Vitamin D and immune system

I won’t know until I have antigen testing done but I’m fairly sure I was infected with the SARS-COV-v2 aka COVID-19 at the starting of March. Last year I was also diagnosed with lymphoma. The connection between the 2 is that the lymphatic system is involved in the defense against infectious pathogens. If you have cancer in your lymph nodes then viruses become a potential aggravating factor. This is something I experienced as the affected lymph nodes became more sensitive during the COVID-19 infection and on the latest PET-scan showed increased activity. Naturally my interest is sparked in this area.

So with that introduction, the main reason to write about vitamin D is because of its role in the immune function and because that is of interest to the general public. For me there is an extra motivation due to the lymphoma. So join me in the exploration and let’s see what we can dig up.

Intro

The research is split up into observational, interventions and cellular studies so that we can have a better overview and see how they relate to each other.

Vitamin D is an enhancer in reactions and helps to either upscale or downscale effects. It affects the expression of over 2000 genes.

In the “Structure” section you’ll see about the half life of the different molecules and at the bottom section you’ll see about supplementation. This makes research tricky if it is not followed up and measured properly. The way the dosing is done can already create no noticeable effect so I won’t be looking into those kind of studies.

If you already know the ins and outs of vitamin D then you can skip the next section but I want to introduce high level the different forms and at the end I’ll show what I consider good supplementation strategies and why. If you don’t do it right, you could be throwing money in the toilet (or donate it to me to keep this site up and running 😉 ).

Structures

As most people know, vitamin D3 (D3) is produced in the skin through UVB exposure. There is also vitamin D2 but I’ll not get into that. D3 is cholecalciferol and is what you find in most supplements. It has a half life of around 24 hours or even a bit shorter.

D3 is converted in the liver to 25-hydroxyvitamin D which I’ll refer to as 25D from now on. This is what is measured in your blood panel by your doctor and it is also known as calcifediol or calcidiol. It has a half life of several weeks. You will see that in most of the scientific literature, this is what is reported on and measured to find association with clinical outcomes etc..

25D is not yet the active form though. It needs to be further converted to 1,25 dihydroxycholecalciferol (1,25D) also known as calcitriol. This is done by both the kidneys and virtually every cell in your body. Its half life is in the range of a few hours at most.

Observations

Important about observational data is that we look at 25D status before disease appears. If 25D is affected by a disease itself then it doesn’t make sense to claim any links on the diseased state due to 25D status.

First too give some idea about blood levels of 25D, a level of 20 ng/mL (x2.5 -> 50 nmol/L) is reported to be adequate by this paper to prevent respiratory infections. Whether this is correct is another question but it allows you to map your own 25D level to this reference.

“Targeted 25-hydroxyvitamin D concentration measurements and vitamin D3 supplementation can have important patient and public health benefits” https://www.nature.com/articles/s41430-020-0564-0

In a group of granulomatosis with polyangiitis patients they find correlation with respiratory infection and 25D level. What the level should be to reduce the risk heavily is not established.

“Serum 25-hydroxyvitamin D levels in patients with Granulomatosis with Polyangiitis: association with respiratory infection” https://www.scielo.br/scielo.php?script=sci_arttext&pid=S1807-59322017001200723

When looking at athletes, measuring 25D before and after and record their incidences of illness and severity symptoms related to respiratory infections during winter. They also measured cathelicidin which we’ll come back to under the “Mechanisms” section. At the start the median 25D (total so including the small fraction of D2) was 57 nmol/L and at the end, after 4 months, dropped to 47 nmol/L. The groups were divided as follows:

  • 12-30 nmol/L (deficient) -> 4.8-12 ng/mL
  • 30-50 nmol/L (inadequate) -> 12-20 ng/mL
  • 50-120 nmol/L (adequate) -> 20-48 ng/mL
  • >120 nmol/L (optimal) -> >48 ng/mL

Everything tracks along their 25D status according to nr of infections, severity and duration.

“Influence of vitamin D status on respiratory infection incidence and immune function during 4 months of winter training in endurance sport athletes.” https://www.ncbi.nlm.nih.gov/pubmed/23977722/

I want to highlight from this study the following 2 graphs because they relate to the ‘cytokine storm‘ that has been mentioned so much in relation to the severity of symptoms during a COVID-19 infection. They took blood samples and tested the cytokine reactions in the lab.

As you can see, those with sufficient vitamin D levels are able to produce more cytokines. Keep this in mind when reading further below the “Immune cells” section.

The next study did a similar thing except they measured 25D every month and had respiratory infections evaluated by the investigators. The investigators were blinded from the 25D status. Again we see a correlation in incidence, severity and duration based on 25D status. The researchers also conclude that levels of 38ng/mL or above should be maintained.

