5. Vitamin D

5.1. Metabolism and Functions
Vitamin D is a fat-soluble hormone that is mainly synthesized in the skin after exposure to ultraviolet rays from sunlight (in the form of vitamin D3), and, to a lesser extent, is derived from dietary intake in the form of either vitamin D2 or D3 (the main sources of vitamin D are fatty fish, fish oils, egg yolks, cheese, and vitamin D-fortified foods). After vitamin D is produced in the skin or absorbed through the gastrointestinal tract, it is transported to the liver by vitamin D-binding protein (VDBP). In the liver vitamin D is converted to 25 hydroxy vitamin D (25(OH)D), which is monitored to evaluate vitamin D status because of its half-life of 2–3 weeks. Next, 25 hydroxy vitamin D is transported to the kidneys, where it is finally converted to its active form, 1,25 dihydroxyvitamin D (1,25(OH)2D).
The actions of 1,25(OH)2D are mediated through ligation with a nuclear vitamin D receptor (VDR), leading to the regulation of the transcription of over 1000 target genes. VDR is widely distributed in many different cells and tissues, including the immune system. VDR gene polymorphisms, located on chromosome 12q13.1, have been associated with higher prevalence of respiratory infections [74,75,76,77]. One of the main roles of vitamin D is to maintain calcium homeostasis by promoting calcium absorption in the intestine and reabsorption in the kidneys and stimulating bone remodeling by increasing osteoclasts number. This effect was the first to be discovered, studying the causes of rickets and osteomalacia, but now it is thought that vitamin D has physiological effects much broader that its role in mineral homeostasis and bone function [78], including regulation of immunity, fetal development [79], and pulmonary function [80]. In addition, vitamin D can also induce cathelicidin in gastrointestinal epithelium [81] and plays a role in controlling gastrointestinal infections [82].
For the purpose of this review, we focused on the effects of vitamin D in modulating the immune system. Several mechanisms have been described [83]: firstly, vitamin D was found to induce the production of antimicrobial peptides such as cathelicidin and human beta-defensin from immune system cells such as neutrophils and macrophages and from epithelial respiratory cells [81,82,83,84,85,86,87]. Vitamin D also enhances the antimicrobial activity of macrophages by increasing TLR and CD14 expression [88], autophagy [89,90], and the activity of NADPH-dependent oxidase [91]; it also promotes the migration of dendritic cells to lymphoid organs where they can present antigens to T cells [92]. On the other side of the coin, vitamin D can also inhibit the production of pro inflammatory cytokines, which might appear counterproductive [93]; it is known, however, that the pathogenicity of respiratory viruses, including SARS-CoV2, can be linked to hypercytokinemia up to the so-called “cytokine storm” [94,95,96,97,98]. This immunoregulatory effect of vitamin D can thus be beneficial to the host while facing a viral infection.
It has been reported during influenza A infection that IFN-beta, tumor necrosis factor (TNF)-alfa, IL-8 and IL-6 in the lungs were reduced in response to treatment with vitamin D [99]; during RSV infection the NfkB inhibitor was induced [100]; similar immunomodulatory effects were also described during Dengue infection [101]. Vitamin D can suppress excessive activity of IFN gamma-activated macrophages [102]; decrease macrophagic cytokines release through upregulation of MKP-1 [103]; reduce the production of metalloproteinase MMP-9 in keratinocytes, whose excessive and potentially harmful activity is induced by TNF alfa during hyperinflammation [104].
Vitamin D can also regulate FOXP3 expression in T cells, thus inducing the differentiation of this cells to FOXP3+ T regulatory cells (T reg), which have an immunosuppressor activity [105,106], and can promote the secretion of anti-inflammatory IL-10 from T cells [107]. It was also reported in a placebo-controlled trial on healthy adults that high dose vitamin D supplementation significantly increased the frequency of circulating Tregs [108].

5.2. Vitamin D Status
There is currently no definitive consensus regarding the optimal concentration of vitamin D in children and adults. Most authors define vitamin D in normal range from 30 to 100 ng/mL, which might be the optimal range to ensure its immunoregulatory effects; insufficient between 20 and 29 ng/mL, and deficient if serum levels are <20 ng/mL (50 nmol/L), since this level is necessary to maintain optimal bone mineralization and calcium homeostasis in 97.5% of the population [109,110]. Severe vitamin D deficiency is defined as <10 ng/mL; below this cut-off, the risk of developing rickets is very high. Concentrations >100 ng/mL may instead be harmful, although toxicity is more commonly seen over 200 ng/mL.
