, which Immunoglobulin (Ig) A has a pivotal role. The Common Mucosal Immune System (CMIS) connects NALT inductive sites with effector areas of the nasal mucosa, for the formation of antigen-specific T Helper 1 (TH1) and Th2 cell, Cytotoxic T Lymphocyte (CTL), B lymphocyte (such as plasma cells and memory cells), and Innate Lymphoid Cells (ILCs) [3, 10, 11, 12, 13]. With other immunological cells like dendritic cells, Microfold (M) cells, and macrophages; nasal epithelial cells and goblet cells involve in initiating local and systemic immune response in contrary to pathogens [1, 12].

Nasal epithelial cells have the function as physical barrier and to eliminate pathogens through the generation of enzymes (lysozyme, peroxidases, phospholipases), complement components, protease inhibitors [Secretory Leukocyte Proteinase Inhibitor (SLPI) and elafin], permeabilizing peptides [β-defensins, cathelicidins, Bacterial Permeability Increasing protein (BPI) and Palate, Lung, and Nasal Epithelium Clone (PLUNC)], collectins (such as SP-A, SP-D, and MBL), binding/neutralizing proteins [mucins, Serum Amyloid A (SAA), and lactoferrin], pentraxins (PTX- 3 and CRP), small oxygen-reactive molecules (reactive oxygen species/ROS and nitric oxide), whose production is regulated by the engagement of Pathogen-Recognition Receptors (PRRs), such as Toll-Like Receptors (TLRs), and NOD-Like Family Receptors (NLR) [1, 2, 3, 4]. In addition to their direct antimicrobial activity, epithelial-derived β-defensins and collectins (such as SP-A, SP-D) can also function as “Damage-Associated Molecular Patterns” (DAMPs). In particular, β-defensin 2 activates dendritic cells (DCs) upon binding to TLR-4, and β-defensin 3 to TLR-1 and TLR-2, while SP-A and SP-D act through the modulation and/or direct binding of TLR-2 and TLR-4 [3].

Exogenous antigens are transported by specialized intraepithelial M cells to antigen-presenting cells, such as macrophages, which in turn proceed and provide antigens to CD4+ T lymphocytes, which polarize in TH2 cells, generating Interleukin (IL)-4, IL-5 and IL-6, which trigger an Ig A-committed B-cell development, associated to the formation of the intrafollicular Germinal Centers (GC) [1, 2, 3, 4]. Human M cells in adenoidal tissues are recognized by cytokeratin 20 (Ck20), and irregular microvilli as well as pocket-like structures, observed by Scanning Electron Microscopy (SEM) (Figure 1) [3, 6].

Figure 1.

Nasal innate immune system [6].

3. The impact of ascorbic acid in immunological system

Ascorbic acid is an important vitamin for health, with numerous benefits associated to its potency as antioxidant and immune-modulator. It is a powerful antioxidant and a co-factor for a family of biosynthetic and gene modulatory enzymes, supporting various cellular functions that contribute to immune defense of both the innate and adaptive immune system [14, 15].

The epithelial defense function against pathogens is improved by ascorbic acid, promoting the oxidant scavenging ability of the skin. Ascorbic acid has the potential to protect against oxidative stress resulting from environmental exposure, through increased collagen formation and stabilization, induction of keratinocyte differentiation and lipid production, increased proliferation and migration of fibroblasts, and accelerated wound healing. The chemotaxis, phagocytosis, and the formation of free radical are elevated in phagocytic cells (like PMN leukocytes and macrophages), which accumulate ascorbic acid, which in turn increases the ability of cells to kill microbes. Apoptosis and elimination of the PMN leukocytes from areas of inflammation by macrophages are also need ascorbic acid, in order to reduce necrosis/NETosis and potential tissue destruction, but unfortunately its role in lymphocytes is insufficient of understanding. Ascorbic acid has been exhibited to increase differentiation and proliferation of B- and T-cells (and increases antibody levels), it’s possible because of its gene modulating impacts. Ascorbic acid also regulates cytokine and interferon formation and also reduces histamine levels [14].

