For a comprehensive overview of vitamin D and the immune system and of the need for proper (e.g. 0.125 mg 5000 IU /day or more, for 70 kg bodyweight) vitamin D3 supplementation, please see: https://vitamindstopscovid.info/00-evi/ .

Vitamin D based intracrine and paracrine signaling are the two primary (perhaps sole) ways most immune cells use the vitamin D compounds.  These signaling systems are crucial to the ability of each individual cell to respond to its changing circumstances. 

The immune system is second only in complexity to the nervous system.  Its operation is not coordinated by neurons.  All coordination is done by individual cells, of multiple types, sensing their surroundings, sending chemical signals to other cells and so changing their behaviour in various ways.  Vitamin D based intracrine and paracrine signaling is a long-evolved, flexible, powerful (can up and down regulate the transcription of hundreds of genes) mechanism which plays a very large role in how immune cells change their behaviour.  This only works to the extent that sufficient 25-hydroxyvitamin D is supplied to these cells, all over the body. 

All current research indicates that 50 ng/mL 125 nmol/L 25-hydroxyvitamin D in the bloodstream provides sufficient 25-hydroxyvitamin D for immune cells.  Without substantial recent UV-B skin exposure or proper vitamin D3 supplementation (or, for emergency repletion, calcifediol, which is 25-hydroxyvitamin D) most people have only 1/2 to 10th of this.  So their immune system does not work very well.

Vitamin D based intracrine signaling is also, incorrectly, known by the rather similar term "autocrine", but to our knowledge, there is no vitamin D based autocrine signaling, since that would only occur if the vitamin D receptor (VDR) molecules were located in the cell membrane with their active site pointing outwards. 

VDR is not a membrane based receptor.  Intracrine signaling is like autocrine signaling but the receptor is in the cytosol.  The previous version of this page referred to "autocrine" signaling, and I made this new "intracrine" version on 2022-05-14.

Terminological note 2022-05-14:

1. Autocrine signaling involves some molecules (acting as an autocrine agent, not a hormone) being made inside a cell, to signal information to another part of the cell. The molecules leave the cell's cytoplasm and activate receptors on the outside of the cell membrane, of the same cell.  The activated receptors cause changes inside the cell, such as changes leading to altered transcription of genes, which leads to different proteins being made and so to altered cellular behaviour.
   
   This does not occur with the vitamin D compounds, in which 1,25-dihydroxyvitamin D (calcitriol) acts as a signaling molecule to activate vitamin D receptor molecules, because these vitamin D receptor molecules are not found on the outside of the cell's membrane.
   
   Autocrine signaling is known to exist with other compounds and the term autocrine signaling has been used, incorrectly, to refer to what is actually intracrine signaling.  This page uses "autocrine" in this way, as does Chauss et al. 2021 in their extraordinarily important work with Th1 lymphocytes from the lungs of hospitalised COVID-19 patients.
   
   
2. Intracrine signaling refers to signaling molecules being generated inside a cell where they activate receptor molecules, also inside the same cell, with those activated receptors altering the cell's behaviour as just described.  These molecules are acting as an intracrine agent, not a hormone.
   
   This does occur with 1,25-dihydroxyvitamin D.  As best I can tell, this signaling system was first discovered by Martin Hewison and colleagues in the mid to late 2000s.  See below for a 1991 article entitled Intracrinology.
   
   The exact location of the receptor molecules is important for molecular biology research, but is a fussy detail from the point of view of most doctors and lay people in understanding the importance of good supplies of 25-hydroxyvitamin D to all such cells, which (in particular circumstances) initiate their intracrine signaling system by converting this to 1,25-hydroxyvitamin D.
   
   #fletcher
   For a recent review article on vitamin D based intracrine signaling:
   
   

   Autoimmune disease and interconnections with vitamin D Jane Fletcher, Emma L Bishop, Stephanie R Harrison, Amelia Swift, Sheldon C Cooper, Sarah K Dimeloe, Karim Raza  and Martin Hewison Endocrine Connections 2022-02-01 https://ec.bioscientifica.com/view/journals/ec/aop/ec-21-0554/ec-21-0554.xml

   
   
   My Twitter-brief appreciation of this: 1, 2, 3, 4, 5 and 6.   (I disagree with the use of "hormonal" in the abstract - this 1,25-dihydroxyvitamin D is acting as an intracrine or paracrine agent, not as a hormone.)
   
   
   
3. Paracrine signaling involves a signaling molecule (in the case of vitamin D based paracrine signaling, 1,25-dihydroxyvitamin D) being produced inside one or typically multiple cells in a particular location in the body, due to particular circumstances being detected by those cells, and these molecules, acting as a paracrine agent, not a hormone, diffuse out of the cell and find their way to nearby cells (typically of a different type, as best I understand it) where those cells' behaviour is changed by the presence of these diffused signaling molecules. 
   
   I guess, in humans, that "nearby" means fractions of a millimetre to a few millimetres.  I am not aware of anyone describing observed or theoretical distances.  A single cell, or multiple cells of the same type, may, when they detect particular circumstances, convert intracellular 25-hydroxyvitamin D into 1,25-hydroxyvitamin D, which acts both as an intracrine agent for the cell in which it was produced, and as a paracrine agent when some of these molecules diffuse out of this cell, and affect the behaviour of other nearby cells.
   

Vitamin D based intracrine and paracrine signaling is unrelated to the one hormonal function of vitamin D, in which a very low, but tightly regulated, concentration of circulating 1,25OHD is produced by the kidneys to control calcium-phosphate-bone metabolism.

Most people - including many doctors, immunologist and virologists - are not familiar with intracrine or paracrine signaling.  To understand vitamin D in general - and especially to understand why population-wide vitamin D repletion targeting at least 50 ng/mL 25(OH)D vitamin D blood levels (125 nmol/L) is the key to improving health - we need a good understanding of vitamin D based intracrine signaling. 

As far as I know, all this is correct, since it is based on some earlier text which met with the approval of a senior vitamin D researcher.  If you spot any errors or can suggest any other improvements, please let me know.

Sections 00 to 04 are preliminaries.  To go straight to the explanation of vitamin D based intracrine signaling: #05-intra .

#00-term	Notes on terminology.  Read this first to reduce later confusion.
#01-compounds	D3 cholecalciferol, 25(OH)D calcifediol and 1,25(OH)2D calcitriol.
#02-nothorm	Hormonal 1,25(OH)2D for calcium-phosphate-bone metabolism is at a much lower level than the 1,25(OH)2D generated as an intracrine agent and/or paracrine agent in immune and other cells, so the hormonal 1,25(OH)2D levels, which are quite stable, have no significant effect on the intracrine and paracrine signaling systems of numerous types of cell.
#03-minlev	Numerous reasons why we should aim for at least 50 ng/mL (125 nmol/L) 25(OH)D blood levels.
#04-quraishi	2014 research which indicates we should aim for at least 50 ng/mL 25(OH)D blood levels.
#05-intra	Description of intracrine signaling with two examples from research articles, the second of which is directly relevant to severe COVID-19.  Paracrine signaling is easily understood as an extension of this to nearby cells, by diffusion.

Here is my best attempt at untangling some contradictory and/or divergent and at least confusing terminological problems.  This includes my own value judgments on how particular terms should be used, and how they are sometimes misused.

The term Vitamin D is generally and properly used to refer collectively to the three compounds best known in mammalian biology:

• Vitamin D3 cholecalciferol. [WP]  Shorter forms: D3.  This never acts as a hormone.
  
  
• 25-hydroxyvitamin D calcifediol. [WP]  Shorter forms: 25(OH)D, 25OHD₃, 25(OH)D₃ and  25D.  This never acts as a hormone.  An alternative name for this is calcidiol - I suggest that this term be avoided entirely because it is redundant, confusing and looks and sounds very much calcitriol (and like calcidol which sometimes used to refer to D2 ergocalciferol, as described below).
  
  
• 1,25-dihydroxyvitamin D calcitriol. [WP]  Shorter forms: 1,25(OH)²D and 1,25(OH)²D₃.  This has a much greater affinity for the vitamin D receptor than D3 or 25OHD or any other vitamin D related compounds.  It is sometimes referred to as activated vitamin D but I regard this as a mistake.  This has one hormonal function.  All its other functions, in probably hundreds of cell types, are not related to hormonal signaling - it is produced and sensed by the same cell in intracrine signaling and diffuses to nearby cells where it is sensed, in paracrine signaling.
  

