The following box repeats my summary from the list of recent research
../#lr
and in the 02-intracrine page (which originally referred to, erroneously,
autocrine signaling - below the text is as updated on 2023-06-29). Below the box is a more detailed
summary of the cellular mechanisms described in this article.
Th1 lymphocytes isolated from the lungs of patients with
severe COVID-19 symptoms have an intracrine (https://vitamindstopscovid.info/02-intracrine/) signaling pathway
which should be
activated by high levels of complement (WP), to turn these cells off their initial hyper-inflammatory
program which produces 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 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 solely to insufficient 25-hydroxyvitamin D = 25(OH)D = calcifediol for each cell's intracrine signaling system to function. (Until 2021-03-01 I mistakenly stated that the Th1 cells initially produced IL-17 - and that the experimenters restored the Th1's anti-inflammatory pathway by adding 25(OH)D in-vitro.)
(The final Chauss et al. article stated that the cause was largely or entirely due to inadequate 25-hydroxyvitamin D)
This is a molecular and
cellular explanation for why people with
low 25-hydroxyvitamin D have self-destructive, wildly dysregulated,
indiscriminate cell destroying, overly-inflammatory 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 ../#2015-Stagi for research which shows KD children have very low 25(OH)D 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- 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 and/or by direct actions of the virus.
However, the determination of
the exact mechanism of failure in Th1 cells, in the context of such failures likely
also occurring in other cell types, is an extraordinarily valuable
contribution which deserves to be very widely known.
Low
circulating 25-hydroxyvitamin D (produced in the liver
from UV-B-generated and/or ingested vitamin D3 cholecalciferol) levels 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 25-hydroxyvitamin 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-dihyroxyvitamin D), to
supply the intracrine / paracrine (inside the cell / to nearby cells)
signaling systems of many or perhaps all types of immune cells. 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 circulating 1,25-dihydroxyvitamin D which regulates
calcium-bone metabolism. https://vitamindstopscovid.info/02-intracrine/#02-nothorm .
See http://aminotheory.com/cv19/#2020-Fabbri [B] for why 40ng/ml or more 25OHD is required for these autocrine signaling systems to function properly. See also the Quraishi et al. graph https://vitamindstopscovid.info/02-intracrine/#04-quraishi
which suggests that innate immune cell responses which fight bacterial and perhaps fungal infections keep improving,
presumably due to faster and stronger intracrine/paracrine signaling, as
25-hydroxyvitaminD levels rise, up to about 55ng/ml.
Please also see ../#25plusD3
for my suggestion of oral calcifediol (25-hydroxyvitamin D) plus
vitamin D3 as the best
treatment for hospitalised COVID-19 patients, since this raises
circulating 25-hydroxyvitamin D to the levels needed for intracrine /
paracrine
signaling in a few hours, rather than in the several days to a week
with, for instance, a 10 mg 400,000 IU bolus dose of vitamin D3.
See this 0.014 mg calcifediol per kg body weight recommendation in
Prof. Wimalawansa's article in Nature, in 2022: https://www.mdpi.com/2072-6643/14/14/2997.
Very strong clinical evidence of the importance of rapidly raising
circulating 25OHD levels hospitalised COVID-19 patients can be found the Cordoba calcifediol (25OHD) RCT: Castillo et al. 2020: ../#2020-Castillo .
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Here is a
more detailed summary of the cellular mechanisms reported in this article - but please remember that this is the
best effort of an electronic technician and computer programmer, trying to summarize a dense cell biology
preprint reporting on an extensive project conducted by 25 researchers. What I have written below
goes beyond a summary and includes elements of
critique (regarding terminology) and some
commentary
- so please parse it carefully and refer to the article itself, rather
than to what I write here, for your final decisions on the veracity and meaning of this research.
!! The graphic below refers to "autocrine" but it should be "intracrine" signaling.
The wide positioning of the
A,
B,
C and
D
on the above graph exaggerates the relative changes in the degree
to which these two cytokines are created. (The horizontal and vertical
dimensions of Fig 1f are for the rates at which the genes for these two
cytokines are transcribed, which is approximately proportional to the
rates at which the cytokines themselves are produced.)