Infection and inflammation mostly related to ascorbic acid level in tissues, that the insufficiency of ascorbic acid involves in disturbance of immune function and the risk of infections. Infections enhanced inflammation and metabolic requirements, so may significantly impact on ascorbic acid levels. However, its supplementation can prevent and medicate respiratory and systemic infections. Intake of ascorbic acid supplementation that provides at least adequate is needed for prophylactic prevention of infection. The saturating plasma concentrations (i.e., 0.1–0.2 g/day) may enhance cellular concentrations, but in the treatment of underlying infections need much greater doses of ascorbic acid to neutralize the inflammatory response and metabolic demand [14, 15].

4. Ascorbic acid and leukocyte function

Leukocytes, like neutrophils and monocytes, contain higher ascorbic acid levels (about a hundred fold) than in plasma concentrations. Leukocytes contain maximal ascorbic acid’s levels at intakes of about 0.1 g/day, although other cells likely need more ascorbic acid levels for saturation. Neutrophils accumulate ascorbic acid through Sodium-dependent Vitamin C Transporter (SVCT) and typically have intracellular concentrations minimal about 1 mM [14]. After releasing ROS, neutrophils elevate cellular level of ascorbic acid via non-specific uptake of the oxidized form, Dehydroascorbate (DHA), through Glucose Transporters (GLUT). DHA is then quickly decreased to ascorbic acid intracellularly, to provide concentrations of about 10 mM, and ascorbic acid exhibits pivotal effects in the related cells. Neutrophils that have sufficient levels of ascorbic acid are postulated to defend from ROS destruction [14]. As a vigorous water-soluble antioxidant, ascorbic acid may also scavenge numerous ROS and rejuvenate the pivotal function of some antioxidants, such as glutathione and vitamin E. Ascorbic acid is depleted from neutrophils in an oxidant-dependent pathway after phagocytosis or activation with soluble inductions [14, 15, 16].

The transcription factor Nuclear Factor кB (NFкB) was a pivotal part in the pro-inflammatory response due to the homeostasis imbalance between oxidant production and antioxidant protections. Oxidants can initiate NFкB, that activates a signaling pathway, directing to generation of ROS and other inflammatory mediators [14]. Ascorbic acid has been exhibited to influence both oxidant production and NFкB induction in dendritic cells in vitro, as well as in PMN leukocytes. The cells, with Thiol-containing proteins, can be specifically susceptible to redox changes, and they are frequently crucial for controlling redox-related cell signaling pathways. T-cells have been used to study the ascorbic acid-dependent regulation of thiol-dependent cell signaling and gene expression pathways [14, 15, 16].

As a conclusion, the regulation of immune function by ascorbic acid through stimulation of redox-sensitive cell signaling cascades or directly defending pivotal cell parts [14]. The exposure of leukocytes (neutrophils and monocytes) to oxidants may suppress cell motility, which is believed to be caused by lipid peroxidation and their effects on the fluidity of cell membranes. Because neutrophils have elevated concentrations of polyunsaturated fatty acids in their plasma membranes, an increase in neutrophil motility observed after ascorbic acid intake may be associated with oxidant scavenging, such as vitamin E rejuvenation [14, 16].

4.1 Neutrophil chemotaxis

A beginning part in non-specific immunity is started by neutrophil infiltration into infected tissues as the effect from pathogen- or host-derived inflammatory signals such as N-formylmethionyl-leucyl-phenylalanine (fMLP), IL-8, leukotriene B4, and complement component (C5a). The chemotaxis’ migration of neutrophils in response to chemical stimuli, may be randomly, is as named chemokinesis [14, 17]. Neutrophils expressed over than 20 different chemokine and chemoattractant receptors to recognize and quickly reciprocate to tissue destruction signals. The scorbutic guinea pigs’ study showed a damaged leukocyte chemotactic response compared with leukocytes isolated from animal models administered with ascorbic acid in the intake. These suggest that ascorbic acid depletion may affected on the function of phagocytes to migrate to inflammatory areas [14, 15, 18].

Neutrophils may get “paralysis,” especially in patients with severe inflammatory condition and associated with compromised neutrophil chemotactic’s dysfunction [14]. These neutrophils are the part of the increased levels of immune-suppressive and anti-inflammatory mediators (such as IL-4 and IL-10) during the compensatory anti-inflammatory response seen after primary induction of the immune system. It is also a possibility, that ascorbic acid deficiency may associated and is common during severe infection. The previous researches showed that recurrent infections had damaged leukocyte chemotaxis, which could be regenerated in return to administration with gram doses of ascorbic acid. Furthermore, neonates with systemic infections injected with 0.4 g/day of ascorbic acid, dramatically increased neutrophil chemotaxis [14, 18].

