Considering that the world is currently entering a new wave involving the more transmissible and more immune evasive SARs-CoV-2 BA.5 variant that has been found to be more pathogenic and exhibits even stronger tropism towards the lungs, the relevance of Vitamin D supplementation is even more important now.
The study findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2022.06.29.498158v1
Low vitamin D status has been associated with poorer outcomes following acute respiratory diseases including influenza . Vitamin D supplements have been touted as being useful in high doses for reducing the severity of seasonal influenza [2-4]. The recent emergence of severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2 and the ongoing pandemic has led to a renewed interest in high dose vitamin D supplements to prevent and treat severe SARS-CoV-2 disease (i.e. COVID-19) .
Infection with SARS-CoV-2 results in local and systemic inflammation that when controlled may enhance survival and clinical outcomes of COVID-19 . Severe respiratory illness can also be caused by influenza viruses or co-infection with influenza and coronaviruses. An association between low vitamin D status and severe COVID-19 has been postulated and accordingly, vitamin D supplementation has been proposed to be beneficial to treat COVID-19 .
A recent systemic review concluded that low circulating levels of vitamin D (serum 25(OH)D, 25D) were associated with more severe symptoms, and higher mortality in patients with COVID-19 . Interventions that control the lung inflammatory response to viruses have the potential to benefit the global population.
Vitamin D, and the active form of vitamin D (1,25(OH)2D, 1,25D) has been implicated to play a role in the anti-viral response; however, this effect may be specific to different viruses. For example, 1,25D treatment of T cells from human immunodeficiency virus (HIV) infected individuals in vitro resulted in a decrease in viral RNA transcription by direct reduction of NF-κB, which reactivates proviral HIV .
Conversely, 1,25D treatment of respiratory syncytial virus (RSV) infected human epithelial cells in vitro did not affect viral replication . Production of antimicrobial peptides such as cathelicidin, β-defensin etc., and production of cytokines such as TNFα, IL-5, IL-1β, IL-6, IL-10 and type I interferons at the mucosal surface are important parts of the innate immune response against viruses [10,11].
Several studies have shown that 1,25D and other vitamin D analogs induced cathelicidin production in response to virus infection . Cathelicidin LL-37 has been shown to bind and kill viruses including influenza viruses in vitro [13-17]. Therefore, it is possible that vitamin D through the induction of cathelicidin could directly target SARS-CoV-2 and influenza. 1,25D also limits inflammatory responses by decreasing IFNγ, IL-6 and TNFα [18,19]. The effects of 1,25D to reduce inflammation could be detrimental for the ability of the host to clear some viruses.
Lung epithelial cells are vitamin D targets since they express the vitamin D receptor (VDR) and are regulated by 1,25D treatments. In mice, 1,25D treatments reduced inflammation following lipopolysaccharide induced lung injury and regulated angiotensin converting enzyme (ACE) expression in the rat lung epithelium .
In mice, infection with an influenza H9N2 virus induced mRNA for the VDR in the lung and 1,25D treated animals had reduced lung inflammation . Treating mice with the 1,25D precursor, 25hydroxyvitamin D (25D), also protected mice from subsequent H1N1 influenza infection . 1,25D treatment of human bronchial epithelial cells suppressed IL-6 and protected the cells from oxidative damage .
VDR knockout (KO) mice had reduced expression of tight junction proteins such as zonula occludens-1, occludin, and claudins (2,4, and 12), suggesting an important role for the VDR in maintaining the integrity of the lung . Vitamin D has direct effects on the lung epithelium and 1,25D suppresses inflammation in the lung.
Extensive association studies in humans have led to the proposal that high dose vitamin D supplements could be beneficial for protecting from severe influenza and SARS-CoV-2 infections. Since cause and effect are extremely difficult to determine in human studies: we sought to evaluate the effects of vitamin D on the lung anti-viral response in animal models.
The data from mice and hamsters suggests that vitamin D supplementation reduces inflammation in the lung following pandemic H1N1 and SARS-CoV-2 infection. D-mice had lung inflammation even without infection. We had shown previously that feeding D-diets to mice that cannot produce 1,25D (Cyp KO) resulted in severe vitamin D deficiency .
Influenza disease was greatest in D- Cyp KO and the least amount of disease was in D+ WT mice. The survival of D+ Cyp KO mice was significantly less than D+ WT mice following an influenza infection. Vitamin D supplementation reduced lung inflammation and Ifnβ expression in the lung of mice following SARS-CoV-2 infection.
Vitamin D treatments had no effect on the expression of viral RNA for either SARS-CoV-2 or H1N1 influenza in the lungs of hamsters or mice. Instead, the data support an important role for vitamin D and 1,25D in controlling the host inflammatory response to viruses in the lungs.
Vitamin D deficiency resulted in lung inflammation in the absence of infection. Infected D-mice had more severe lung inflammation and respiratory symptoms than D+ or D++ mice when infected with either H1N1 influenza or SARS-CoV-2. The data point to shared effects of vitamin D to control inflammation in the lung following influenza or coronavirus infection.
D-mice had significantly more inflammation than D+ mice following H1N1 influenza infection (Fig. 1). High dose vitamin D treatment (D++) resulted in some protection of mice from SARS-CoV-2 (Fig. 3). In addition, 1,25D treatment showed a trend toward faster recovery of surviving mice from SARS-CoV-2 (Fig. 3).
