Role of vitamin D in pathogenesis and severity of COVID-19 infection

Abstract Coronavirus disease (COVID-19) is an infectious disease caused by a new virus that causes respiratory illness. Older adults and individuals who have pre-existing chronic medical conditions are at higher risk for more serious complications from COVID-19. Hypovitaminosis D is attributed to the increased risk of lung injury and acute respiratory distress syndrome (ARDS) as well as diabetes, cardiovascular events and associated comorbidities, which are the main causes of severe clinical complications in COVID-19 patients. Considering the defensive role of vitamin D, mediated through modulation of the innate and adaptive immune system as well as inhibition of the Renin Angiotensin System (RAS), vitamin D supplementation might boost the immune system of COVID-19 patients and reduce severity of the disease in vitamin D deficient individuals.


Introduction
An increase in the incidence of infections caused by various human respiratory pathogens emerges each winter, however, the timing and magnitude of the infection are widely variable (Dowell and Ho 2004). Seasonality and persistence are the two main characteristics of respiratory viruses, such as influenza, Respiratory syncytial virus (RSV) and the two previously described human coronaviruses (CoV 229E and CoV OC43) (Dowell andHo 2004, Grant et al. 2020).
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) was first identified in December 2019 in China. SARS-CoV-2-infected individuals suffer from a variety of symptoms, ranging from tiredness, fever and cough to severe pneumonia, acute respiratory distress syndrome, sepsis, septic shock and death (Wang et al. 2020, Zhang et al. 2020. It has been found that entry of SARS-CoV-2 into the human cells is via Angiotensin Converting Enzyme 2 (ACE 2), a membrane exopeptidase that converts Angiotensin I to angiotensin (Xu et al. 2020). The Renin-Angiotensin (Ang) System (RAS) regulates blood pressure through conversion of angiotensinogen to angiotensin I and ultimately angiotensin II, catalysed by ACE2. Moreover, fluid and electrolyte balance, in addition to, systemic vascular resistance are regulated by the RAS ( Figure  1) (Xu et al. 2017). Although ACE2 counteracts the effects of RAS activation, bindingofcoronavirusto ACE2 results in decreased level of active form of ACE2 on the cell membrane that in turn activates local RAS in the lung tissue leading to acute lung injury which can progress to adult respiratory distress syndrome (ARDS) (Augoustides 2020).
ACE2 is expressed in human airway epithelia, lung parenchyma and especially in the epithelial lining of the oral cavity, making it the portal of entry for the SARS-CoV-2 infection (Jia et al. 2005, Xu et al. 2020). More interestingly, expression of ACE2 was also detected in lymphocytes (B and T cells) within the oral mucosa as well as other organs of the digestive system (Xu et al. 2020). This expression pattern of ACE2 might be associated with the severity of the SARS-CoV-2 -induced disease, COVID-19 (Dijkman et al. 2012, Xu et al. 2020. Furthermore, there is growing evidence of a correlation between COVID19-related mortality rate and latitude, so that the mortality rate of COVID-19 is significantly lower in the countries that lie below 35 degrees north (Rhodes et al. 2020). These distinct mortality rates might be due to the greater extremes of daylight during winter in these regions which might consequently result in lower rate of vitamin D deficiency (Rhodes et al. 2020). This suggests a possible role for vitamin D in severity of complications of COVID-19. Since vitamin D plays an important role in regulation of immune response against various pathogens, such as respiratory viruses, it might induce a protective effect against SARS-CoV-2 infection by preventing the cytokine storm, which is the common cause of the disease severity and mortality.
The aim of the present study is to discuss the possible protective role of vitamin D against COVID-19 pathogenesis and severity in order to prevent further serious complications in the high-risk general population. level remains unchanged. Therefore, angiotensin II level is increased in the lung tissue followed by dominance of the ACE/angiotensin II, that consequently results in acute lung injury (ALI) (Kai and Kai 2020).