A 2016 study [111] combined data from 14 European population studies, including children, adolescents, and adults, and found an estimated prevalence of insufficient vitamin D levels of 13% in the general population. The prevalence according to age in pediatric populations varied from 4%–7% (1–6 years), 1%–8% (7–14 years) and 12%–40% (15–18 years). Italian data usually regards smaller populations and reports a high prevalence of vitamin D deficiency and insufficiency. A 2014 study from Stagi and colleagues found a 30% prevalence of vitamin D insufficiency in Italian children and adolescents and a 58.7% prevalence of vitamin D deficiency [112]. In the same year, Vierucci and colleagues reported a prevalence of 32.3% insufficiency and 49.9% deficiency [113]. Cadario et al. [114] described in Italian newborns a high frequency of vitamin D deficiency (40.1%) and severe deficiency (38%).

5.3. Recommended Daily Allowance and Supplementation
In Italy, the currently recommended daily allowance for vitamin D is 10 mcg (400 IU) for infants up to 12 months of age, and 15 µg (600 IU) for children and adolescents [115]. Vitamin D prophylaxis is recommended to all newborns and infants up to 12 months of age, regardless of their being formula or breast-fed [110,116], as also recommended by various others international Scientific Societies [117,118,119,120]. A higher dose is recommended for preterm infants over 1500 gr of weight (600–800 IU/day); for very low birth weight (VLBW) newborns below 1500 gr an intake of 200–400 UI is recommended.
Vitamin D supplementation is also recommended for children and adolescents with risk factors (obesity, reduced sunlight exposure, intestinal malabsorption, chronic hepatic or kidney disease, chronic therapies such as anticonvulsants, ketoconazole, etc.), at the dose of 600 IU/die up to 1000 IU/die in the presence of multiple risk factors [110]. Other societies recommend systematical supplementation of vitamin D during winter months [121,122,123]. In Italy, despite a high prevalence of hypovitaminosis D, there is currently no indication to conduct routine testing in healthy children and adolescents without known risk factors, nor to routinely supplement vitamin D. In case of detection of vitamin D insufficiency (<20 ng/mL), it is recommended to administer a higher dose of vitamin D (2000 IU/day for 6–8 weeks).

5.4. Vitamin D against Lower Respiratory Tract Infections
Considering the above-mentioned role of vitamin D in modulating the immune response, many studies focused on the link between vitamin D and viral infections. In this review, we focused on the present knowledge on the relationship between vitamin D and lower respiratory tract infections (LRTI) in children, since they are a leading cause of morbidity and mortality worldwide, especially in developing countries and in children younger than 5 years. We searched PubMed using keywords such as “vitamin D” and “lower respiratory tract infections” or “viral infections,” focusing on studies on pediatric populations, including both observational studies and clinical trials. Numerous studies investigated the association between low levels of 25-hydroxyvitamin D and increased susceptibility to LRTI in childhood, as listed in Table 4 [124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146].
Since 1997, it has been shown in developing countries, where malnutrition and micronutrient deficiency were more common, that the incidence of pneumonia was higher in children with rickets [124,126,131], and treatment failure was seen more frequently in rachitic children [132]. Similar studies were conducted worldwide, evaluating the circulating levels of vitamin D in children with LRTI and in controls: several studies found that lower vitamin D levels were associated with higher risk of developing an acute respiratory tract infection [125,130,132,133,135,139,144], or were linked to a more severe course of illness [136,142], with more frequent need for oxygen supplementation, ventilation support [134], or increased risk of intensive care unit (ICU) admission and longer hospital stay [143].
Some studies showed contrasting evidence and found no difference in vitamin D status in LRTI patients vs. controls [129,137,141]. A 2016 study conducted in Hong Kong on children and adults found no significant association between lower levels of vitamin D and higher incidence of influenza virus infections [140]. McNally and colleagues [127] found no significant difference in the frequency of vitamin D deficiency between the LRTI group and healthy controls but evidenced that vitamin D levels were significantly lower in patients admitted to PICU. A 2015 American study [138] found no difference in the duration of hospitalization and in the severity of the disease between deficient and nondeficient children. As highlighted by the authors themselves, some of these findings might be explained by the different vitamin D status in different countries around the world, some implementing extensive vitamin D supplementation programs, and some still struggling with malnutrition and micronutrients deficiencies. Roth and colleagues [130] underlined how average vitamin D concentration varies through studies from different regions of the world, ranging from 22.8 nmol/L–equal to 9.12 ng/mL-in India and Turkey [125,145], 29.2 nmol/L in Bangladesh [128], to 77.2–81 nmol/L–equal to 30.8–32.4 ng/mL-in Canada [127,129].
Several studies also focused on the link between maternal vitamin D status during pregnancy and/or cord blood vitamin D levels and respiratory tract infections in infants. Higher vitamin D levels were found consistently associated with reduced risk of LRTI in infants worldwide [145,146,147,148,149,150,151,152,153,154], as described in Table 5.
These observational findings have laid the foundation for clinical trials of vitamin D supplementation for treatment or prevention of childhood respiratory tract infections, as shown in Table 6a,b [155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170].