I wanted to know the relative concentrations of the two cytokines
produced by the cells between the
B activated, inflammatory, state and the
D
shutdown, anti-inflammatory state. This can be estimated from each of two sets of data.
Firstly, the graphs in Fig 2C show the levels of the two cytokines
produced when there is zero 1,25-dihydroxyvitamin D (hereafter 1,25(OH)
2D) supplied to the cells, and then the
levels when three different concentrations of 1,25(OH)
2D are
supplied. I judge the 10nM supply to be sufficient to fully
activate the VDRs as they would be by normal, full, activation of the
autocrine signaling system with sufficient 25-hydroxyvitamin D (hereafter 25(OH)D) supplied. So,
for instance, for the top IFNγ curve of Fig 2c, I can divide the ~5900
pg/ml shutdown
D level (4th red dot from the left) by the ~9,800 pg/ml
B initial, inflammatory, level and get a
D/
B shutdown/inflammatory ratio of 0.6.
Secondly I can do the same for Fig S5d (in the separate Supplementary
Materials PDF) which represents the same things, but this time in
response to the experimenters supplying 25(OH)D so the real, already
activated, intracrine signaling system can operate, producing its own 1,25(OH)
2D to activate the cell's VDRs. Here, I chose the 3rd dot
from the right, because I assumed, perhaps wrongly, that the 50nM
(20ng/ml) 25(OH)D concentration was sufficient for the autocrine
signaling system to operate fully. For IFNγ in this graph I
estimate 7100 / 9100 (why not 9800 as before adding 1,25(OH)
2D?) = 0.78.
I estimate that
the
D/
B shutdown/inflammatory ratios are, from the two techniques respectively (first added 1,25(OH)
2D and second added 25(OH)D) to be
IFNγ 0.6 & 0.78, and
IL-10
2.5 & 1.4 (ignoring the vertical scale discrepancy, which I assume
is a typo). Neither of these experimental techniques tell us
exactly what quantities of the two cytokines Th1 cells produce in-vivo,
but it is the best we can do.
The
normal, healthy, behaviour of Th1 lymphocytes to SARS-CoV-2 infection is as follows.
(Those isolated from the lungs of severe COVID-19 sufferers did not
transition to the anti-inflammatory program, and kept producing the
pro-inflammatory IFNγ, presumably due to lack of sufficient 25(OH)D in
those patient's bloodstream and therefore interstitial fluid, or
whatever fluids the cells were bathed in.)
This discussion concerns T helper lymphocytes (
WP)
which developed from Th0 program cells into Th1 types, were attracted
to a site of infection - in this case the lungs of hospitalised
COVID-19 patients - and there multiplied (AKA, I think, "differentiated") so they
are present in much higher than numbers at this site than when they first arrived.
These Th1 cells have been activated (described below, perhaps before
they multiplied in number) and so are in their pro-inflammatory
B state (Fig 1f in the McGregor et al. preprint and in my infographic above), producing their higher level of IFNγ (
WP) and their lower level of anti-inflammatory IL-10 (
WP).
We are interested in understanding how they transition from this state, firstly to a temporary state
C in Fig 1f, in which they produce high levels of both IFNγ and IL-10, and then to their final, anti-inflammatory, state
D
of low IFNγ production and high anti-inflammatory IL-10. This is
the anti-inflammatory "shutdown program" referred to in the
title of the preprint.
Messenger RNA analysis (scRNA-seq
WP)
of Th1 cells from the lungs of hospitalised COVID-19 patients and
healthy controls revealed, in the patient's Th1 cells, elevated levels
of mRNAs associated with the production of both IFNγ and complement.
Compared to the mRNA expression patterns in the T cells found in BALF
(bronchoalveolar lavage fluid) of controls, patients' patterns were
skewed to Th1 as opposed to the Th2 and Th7 lineages. However, no
such skew was observed in T cells from peripheral blood:
suggesting
that expression of the Th1 program is a specific feature of Th cells at
the site of pulmonary inflammation where virus-specific T cells may be
concentrated.
