Abstract

Impaired immune function may increase the risk of infection of pathogenic microbes; therefore, improving immune function may reduce the risk of such infection. Among viruses, COVID-19, which causes acute respiratory distress syndrome (ARDS), has caused a pandemic worldwide because of its highly infective behavior. One of the reasons for its highly infective nature is that it has high binding affinity with the host’s angiotensin-converting enzyme-2 (ACE-2) receptors. Impaired immune function in older people and individuals with pre-existing diseases may account for increased vulnerability to this infection. High levels of oxidative stress and inflammation in these people contribute to impaired immune function. Asymptomatic individuals have a low viral load in their nasal cavity but can infect others. Symptomatic individuals show infection in the lung, which has dense ACE-2 receptors. Following binding with the ACE-2 receptors and fusion with the host’s cell membrane, the virus is engulfed inside the cell and starts replication. The virus infected cells release cytokines, which activate immune response. If the immune response is strong, individuals may fully recover with or without supportive care; however, if the immune response is weak, the symptoms of ARDS may become severe and in some cases may progress to death. Recommended guidelines are effective in preventing infection; however, they are ineffective for individuals who are tested positive for COVID-19 infection. Improving immune function remains one of the best options for them to eliminate this infection. A hypothesis is proposed that a mixture of micronutrients containing dietary and endogenous antioxidants may be more effective than a single micronutrient in improving immune function by reducing oxidative damage and inflammation and by stimulating fighting ability of immune cells.

Keywords: micronutrients, oxidative stress, inflammation, innate and adaptive immunity, COVID-19 infection

Introduction

During evolution, humans have been exposed to pathogenic microbes. Some of them have died, while others have recovered with or without treatments. The survivors may have developed specific immunity that prevents future infection with that microbe. The development of specific antibodies against certain viruses has prevented the infection of such viruses in people irrespective of their age, gender, or pre-existing diseases.

At the end of 2019, a novel corona virus causing pneumonia was identified in Wuhan city, Hubei province, China [1,2]. In February 11, 2020, World Health Organization (WHO) named this virus COVID-19 (corona virus disease-19). This virus appears to be different from other corona viruses in its highly infective behavior and it is now causing a pandemic throughout the world. The exact reasons for its highly infective behavior are unknown. One of the reasons could be that its spike protein has a greater affinity to bind with the host’s angiotensin-converting enzyme-2 (ACE-2) receptor than other corona viruses. Rates of infection and mortality are higher in older individuals and those with pre-existing diseases than younger individuals and those without pre-existing diseases [3-5]. Such individuals may have impaired immune function because of the presence of high levels of oxidative stress and inflammation. Current guidelines, which include facemask, social distancing, and contact tracing, are effective in preventing infection. However, they are not effective for those individuals who are tested positive for COVID-19. For such people, improving immune function remains one of the best options for eliminating the infection and, increasing the recovery and survival rates. Therefore, it is essential to develop a non-toxic biological strategy to improve immune function.

This review briefly describes (a) structure of COVID-19 virus, (b) death rates, (c) steps involved in entry of this virus into the host cell, (d) innate and adaptive immune responses to infection, (e) differentiation of cytokine storm, and (f) effects of individual antioxidants in improving immune function. This review proposes a hypothesis that a mixture of micronutrients containing dietary and endogenous antioxidants may be more effective than a single micronutrient in improving immune function by protecting it from oxidative and inflammatory damages and by increasing the ability of individual immune cell to fight infection.

Structure of COVID-19 virus

Highly infectious COVID-19 virus is a positive-sense single-stranded RNA of about 27-32 Kb, which acts like a mRNA, and serves as a template to make new single-stranded RNA for replication within the host cells, using their synthetic machinery. COVID-19 appears spherical with a diameter of 100-160 nm. This virus is characterized by the presence of many spikes, which give the appearance of a crown. Viral RNA genome encodes proteins, including the spike (S) glycoprotein, the nucleocaspid (N) protein, the matrix (M) protein, and the small envelop (E) protein. Each of them has specific function in viral entry and replication within the host cells.