Vitamin D3 (1,25-dihydroxycholecalciferol;1,25(OH)2D3), the active form of vitamin D and a lipophilic micronutrient, is either obtained through the conversion of 7-dehydrocholesterol by the skin when exposed to ultraviolet B radiation or via food intake (Pilz et al. 2009, Martineau et al. 2017. It is involved in several cellular processes, maintenance of calcium homeostasis, and phosphorus absorption. Recent studies have recognised vitamin D as one of the most important modulators of the RAS (Li 2011), however, the molecular mechanism underlying the vitamin D-induced down-regulation of the Renin-Angiotensin System (RAS) is yet to be fully understood. Vitamin D, is a central negative regulator of renin biosynthesis. The repressive activity of 1,25(OH)2D3 is mediated by the vitamin D receptor (VDR) (Yuan et al. 2007). It has been reported that the formation of the cyclic adenosine monophosphate (cAMP) response element-binding protein 1 (CREB1) and nuclear receptor corepressor 1 (NCOR1), as the enhancer of rennin protein expression, is blocked by1,25(OH)2D3 (Andersen et al. 2015). Therefore, vitamin D may suppress the RAS activity through down-regulation of renin transcript and consequently inhibition of conversion of angiotensinogen to angiotensin I and ACE/Ang II cascade (Xu et al. 2017) (Figure 1).

Vitamin D as an immunemodulator factor
Vitamin D deficiency leads to the development and progression of several chronic diseases, as well as susceptibility to infectious diseases and is particularly associated with an increased occurrence of respiratory viral infections (Pilz et al. 2009, Yamshchikov et al. 2009, Bryson et al. 2014, Lee 2020. Vitamin D and VDR polymorphisms activates the innate immune response whereas it reduces the adaptive immune response (Beard et al. 2011, Hewison 2011. Moreover, vitamin D shifts the adaptive immune system from Th-1 to Th-2 responses through increasing the levels of the TLR co-receptor in monocytes and decreasing of viral induction of inflammatory genes (Beard et al. 2011). The toll-like receptors (TLRs) are fundamental to the innate immune response and can recognise viral proteins and nucleic acids. Activated TLRs induce reactive oxygen species (Beard et al. 2011). The active form of vitamin D, 1,25(OH)2D3, is a steroid hormone that acts as an immune system modulator by down-regulating the expression levels of inflammatory cytokines and enhancing M2 macrophage function. Furthermore, it induces the expression of potent antimicrobial peptides (AMPs), which are present in natural killer cells, monocytes, neutrophils, as well as the epithelial cells lining the respiratory tract (Mawson 2013). Vitamin D triggers development of suppressive regulatory T cells and inhibits development of proinflammatory Th17 cells (Yang et al. 2016), which consequently may help the host cells to maintain the balance between COVID-19 induced tissue-damage and protective effects of the immune response (Keynan et al. 2008). Moreover, the pro-inflammatory cytokines of the adaptive immune system (such as IL-1, IL-6, etc.) are modulated by vitamin D, particularly those involved in acute inflammation cytokine storms (Mawson 2013, Chen et al. 2013, Andersen et al. 2015, Martineau et al. 2017). It has also been found that metabolites of vitamin D induce other innate  (1 7) acts against the effects of Ang II. Therefore, the endogenous ratio of Ang II: Ang (1 7) is affected by the balance between ACE and ACE2 levels [8]. Furthermore, vitamin D may suppress RAS activity through inhibition of renin.