Sometimes "vitamin D" is used to refer just to D3, and "vitamin D metabolites" to the other to compounds, as well as to other compounds not mentioned here such as those which result from the breakdown of any of the above three compounds or their hydroxylation, such as at the 24th carbon atom.

The vitamin D receptor AKA VDR.  According to the Wikipedia page [WP] an alternative term for this is calcitriol receptor.  I have never seen this term, but it is substantially more correct and would ideally be widely used.  However, I suspect that we are stuck with the current terminology, and "VDR" is short and distinctive.

D3 and 25-hydroxyvitamin D have a very low affinity for the VDR, so it is generally wrong to think of it as a receptor for these compounds.  1,25-dihydroxyvitamin D (calcitriol) has a far greater affinity for the VDR, but the lower affinities should not be forgotten in scenarios where there is little 1,25-dihydroxyvitamin but very high levels of D3 and 25-hydroxyvitamin D. 

There are an alternative set of three compounds to those above, based on Vitamin D2 ergocalciferol which is produced industrially by UV-B irradiation of ergosterol, from fungi or yeast.  This is not a normal part of mammalian biology.  Here is a rare diagram showing both the molecular differences and the carbon numbers, from this site.



Vitamin D2 and its 25- and 1,25 hydroxylated forms have no advantages over the D3 compounds, so generally I don't discuss them.  For some obscure historical reasons doctors in the USA were only able to prescribe D2, and often still do - I have been told that this is no longer mandated.  The definitive article on the D3 vs. D2 seems to be:


The term vitamin is questionable.  From a book chapter cited here , where the milk (at least in the USA in the 2020s) may be fortified with D2.


It is common to find the phrase "vitamin D is a hormone" (Google Scholar 711 hits) as if this is technically more accurate than the questionable term "vitamin" - or as if this important group of compounds deserves additional gravitas not conferred by "vitamin".  For additional oomph, there is also "Vitamin D is a secosteroid hormone" (328 hits). 


It is common for the term vitamin D to be used both for referring collectively to the compounds listed above, and potentially other related compounds, which I think is a correct use of the term.  "Vitamin D" is also often used it when the author is actually referring to one of these compounds, but does not refer to it specifically.  The reader is left to infer exactly which compound is meant, which is a terrible mistake in this already difficult field.

#2004-vieth
I was very happy to find a 2004 article concerning common terminological mistakes.  This is from Prof. Reinhold Vieth, who has researched vitamin D since 1975, and is still active in 2022.  Google Scholar reports 325 articles.





As best I can tell, the term hormone [WP] originally meant signaling molecule with the unstated assumption that the signaling path was from one type of cell (or at least an organ somewhere) to one or more other cell types (such as in one or more other organs), via the bloodstream (in animals) or by fluids which are transported within plants.  

This signaling is the conveyance of information by way of the level (concentration) of the compound.  This is detected by the recipient cells. 

In plants (which lack organs) the long distance, within the plant itself, transport of the hormone molecules is by the plant's fluid transport system.  In vertebrates, the signal is conveyed by the level (concentration) of the substance in the bloodstream and potentially the CSF (Cerebrospinal Fluid [WP]) and interstitial fluid [WP]. 

Unfortunately the term hormone is sometimes also applied to any signaling molecule - including those which operate over short distances such as within cells and to nearby cells, without involving the bloodstream at al. 

Today, the only proper use of the term hormone in vertebrates is as described above, and not to refer to signaling molecules which operate within cells or between nearby cells. 

Endocrine signaling [WP] is hormonal signaling for vertebrates.  The Wikipedia page concentrates on this, but also includes in the field of endocrinology three other signaling modalities, while specifically excluding neurotransmitter signaling: autocrine, paracrine and juxtacrine.  However, I am not at all sure that endocrinologists in general, or those who write endocrinology textbooks, are really interested in these fields, since they have nothing in common with hormonal signaling.


Here are the number of Google search hits within the English Wikipedia for:


These are my notes on  Hector F DeLuca's 2014 History of the discovery of vitamin D and its metabolites  PMC3899558: 


Here is my current (2022-05-14) understanding of the terminology.  The Wikipedia references are for general information and an indication of current usage, not to indicate that these definitions are authoritative.  I am not sure that there is a single source of authoritative definitions of scientific terms such as these, since usage and definitions change as research progresses.


#chauss-1
This is quite messy.  The preprint of arguably the most important article ever written on the etiology of severe COVID-19:


uses autocrine in the title, but refers to the vitamin D based autocrine signaling as also potentially involving paracrine signaling.  The molecular mechanisms all involve VDR in the cell, which according to the 1991 Labrie diagram, is intracrine signaling.  There is no mention of intracrine.   My summary of this dense cell-biology preprint is at: https://aminotheory.com/cv19/icu/#2021-Chauss .

"Autocrine" also features in the title of the final article:


It is difficult enough getting doctors,  immunologists etc. to understand vitamin D's role in the immune system without these terminological complications.  Two recent immunology textbooks Janeways 9th 2017 and Abbas 10th 2021 total 1500 pages and do not mention vitamin D in their indexes.  The only mention of autocrine and paracrine signaling is in Janeways 9th, regarding cytokines in which autocrine is defined as affecting the behaviour of the cells which release the cytokine, without reference to the location of the receptor. 

Knowledge of vitamin D intracrine signaling has been developed since the mid-2000s.  As far as I know, there are no accurate estimates of how many types of cell use vitamin D intracrine or intracrine-paracrine signaling.  See #cells below.

For more in-depth material, a good place to start might be articles which cite a 2010 article,  Autocrine and Paracrine Actions of Vitamin D by  Howard A Morris and Paul H Anderson. Also: Vitamin D metabolism and signaling in the immune system (2012), Vitamin D and immune function: autocrine, paracrine or endocrine (2013)  and Vitamin D and immune function (2013).  These processes were not always described as "intracrine" or "paracrine", but these are the proper terms to use now.

As far as I know, there is no research article which presents vitamin D based intracrine and/or paracrine signaling in an easy-to-understand manner.  So I created this web page.


Here are two other terminological and conceptual failings frequently found in vitamin D research articles:

• Many MDs and researchers have no idea what intracrine (to some, autocrine) and paracrine signaling are.   They think that vitamin D (the collective definition, or at least 1,25(OH)²D) "modulates" immune cells' behavior solely by way of the 1,25(OH)²D produced in the kidneys and which circulates in the bloodstream as a hormone.  This is absolutely not the case, and it is a very serious misconception.  See, for instance, Figure 1 of Newmark et al. 2017 https://www.frontiersin.org/articles/10.3389/fimmu.2017.00062/full This is an interesting article (I could follow it to about page 6) about the evolution of the vitamin D systems.  However, the diagram showing 1,25(OH)²D going from the kidneys to the immune cells is just plain wrong.  They surely do reach these cells and probably diffuse into the cytosol, after which they probably do bind with a VDR molecule.  However, the level of this1,25(OH) ²D is very low, and is very stable and tightly controlled for the purpose of regulating calcium-phosphate-bone metabolism.  So how could it signal information of use to immune cells?  Furthermore, its level is much lower than the localised levels of1,25(OH) ²D produced by intracrine and presumably paracrine signaling.  See #02-nothorm below.
  

• It is common to write of people being vitamin D sufficient, insufficient and deficient, as if there is consensus on what this means.  Generally it means:
  
  Sufficient: Greater than 30 ng/mL 75 nmol/L.
  
  Insufficient: Between 20 and 30 ng/mL 50 and 75 nmol/L.
  
  Deficient:  Less than 20 ng/mL 50 nmol/L.
  
  The question of healthy 25(OH)D levels is a matter of debate - and it can't be assumed that there is a single healthy level for all people.  Maybe some individuals need more, for instance people suffering from multiple sclerosis, rheumatoid arthritis, psoriasis, cluster headache and migraine https://vitamindstopscovid.info/06-adv/.   Perhaps 25(OH)D levels are only part of what constitutes good health and other factors, such as Vitamin D Binding Protein (VDBP [WP]) characteristics and concentrations really matter too.   Ordinary blood tests are for total circulating 25(OH)D, and most of it is bound tightly to VDBP or loosely to albumin, which reduce its availability for diffusion to many cell types.  See: #vdbp.
  
  Researchers should report the proportion of people whose 25(OH)D levels are below 30 ng/mL and above 20 ng/mL or whatever, not just that they "are vitamin D insufficient".
  