Others have shown that 1,25D treated mice had reduced lung inflammation [20,21], and treating D+ mice with 25D had a small protective effect on weight loss and lethality following H1N1 infection . A recent clinical trial that used 25D in humans showed reduced mortality in hospitalized patients with COVID-19 [37,38]. However, it is unclear whether 25D treatment would be effective in mouse or hamster models of SARS-CoV-2.
This is the first study that investigated the effects of vitamin D in animal models of SARS-CoV-2. The data suggest that vitamin D and 1,25D may be effective to protect the lung from SARS-CoV-2. SARS-CoV-2 infection induced Cyp27B1 and Cyp24A1 in the lung of mice suggesting a role for vitamin D metabolites in the lung response to SARS-CoV-2 infection (Fig. 2). There are likely shared and unique mechanisms by which vitamin D regulates the host response to influenza versus SARS-CoV-2.
A better understanding of the mechanisms by which vitamin D regulates the anti-viral response in the lung to both influenza and coronaviruses is needed to inform clinical studies. Cyp27B1 KO mice cannot produce 1,25D, which induces Cyp24A1 and degrades 25D and 1,25D. Feeding Cyp27B1 KO mice D+ diets results in the accumulation of 25D (Fig. 1A, [25,39]). 25D is a low affinity ligand for the VDR and it has been shown that high amounts of 25D can replace the need for 1,25D for the regulation of calcium homeostasis and osteomalacia .
Previous experiments showed that D+ Cyp KO and D+ WT mice cleared a bacterial infection in the gut with similar kinetics . Conversely, D+ Cyp KO mice had higher lethality and more severe inflammation than D+ WT mice when infected with H1N1 influenza (Fig. 1). The effects of Cyp27B1 expression on host-resistance to a bacteria could be different than the effects on host-resistance to a virus. It would be interesting to determine the effect of the Cyp27B1 deletion on host resistance to other respiratory viruses including SARS-CoV-2.
Conversely the differential effect of Cyp27B1 could be due to the location of the infection in the gut versus the lung. Regardless, it seems that the ability of the host to produce Cyp27B1 is important for the mice to survive an H1N1 lung infection.
The dietary interventions to generate D-, D+ and D++ K18hACE2 mice resulted in D++, but not D+, mice having higher serum 25D than D-mice (Fig. 4). Interestingly, the hamster studies suggest that the commercially available chow may not be adequate to raise serum 25D levels (Fig. 5A).
The data suggest that the adequacy of vitamin D should be considered in evaluating studies that infect chow fed hamsters with SARS-CoV-2. At d6 post-SARS-CoV-2 infection, the D++ K18-hACE2 mice had less inflammation and lower IFNβ in the lung than the D-mice (Fig. 3).
The results are consistent with the anti-inflammatory effects of vitamin D. Suppression of type-1 inflammatory cytokines by vitamin D underlie the effects of vitamin D and 1,25D to suppress immune mediated diseases [41-43]. The benefits of 25D from influenza infection was associated with a reduction in IFN-γ in the lung .
Recently, Chauss et.al showed that 1,25D promotes anti-inflammatory responses by switching off IFNγ production from Th1 cells and upregulating IL-10 . IFN-γ and IFNβ production is essential for effective viral clearance, viruses have mechanisms to evade the IFN responses, and severe COVID-19 is associated with dysregulation of IFN responses [45-47].
Down-regulation of IFNβ by vitamin D is associated with protection from inflammation in the lung following a virus infection with either influenza or SARS-CoV-2.
Importantly, there was no effect of vitamin D on SARS-CoV-2 N gene or H1N1 M gene expression in the lungs of mice or hamsters. This indicates that vitamin D did not reduce or inhibit viral replication in the lung. We found SARS-CoV-2 N gene expression in the hamster colons.
Interestingly, D-colons had relatively more SARS-CoV-2 N gene expression than D+ colons. The implications of having SARS-CoV-2 in the colon but not the lung would need to be determined and it would be important to quantitate live virus in the tissues. Unfortunately, we did not save colons from our SARS-CoV-2 mouse studies. Vitamin D has been shown to be a strong inducer of cathelicidin LL-37 in human cells .
There has been some evidence that LL-37 can directly kill some viruses including influenza viruses [13-17]. Treating mice with a high dose (500µg/d) of human LL-37 peptide protected from lethal influenza infection and significantly reduced viral titers at d3 post-H1N1 infection in the lung . LL-37 inhibited binding of SARS-CoV-2 spike protein containing pseudo viruses both in vivo and in vitro blocking entry via ACE2 .
There were no effects of vitamin D in vivo on the expression of viral genes for SARS-CoV-2 or H1N1 influenza in the lung. The cathelicidin peptides found in mice are not the same as the LL-37 in humans and the mouse cathelicidin is not regulated by vitamin D . The lack of a vitamin D effect on SARS-CoV2 was shown in mice and hamster lung.
It is unclear whether the hamster cathelicidin gene has vitamin D response elements. Furthermore, no studies have been done to test the effect of vitamin D on SARS-CoV-2 in vitro. Therefore, an effect of vitamin D through the induction of anti-bacterial peptides, like LL-37, that reduces viral titers cannot be ruled out.