antimicrobial effect or mechanisms, including autophagy that is a powerful defense mechanism against viral infection and synthesis of reactive oxygen (ROS) and nitrogen intermediates, through activation of monocytes and macrophages Pawliczak 2016, Martineau et al. 2017). Autophagy controls viral infections through viral destruction, inflammatory responses regulation and antigen presentation augmentation (Choi et al. 2018). Moreover, ROS, as a secondary messenger, mediates death in viral infected cells through modulating the expression levels of a number of apoptotic, antiapoptotic, and proapoptotic genes (Reshi et al. 2014, Yang et al. 2016, Umar et al. 2018. Therefore, induction of autophagy and activation of ROS signalling pathway can be considered as the underlying mechanisms that contribute to the potential role of vitamin D against coronavirus infection. Furthermore, respiratory epithelium, as one of the major players in innate immunity, is able to generate 1,25(OH)2D3 that is involved in phagocytosis through alveolar macrophages and dendritic cells, release of cathelicidin and chemokines, reduction of IL-12, Th1 and Th17 cytokines levels and increase of IL-10 regulatory T cells during viral infection (Hansdottir and Monick 2011). These characteristics of respiratory epithelium may result in reduced severity of COVID-19 complications in patients with optimal levels of vitamin D concentration.
Therefore, it can be assumed that the active form of vitamin D (D3) induces its possible beneficial effects on COVID-19 by and down-regulation of pro-inflammatory cytokines which in turn might prevent or ameliorate cytokine stome in COVID-19 patients (Hashemi et al. 2018, Sassi et al. 2018, Szymczak-Pajor and Sliwi nska 2019, Grant et al. 2020).

Potential mechanisms underlying anti-viral effects of vitamin D
Using vitamin D supplementation to prevent respiratory tract infection is not a common practice. Effectiveness of this intervention relies on its continuity prior to the onset of the infection (Khan and Sellen 2011).
The association between hypovitaminosis D and increased risk for serious complications from the influenza and other respiratory tract infections, especially among patients with HIV infection, has been proven by several interventional and observational epidemiological studies (Beard et  The hypothesis that vitamin D directly affects viral infections, particularly infections caused by enveloped viruses, has been supported by results of several cell culture experiments (Beard et al. 2011). These functions of vitamin D may be linked to its up-regulatory effects on the cathelicidin family, that are a group of antimicrobial peptides which is released from neutrophil, macrophages and lung epithelial cells (in the form of LL-37), and human beta defensin 2 and may also be induced through releasing of reactive oxygen species (Beard et al. 2011). Cathelicidins modulate innate and adaptive immunity, predominantly by influencing monocytes, dendritic cells and T cells (Sousa et al. 2017).
The anti-bacterial effect of LL-37 is linked to its ability to disrupt bacterial membranes via electrostatic interactions. It may also induce similar effect on the lipid envelopes of viruses (Beard et al. 2011, Teymoori-Rad et al. 2019. The ability of LL-37 to inhibit these viruses is resulted mostly from its direct effects on viral envelope (Tripathi et al. 2013). It is probable that entry of viruses into the cell is blocked by LL-37 in a similar manner to what is observed in other antimicrobial peptides, mainly in enveloped viruses (Beard et al. 2011). LL-37 has been demonstrated to have anti-viral effects, including inhibition of replication of herpes simplex virus type one (HSV-1), vaccinia virus (VACV), retroviruses, respiratory syncytial virus (RSV) and some non-enveloped viruses, such as adenovirus, rhinovirus and Human papilloma virus (Beard et al. 2011, Sch€ ogler et al. 2016. These findings support the hypothesis that the anti-viral effects of LL-37 may be partially mediated via envelope disruption (Beard et al. 2011). Since SARS-CoV-2 is an enveloped virus, adjusting the circulating vitamin D level might help treating the newly emerged COVID-19 through LL-37, which is present in bronchoalveolarlavage, via envelope disruption or modulation of apoptotic and immune pathways (Sousa et al. 2017). Furthermore, the cytokine storm that occurs in sever phase of COVID-19, may be suppressed by vitamin D throughsuppression of pro-inflammatory cytokines in PBMCs and CD14þ monocytes (Hoe et al. 2016, Daneshkhah et al. 2020) (Figure 2).