These studies differ in many aspects, such as geographical location, baseline vitamin D level before the intervention, and dose and timing of vitamin D supplementation, and this heterogeneity leads to sometimes contradictory results.
A few studies investigated the effect of a short-term, high-dose vitamin D supplementation, which is the most practical administration scheme, but mostly found that these regimens were not significantly beneficial. Manaseki-Holland and colleagues evaluated the administration of a single high-dose (100,000 IU) vitamin D supplementation in addition to routine pneumonia treatment in children in Kabul, and evidenced a lower risk of recurrence in the intervention group but no difference in the time needed to recovery from the first infection [155]. Furthermre, a smaller study from Pakistan described a lower recurrence of pneumonia in children supplemented with a single dose of vitamin D [160]. Similar trials from Choudhary et al. [157] and Gupta et al. [165] tried a short-term vitamin D supplementation in Indian children with severe pneumonia: the first study supplemented vitamin D (1,000–2,000 UI/day) for 5 days, and the second 100,000 UI in a single dose; in both studies the authors did not evidence significant beneficial effects in the resolution of pneumonia in the intervention group. Gupta et al. evidenced only a slightly quicker resolution of the severe respiratory distress (1 h) in the intervention group, which might not be clinically relevant. Similar results were reached in 2017 by Somnath and colleagues [166], who investigated the efficacy of a single high dose of vitamin D in the treatment of children hospitalized with ALRI, and found it did not influence the duration of hospital stay nor the secondary outcomes (mortality, PICU admissions, complications, etc.). A supplementation of 50,000 IU/day for 2 days was tried in Iran in children with pneumonia and it did not influence the severity of symptoms, however the study reported a lower duration of antibiotic use in the intervention group [166]. Contrasting evidence was found in a 2015 Egyptian trial on children hospitalized for bronchiolitis [161], where the administration of vitamin D 100 IU/kg/day for 5 days was associated with a significant improvement in the duration of hospitalization and time taken to improve oral feeding. The efficacy of a high dose, short-term supplementation of vitamin D in preventing respiratory tract infections was also analyzed in 2012 by Manaseki-Holland and colleagues [157], who found 100,000 IU supplementation every 3 months ineffective in reducing the incidence of pneumonia, and later in 2019 by Singh et al. [169], who achieved similar results with a 300,000 IU supplementation every 3 month. Overall, the administration of a bolus dose or short-term supplementation of vitamin D did not demonstrate a consistent efficacy in treating nor in preventing LRTI [171], although there is, at times, conflicting evidence on the matter.
More promising results were reached using daily or weekly administration of vitamin D for longer periods of time. A 2010 Japanese study found that daily administration of vitamin D (1,200 IU/die) to schoolchildren during winter months reduced the incidence of influenza A infections [156]. In 2012, Camargo and colleagues [159] investigated the administration of vitamin D-fortified milk during winter months in Mongolian children, and reported significantly lower RTI episodes during the study period. A Chinese 2019 prospective study analyzed a cohort of infants recording whether they received vitamin D daily supplementation (400–600 UI/die) up to 6 months of age, and reported the median time of the first RTI episode, which ended up being 60 days in infants without supplementation and longer than 6 months in infants with supplementation [168].
The right dosage to achieve a protective effect on respiratory infections is yet to be established. Different studies evaluated different doses, ranging from 400 to 2000 UI/die. We found two studies that compared a lower vs. a higher dose of daily vitamin D supplementation, both reporting better results in the higher dose group.
In 2015 Grant and colleagues analyzed the supplementation of vitamin D to pregnant women and to their infants up to 6 months of age, comparing two regimens: 1000 IU to the mothers and 400 IU to the infants vs. 200–800 IU, and found a lower proportion of children made a primary care visit for respiratory infections up to 18 months of age in the higher dose group [163]. A 2018 Chinese study tested the efficacy of vitamin D in preventing influenza A, comparing a low dose scheme (400 IU/day) vs. a high dose one (1200 IU/day) for 4 months, and reported less frequent infections in the high dose group [167].
For the purpose of this review, we focused on studies on LRTIs, even though similar studies were also conducted on the prevention of upper airways infections; a large study conducted on the TARGet kids! research network in Toronto (Canada) led to different results, reporting that a high dose (2000 IU/day) was not more effective than a standard dose (400 IU/day) in preventing upper respiratory tract infections in children [172]. We also found two studies reporting negative results with vitamin D daily/weekly supplementation: the first, conducted in 2014, tested a daily supplementation of vitamin D 2000 IU/day for 2 months to Japanese high school students, and found no efficacy in lowering the overall incidence of influenza A [160]; the second, conducted in 2019 in Vietnam, analyzed a 14,000 IU/week supplementation of vitamin D to children and adolescents for 8 months, which was unable to prevent influenza infection during the flu season, but moderately reduced the incidence of other respiratory viral infections [172]. In this population, the authors reported a mean baseline vitamin D of 65 nmol/L (26 ng/mL), which might be one of the reasons why a further vitamin D supplementation did not lead to the expected results. In the previously cited studies, the baseline vitamin D status is not always reported; where it is known, it is usually lower, from 7 ng/mL, equal to 17.5 nmol/L [153], to 43 nmol/L, equal to 17.2 nmol/L [167].