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There were only 18 subjects >= 38 ng/mL so the sample is relatively small compared to the 180 who were below. Note though that the 18 subjects were the ones who still had a high 25D status at the end of the study. At the beginning there were 32 people. So at the end of those 18, 83.3% in the high 25D group survived the observation period without infection compared to 55% in the low 25D group.

“Serum 25-hydroxyvitamin d and the incidence of acute viral respiratory tract infections in healthy adults.” https://www.ncbi.nlm.nih.gov/pubmed/20559424

One more follow up study this time in Canadian children shows us again the same observation. They found 25D status to be correlated with respiratory infection (and age). Levels of <70 nmol/L (28 ng/mL) increased the risk with 50% and levels <50 nmol/L (20 ng/mL)increased the risk with 70%.

“Low Serum 25-Hydroxyvitamin D Level and Risk of Upper Respiratory Tract Infection in Children and Adolescents” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3888147/

Interventions

For interventions we can look at what the effect is of supplementation (either supplementation or increased sun exposure) and how that prevents disease but we can also look at a diseased state and see if supplementation helps you recover more quickly. It is very well possible that only one of these scenario’s is effective. We’ll see.

A major review finds a reduction in death when D3 is administered together with calcium. That is interesting but is that due to improvement in immune function and thus prevention from infection-caused death or more due to reduction in fracture etc.. so no conclusions from this one yet.

The task would be too big but you would have to go through all the trials in the review and see what dosage was used and how frequently administered. Not only intake but you’d also have to assess to what levels the subjects their 25D was improved. There is a lot of variation and that influences the reported results.

“Vitamin D with calcium reduces mortality: patient level pooled analysis of 70,528 patients from eight major vitamin D trials.” https://www.ncbi.nlm.nih.gov/pubmed/22605432/

“Vitamin D supplementation for prevention of mortality in adults.” https://www.ncbi.nlm.nih.gov/pubmed/21735411/

A first RCT shows quicker recovery when supplementing with 25D. Interesting as usually D3 is supplemented. This is probably done to overcome issues with the liver in the conversion of D3 to 25D. Both the duration and the severity were reduced with a supplementation of 10 microgram/day (400 IU/d). Because it was double-blind placebo controlled, we don’t know by how much this raised patients’ 25D levels. They did report that about 59% started with a deficiency of levels below 30 ng/mL.

“Intake of 25-hydroxyvitamin D3 reduces duration and severity of upper respiratory tract infection: A randomized, double-blind, placebo-controlled, parallel group comparison study” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866826/

The next intervention was in school children (6-15 y) and noticed a reduction in Influenza infections versus the placebo group. They were given 1200 IU/d and the trial was run from December to March. Although a reduction is noted, we have no clue on their starting and ending 25D status.

“Randomized trial of vitamin D supplementation to prevent seasonal influenza A in schoolchildren.” https://www.ncbi.nlm.nih.gov/pubmed/20219962/

In a special group of patients with frequent respiratory infections or antibody deficiency they gave 4000 IU/d for 1 year. Their baseline level of 25D was around 50 nmol/L (20ng/mL). The daily intake resulted in an increase towards 133 nmol/L (53,2 ng/mL). They went into the details to detect differences in bacteria and fungi and found primarily Staphylococcus aureus and fungi to be reduced. The end result is a reduction in infection burden as we see throughout all the studies.

“Vitamin D3 supplementation in patients with frequent respiratory tract infections: a randomised and double-blind intervention study” https://bmjopen.bmj.com/content/2/6/e001663

Mechanisms

As a third category we can see what we find in the lab. What do we find as effect at cellular level and does that fit within our observations and interventions?

For the actual mechanisms we’ll need to look at the active form 1,25D.

Endothelial/Epithelial barrier

The active form 1,25D keeps the epitehlial cells and endothelial cells better clustered together, decreasing permeability so keeping a tight junction. We see this reflected in the blood-brain-barrier and also in the gut.

“Effect of vitamin D on blood–brain barrier function in multiple sclerosis” https://onlinelibrary.wiley.com/doi/abs/10.1111/cen3.12410

“Active form of vitamin D directly protects the blood–brain barrier in multiple sclerosis” https://onlinelibrary.wiley.com/doi/10.1111/cen3.12398

In the gut they tested the effect using lipopolysaccharides (LPS). LPS causes an increase in gut permeability allowing pathogens, from which the LPS originates, to enter the body. We see here a fight between LPS trying to downregulate the vitamin D receptors (VDR) while 1,25D restores it.