Vitamin D3 cholecalciferol. [WP]   This is produced by 295 to 297 nanometre wavelength UV-B light acting on 7-dehydrocholesterol in the skin.  It can also be ingested in food or supplements.  While this plain D3 directly protects the endothelial cells which line our blood vessels [Gibson et al. 2015], all its other currently known roles in the body rely on it being converted in the liver (there may also be some conversion in cells out side the liver), over a period of days to a week, by the enzyme vitamin D 25-hydroxylase (encoded by the CYP2R1 gene, a name sometimes given to the enzyme itself) to 25(OH)D.  (Another enzyme encoded by the CYP27A1 gene does the same thing and so produces some of the 25(OH)D.)

The numbers indicate carbon positions.  Most hydrogen atoms are not shown.  The special trick to producing this from 7-dehydrocholesterol is to use 295 to 297 nanometre UV-B light to break the second carbon ring between carbon 9 and 10.  The resulting molecule is not quite the shape of D3, but thermal motion over a period of minutes or hours into the correct shape.



25(OH)D calcifediol = 25 hydroxyvitamin D3 = calcidiol [WP].  This has an OH oxygen-hydrogen hydroxyl group at the 25 position, in place of the H (not shown) which was there in D3.  25(OH)D circulates in the blood, mainly bound strongly to the vitamin D carrier protein and more weakly to albumin proteins.  It is also absorbed into fatty tissue, as is D3.  Vitamin D blood tests measure the total bound and unbound level of 25(OH)D.  This level is the most important part of the whole vitamin D system, however, depending  the amount which is unbound to the vitamin D carrier protein and perhaps the albumin proteins may affect the amount of 25(OH)D which diffuses from the bloodstream into the interstitial fluid between the cells, and through their cell membranes into the cytosol of the cells. .

Neither D3 nor 25(OH)D bind strongly to the vitamin D receptor [W] which is a complex protein far bigger than these molecules.



1,25(OH)²D calcitriol [WP] = 1,25 dihydroxy vitamin D = 1,25(OH)² vitamin D, is produced by a second enzyme, 1-hydroxylase, encoded by the CYP27B1 gene, which adds an OH hydroxyl group at the 1 position to 25(OH)D.   This happens in the kidneys and inside many types of cells, including immune cells.

1,25(OH)²D binds strongly to, and so activates, the vitamin D receptor.

There is another enzyme CYP24A1 which can add an OH hydroxyl group to the 24 position of 25(OH)D and 1,25(OH)²D, which is an irreversible process.  The resulting molecules are degraded and excreted.  The activity of this enzyme scales up with increasing circulating 25(OH)D levels, and so gives rise to a strong self-limiting process which reduces high 25OHD levels.  This accounts for the curves in the 25(OH)D by bodyweight and D3 intake graph from Ekwaru et al. 2014 at 01-supp/a-ratios/ .  This self-regulation makes it very hard to attain potentially toxic 25OHD levels.  Above 150 ng/mL (375 nmol/L) there is a risk of hypercalcemia [WP]. 

There is quite a lot of research into this self-imitating system.   I have not tried to understand the details and I don't  know  to what extent there is consensus on how it works, or even whether any researchers have reliably established the mechanisms.  This is a very important process and ideally I would be able to understand and explain it better.

Vitamin D based intracrine signaling and paracrine signaling, at least in immune cells, is not a continual process.  The signaling system is activated in a particular cell when certain conditions are detected.  As described in a section below #05-intra, this causes both VDR (vitamin D receptor) and the 1-hydroxylase enzyme to be produced in the cytosol, whereupon the enzyme converts the 25(OH)D which should already be there (and be supplied from outside the cell, as it is consumed) into 1,25(OH)²D, which immediately binds to a VDR molecule.  The bound complexes find their way to the nucleus where they alter gene transcription, typically upregulating and downregulating the transcription of dozens or hundreds of genes. 

Vitamin D based intracrine/paracrine signaling is the same general system in multiple cell types.  Tens or hundreds of millions of years of evolution have used these systems for a variety of purposes - a different purpose in each type of cell - with the activating conditions and the changed cell behaviour being entirely cell-type specific. So there is no generalised way of describing these signaling systems in terms of what activates them and what changes they create in cell behaviour - since these vary widely from one cell type to the next.

In this section I explore something I haven not seen explicitly tackled in the research literature, though the common misconception of "vitamin D" being a hormone was tackled by Reinhold Vieth in 2004, as mentioned above: #2004-vieth :

The question is:


As far as I can see, this hormonal level is too low to significantly affect intracrine signaling, so moderate changes in that hormonal level (within whatever range it might healthily change, in order to maintain the correct balance between calcium absorption, excretion and blood levels) would also have no significant effect on individual immune cells or on the whole immune system.

A related question is the same regarding paracrine signaling.  I don't know of any measurements of the levels of 1,25(OH)²D diffusing to nearby cells, but these levels will be somewhat or perhaps a lot lower than the levels at which 1,25(OH)²D is generated intracellularly.  For paracrine signaling to work at all, it needs to be sensitive to these diffused levels, and not significantly affected by low levels of hormonal 1,25(OH)²D which presumably diffuse from the bloodstream into the interstitial fluid between the cells, and from there diffuses across the cell membrane into the cytosol of the cell.  Below, I attempt to answer this question quantitatively.  It seems likely that paracrine signaling involves diffused levels of 1,25(OH)²D which are well above the hormonal level of 1,25(OH)²D.  

I don't have references handy for this, but I recall that some or many cells have a 24-hydroxylase enzyme which irreversibly degrades 25(OH)D, 1,25(OH)²D and think D3.   It would make sense for this to be active, to some probably small degree, in cells which are involved in vitamin D based intracrine / paracrine signaling since these are time-sensitive, rather than long-term, relatively static, processes.  For the gene transcription changes, which occur when the intracrine / paracrine system is operating, to be reverted back to normal patterns of gene transcription, it would make sense for the cell to have a certain degree of 24-hydroxylase enzyme, to mop up 1,25(OH)²D rather than let it float around for tens of hours or more.  (I guess the activated complexes of the 1,25(OH)²D bound to the VDR are degraded in due course, for the same reason.)   To the extent that there is a non-trivial level of 24-hydroxylase enzyme activity in the cell, it is reasonable to think that this would be continually degrading any hormonal 1,25(OH)²D which diffused into the cell.

Here is a description of the one hormonal function of the vitamin D compounds.  All doctors understand this mechanism.  A hormone is a compound in blood circulation (or perhaps the cerebrospinal fluid), whose level (concentration) is controlled, with that level affecting the behaviour of one or more cell types which could be anywhere in the body.

While 1,25(OH)²D (discovered in 1972) is the best known has a hormone for its role circulating in the blood, its production (in the kidneys, as just described) is not where most of the 25(OH)D (produced from D3) is used.  Until about 1979, kidney conversion was the only known source of 1,25OHD.  Then, extra-renal (outside the kidneys) conversion to 1,25(OH)²D was first discovered (Gray et al. 1979).  In 2007 an important article was published, discussing vitamin D intracrine and paracrine signaling:


#1ng
These researchers used some macrophages and monocyte derived dendritic cells, both with their intracrine / paracrine signaling systems turned on, to find out how their conversion of 25(OH)D to 1,25(OH)²D was affected by differing levels of 25(OH)D: 2, 20 and 60 ng/mL.  (I am not sure that this is directly equivalent to such levels in the bloodstream plasma, in vivo, where the 25(OH)D is mainly strongly bound to the circulating vitamin D binding protein, and to e lesser extent to albumin proteins, with only a small fraction available for diffusion into tissues and/or immune cells.)   The levels of 1,25(OH)²D produced, after 48 hours, were (Fig 1 levels divided by 2.5 to give ng/mL) approximately 0.013, 0.12 and 1 ng/mL respectively.

The 0.12 ng/mL 1,25(OH)²D (resulting from 20 ng/mL 25(OH)D supply to the cells) only marginally affected the gene transcription and protein synthesis which intracrine signaling in the macrophages drives.  This is upregulation of CD14 [WP] and downregulation of three other proteins (Fig. 2).  The 1 ng/mL 1,25(OH)²D level, produced when 60 ng/mL 25OHD was supplied to the macrophages) fully upregulated CD4 and downregulated the other three proteins - the effect was just as strong as when 40 ng/mL 1,25(OH)²D was added to the cells.