Symptoms and death rates among infected persons

Symptoms

Initially, majority of COVID-19 infected persons exhibit clinical symptoms, such as dry cough, high fever, myalgia (muscle pain), fatigue, dyspnoea (difficulty in breathing), headache, vomiting, and diarrhea. Among all COVID-19 infected patients, approximately 32.8% developed ARDS, 20.3 % required critical care and admitted to ICU care, 6.2% developed shock, and 3.9% died [6]. Patients with COVID-19 infection at an advanced state of the disease exhibit venous thromboembolism and pulmonary emboli. Cytokine storm contributes to hypercoagulability due to damage to endothelial cells, microvessels, and development of prothrombotic anti-phospholipid antibodies [7-9]. Hypercoagulation increased mortality rates in COVID-19 infected patients [10].

Death rates

An early estimate of death rates is 5.25 % in Wuhan city, 1.41 % in Hubel province excluding Wuhan city, and 0.15 % in Mainland China [11]. The death rates in the USA are 5.9 % and it is 6.3 % worldwide as of May 26, 2020. These death rates are lower than reported for other corona viruses [12].

Viral entry in the host cell

The spike (S) of COVID-19 promotes its entry into the cells by a high-affinity receptor binding (RBD) protein, which fuses with the angiotensin-converting enzyme 2 (ACE2) receptors located at the host’s cell surface. ACE2 receptors are densely populated in cells lining the alveoli and small intestine. The S protein is cleaved by the host cell furin-like protease into two separate polypeptides S1 and S2 [13,14]. S1 polypeptide acts as the receptor-binding domain (RBD), whereas S2 polypeptide forms the stalk of the spike [15]. The spike RBD has much higher binding affinity with the human ACE2 receptor than other corona viruses allowing easy entry in the cells [16]. Following binding to the ACE2 receptor, S2 protein is cleaved by a cathepsin, TMPRRS or another protease at two sites. The first cleavage separates the RBD and the fusion domains of the S2 protein, while the second cleavage exposes the fusion peptide to cell membrane [17]. This facilitates the fusion process between the spike and ACE2 receptor [18-21]. Following fusion with the host’s cell membrane, the virus is taken inside the cells by endocytosis and starts replication, using the host cell synthetic machinery.

Innate and adaptive immune responses to infection

Innate immune responses

The cells infected with COVID-19 release cytokines, which signal the immune system to send immune cells to the site of infection for the eliminating the viruses as well as for facilitating the healing of the injured tissue [22]. The innate immune cells are the first to respond to defend the host against any pathogenic microbes [23]. This response is activated as soon as infected cells release cytokines and complement proteins, which signal the innate immune system, to send immune cells at the site of infection [24,25]. While the innate immune responses do not confer long-lasting immunity against pathogenic microbes, they promote activation of the adaptive immunity, which provides such immunity [26,27]. A similar mechanism of alerting the immune system may exist after COVIF-19 infection. The function of specific immune cells of the innate immunity, which are recruited to fight infection, is described here.

Neutrophils

Neutrophils are the first immune cells that are recruited to fight infection. These cells engulf pathogens by phagocytosis where they are killed by proteolytic enzymes aided by reactive oxygen species.

Monocytes

Monocytes are precursors of macrophages and dendritic cells and produce full set of complement proteins [28]. Macrophages exhibit phagocytic activity and play an important role in killing infective agents as well as cleaning the debris of dead cells. Dendritic cells do not exhibit phagocytic activity, but they play an important role in presenting antigen to the T-cells. Complement proteins are the major humoral components of the innate immune response [29] and are toxic to the pathogens and infected cells. They regulate the production of antigen presenting cells, activate main immune cells of innate immunity [30], and regulate B-cells and T cells responses of adaptive immunity [31].

Natural killer (NK) cells

NK cells of the innate immunity represent about 10 to 15 % of the peripheral blood lymphocytes. They kill infected cells and any foreign cells such as cancer cells [32].