The genetic disorders of neutrophils’ function may also contribute to recurrent infection. The cases of Chronic Granulomatous Disease (CGD), an immunodeficiency disorder, resulting in the reducing of leukocyte production of ROS, and Chediak-Higashi Syndrome (CHS) as a rare autosomal recessive disease, is associated with vesicular transport trafficking and decreased neutrophils’ mobility [14]. Ascorbic acid may promote the action of unnecessary antimicrobial mechanisms in these cells, although ascorbic acid supplementation would not be expected to influence the elemental disorders of these genetic diseases. Patients with CGD showed increased leukocyte chemotaxis following ascorbic acid intake were related with reduced infections and clinical improvement. The improvement of neutrophils’s chemotaxis was reported in a mouse model of CHS following ascorbic acid administration. Besides, neutrophils isolated from two children with CHS exhibited enhanced chemotaxis after administration with 0.2–0.5 g/day ascorbic acid. The ascorbic acid-dependent improvement of chemotaxis was assumed to be treated in part through the results on microtubule assembly, and has showed that intracellular ascorbic acid may repair microtubules [14, 15, 18].

The healthy persons that take ascorbic acid have also been recorded to boost neutrophils chemotactic function, that associated with the antihistamine effect of ascorbic acid [14]. A dietary source of ascorbic acid (providing 0.25 g/day) supplementation, to inadequate ascorbic acid status (i.e., <50 μM) persons, resulted in a 20% elevate in neutrophils chemotaxis. A combine administration of elderly women with 1000 mg/day of ascorbic acid and vitamin E increased neutrophils functions. Thus, ascorbic acid administration provides benefits in boosting the immune system, especially for an inadequate ascorbic acid status, and can be more frequent in the aged. Although, it should be notable that it is not yet convinced to what extent increased ex vivo leukocyte chemotaxis results into enhanced in vivo immune activity [14, 18].

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, 15, 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, 15, 16].

4.3 Neutrophil apoptosis and clearance

Apoptosis is the process of programmed cell death after pathogen phagocytosis and eliminating. Apoptosis facilitates following phagocytosis and elimination of the spent neutrophils from areas of inflammation by macrophages, thus helping resolution of inflammation and avoiding excessive tissue destruction [14, 19]. When phosphatidyl serine is exposed during the apoptotic process, which culminates in caspase activity, the cells are marked for uptake and elimination by macrophages. Fascinatingly, since caspases are thiol-dependent enzymes, ROS generated by active neutrophils can inactivate them. As a result, ascorbic acid may be expected to prevent the oxidant-sensitive caspase-dependent apoptotic process after activation of neutrophils. This idea is supported by in vitro experiments that show ascorbic acid can enhance the amount of Escherichia coli-mediated neutrophil apoptosis in human neutrophils. Reduced apoptosis was seen in peritoneal neutrophils obtained from ascorbic acid-deficient Gulo mice, who instead experienced necrotic cell death. These ascorbic acid-deficient neutrophils were not phagocytosed by macrophages in vitro, and remained at inflammatory sites in vivo. In addition, supplementation of ascorbic acid to severe infection animals reduced the levels of neutrophils in the lungs of these animals [14, 20].

Several researches have explained about reduced neutrophil apoptosis in subjects with sepsis compared with healthy individuals. The greater tissue destruction seen in individuals with severe infection is assumed to be associated to the delayed apoptosis, which seems to be linked to disease severity. Additionally, it was shown that immature “band” neutrophils produced during sepsis had extended life spans and were resistant to apoptosis. Apoptosis in healthy neutrophils was shown to be inhibited by plasma from patients with severe infections, indicating that pro-inflammatory cytokines were crucial for the elevating in vivo survival of neutrophils under inflammatory situations [14]. Interestingly, high-dose ascorbic acid supplementation has been exhibited to regulate cytokine concentrations in individuals with carcinoma and, although this has not yet been evaluated in subjects with sepsis, it is conceivable that ascorbic acid may regulate neutrophil function in these subjects through a different mechanism. The role of ascorbic acid administration on neutrophil apoptosis in severe infection subjects has only been described in one study. Ascorbic acid 0.45 g/day administered intravenously to abdominal surgery subjects with severe infections was shown to reduce caspase-3 protein concentrations and thus was thought to have an anti-apoptotic impact on peripheral blood neutrophils. However, caspase activity and apoptosis of the neutrophils after activation was not examined. Additionally, neutrophils in circulation could not accurately represent their activation level at inflammatory tissue areas. Clearly, further researches are required to determine how ascorbic acid affects neutrophil apoptosis and elimination from inflammatory sites [14, 15, 20].