Worldwide prevalence of vitamin D deficiency
Vitamin D deficiency is a major public health problem worldwide. In 2011, the Endocrine Society considered the serum circulating 25-hydroxyvitamin D concentration (25(OH)D; major form of Vitamin D found in the blood) to define vitamin D status in the population: >30 ng/mL is considered "optimal", 20-30 ng/mL is "insufficient", and <20 ng/mL is "deficient" (Hernando et al. 2020). It has been estimated that the global prevalence of vitamin D deficiency is approximately 30-50% (Kheiri et al. 2018). The highest prevalence of vitamin D deficiency belonged to the Middle Eastern population of all the age groups. In adults, the number of women who suffered hypovitaminosis D was higher than that of men. Therefore, hypovitaminosis D in the Middle East, particularly in women, is an important health issue (Palacios andGonzalez 2014, Aghili et al. 2020). Cannell et al. (2006) have argued that vitamin D status may contribute to susceptibility of the population to seasonal infections as well as the degree of associated morbidity and mortality, (Cannell et al. 2006). Furthermore, the results of a recent meta analysis demonstrated a possible connection between vitamin D repletion, susceptibility to infection, and clinical outcomes in a variety of infectious, such as influenza, and upper respiratory tract viral infections.Moreover, authors of the abovementioned meta analysis have claimed that adequate concentrations of vitamin D may decrease the rates of all-cause infection in their analysed populations (Yamshchikov et al. 2009).

Health risks of vitamin D deficiency
Several health implications are attributed to hypovitaminosis D, including respiratory disorder-related mortality, susceptibility toviral infections, cardiovascular diseases (CVD), diabetes, hypertension and osteoporosis (Hughes and Norton 2009, Akhtar 2016, Kheiri et al. 2018. Vitamin D deficiency induces inflammation in epithelial cells, dysregulates the expression levels of over 600 genes and contributes to the development of numerous diseases, including musculoskeletal, and respiratory systems diseases, as well as cancer (Gatera et al. 2018). Several studies have indicated that people with diabetes, CVD, pulmonary diseases and hypertension as well as aged people (all conditions are severe comorbidites in COVID-19) have lower vitamin D concentrations than healthy individuals (Hughes and Norton 2009, Andersen et al. 2015, Mozos and Marginean 2015, Nakashima et al. 2016, Berridge 2017, Kheiri et al. 2018, Grant et al. 2020.

Vitamin D-ACE2 interaction and respiratory illness
Acute lung injury (ALI)andacute respiratory distress syndrome (ARDS) are the main causes of severe lung damage and respiratory failure in COVID-19 patients and vitamin D deficiency could be considered as a contributing factor (Palacios andGonzalez 2014, Xu et al. 2017).
Local RAS is present in lung tissue (Chen et al. 2013). Both enhanced expression levels of ACE/Ang II and decreased expression of ACE2/Ang-(1-7) occur in ALI (Chen et al. 2013). While Ang-II contributes to the development of lung fibrosis, ACE2/Ang-(1-7) may have a protective function in ALI (Chen et al. 2013, Wang et al. 2018. Enhanced expression levels of ACE2 and Vitamin D receptor (VDR) play a protective role against the development of ALI (Yang et al. 2016). It has been reported that over-expression of ACE2 resultes in improvement of dysregulated expression of ACE/ACE2 and Ang II/Ang-(1-7) and alleviated lung injuries, whereas ACE2 knockout further attenuated the imbalance of ACE/ACE2 and Ang II/Ang-(1-7) expression levels, resulting in exacerbated lung injuries. Maintaining RAS homeostasis by enhancing the expression level of ACE2 may reduce lung injury (Figure 3) (Chen et al. 2013).
Association between hypovitaminosis D and susceptibility to acute respiratory tract infections has been reported by several observational studies . Results of an individual participant data (IPD) meta-analysis of randomized-controlled trials revealed that the risk of experiencing at least one acute respiratory tract infection is significantly reduced by taking vitamin D supplementation (Martineau et al. 2017). Furthermore, one step analyses of acute respiratory tract infection rate indicated the significant role of vitamin D (adjusted incidence rate ratio 0.96, 95% confidence interval 0.92 to 0.997, p ¼ .04; p for heterogeneity <.001; 10 703 participants in 25 studies) in reducing the risk for acute respiratory tract infections. Hence, vitamin D supplementation induces a defensive effect in patients with baseline circulating 25(OH)D concentrations less than 25 nmol/L (adjusted odds ratio 0.58, 0.40 to 0.82, NNT ¼ 8, 5 to 21; 538 participants in 14 studies; within subgroup p ¼ .002) (Martineau et al. 2017).