Some important points arise from the abovementioned research:

• Researchers in 2007 determined that 25(OH)D levels at the cells needed to be more like 60 ng/mL than 20 ng/mL for intracrine signaling to work properly.  25(OH)D is consumed in those cells by intracrine / paracrine conversion to 1,25(OH)²D - at the times when this signaling system is activated - and slowly, at all times, by 24-hydroxylase which consumes a little of it, degrading 25(OH)D to 24,25(OH)²D.  Since 25(OH)D diffuses to all cells from the bloodstream, it follows that blood 25(OH)D levels need to be more like 60 ng/mL than 20 ng/mL for good health. 
  
  #vdbp
  As far as I know, the exact concentrations of 25(OH)D inside cells such as immune cells is not known.  There is some uncertainty or debate about how 25(OH)D molecules get from the bloodstream (the plasma, fluid, part of the blood, not from blood cells) to the interstitial fluid and then into the cells, as discussed in the next cited article.  There is no evidence of active transport of 25(OH)D into immune cells, though I recall reading that the kidney cells which need 25(OH)D transport it across their cell membranes with the 25(OH)D bound to the much larger Vitamin D Binding Protein VDBP molecule.  Most of the 25(OH)D in the bloodstream is in these bound complexes, so this implies a high-efficiently transport system compared to relying on the much smaller amount of unbound 25(OH)D.
  
  According to:
  
  Vitamin D Binding Protein, Total and Free Vitamin D Levels in Different Physiological and Pathophysiological Conditions
  Daniel David Bikle and Janice Schwartz
  Frontiers in Endocrinology, Bone Research 2019-05-28
  https://www.frontiersin.org/articles/10.3389/fendo.2019.00317
  
  85% of serum 25(OH)D is strongly bound to VDBP.
  15% is more loosely bound to albumin proteins.
  0.03% is unbound, freely in solution in the plasma.
  
  There is a question of how, when the great majority of 25(OH)D in the bloodstream is bound to VDBP or albumin proteins, can more than the very small fraction of it which is in solution (unbound to any molecule) diffuse into the interstitial fluid and so into cells, where the final concentration in those cells must, as best we know, be no greater than the concentration of unbound 25(OH)D in the bloodstream, which is very low.
  
  Fletcher et al. 2022 cited above #fletcher states (pp 4, middle of right column), that reduced levels of VDBP with a given total concentration of 25(OH)D enhanced the ability of that 25(OH)D to facilitate antibacterial responses by monocytes and dendritic cells.  This implies strongly that the cell's supply, by passive diffusion, of 25(OH)D from the bloodstream depends largely or wholly, for any given concentration of 25(OH)D on the proportion of 25(OH)D which is not bound to VDBP.
  
  
• Now, in 2020, we reasonably assume* that many or most types of immune cell need 25(OH)D, which they consume by hydroxylating it to 1,25(OH)²D when their intracrine  / paracrine signaling systems are activated - and that an unknown number of other cell types also need for the same reason.  The cells of the types which sense 1,25(OH)²D as a paracrine agent don't necessarily produce it themselves.  If they don't produce it, they presumably don't need 25(OH)D, but the nearby cells, probably of different types, which produce this paracrine 1,25(OH)²D do need 25(OH)D - and they may well produce 1,25(OH)²D for their own use as an intracrine agent as well.
  
  #cells
  * The Fletcher et al. 2022 article cited above #fletcher reports on Martin Hewison's team's research with macrophages [WP] and dendritic cells [DCs WP], both types of cell which are derived from monocytes [WP].  They mention (start of pp 5) that both macrophages and DCs may emit 1,25(OH)²D as a paracrine agent to affect the behavior of nearby T lymphocytes.[WP] and that cytotoxic T cells may also emit 1,25(OH)²D as a paracrine agent.  They cite Chauss et al. 2021's #chauss-1 beautiful and extensive (my value judgment) research with intracrine signaling in Th1 regulatory lymphocytes (though, as noted above, the title of this article uses the term "autocrine").  I don't have a reference handy, but the genes for both VDR and the 25-hydroxylase enzyme are expressed in many types of immune cell and in other cell types which likewise are not involved in calcium-phosphate-bone metabolism. While researchers have not delved into all these cell types regarding their vitamin D based intracrine and paracrine signaling activity, it is reasonable to assume that any cell types such as these express these genes for the purposes of vitamin D based intracrine and/or paracrine signaling.
  
  
• Yet still, despite increasingly desperate protests from some MDs and researchers, in the midst of the COVID-19 pandemic, many government official guidance documents on vitamin D3 supplementation are still based on the outdated (and even at the time, mistaken) 2010 conclusions of the US Institute of Medicine ../01-supp/#iom and https://nutritionmatters.substack.com/p/government-vitamin-d3-supplementation , which seek only to achieve 20 ng/mL as needed for bone health, with no regard to the higher levels needed for good immune system health.
  
  Please remember  these numbers.  In other pages here (see https://vitamindstopscovid.info/00-evi/) you can read arguments that if everyone supplemented vitamin D3 to attain, on average, around 50 ng/mL 125 nmol/L 25(OH)D, that SARS-CoV-2 would only rarely cause severe symptoms, with those infected shedding much fewer viruses on average, causing transmission to be much lower than today - so there would be no COVID-19 pandemic, or at least not one to worry much about.
  
  
• The hormonal, circulating, 1,25(OH)²D level of around 0.045 ng/mL can be expected to diffuse into cells which use 1,25(OH)²D as part of their intracrine signaling systems.  (What level this occurs at in those cells depends on how much of that serum, circulating, hormonal 1,25(OH)²D is bound to vitamin D binding protein and albumin proteins - I haven't read the details, but I guess only a fraction is free to diffuse into the interstitial fluid and cells.)
  

•  This is not enough to significantly activate the gene transcription changes of the intracrine / paracrine signaling systems, since they are only marginally activated by 0.12 ng/mL 1,25(OH)²D - and it takes about 1 ng/mL to fully activate them.
  
  So while all these cells are bathed in hormonal 1,25(OH)²D, the level of this - which is generally stable - is too low to significantly affect their intracrine or paracrine signaling systems.
  

The importance of proper (at least 50 ng/mL = 125nmol/L = 1 part in 20 million by mass) levels of 25(OH)D is not widely enough known.   While lower values may be sufficient for the kidneys to maintain the proper level of hormonal 1,25(OH)²D, we need at least this level of 25(OH)D for numerous types of cell - especially immune cells - to function correctly.  These cell types (as noted above #cells, of which only a few have been properly researched) need good supplies of 25(OH)D for their their intracrine / paracrine signaling systems, which play a likely major role in the ability of each individual cell to respond to its changing circumstances . 

Cannell et al. 2006 proposed that 50 ng/mL (125 nmol/L) be the target 25-hydroxyvitamin D level, all year round:


The target range of 40 to 60 ng/mL (100 to 150 nmol/L) was stated in 2008 by 48 leading researchers and MDs in the Call to D*Action:


This approximately 50 ng/mL level was fully justified by the research of Quraishi et al. 2014, mentioned in a section below: #04-quraishi

Yet the Institute of Medicine (IOM) chose 20 ng/mL in 2011, in a major report, which forms the basis of most of today's (2022) government recommendations regarding 25-hydroxyvitamin levels and D3 supplementation quantities to attain this.   This was a huge mistake, including their botched calculation for how much D3 people should be taking. 

See my article on this:

This 2020 review article, co-authored by the world's leading vitamin D researcher, (Prof. Michael Holick) also calls for 40 to 60 ng/mL 25-hydroxyvitamin D:


This article and another one:


report on hundreds of genes which are upregulated or downregulated by vitamin D in a sample of white blood cells.  Below, I explain how the upregulation occurs, but not the downregulation since I don't yet understand the molecular mechanisms, which are very complex and involve the exact way in which DNA is uncoiled and formed around histones, so it is exposed to enzymes which copy its information into messenger RNA. All these genes are affected as part of intracrine / paracrine signaling in an unknown number of cell types #cells, including probably most or all immune cell types. 

So there seems to be a large and so-far undefined number (I guess dozens to hundreds) of cell types who respond to their circumstances in part, at least, via vitamin D based intracrine / paracrine signaling.

This means vitamin D (the three compounds in general, but in the cells themselves, just 25(OH)D and 1,25(OH)²D) are extraordinarily important for most or all systems of the body.  The scope of vitamin D's role in the body extends beyond the proteins for which these specific genes provide the instructions, because some of these genes involve proteins which affect histones [WP].   Histones are proteins which physically organise the long DNA molecules of the chromosomes, 1.8 metres in total.  An important role of the histones is to unwind particular regions of the DNA so its genes can be copied into messenger RNA molecules and so direct the cell's protein making machinery.  To whatever extent vitamin D intracrine / paracrine signaling affects histones, it therefore affects numerous other aspects of the cell's ability to perform its functions.