Adaptive immune responses

The adaptive responses to an antigen are strong and are responsible for continuously storing and re-calling immunologic memory for recognizing and eliminating a specific antigen.

Lymphocytes

The lymphocytes (T-cells and B-cells) are responsible for the adaptive immune response. B-cells mature to plasma cells that secrete specific antibody in response to a particular pathologic microbe. The immune cells contain three types of antigen presenting cells: macrophages, dendritic cells, and B-lymphocytes. These cells break down antigen of infective agent in smaller peptides, which combine with major histocompatibility complexes I and II, and then locate themselves on the surface of antigen presenting cells [33].

T-lymphocytes

CD4+ (T-cells) are divided into regulatory T cells (Treg) and T helper cells (TH) [34]. Helper T-cells become activated when they are presented with peptide antigens associated with MHC class II molecules, which are expressed on antigen presenting cells. Activated helper T cells rapidly divide and secrete cytokines. T cells can detect the presence of intracellular pathogen because infected cells display on their surface peptide fragments-derived from the protein of invading pathogens on their cell surface. Helper T cells also activate B-cells to secrete antibodies. These cells assist macrophages in destruction of ingested pathogenic microbes. Helper T-cells also activate cytotoxic T-cells [33].

CD4+ (T-cellsBoth T-cells and B-cells carry receptors that recognize specific antigens. T-cells can recognize only membrane bound antigens. The cell surface major histocompatibility complex (MHC) molecules bind peptide fragments of foreign proteins for presentation to appropriate antigen-specific T-cells. There are two major subtypes of T-cells: the killer T-cells and the helper T-cells. The killer T-cells can recognize antigens bound to Class I MHC molecules, whereas the helper T cells recognize antigens bound to Class II MHC molecules. A minor subtype of T-cells is γδ T-cells, which recognize intact antigens that are not bound to MHC receptors.

Development of cytokine storm in COVID infected patients

The development of “cytokine storm” due to uncontrolled production of cytokines occurred in critically ill COVID-19 patients, leading to multiple organ failure and death [35-37]. In patients with pre-existing diseases, such as diabetes, hypertension, cardiovascular disease, and chronic obstructive pulmonary disease, who are infected with COVID-19, cytokine storm is further aggravated to super cyclone that contributes to enhanced mortality rate in these patients [38].

Uncontrolled viral replication causes delayed anti-viral IFN response, which initiates migration of increased number of neutrophils and macrophages at the site of injury with a simultaneous rise in the levels of cytokines, which causes damage to the respiratory system [39]. Monocyte-derived macrophages were present in large number in the bronchoalveolar lavage fluid (BALF). These macrophages produce extensive amounts of inflammatory cytokines that contribute to cytokine storm [40]. The pro-inflammatory cytokines include IL-2, IL-6, IL-7, IP-10, TNF-alpha, G-CSF (Granulocyte-colony stimulating factor), and chemokines such as MCP-1 (monocyte chemoattractant protein-1), MIP-1alpha (macrophage inflammatory protein-1 alpha) were higher in patients requiring ICU care compared to those not needed ICU care [37]. Among these cytokines, only the levels of 3 cytokines, IL-6, IP-10 (induced protein-10) and IFN-gamma were markedly increased in COVID-19 patients [41]. Induction of IP-10 (induced protein-10) in the lung causes increased influx of immune cells leading to apoptosis and lung damage [42].

Health risks among survivors of COVID-19 infection

Many survivors of COVID infection are exposed to very high levels of reactive oxygen species (ROS) and pro-inflammatory cytokines; therefore, the risk of developing adverse health effects among them is high. Studies are in progress to define these health effects. Reported adverse health effects include lung fibrosis, cardiomyopathy, and damage to kidney and brain.