Effects of vitamin D deficiency in high risk groups for COVID-19
Individuals with associated comorbidities, such as obesity, diabetes, hypertension, cardiovascular diseases, respiratory diseases and cancer, are at higher riskof severe illnesses and death (Kheiri et al. 2018).
It has been reported that the case fatality rate (CFR) in COVID-19 patients with no comorbidity is 1.4%, while it is 13.2%, 9.2%, 8.4% and 8.0% in those with cardiovascular disease, diabetes, hypertension and chronic respiratory disease, respectively ( Figure 3). Increased susceptibility to respiratory infection in patients with diabetes and cancer is the result of reduced effects of antibodies, decreased amounts of inflammatory cytokines (such as IL-1, IL-6, IL-10, TNF and IFNs) and immunodeficiency (Klekotka et al. 2015, Liang et al. 2020. Vitamin D directly affects smooth muscle cells by inhibiting proliferation that leads to calcification, which results in cardiovascular diseases (CVD) (Mozos andMarginean 2015, Kheiri et al. 2018). In an analysis of the Third National Health and Nutrition Examination Survey (NHANES III 1988-1994, low level of vitamin D was introduced as CVD risk factor along with hypertriglyceridaemia, obesity and diabetes mellitus (DM). Furthermore, results of a prospective nested casecontrol study revealed that hypovitaminosis D enhanced the risk of myocardial infarction compared to sufficient 25(OH)D level following multivariate adjustment.
The results of a meta-analysis of 19 prospective studies revealed a linear and inverse association between circulating vitamin D level and risk of CVD (Kheiri et al. 2018).
Resistant hypertension, is considered as a major risk factor for CVD (Pilz et al. 2009, Andersen et al. 2015, Mozos and Marginean 2015, Kheiri et al. 2018. Prolonged vitamin D3 deficiency or even short-term severe vitamin D deficiency can result in development of hypertension via modulation of the RAS system (Li 2003, Andersen et al. 2015. Using animal models, vitamin D3 has been found to down-regulate renin Ace and Agn genes, whereas it up-regulates the expression level of Ace2 (Andersen et al. 2015). Results of several human cross-sectional studies revealed an association between the lower vitamin D levels and higher RAS activity in vascular tissue, higher plasma rennin activity (PRA), higher Ang II concentrations, and altered responses to Ang II (Andersen et al. 2015, Mozos and Marginean 2015, Kheiri et al. 2018. Oral vitamin D supplementation has been found to notably reduce diastolic blood pressure (BP) while it slightly, but significantly, reduces diastolic BP, in patients with pre-existing cardiovascular risk (Pilz et al. 2009, Andersen et al. 2015, Mirhosseini et al. 2017.
As the other risk factors of CVD, insulin resistance and risk of diabetes are linked to vitamin D status (Kayaniyil et al. 2010, Nakashima et al. 2016, Berridge 2017. Results of an observational study performed on 494 women undergoing serial metabolic characterisation indicated that hypovitaminosis D along with increased Parathyroid hormone' (PTH) levels were independent predictors of b-cell dysfunction, insulin resistance, and hyperglycaemia (Kayaniyil et al. 2010, Nakashima et al. 2016.