Also, since in the case of Th1 regulatory lymphocytes at least #chauss-1, vitamin D based intracrine / paracrine signaling affects the production of cytokines (both pro- and anti-inflammatory), the actions of other cell types and in this case the destruction of pathogens and the body's own cells are also affected by vitamin D based intracrine / paracrine signaling.

40 to 60 ng/mL (100 to 150 nmol/L) was also suggested as the proper target range in this 2019 article (68 citations):


This article also discusses the benefits some people find from much higher 25(OH)D levels, for suppressing inflammatory disorders such as psoriasis and rheumatoid arthritis.  Please see https://vitamindstopscovid.info/06-adv/ for more on this and how it relates to our lack of helminths (intestinal worms).

Please also see the recent article from MDs in Dubai who had great success with COVID-19 patients by either previously raising their 25(OH)D levels to the 40 to 90 ng/mL 100 to 225 nmol/L levels or by using the same bolus D3 and then body-weight ratio continuing supplemental D3 intakes on newly diagnosed hospitalised COVID-19 patients.  The link and my summary is at: https://aminotheory.com/cv19/#2020-Afshar .

Here is another recent research article:

Editorial – Vitamin D status: a key modulator of innate immunity and natural defense from acute viral respiratory infections A. Fabbri, M. Infante, C. Ricordi Eur Rev Med Pharmacol Sci 2020; 24 (7): 4048-4052 2020-04-05 https://www.europeanreview.org/article/20876

They mention that 40 to 60 ng/mL circulating 25OHD is required for the autocrine signaling system of immune cells to function properly.  The proper term for this is intracrine signaling.

In this article's title, the term "modulator of . . . " is potentially misleading, especially when it is used with the overly general term "vitamin D".   In-vitro addition of 1,25OHD (calcitriol) to immune cell cultures will change their behaviours in ways which resemble healthy responses, so it is reasonable to state this experimental addition of 1,25(OH)²D "modulates" immune responses.   However, this does not resemble the natural process of intracrine / paracrine signaling, in which 11,25(OH)²D is locally (intracellularly or in a nearby cell) produced only in particular circumstances.

Similarly, if an in-vitro cellular system or an in-vivo mammal's immune system changes its behaviour upon the experimental addition of extra 25(OH)D, this does not mean that in Nature, the level of 25(OH)D modulates anything, like a level of a hormone "modulates" some cells' behaviour.  All that is happening is that the immune cells or immune system are functioning better than before, due to proper supplies of 25(OH)D rather than baseline state of being unable to work properly due to insufficient 25(OH)D for their intracrine / paracrine signaling.  (Also, if the level of 25(OH)D is excessive, the functioning of the immune system may be degraded, but this is over-supply, exceeding the proper operating conditions of that system, not signaling anything as the level of a hormone does.
 
The text (in the quote below) "the beginning point of the plateau where the synthesis of the active form calcitriol becomes substrate-independent" requires some explanation for non-specialists.  The 1-hydroxylase [WP] enzyme (molecular diagram below) is a large, complex protein, whose actions are powered by some other molecules which are changed in the process.  The authors are discussing the hydroxylation of 25(OH)D to 1,25(OH)²D, which is a crucial early step in intracrine signaling.  There are multiple 1-hydroxylase enzyme molecules in the cell, and each converts one 25(OH)D molecule at a time to 1,25(OH)²D.  The speed of this conversion is important, since if it is too slow, then the 1,25(OH)²D levels in the cytosol (main body of the cell, where this happens - not in the nucleus) will not raise to a high enough concentration (as noted above #1ng), around 1 ng/mL (1 part per billion by mass) that a sufficient number of these 1,25(OH)²D molecules will bind with vitamin D receptor molecules, after which some of these bound complexes will migrate (or at least diffuse) to the nucleus, as I will describe properly below.


If there is no 25(OH)D, the intracrine signaling system cannot work.  If there is too little, then it will work too slowly, or not work properly - so the cell will not respond fully to its new circumstances and our health will suffer.

The enzyme itself is not changed - it is a catalyst.  When, by random thermal motion, a molecule of 25(OH)D is in the right position in the enzyme's active site, the enzyme replaces the hydrogen H which is bound to the number 1 carbon C atom with an oxygen-hydrogen OH hydroxyl group, after which. the newly-formed 1,25(OH)²D is no longer so attracted to the enzyme's active site, and floats away.  

The 25(OH)D molecule, up to the point where it is converted to 1,25(OH)²D, is the substrate.  The authors imagine a graph with 25(OH)D concentration being the horizontal axis and the total rate of conversion to 1,25(OH)²D being the vertical.  The plateau they refer to is where the rate of conversion no longer rises linearly (upwards and to the right) with 25(OH)D concentration, due to the limiting factor being mainly the enzyme's own intrinsic speed of conversion, when it has it hardly has to wait for a fresh 25(OH)D molecule to arrive in its active site.  


The authors mean that if 25(OH)D levels (in the blood) are around 40 ng/mL or more, then this leads, via diffusion - there being no active transport of 25(OH)D from the bloodstream into the fluid between the cells and across the cell's membrane - to a concentration of 25(OH)D in the cell to start with which, after the cell's intracrine signaling system is activated (by the creation of 1-hydroxylase enzyme and VDR molecules in the cytosol) which enables each such 1-hydroxylase enzyme molecule to work at close to its full speed hydroxylating these 25(OH)D molecules to 1,25(OH)²D.  Also, this 25(OH)D level in the blood is required to maintain the 25(OH)D levels in the cell as some of the 25(OH)D is consumed by the conversion process.  So this 40 ng/mL or so level in the bloodstream is required so that passive diffusion (probably from the relatively small proportion (15%) of 25(OH)D which is not bound to the vitamin D binding protein #vdbp) results in enough 25(OH)D diffusing into the cells as the intracrine hydroxylation process continues, so the enzyme is not slowed down by too low a level of 25(OH)D in the cell.  If there was too low a level of 25(OH)D in the cell, each enzyme molecule would need to wait, on average, an excessive amount of time before a fresh 25(OH)D molecule to arrived at its active site in the correct orientation. 

The key thing to remember is that 25(OH)D levels are very low.  A healthy level is 50 ng/mL, (as explained in the next section #04-quraishi)  but many people, without supplements, never achieve this. So for many people, average levels are 1/2 or even as low as 1/10th of this.  50 ng/mL (50 parts per billion) is only one part by mass of 25(OH)D per 20,000,000 parts by mass of all the water and other compounds in the cell.  So these are quite rare molecules.   A 70kg person only needs a gram of D3 every 22 years, about 1/3  to 1/4 of which is converted to 25(OH)D in the liver, to maintain this healthy level.  (50 parts per billion is like a 3.7mm cube of water in a cubic metre of water.) 

Here I am assuming that the concentration of 25(OH)D in the cell is about the same as that in the bloodstream, but as noted above #vdbp it is probably a lot lower than this, since it seems to derive, by diffusion, from the 15%  which is not tightly bound to the vitamin D binding protein, almost all of which is more loosely bound to albumin proteins.

#thermal
You probably began reading this page thinking of the COVID-19 crisis, the influenza crisis and perhaps the sepsis crisis.  Now you are contemplating lonely 25(OH)D molecules being jostled around by the thermal vibrations of surrounding molecules (mainly water) until one of these molecules:

1. Arrives very close to the active site of the much larger enzyme molecule.  This is 3 dimensions (X, Y and Z) of movement over large distances (one such molecule on average per ~320 nanometres cubed) compared to the size of the 25(OH)D molecule (~0.2 nanometers) and the enzyme molecule:
   
   
   
   Adapted from paywalled article https://www.degruyter.com/document/doi/10.1515/jpem-2013-0183/ Sci-Hub: https://sci-hub.se/10.1515/jpem-2013-0183 Wei-Wei Hu et al. A novel compound mutation of CYP27B1 in a Chinese family with vitamin D-dependent rickets type 1A 2013-11-07 .
   
   
2. Is pointing in exactly the right direction for it to fit.  This needs to be correct in 3 rotational dimensions in order to align the end-to-end axis of the molecule with the axis of its  position in the enzyme's binding site.
   
   
3. Is rotated correctly along its axis - this is 1 axial rotational dimension - so the 25(OH)D molecule is precisely aligned with the matching outer electron orbitals of the atoms of the enzyme's binding site..
   