“Protective effect of 1,25-dihydroxyvitamin d3 on lipopolysaccharide-induced intestinal epithelial tight junction injury in caco-2 cell monolayers.” https://www.ncbi.nlm.nih.gov/pubmed/25344656

“1,25-Dihydroxyvitamin D3 preserves intestinal epithelial barrier function from TNF-α induced injury via suppression of NF-kB p65 mediated MLCK-P-MLC signaling pathway.” https://europepmc.org/article/med/25838204

Antibiotics

Both the D3 supplementation and production in the skin from the sun causes the skin to produce cathelicidin. This is like our endogenous antibiotics production but the effects are more wide and modulate our immune system as shown in the image. For more information you can check the reference but I wanted to highlight specifically the antimicrobial function.

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image source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161271/

“Cathelicidins: Immunomodulatory Antimicrobials” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161271/

“The human cathelicidin antimicrobial peptide LL-37 and mimics are potential anticancer drugs” https://www.frontiersin.org/articles/10.3389/fonc.2015.00144/full

Very relevant to COVID-19 and any other respiratory infection is the conversion of 25D to 1,25D in lung epithelial cells which helps to increase cathelicidin production.

“Respiratory epithelial cells convert inactive vitamin D to its active form: potential effects on host defense.” https://www.ncbi.nlm.nih.gov/pubmed/18981129/

Immune cells

First a quick word of explanation. What we have seen with COVID-19 is that the immune response is prolonged in the more severe cases. The so called “cytokine storm” is a severe and prolonged response of the immune cells which are tasked to destroy infected cells. In the lungs, the endothelial cells are the first in line to be infected and destroyed by the immune cells but that leads to leakage of plasma into the aeveoli, restricting the ability to breath.

What we want is an immune system that can respond quickly by very fast proliferation so that enough immune cells are created to handle the infected cells but also fast to stop the virus from spreading and infecting many cells. This is the defense that is needed. We also want a quick turn-down of this proliferation once the threat has been handled to avoid excessive damage.

“Acute Lung Injury, Repair, and Remodeling: Pulmonary Endothelial and Epithelial Biology” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5368364/

If the initial response is not fast and strong enough, we risk a more prolonged fight because the virus is able to spread more, causing a prolonged increase in immune cells and destruction of infected cells increasing the overall damage.

“Dysregulated Type I Interferon and Inflammatory Monocyte-Macrophage Responses Cause Lethal Pneumonia in SARS-CoV-Infected Mice.” https://www.ncbi.nlm.nih.gov/pubmed/26867177

Vitamin D receptor (VDR)

A first good indication of modulation by vitamin D is the presence of a receptor for it. 2 review paper shows us that the immune cells (monocytes, macrophages, dendritic cells (DCs), T-lymphocytes and B-lymphocytes) have VDR’s to produce their own 1,25D showing that vitamin D is an active player in these cells.

“Vitamin D: modulator of the immune system.” https://www.ncbi.nlm.nih.gov/pubmed/20427238/

“Vitamin D regulation of immune function.” https://www.ncbi.nlm.nih.gov/pubmed/21419265/

Differentiation

In order to replicate fast, the immune cells require ATP via the cytosolic glycolysis. This happens in T-cells when an anti-gen is presented. Glycolysis means that the ATP has to come from glucose.

Unfortunately I could not find tests on immune cells but we can have a look at cancer cells and embrionic cells, which both are cells that proliferate at a rapid rate, to have an idea what 1,25D may mean. We see in a cancer-specific cell line that the active 1,25D is able to reduce glycolysis. In an embryonic kidney cell line it is able to modulate the reductive state. I’m interpreting here but what it means, according to my insight, is that it will support the capability to switch back from glycolysis to oxygen phosphorilation. This is important for a cell to stop the proliferation.

“1, 25 Dihydroxyvitamin D Regulation of Glucose Metabolism in Harvey-ras Transformed MCF10A Human Breast Epithelial Cells” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4009997/

“Metabolomic Analysis Reveals Vitamin D-induced Decrease in Polyol Pathway and Subtle Modulation of Glycolysis in HEK293T Cells” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5573350/

“1,25-Dihydroxyvitamin D3 stimulates odontoblastic differentiation of human dental pulp-stem cells in vitro” https://www.tandfonline.com/doi/abs/10.1080/03008207.2016.1264395

Not only can it help to induce differentiation, it can also help to prevent differentiation. This is an example of how 1,25D provides a supportive modulatory role. In the paper below, the preventive action improves encephalomyelitis, an autoimmune disorder potentially linked to T17 cells.