Hypovitaminosis D induces its effect on insulin secretion, insulin resistance, and b-cell dysfunction via RAS in the pancreas, so that it can enhance the production of ROS and G protein RhoA via increasing renin and angiotensin II synthesis, leading to inhibition of the pathways essential for intracellular glucose transportationand consequently the development of insulin resistance and metabolic syndrome (Mozos andMarginean 2015, Kheiri et al. 2018). Vitamin D induces its regulatory effect on insulin secretion through regulation of intracellular calcium concentration (Sung et al. 2012, Nakashima et al. 2016. Moreover, vitamin D is indirectly associated with insulin synthesis and secretion in the pancreas via regulation of PTH concentration. It also regulates insulin sensitivity (Sung et al. 2012, Berridge 2017) by up-regulating the expression levels of insulin receptors (Figure 3) (Sung et al. 2012, Nakashima et al. 2016. Furthermore, inflammation, which is a main process in inducing insulin resistance, is reduced by vitamin D (Kayaniyil et al. 2010, Sung et al. 2012, Nakashima et al. 2016. Vitamin D adjusts the resting levels of both Ca 2þ and ROS, that are elevated in the b-cells during diabetes, to their normal levels (Berridge 2017, Pittas et al. 2019. Chronic kidney disease (CKD), as a complication of type 2 diabetes, is associated with reduced concentrations of 1,25(OH)2D3 (Nakashima et al. 2016). CKD patients with low vitamin D levels are at higher risk of end stage renal disease, all-cause mortality and cardiovascular diseases (Nakashima et al. 2016). Although a recent study revealed that vitamin D has renoprotective effects through regulation of tubular ACE and ACE2 (Lin et al. 2016), the effectiveness of vitamin D supplementation for protection of kidney function requires further investigated.

Discussion
Patients with chronic diseases have significantly higher risk of death from respiratory tract infections. On the other hand, deficient vitamin D concentration isassociated with increased risk of various diseases, including cardiovascular disease, diabetes mellitus, and hypertension (Grant et al. 2020, YAP et al. 2020. Observational studies suggest that serum 25(OH)D concentrations are generally low in many populations, especially in elderlies (Martineau et al. 2017, Grant et al. 2020. It has been shown that having comorbid conditions significantly increases the case fatality rate, especially with aging. This could be explained by compromised adaptive immune response (Grant et al. 2020 Therefore, it would be reasonable to postulate that the seasonality of many viral infections is associated with low 25(OH)D concentrations especially population with associated co-morbid conditions. Vitamin D reduces viral infections by its diverse immunomodulatory effects, including strengthening of epithelial cell junction integrity, up-regulatory effect on the cathelicid family, recruitment of immune cells to the site of infection, and reducing the cytokine storm induced by the innate immune system as well as adaptive immune response (Grant et al. 2020).
The entry of SARS-CoV-2 into the human cells is via ACE 2, a membrane exopeptidase that converts Angiotensin I to the nonapeptide angiotensin. ACE2 negatively regulated the RAS by converting Ang II to Ang-(1-7). It is expressed in human airway epithelia. The RAS, which includes ACE and ACE2, is a complex network that has a major role in various biological functions (Li 2003). Chronic vitamin D deficiency may induce lung fibrosis through activation of the RAS (Zhou et al. 2008). Increasing evidence indicates that 1,25(OH)2D3 may also be a negative endocrine regulator of the RAS. Vitamin D inhibits renin, ACE and Ang II expression, and induces ACE2 over-expression in ALI. Therefore, vitamin D may attenuate ALI by inducing ACE2/Ang-(1-7) axis and inhibiting renin and the ACE/Ang II/AT1R cascade (Yang et al. 2016, Xu et al. 2017. Although the positive effect of using Vitamin D supplementation in supporting the immune system and preventing different chronic and infectious diseases, such as acute respiratory tract infection, has been claimed by numerous researchers, its reference range and recommended intake dosage in disease state need to be adjusted (Martineau et al. 2017, Ghanaati et al. 2020, Laird et al. 2020.
In conclusion, considering the defensive function of vitamin D in ALI, supplementing vitamin D deficient individuals may boost the immune system to fight COVID-19 infection and reduce its severity, especially in patients with associated co-morbidities.

Disclosure statement
There is no potential conflict of interest.