When this happens, the positive and negative charges (due to some negatively charged outer electron orbitals being offset from the positively charged nucleus they surround) on particular parts of the two molecules will draw them closer, the 25(OH)D molecule will be fully docked, and the enzyme and its co-factor molecules will do their work of attaching the OH to the number 1 carbon atom.

This probably seems a long way from the immune system and COVID-19, but it is absolutely germane.  If everyone in the world had 50 ng/mL or more 25(OH)D in their blood, then:

1. The enzymes in all their cell types which use vitamin D for intracrine / paracrine signaling would not be waiting long for another 25(OH)D molecule to dock in their active sites.
   
   
2. So they would produce 1,25(OH)²D at a perfectly healthy rate whenever the cell's intracrine signaling system is activated.
   
   
3. The intracrine signaling systems of all cell types (including many types of immune cell) would work correctly, making then respond fully and rapidly to their changing circumstances.
   
   
4. Although there are numerous other factors affecting total immune system performance, this would mean that the current vitamin D deficiency epidemic would not exist - and it is low vitamin D which is the primary cause of some immune responses being weak, while others are dysregulated - meaning overly-aggressive, hyper-inflammatory and self-destructive.   These weak and dysregulated immune responses are the primary or sole reason why some people who are infected with SARS-CoV-2 develop severe COVID-19.  (The dysregulated, hyper-inflammatory responses are also caused by lack of helminths: https://vitamindstopscovid.info/06-adv/#02-helminths .)
   
   
5. So almost all people would fight off the SARS-CoV-2 infection without serious symptoms.  Likewise flu.  Also, very few people would develop sepsis, Kawasaki disease or Multisystem Inflammatory Syndrome.  Likewise pre-eclampsia, which is a dysregulated, hyper-inflammatory, immune disorder of pregnancy.
   
   (Note: there is a lot of interest in the idea that high vitamin D levels will substantially reduce the chance of being infected with COVID-19 for any given vital insult.  I see no evidence that this is more than a marginal effect.  The most important point, for all society, is the next one, followed by the just-mentioned great reduction in average severity.)
   
   
6. For those infected, average total quantities of viral shedding would also be greatly reduced, so fewer people would become infected.  COVID-19 would not spread very much at any time of year.  Likewise flu.
   
   
7. So there would be no COVID-19 crisis, with no need for lockdowns, social distancing, vaccines or masks.  The few who did become seriously ill could be treated with oral 25(OH)D (calcifediol) and D3 ../04-calcifediol/  as well as other early treatment techniques: https://c19early.com.)
   

You now have an understanding of a crucial part of the current global crisis down to a molecular level - and if you want to, you can look up the gory details of the virus, the ACE2 receptor, the destruction of the endothelium, the hypercoagulative state of the blood and the microembolisms and larger clots in the lungs, brain, heart, spinal cord, liver kidney etc.

None of those gory details would matter as much as they do now, because they would not exist to anything like the current extent, if everyone had enough vitamin D.   70kg adults, on average, to attain about 50 ng/mL (125 nmol/L) 25(OH)D, without relying on UV-B skin exposure, or the small amounts of D3 in food, need to ingest 45 milligrams of D3 year = a gram every 22 years.  This is 0.125mg 5000 IU a day.   Pharma grade D3 costs about USD$2.50 a gram, ex-factory. 

Please see https://vitamindstopscovid.info/01-supp/ for D3 supplemental intakes, as ratios of bodyweight, which I derived from the work of Ekwaru et al. 2014, and for an article by Iranian doctors working in Dubai who found similar ratios worked really well.

Here is another way of understanding the need for proper 25(OH_D levels around or above 50 ng/mL (125 nmol/L).  The following graph comes from research into the risk of infections in people (all morbidly obese) who had just been operated on for Roux-en-Y gastric bypass [WP].  weight loss surgery.



This PNG is from my Inkscape version combining two similar graphs, made from the vectors in the PDF.  So the red and purple lines, and the scales, are direct from the article's graphs, not a result of me trying to copy them by some approximate method.


Please think of these graphs whenever you read of individual and average 25(OH)D levels in people who are not adequately supplementing D3 and who do not get very substantial UVB skin exposure (which damages DNA and increases the risk of skin cancer).  Their levels are typically between 5 and 20 or 25 ng/mL.

The graphs depict how the risk of infections in hospital - either directly resulting from the surgery or due to other reasons - vary with vitamin D 25(OH)D levels, for 770 patients.

Low rates of infections occur when the immune system's innate [WP] and adaptive ([WP] antibodies etc. ) responses are functioning properly.  The high rate of infections at the left of the graph are due to weak immune responses which directly combat the (primarily bacterial) pathogens which cause these infections, as in the first intracrine signaling example below.  (The second Chauss et al. example below concerns innate immune system regulatory lymphocytes which, when their intracrine signaling fails due to lack of 25(OH)D, cause trouble by producing pro-inflammatory cytokines for longer periods than they should.  This failure causes other immune cells to destroy healthy cells, especially in the blood vessels of the lungs.  This causes or at least strongly drives severe COVID-19, but is not likely to be important in the infections in hospital which are the subject of Quraishi et al.'s research.)

With one potential exception, wherever the graphs rise above about 2.5%, this is due to vitamin D based intracrine / paracrine signaling not working properly in some - probably many - types of immune cell.  A potential exception is that that the higher D3 levels which give rise to the higher 25(OH)D levels are also directly useful (without involving intracrine / paracrine signaling) in the protection of endothelial cells [Gibson et al. 2015].  I have not been able to quantify how important this is, and I suspect the main cause of these infections is the failure of the innate immune system to rapidly defeat bacteria.

The raised risks of infection indicate dysfunction of intracrine / paracrine signaling due to inadequate 25(OH)D.   Eyeballing this we see that the 40 ng/mL minimum recommendations mentioned above don't go quite far enough.  The evidence of this substantial research (770 subjects, in one hospital, with all the researchers being from Harvard Medical School) indicates that we should be aiming for at least 55 ng/mL, at least in these obese adults.  There is some scatter in the measurement of 25(OH)D levels.  For this reason, and to simplify things a little, I write of this research as if it suggests that at last 50 ng/mL is required for proper immune system function.

This research does not tell us directly what 25(OH)D levels are required for Th1 regulatory lymphocytes to avoid the pattern of being stuck in their pro-inflammatory startup program indefinitely, as described in Chauss et al. above #chauss-1 above and #chauss-2 below.  There are surely other regulatory immune cells which behave in a similar fashion - weakening direct innate and adaptive responses or pathologically over-strengthening inflammatory responses - if they don't have enough 25(OH)D.  However, it is reasonable to guess that these Th1 cells would generally work reasonably well only when 25(OH)D was also at or above 50 ng/mL.
 
The exact 25(OH)D levels required to suppress hyper-inflammatory immune responses surely vary considerably from one person the next, according to individual genetic variation, in a context in which many people have a problem with these responses, by way of auto-immune inflammatory conditions, due to no longer having helminths.  Please see https://vitamindstopscovid.info/06-adv/ for more information on this.   I expect that if we all had helminth infections, we would generally not need such high 25(OH)D levels to suppress these autoimmune problems.   This page links to mid-2021 research from Ethiopia which indicates that active helminth infections reduce the risk of severe COVID-19 by 77%.

Helminths are known to suppress inflammatory responses, which are primarily directed at helminths and other multicellular parasites.  They are not known to substantially suppress the innate and adaptive responses to viruses, fungi or bacteria - and it is primarily these anti-bacterial innate and adaptive responses which are directly weakened by the lower than 50 ng/mL 25(OH)D levels which are indirectly measured in Quraishi et al. by way of their weakening increasing the risk of infection.

The diagrams below are adapted from a diagram in this 2011 article which has a good description of vitamin D intracrine signaling in a particular type of immune cell, although the term intracrine is not used:

Antibacterial effects of vitamin D Martin Hewison,  Nature Reviews Endocrinology v7 2011-01-25 https://www.nature.com/articles/nrendo.2010.226 (Paywalled.) https://sci-hub.se/10.1038/nrendo.2010.226  415 Google citations.

Here is a description of intracrine signaling which assumes an interest in cell biology, but little prior knowledge.

In this example from Prof. Martin Hewison, we learn how toll-like receptors [WP] on the cell membrane of some types of monocytes [WP] - in this case a macrophage [WP] - respond to bacterial infections.  The same principles of vitamin D intracrine signaling apply to other types of cell, including the Th1 regulatory lymphocytes discussed below (Chauss et al. 2021), although the stimulus for activating intracrine signaling is totally different to the bacterial fragments in the current example, and the response of the lymphocyte is also entirely different.