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 neutrophil-specific 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 ability, and both natural killer cell function and lymphocyte blastogenic responses to T- and B-cell mitogens were returned to normal concentrations after ascorbic acid administration. Although the human researches are supported the effect of ascorbic acid to lymphocyte, it is clear that more human experimental researches are required to validate these researches [14, 22].

Recent study in wild-type and Gulo knockout mice showed that parenteral supplementation of 0.2 g/kg ascorbic acid increased proliferation of regulatory T-cells (Tregs) and suppressed the negative immunoregulation of Tregs (by suppressing the expression of specific transcription factors, antigens, and cytokines) observed in severe infection. The mechanisms depend on the gene regulatory impacts of ascorbic acid. For instance, recent study has involved ascorbic acid in epigenetic modulation via its function as a cofactor for the iron-containing dioxygenases which hydroxylate methylated DNA and histones [14]. The Ten-eleven Translocation (TET) enzymes hydroxylate methylcytosine residues, which may act as epigenetic marks in their own right, and also facilitate removal of the methylated residues, a pivotal step in epigenetic modulation. Preliminary data shows that ascorbic acid can modulate T-cell maturation through epigenetic pathway including the TETs and histone demethylation. It is believed that ascorbic acid function as gene modulatory functions and cell signaling, through modulation of transcription factors and epigenetic marks, have important parts in its immune-regulating functions [14, 15].

5.2 Inflammatory mediators

In response to infection and inflammation, a variety of immune cells, both non-specific and specific, generate crucial cell signaling mediators called cytokines. They contain a wide scale of mediators, including chemokines, Interferons (IFNs), ILs, lymphokines, and TNFs, that regulate both humoral and cellular immune responses, and modulate the maturation, growth, and responsiveness of specific cell populations [14]. Cytokines can induce pro-inflammatory or anti-inflammatory responses, and ascorbic acid seems to regulate systemic and leukocyte-derived cytokines in a complex system [14, 15, 23].

Ascorbic acid reduced Lipopolysaccharide (LPS)-induced production of the pro-inflammatory cytokines TNF-α and IFN-γ, and elevated anti-inflammatory IL-10 generation, while having no impact on IL-1β levels. Additionally, in vitro administration of ascorbic acid to peripheral blood monocytes isolated from subjects with pulmonary infection reduced the production of the pro-inflammatory cytokines TNF-α and IL-6 [14]. However, another research initiated that in vitro supplementation of peripheral blood monocytes with ascorbic acid and/or vitamin E increased LPS-stimulated TNF-α production, but had no effect on IL-1β production. In addition, incubation of ascorbic acid with virus-infected human and murine fibroblasts increased production of antiviral IFN. Administration of control subjects with 1 g/day ascorbic acid (with and without vitamin E) was exhibited to increase peripheral blood mononuclear cell-derived IL-10, IL-1, and TNF-α after induction with LPS. Thus, the role of ascorbic acid on cytokine production seems to depend on the cell type and/or the inflammatory inducer. Recent study has showed that ascorbic acid supplementation of microglia and resident myeloid-derived macrophages in the central nervous system changes activation of the cells and generate of the pro-inflammatory cytokines TNF, IL-6, and IL-1β. This is indicative of an anti-inflammatory phenotype [14, 23].