How to improve immune function by micronutrients

It has been known that individuals with impaired immune function are very susceptible to infection with pathogenic microbes. Factors, which impair immune function include advancing age, poor diet that lacks fresh fruits and vegetables, obesity, environmental pollution, tobacco smoking, alcoholism, chronic stress, and lack of exercise [34,43]. Deficiency of one or more micronutrients, pre-existing disease, and substance abuse can also impair immune function [44,45]. Increased oxidative stress and chronic inflammation are found under above environmental, dietary, and lifestyle related factors as well as health conditions. These cellular deficits can impair immune function. Therefore, attenuation of oxidative stress and chronic inflammation at the same time may improve immune function. Since antioxidants are known to reduce oxidative damage and persistent inflammation, the effects of individual antioxidants and other micronutrients on immune functions were evaluated. These studies are described here.

Vitamin A

Vitamin A maintains normal antibody-mediated TH2 response by suppressing the production of IL12, TNF-alpha, and INF-gamma [46]. Vitamin A also helps normal functioning of B cells, which is needed for generation of antibody responses to antigen [46]. Thus vitamin A is required for B cell-mediated IgA antibody responses to bacterial polysaccharide antigens [47].

Vitamin C

Vitamin C treatment enhances proliferation of lymphocytes leading to increased production of antibodies [48].

Vitamin D3

Immune cells express vitamin D receptor (VDR). Vitamin D treatment influenced both innate and adaptive immunity as well as antigen presenting cells that links them. Vitamin D3 at a physiological concentration stimulated proliferation of human monocytes in culture [49], chemotactic and phagocytic activity of macrophages [50], and increased production of several antimicrobial peptides in monocytes, neutrophils, and epithelial cells lining the respiratory tract [51].

In contrast to the effect of vitamin D3 on innate immune cells, vitamin D3 inhibited both T-cell and B-cells of adaptive immunity [52]. It also inhibited maturation of dendritic cells (DC), one of antigen presenting cells [53]. These function of vitamin D may be useful in alleviation some of the symptoms of autoimmune disease and inflammatory disease [34].

Vitamin E

In animal model, supplementation with vitamin E enhanced T-cell mediated function including delayed-type hypersensitivity (DTH) response, lymphocyte proliferation, and IL-2 production, and reduced prostaglandin E2 (PGE2) production [54]. In healthy older individuals, supplementation with vitamin E increased DTH response, T-cell proliferation, and IL-2 production, and reduced lipid peroxidation and PG2 [55].

Vitamin B6, B12, and Folate

All these vitamins are involved in the production of antibodies [46,47].

Zinc

Zinc supplementation increased the number of T-cell lymphocytes and NK cell activity [56,57]. Zinc induced the development of regulatory T-cell (Treg) population [58,59], and reduced levels of pro-inflammatory T helper cell (TH 17 and TH9) differentiation [60,61]. Zinc is also involved in the production of antibodies, particularly IgG [62].

Selenium

Selenium is important in the maintenance of antibody levels [63].

Limitations of using single antioxidant in improving immune function in humans

The role of micronutrients in improving immune function to reduce the risk of infection was discussed in detail [43]. Although individual micronutrient has improved some functions of innate and adaptive immune cells in experimental systems, it is unlikely that such an approach would be useful in improving immune function in humans.

The potential reasons include (a) different antioxidants are distributed differently in the sub-cellular compartments of cells; therefore, a single antioxidant cannot protect all parts of the cell; (b) administered single antioxidant in a high internal oxidative environment of high-risk populations for COVID-19 infection becomes oxidized and then acts as a pro-oxidant; (c) an elevation of the levels of antioxidant enzymes and dietary and endogenous antioxidants is essential for reducing oxidative stress and inflammation, a single micronutrient cannot achieve this; (d) the affinity of different antioxidants for free radicals differs, depending upon their solubility; (e) both the aqueous and lipid compartments of the cell need to be protected together; a single antioxidant cannot meet this goal; (f) vitamin E is more effective in quenching free radicals in a reduced oxygenated cellular environment, whereas vitamin A and beta-carotene are more effective in a higher oxygenated environment of the cells [64]; (g) vitamin C is important for recycling the oxidized form of alpha-tocopherol to the antioxidant form [65]; (h) different antioxidants alters the expression of different microRNAs each of which guides its respective mRNA to produce only protective proteins [66]. For example, some antioxidants can activate Nrf2 by upregulating miR-200a that inhibits its target protein Keap1, whereas others activate Nrf2 by downregulating miR-21 that binds with 3-UTR Nrf2 mRNA [67].