In this cell type, fragments of pathogens activate toll-like receptors which are embedded [WP] in the cell membrane, with their detector section facing outwards and other parts of the molecule facing inwards, immersed in the cytosol [WP].  When the receptor is activated, it changes its shape so the part of the molecule inside the cell causes some other signaling molecules to migrate to the nucleus and upregulate the transcription [WP] of two genes: for the 1-hydroxylase enzyme and for the vitamin D receptor (VDR) protein.

Those signaling molecules are cell-type specific, and somehow cause transcription enzymes to make mRNA (messenger RNA [WP]) copies of the information in those genes.  These multiple mRNAs migrate out of the nucleus, to the cytosol, where they are found by ribosomes [WP] which work along each mRNA molecule, following its instructions of which amino acids to assemble into the protein chain.  This is called translation. 

When each ribosome reaches the other end of the mRNA molecule, it has produced one chain, which folds of its own accord to become a complete single molecule of protein.  This creates some number (I guess hundreds or thousands) of complete, operational, 1-hydroxylase enzyme molecules and likewise vitamin D receptor molecules.  (There can be other details, such as editing the mRNA and modifications to the proteins.)

25(OH)D is carried in the blood plasma primarily bound to vitamin D binding protein molecules  [WP], with a lower proportion bound less strongly to albumin [WP] proteins. (#vdbp) A small proportion of these 25(OH)D molecules (red discs) are free to diffuse from the plasma, into the interstitial fluid [WP] between cells (in the case of cells which are not in the bloodstream or in the walls of blood vessels) and then they  diffuse across the cells' lipid bilayer [WP] plasma membrane into the cytosol of the cell.  

Vitamin D3 cholecalciferol has only one hydrophilic hydroxyl group, in the 3 position with all its other sides made up of hydrophobic hydrogen atoms.  So it is soluble in oils but not much in water.  25(OH)D has two hydroxyl groups and so is more soluble in water.


Assuming there is an adequate concentration of 25(OH)D molecules (red discs) - which there will be if blood levels are 50 ng/mL or more, then it doesn't take long for one of these 25(OH)D molecules to find its way to the active site of the newly created 1-hydroxylase enzyme molecules, be hydroxylated at the 1 position, and be ejected back into the cytosol as 1,25(OH)²D molecules, (green discs).

By now there will be some number of vitamin D receptor VDR molecules and the freshly made 1,25(OH)²D molecules find their way (as described above #thermal, with random thermal motions and rotations) into the active site of one of these receptors, where the two are strongly attracted and stick together as an activated receptor complex.

This newly produced 1,1,25(OH)²D is functioning as an intracrine agent which binds to these vitamin D receptor molecules, inside the cell in which the 1,25(OH)²D was produced.

This step is identical for the vitamin D based intracrine signaling systems of all cell types.

When a 1,25(OH)²D molecule binds to the receptor molecule, this changes the shape of the receptor molecule and causes some of them to migrate into the nucleus.  (I guess only a subset of them migrate to the nucleus, so perhaps is is diffusion, rather than them all marching off in the direction of the nucleus.)  Articles mention them "translocating" to the nucleus, but this just means "move " and I know of no active transport or guidance system for them doing this, so for now I assume that the activated 1,25(OH)²D-VDR complexes simply move around at random, due to thermal motion, and that some subset of them diffuse into the nucleus.

There the activated receptor complexes find their way (by diffusion, I guess) to another molecule (retinoid X receptor [WP], which is related to vitamin A and is not shown in these diagrams) with binds to them as well and the entire heterodimer [WP] complex then finds its way to particular patterns of DNA which are exposed (according to how the DNA of the various chromosomes are wrapped around and otherwise organised by histones [WP]) and ready to accept them.  These are the VDRE (Vitamin D Response Elements [WP]) and they are upstream of a particular gene which this process is intended to increase or decrease the copying of.   (The whole human genome has thousands of such genes, with one or more VDREs upstream.  By various means, each cell type exposes only these to being bound by the heterodimer complex - the particular genes which this cell needs to be copied in order to respond to its circumstances properly.)


Once the VDRE section of DNA has the heterodimer attached, in some circumstances this signals DNA copying enzymes to start work there, copying the data in the downstream gene into messenger RNA molecules.  In others, it reduces the amount of copying of this gene.  (I have found some explanations of these mechanisms but they are exceedingly complex and require a strong understanding of DNA, histones and the like.  I think these low level details don't need to be understood in order to have a good general understanding of vitamin D based intracrine and paracrine signaling.)

In principle, if this process of activated receptor complexes finding their way to these transcription regulator molecules was highly guided, then there would only need to be a handful of 25(OH)D molecules converted to 1,25(OH)²D, perhaps by a single or a few enzyme molecules, and likewise there would only need to be a handful of vitamin D receptor molecules.

However, since (as far as I know) the processes are unguided (diffusion) or at least not very efficient, and since the activated complexes and probably the 1,25(OH)²D molecules would have relatively short half-lives (of their own accord, or by 24-hydroxylase enzymes changing them into molecules which are broken down, to get rid of them once the conditions which activated intracrine signaling no longer occurred) then there needs to be quite a quantity of both 1,25(OH)²D and vitamin D receptor molecules ready to bind together in order to make a substantial and sustained change in gene transcription, which is required for the behaviour of the cell to change significantly.  This requires continual conversion of 25(OH)D to 1,25(OH)²D, since the 1,25(OH)²D molecules have relatively short half-lives.  As noted above, to fully alter the gene translation process to change the cell's behavior, it seems there needs to be around 1 ng/mL (1 part per billion by mass) 1,25(OH)²D in the cytosol of the cell.

Considering that:

• There an unknown number of cell types using vitamin D for intracrine signaling.
  
  
• There are likely countless billions of cells of each such cell type.
  
  
• The intracrine signaling systems of each cell of these types is activated at least some of the time .
  
  
• Only a subset of the 25(OH)D is actually used by intracrine / paracrine signaling, one might expect the total vitamin D3 requirements per year to supply this 25(OH)D to be substantial.  But 0.045 grams per year is all it takes for 70 kg adults to maintain, on average, about 50 ng/mL 25(OH)D - and only about 1/2 or 1/4 of ingested or UV-B produced D3 molecules wind up as circulating 25(OH)D molecules after hydroxylation in the liver. 
  

With upregulated gene copying, the newly copied mRNA molecules leave the nucleus and go into the cytosol, where ribosomes run along them, making the proteins they contain the instructions for.  (For downregulation, fewer of these mRNA molecules are produced than previously.)  These proteins are the ones which make the cell respond to its changed circumstances.  In some cells, these may be exported to kill pathogens, or to kill infected cells.  In others, the proteins may cause the release of pro-inflammatory or anti-inflammatory cytokines [WP] - signaling molecules which control activities of other types of immune cell which are nearby.

The alterations to gene transcription alter the mix of mRNAs in the cytosol and so (translation) the quantities of proteins produced by the ribosomes which run along them.  (mRNAs have quite short lives, so for continual protein production a continual supply of them via transcription is required.)

This altered set of protein products is what drives the cell to alter its behaviour.  In this example, the altered behaviour sets the cell up for engulfing and digesting bacteria.

In the Chauss et al. 2021 example below #chauss-2, when the intracrine signaling systems of a Th1 lymphocyte is activated and works properly, the lymphocyte switches from its pro-inflammatory startup program to its shutdown program in which it produces less of a pro-inflammatory cytokine and more of an anti-inflammatory cytokine.

One part of paracrine signaling is depicted at the bottom left of the above diagrams: some of the newly produced 1,25(OH)²D diffuses out of the cell and reaches nearby cells. 

As noted above #02-nothorm, the concentration of this 1,25(OH)²D inside the cell in which it is produced is probably around 1 ng/mL (1 part per billion by mass) when the intracrine signaling system is is fully activated and there is sufficient 25(OH)D to feed the hydroxylation process.  This 1 ng/mL is much higher than the very low levels of 1,25(OH)²D present in the bloodstream as a hormone to regulate calcium-phosphate bone metabolism: around 0.045 ng/mL (0.045 parts per billion).