Ascorbic acid’s roles in modulating cytokines have been identified in preclinical studies utilizing Gluo knockout mice. In ascorbic acid-deficient Gulo knockout mice infected with influenza virus, pro-inflammatory cytokines TNF-α and IL-1α/β in lungs were elevated, while antimicrobial cytokine IFN-α/β was reduced. In addition, when ascorbic acid was given to Gulo mice that had polymicrobial peritonitis, isolated neutrophils’ production of the pro-inflammatory cytokines TNF-α and IL-1β was reduced [14]. Another research in septic Gulo mice supplemented with 0.2 g/kg parenteral ascorbic acid has shown reduced production of the inhibitory cytokines TGF-β and IL-10 by Tregs. In this research, the change in IL-4 production and augmented IFN-γ production was also assessed, indicating immune-regulating roles of ascorbic acid in severe infection. In general, ascorbic acid seems to normalize cytokine production, likely via its gene-modulating roles [14, 15, 23].

Pathogens and stress will induce basophils, eosinophils, and mast cells to produce immune mediator, histamine. Histamine induces vasodilation and elevated capillary permeability, resulting in the classic allergic symptoms of runny nose and eyes [14]. The deficiency of ascorbic acid was related with increased plasma histamine concentrations and ascorbic acid would reduce histamine concentrations in researches using guinea pigs, an ascorbic acid-requiring animal model. Increased histamine production was shown to elevate the utilization of ascorbic acid in this research. Consistent with the animal researches, human experimental researches with oral ascorbic acid (125 mg/day to 2 g/day) and intravenous ascorbic acid (7.5 g infusion) have reported reduced histamine concentrations, particularly in subjects with allergic diseases compared with infectious diseases. The precise mechanisms responsible for the in vivo reduce in histamine concentrations after ascorbic acid supplementation are currently inconclusive although ascorbic acid has been suggested to “detoxify” histamine. Furthermore, the effect of ascorbic acid administration on histamine concentrations are not evaluated in all researches [14, 23].

6. The role of ascorbic acid in sinonasal immune system

As an immunomodulatory, antioxidant, and anti-inflammatory agent, ascorbic acid has a potency in sinonasal immune system. Ascorbic acid is known to treat respiratory diseases, including allergic and infectious diseases. The antioxidant effect of ascorbic acid is through scavenging ROS by quick aqueous phase electron transfer and inhibiting lipid peroxidation [24]. The mechanism of ascorbic acid modulates the immune system by influencing the roles of phagocytes, proliferation of T lymphocytes, generation of interferon and cytokine, and gene expression of monocyte adhesion molecules. Besides, it has been proposed that ascorbic acid acts as a biological modulator of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), which is a cAMP-dependent chloride channel that modulates respiratory tract epithelial surface fluid production, as well as directing the hydration of mucosal surfaces and providing effective mucociliary clearance. Ascorbic acid was shown to increase chloride generation when applied topically to newly excised sinus and nasal mucosa, indicating a potential application as a pharmaceutical agent to enhance mucociliary clearance [24, 25, 26, 27].

7. The possible role of inhalation ascorbic acid in sinonasal immune system

Ascorbic acid can be dangerous at high dosage. Therefore, inhalation route can be a substitute for getting rapid topical elevated level of ascorbic acid (if tested as drug) [15]. Drug/vitamin with inhalation route has advantage as a non-invasive route, minimal side-effect, fast-onset, no first-pass metabolism, and more potent pharmacodynamics drug, so the patient’s compliance and convenience can be maximized [24]. The previous researches have been reported about the role of inhalation vitamin in some diseases. Bhat et al. (2020) reported about the role of inhaled vitamin E in electronic-cigarette, or vaping, product use-associated lung injury (EVALI), while Gelfand et al. (2020) reported about the role of inhaled vitamin A was more potent than intramuscular route in preventing hyperoxia-induced lung injury in a neonatal rat model of bronchopulmonary dysplasia [28, 29].

There was one research about the effect of intranasal ascorbic acid in allergic condition. Intranasal ascorbic acid reduced ROS parameter (intracellular ROS and 8-isoprostanes), NFкB, IL-4 and IL-5, IgE and IgG1, lymphocyte proliferation, Airway Hyper Responsiveness (AHR), and lung inflammation. Intranasal ascorbic acid also increased glutathione peroxidases (GPx) activity, IL-10 level, and induced FOXP3+ cells [30]. There is a potency about the use of ascorbic acid in the inhalation route, but until now, there is no the research about it.