Failure of single antioxidant to produce expected benefits in human diseases

Because of limitations of using individual antioxidant discussed above, supplementation with a single antioxidant in humans did not produce those benefits that were observed in animal models. For examples, administration of beta-carotene alone increased the risk of lung cancer in male heavy smokers [68]. Vitamin E treatment was ineffective in patients with Alzheimer’s disease, but it reduced the rate of decline in cognitive function in early phase of this disease [69]. Vitamin E was ineffective in heart disease on primary and most secondary outcomes [70]. Thus, it is unlikely that the use of single antioxidant would improve immune function. A comprehensive mixture of micronutrients that can protect immune system from oxidative and inflammatory damage as well as stimulate the function of innate and adaptive immunity is needed. This mixture must elevate the levels of antioxidant enzymes as well as dietary and endogenous antioxidant compounds. The usefulness of a mixture of micronutrients containing dietary and endogenous antioxidant compounds for the management of chronic neurological diseases has been proposed [71].

Proposed mixture of micronutrients for improving immune function

A mixture of micronutrients containing vitamin A, mixed carotenoids, vitamin C, alpha-tocopheryl acetate, alpha-tocopheryl succinate, vitamin D3, alpha-lipoic acid, n-acetyl- cysteine, coenzyme Q10, omega-3-fatty acids, curcumin, resveratrol, all B-vitamins, selenomethionine, and zinc for improving immune function is proposed. This mixture would increase the levels of antioxidant enzymes by activating a nuclear transcriptional factor Nrf2 and the levels of dietary and endogenous antioxidant compounds. Therefore, a hypothesis is proposed that such a micronutrient mixture may optimally improve immune function by protecting it from oxidative and inflammatory damages and by increasing the ability of individual immune cells to fight COVID-19 infection. Preclinical and clinical studies should be performed to test the validity of the proposed hypothesis.

Evidence supporting the effectiveness of a mixture of micronutrients in human diseases

It is highly likely that the proposed mixture of micronutrients would be effective in improving immune function in humans as well as may reduce potential adverse health effects among survivors of COVID-19 infection. This possibility is supported by two clinical studies with a mixture of micronutrients. For example, administration of multiple micronutrients reduced the risk of cancer in men [72] and prolonged the time period for initiating the anti-viral therapy in HIV infected patients [73].

Conclusions

Highly infectious COVID-19, which causes acute respiratory distress syndrome, has created a pandemic worldwide. Older people and individuals with preexisting diseases are very vulnerable to COVID-19 infection and death. This virus is highly infectious because of very high binding affinity of spike (S) protein of COVID-19 with the host’s angiotensin-converting enzyme-2 (ACE-2) receptors compared to other corona viruses. Following binding and fusion with the host cell membrane, the virus is engulfed inside the cells and begins replication using the host cell synthetic machinery. The virus infected cells release cytokines, which signal the immune system to send immune cells to the site of infection for eliminating the viruses and facilitating the healing of the injured tissue. Most individuals recover from this infection, but about 5-6% may die despite supportive treatment. Recommended guidelines are effective in preventing infection. These guidelines are ineffective for those who are tested positive for COVID-19 infection. Improving immune function remains one of the best options for them for eliminating the infection. Increased oxidative stress and inflammation contribute to impaired immune function, and individual micronutrient improves immune function somewhat in experimental models. Because of limitations of effectiveness of a single micronutrient in human, a hypothesis is proposed that that a mixture of micronutrients may be more effective than a single micronutrient in improving immune function by reducing oxidative stress and inflammation, and thereby, eliminating of COVID-19 infection. This mixture of micronutrients may also reduce the risk of developing adverse health effects among survivors of COVID-19 infection.

Conflict of interest

The author is Chief Scientific Officer of Engage Global of Utah.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sector.

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