“1,25-Dihydroxyvitamin D3 inhibits the differentiation and migration of T(H)17 cells to protect against experimental autoimmune encephalomyelitis.” https://www.ncbi.nlm.nih.gov/pubmed/20886077

I was able to find the modulating role 1,25D plays in B cells where the authors suggest this role may help in improving B cell autoimmune related disorders by correcting vitamin D deficiency.

“Modulatory Effects of 1,25-Dihydroxyvitamin D3 on Human B Cell Differentiation” https://www.jimmunol.org/content/179/3/1634.short

One of the reasons vitamin D could be improving autoimmune diseases is because it modulates dendritic cells (DCs) in such a way that it reduces T cell response. DCs are the cells that present antigens to T cells who then respond to this antigen by fast proliferation.

“1 Alpha,25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation.” https://www.ncbi.nlm.nih.gov/pubmed/10679076

This effect on DCs seems contrary to the fast and strong (but short) response needed from the immune system but what I suspect is going on is that it doesn’t disable DCs ability overall but is probably fine tuning DCs more towards pathogens somehow.

We find improvement in immune function with higher 25D levels so it must have a beneficial effect somehow.

Although not directly about vitamin D, I found a presentation that talks about how incorrect adaptation in metabolism of the T cells can lead to several diseases.

Taken together, it shows that vitamin D is important in potentiating a correct adaptation in immune cell metabolism to provide the right type of response.

Finally

The cell cultures reveal interesting modulatory roles primarily in the differentiation, possibly by regulating the way energy is metabolised. The papers show the highly complex world of immunology.

Not only is there direct effect in the way immune cells behave but also in the protective barriers such as the epithelial lining in the gut and vasculature do we see its involvement together with an antibiotic production.

The observation and intervention sections show us, in line with the cell cultures that vitamin D status, measured through 25D, does make a difference and is important to maintain an optimal state. What that level should be is debated but it is clear that there is no upper limit defined as of which there are no more positive effects observed.

There is toxicity possible but for that you need to go higher than what you would receive from the sun. Our skin darkens in response to sun exposure and this lowers the D3 production but with supplementation we don’t have this automatic protection. But we are talking about regular intake of 500 000 IU for a longer period whereby the toxic effects are easily reversed by stopping the supplementation.

Dosing

The exercise has been done reviewing the dosage used and timings. The best approach seems to be on a daily basis. The reason for that is because you are normally supplementing with D3 which has a half life of 24 hours roughly. This means that if you take a big bolus once a month of say 100 000 IU, the next day your body has 50 000 IU left, the day after 25 000 IU etc.. After 7 days you have about 1562 IU left. In contrast if you take 2000 IU every day then you only get 60 000 IU per month but it will be much more effective.

This is also reflected in a review of randomized control trials regarding respiratory infections:

There is evidence that daily administration is more effective than high-dose bolus administration [OR = 0.48 (95 % CI 0.30–0.77) vs. OR = 0.87 (95 % CI 0.67–1.14)]

“Vitamin D and airway infections: a European perspective” https://eurjmedres.biomedcentral.com/articles/10.1186/s40001-016-0208-y

Personally I decided to take multiple dosages of 1 000 IU spread across the day. Whatever the dosage you take, I would suggest to spread it from early morning to late afternoon. This is 1) to anticipate on the liver conversion capacity of D3 to 25D and 2) to avoid any negative effect on sleep.

I would suggest not to take D3 in the evening. I could not find any papers showing an immediate influence on melatonin production but out of precaution I think it is better to mimick the natural production we would have from the sun. A light dose in the morning, the heaviest dose at noon when the sun is the highest and a light dose again in late afternoon.

There are papers that looked at 25D and sleep and find correlation between bad sleep and 25D deficiency. There is also a paper looking at MS patients reporting reduction in sleep. However they looked at metabolites in urine rather than sleep or direct melatonin production so we can’t really derive much from those results. Due to the long life I don’t consider 25D having an immediate effect related to intake and melatonin.

Anecdotes and even a very thorough n=1 experiment indicate it is best not taken at night. My way of intake likely will be synergistic with the circadian rhythm.

Exercise

If I know I will exercise then I’ll take an extra dose. It has been shown that exercise increases 25D production during and post exercise. I suspect that this is due to the increase in blood flow pushing more D3 through the liver. Whatever the cause, we want our 25D status up so this is a good way to make most use of your D3 intake (or sunshine).

“Effect of an Acute Bout of Endurance Exercise on Serum 25(OH)D Concentrations in Young Adults” https://www.ncbi.nlm.nih.gov/pubmed/28973380

That is it, I hope you are able to recover well when you get affected and hopefully you can correct your deficiency in 25D in time if you have one.

— THE END —

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