In conclusion, there is evidence on the role of vitamin D in regulating the immune response to viral infections, and data from most observational studies confirm an association between lower vitamin D levels and increased susceptibility to respiratory infections. Clinical trials overall show that daily or weekly supplementation of vitamin D is more beneficial in preventing LRTI than bolus or short-term administration, as confirmed by a 2017 meta-analysis by Martineau and colleagues [171], though more research will be needed to fully determine when and how vitamin D should be supplemented. Vitamin D supplementation did not appear to be effective in treating existing infections in pediatric trials, as also described in a 2018 review from Das and colleagues [173]. The different results reached in the above-mentioned studies might be due to the heterogeneity in the baseline vitamin D status of the observed populations; it is also possible that vitamin D receptor’s polymorphisms affect the daily vitamin D requirements of different individuals. Future studies might better clarify which patients will benefit from vitamin D supplementation and which ones will not, which is the best dose to administer in each case, and whether vitamin D status should always be tested before intervention.

5.5. New Perspectives: Is There a Potential for Vitamin D Supplementation in Preventing COVID-19?
At the time of writing (3 July 2020), the COVID-19 pandemic has claimed over 500,000 lives worldwide with over 11 million confirmed infections. Different regions of the world have been differently affected by the pandemic, with Northern Italy setting an unfortunate record for incidence and mortality. Different factors might explain these geographical variations, such as the earlier spread of the virus in certain countries or the different preventive measures adopted, the different climates and air-pollution levels, or the different age-composition and social proximity of the communities. A North–South gradient in COVID-19 distribution has been noticed [174,175,176]. Areas along a latitude of 30–50° N with similar low-humidity, temperate weather, showed significant community spread of COVID-19 [177]. Marik and colleagues calculated the case-fatality rate in each state of the US and found increasing mortality with increasing latitude (>40° N) [178]. More recently, another study reported a highly significant, positive correlation between lower death rates and a country’s proximity to the equator [179]. Rhodes et al. described that more northerly countries are currently showing relatively high COVID-19 mortality, with an estimated 4.4% increase in mortality for each 1-degree latitude north of 28 degrees North [180].
Vitamin D deficiency is less common in countries where the sun exposure is consistent throughout the year or where the use of vitamin D fortified food is widespread. Various authors suggested that vitamin D deficiency might play a role in the variability of COVID-19 impact on different countries [175,176,177,181]. Ilie and colleagues searched literature for mean vitamin D level in each country and observed a negative correlation between vitamin D levels and number of COVID-19 cases and deaths [181]. Ali described a significant negative correlation between mean vitamin D levels and COVID-19 cases per one million population in European countries, as of 20 May 2020 [182].
Moreover, a wide variation in the severity of SARS-CoV2 infection’s clinical presentation has been noticed, ranging from absent or minimal symptoms to critical conditions and death. To date, although some risk factors have been identified (age, co-morbidities, etc.), it is not yet completely understood why some patients develop more severe symptoms than others. Considering our knowledge on the role of vitamin D in modulating the immune system and in inhibiting a hyper activation of the inflammatory response, together with data from observational and clinical studies on vitamin D supplementation, various authors have also suggested a potential role of vitamin D in reducing the severity of the disease [183,184,185,186,187]. Vitamin D is especially known for its ability to reduce the “cytokine storm” that contributes to the pathogenesis of various viral infections, including COVID-19 [188].
To date, we only have preliminary observations regarding the association of vitamin D deficiency and frequency and severity of COVID-19; the above mentioned study from Ilie and colleagues found a correlation between mean vitamin D levels in each country and COVID-19 cases and deaths [181]; D’Avolio and colleagues investigated vitamin D concentrations in a small cohort of 107 patients with a positive naso-pharyngeal swab for SARS-CoV2 in Switzerland, and found significantly lower vitamin D levels in patients than in controls with negative swabs [187]; Lau et al. described a high frequency of vitamin D insufficiency (84.6%) in COVID-19 patients admitted to ICU in New Orleans, with a 100% frequency in patients younger than 75 years [188].
Interestingly, a recent pilot study demonstrated that administration of a high dose of 25-hydroxyvitamin D significantly reduced the need for intensive care unit treatment of patients requiring hospitalization due to proven COVID-19 [189]. Calcifediol seems to be able to reduce severity of the disease, but larger trials with groups properly matched will be required to show a definitive answer.