The newly produced 1,25(OH)²D is functioning as a paracrine agent when it diffuses out of the cell, and makes its way to other nearby cells where - by one means or another - this increased local level of 1,25(OH)²D is detected in a way which alters the behaviour of those nearby cells.  As far as I know, VDR is only found in the cytosol and nucleus of the cell - it is not located in the cell membrane, ready to detect 1,25(OH)²D outside the cell.  So, as far as I know, paracrine signaling works by extracellular 1,25(OH)²D, diffused from where it was produced in nearby cells, diffusing into the cytosol and binding to VDR molecules there.  Then, some of the bound complexes diffuse into the nucleus and alter cell behavior as just described for intracrine signaling.

I have not read a detailed account of vitamin D based paracrine signaling.  I guess the distances might be 1 mm to 10 mm or so, but if there was a particular part of the body, such as a 5 cm sphere, in which a population of some immune cell of type A generally has all its cells in a state of activated and successful paracrine signaling (or at least producing 1,25(OH)²D purely for paracrine purposes) than these could collectively flood the area with 1,25(OH)²D as a paracrine agent and so alter the behaviour of a population of cells of some other type B in that area.  So while a lone cell releasing 1,25(OH)²D as a paracrine agent into the bloodstream or fluid between cells in an organ wouldn't be able to raise the concentration very much except over a very short distance from itself of (tens of microns??) with a large number of such cells, their collective output could use paracrine signaling over a greater distance.

If most or all of the above makes sense to you, then you are in a good position to either read the entire Chauss et al. 2021 article, or at least my summary and discussion of its preprint, at:

The basic summary is below.  The above page has a more extensive summary and discussion.

Then, you will have a real, cellular and molecular level understanding of some of the most important reasons why the world is going to hell in a handbasket at present (first written in November 2020, and still en-route in May 2022, though Omicron has been a blessing due to its significantly reduced virulence compared to Delta and previous variants).   Some people are still getting sick from COVID-19, and I assume that 10 million people a year, at least, are still being killed by sepsis https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(19)32989-7.

It would be much easier if everyone took vitamin D supplements to raise their 25(OH)D levels to the 50 ng/mL or more  levels which enable our vitamin D intracrine and paracrine signaling systems to work properly.

I regard this article as the most important article in the entire COVID-19 literature.

This is my best attempt to describe some complex processes I have no expertise in.

Th1 lymphocytes isolated from the lungs of patients with severe COVID-19 symptoms have an intracrine signaling pathway (in this article, referred to as "autocrine") which should be activated by high levels of complement (WP), to turn these cells off their initial hyper-inflammatory startup program which produces primarily pro-inflammatory IFNγ (interferon_gamma WP which has antiviral and anti-bacterial activity as well as stimulating inflammation: cell destruction such as by natural killer cells WP) and instead cause them to produce primarily the anti-inflammatory cytokine IL-10.  (The cells always produce both these cytokines, but this transition to a shutdown, anti-inflammatory program, involves less IFNγ and a lot more IL-10.)

However, this anti-inflammatory pathway is not working in the Th1 cells from patients with severe COVID-19, due largely or solely to insufficient 25,hydroxyvitamin D3 == 25(OH)D == calcifediol for each cell's intracrine signaling system to function. 

This is a molecular and cellular explanation for why people with low vitamin D have wildly dysregulated, overly-inflammatory (cell killing), self-destructive immune responses.  Such responses drive sepsis, severe influenza, Kawasaki disease (KD WP), Multisystem Inflammatory Syndrome (MIS discussion) and of course severe COVID-19.  (See Paul Marik's explanation https://www.evms.edu/covid-19/covid_care_for_clinicians/ of how it is the immune response, not the virus, which causes the escalation to severe symptoms and death.  See https://aminotheory.com/cv19/#2015-Stagi for research which shows KD children have very low 25OHD vitamin D levels.)

In severe COVID-19, severe inflammation in the lungs damages endothelial cells (the inner lining of blood vessels and capillaries WP) leading to hypercoagulative blood, causing microembolisms and larger clots all over the body, which cause most of hypoxia, lasting harm and death.

It is not known whether the cause of all the hyper-inflammatory immune system dysregulation - which causes some COVID-19 sufferers people to develop severe symptoms - is primarily the failure of these Th1 lymphocytes to switch from being pro-inflammatory to anti-inflammatory, or whether this endothelial cell destruction etc. is also driven to a significant degree by similar failures in the intracrine signaling systems of many other types of regulatory and/or directly anti-pathogen immune cell.  However, the determination of the exact mechanism of failure in Th1 cells, in the context of such failures likely also occurring in other types of immune cell, is an extraordinarily valuable contribution which needs to be very widely understood.

No-doubt the virus on its own is causing destruction in the lungs and elsewhere, so I don't want to portray all harmful outcomes of COVID-19 as resulting from the immune system's overly inflammatory response.  For instance, Wenzel et al. 2021 describes a viral enzyme cleaving a human protein NEMO which is essential to brain endothelial cells, resulting in blood brain barrier disruption and the death of capillaries, to become "string vessels".  This seems not to be related to inflammatory responses, but is a good argument for proper 25(OH)D levels and early treatments to enable the body to tackle the virus ASAP, hopefully before such terrible things happen to our brains.

Low vitamin D levels (low circulating 25(OH)D, produced in the liver from UV-B-generated and/or ingested vitamin D3 cholecalciferol) are well known to reduce the effectiveness of numerous direct, anti-pathogen, responses by the innate immune system cells and to hinder the creation of antibodies for adaptive immune responses.  These immune functions of vitamin D 25(OH)D are due to it being needed, in the circulation, at higher levels than are sufficient for bone health (sufficient for the kidneys to produce their much lower concentration of circulating - and so hormonal - 1,25(OH)²D), to supply the intracrine / paracrine (inside the cell / to nearby cells) signaling systems of all types of immune cells.  Most or all types of immune cell can express the vitamin D receptor - and this is for intracrine / paracrine signaling - not for responding to the much lower levels of circulating1,25(OH) ²D which regulates calcium-bone metabolism. #02-nothorm .

This McGregor et al. article is a beauty.  It gets down to brass tacks with the molecular processes of one aspect of the cytokine storm of immune dysregulation which is crucial to the development of severe COVID-19.  This failure may be the the biggest single pathological process which causes some people to develop severe COVID-19 symptoms.  If not, then the similar failures of intracrine signaling systems in all other immune cells would be the primary explanation.  Surely the same dysfunction drives sepsis and many other conditions involving excessive inflammation.

This is one example of a specific vitamin D intracrine signaling system in one particular type of immune cell.  The following article discusses the evolutionary basis and other details of 189 human genes know to be regulated in an intracrine / paracrine manner by vitamin D in monocytes [WP], which are a subset of leukocytes [WP], which are a subset of immune cells which are a subset of the cell types in the body which use their own particular version of vitamin D intracrine / paracrine signaling. 

Primary Vitamin D Target Genes of Human Monocytes  Veijo Nurminen, Sabine Seuter and Carsten Carlberg Frontiers of Physiology 2019-03-15 https://www.frontiersin.org/articles/10.3389/fphys.2019.00194/full

I don't want to imply that I have read this, or that I would be able to understand it without weeks of work.  I cite it as an easy way of expanding upon this one concrete example which surely plays a crucial role in severe COVID-19 (and see https://aminotheory.com/cv19/#2020-Castillo for how oral 25(OH)D == calcifediol for hospitalised patients causes most of them to get much better, very quickly) to indicate how important this general principle of vitamin D intracrine signaling is.  

Intracrine signaling is quite complex.  However, there are plenty of other biochemical processes which are more complex - for instance the citric acid cycle [WP] 

Inquiring minds want to know why this evolved and is used for so many cell types.  This is a Mouse Trap (video) approach to biology - Rube Goldberg [WP] engineering when we can imagine something simpler would do the job.  This wacky complexity is a feature of biology - and some or many of the idiosyncratic features of the evolved systems give rise to valuable mechanisms.  


Why, for instance, doesn't the sensing of the changed condition lead to direct upregulation and downregulation of whatever genes the cell needs to produce (or no longer produce) the proteins which will make respond as it is meant to? 

Sidebar for the really curious:


Please also see this article suggesting some hypotheses about the long-term evolutionary history of the vitamin D compounds and their receptors, enzymes and  binding proteins.

Evolutionary Origin of the Interferon–Immune Metabolic Axis: The Sterol–Vitamin D Link Harry Newmark, Widad Dantoft and Peter Ghazal, Frontiers in Immunology, Molecular Innate Immunology 2017-02-09 https://www.frontiersin.org/articles/10.3389/fimmu.2017.00062

I roughly understood it up to about page 6.