High levels of complement production in Th1 cells has been observed in
lung infections and specifically with SARS-CoV-2, which is known to
particularly induce the production of complement factor C (C3). A
fragment of this,
C3b, binds to the CD46 receptors (
WP)
found on the plasma membranes of human (but not mouse) T cells.
(2023-06-29: or is it just Th1 cells?)
When all is working well, with sufficient 25(OH)D, the activation of
the CD46 receptors causes the cell to shutdown from the initial
pro-inflammatory
B state to the final anti-inflammatory
D state.
The switch from effector [such as these Th1 cells in state
B,
which produce complement and IFNγ, which is an antiviral compound which
also stimulates ideally healthy cell destruction AKA inflammation]
T
cells, important for pathogen clearance, into IL-10 producing cells
reduces collateral damage and is a natural transition in a T cell’s
life-cycle. This suggests that IL-10 is produced by cells that are
successfully transitioning into the Th1 shut program. Indeed, in
models of T. gondii [WP] and T. cruzi [Chagas disease WP] infections, mice unable to produce IL-10 clear infection more rapidly but die of severe tissue damage from uncontrolled Th1 responses.
IL-10 mRNA in patients' BALF was found at about 1/4 the level it was
found in controls, consistent with the hypothesis that Th1 cells in the
lungs of hospitalised COVID-19 patients indeed did not initiate - or, at least, did not complete - their
shutdown program.
Activation of CD46 receptors by C3b upregulated 24 transcription
factors (TFs). (Genes for these 24 were found to have been
transcribed to mRNAs at higher levels than without this CD46
activation.) One of these TFs was for the vitamin D receptor
(VDR) gene and another was the CYP27B1 gene for the 1-hydroxylase enzyme,
which converts intracellular 25(OH)D AKA calcifediol (diffused there from
circulating 25(OH)D) into intracellular 1,25(OH)
2D AKA calcitriol which
binds to and activates the VDR.
So we see that this CD46 activation initiates the first steps of vitamin D intracrine signaling
https://vitamindstopscovid.info/02-autocrine/
, while the other transcription factors drive other cellular responses
in parallel to (if all works well) the effects of this intracrine
signaling process, which might also involve this locally produced 1,25(OH)
2D diffusing to nearby cells as a paracrine agent.
Although both vitamin D based intracrine and paracrine signaling had
been previously described in Th1 cells, this research project set out
to elucidate the molecular mechanisms and functional consequences of
this, which were previously unknown. They did a great job!
(Page 5 starts with some
descriptions of CD3 CD28 activation driving internal production of C3b
which binds to CD46 on the Th1 cell's surface, which is an autocrine
signaling process, quite separate from the vitamin D intracrine
signaling process we are most interested in - in which the VDR receptors are in the cytosol.)
Sidebar on vitamin D terminology and "vitamin D is a hormone":
As is common in many vitamin D articles, the article states that "vitamin D is a steroid hormone". Here we get into
terminological and factual difficulties, which I discuss at:
https://vitamindstopscovid.info/02-intracrine/#02-nothorm .
In some articles, "vitamin D" means specifically vitamin D3
cholecalciferol, with 25(OH)D and 1,25(OH)
2D being classed as "vitamin D
metabolites". However, this makes no sense since the receptor
universally known as the "vitamin D receptor", for all (or at least
most) practical purposes, is only activated by 1,25(OH)
2D.
Some other articles use "vitamin D" to refer collectively to the three
compounds: D3 cholecalciferol, 25(OH)D calcifediol and 1,25(OH)
2D
calcitriol. This makes sense to me, and this is how I use the
term (2023-06-29: not any more!). Some articles use "vitamin D" to mean both these incompatible definitions, without
any indication that this is invalid - which is very confusing.
This article classes the artificial compound 1,OHD alfacalcidiol [
WP],
which can be converted to 1,25(OH)
2D by the 25-hydroxylase enzyme normally
found in the liver (which normally converts D3 to 25(OH)D) as both an
active
metabolite of vitamin D, which I am pretty sure it is not, and simply
as vitamin
D, which it is not. Alphacalcidol is an analogue of
"vitamin D", where this is the collective use of the term, since it is
similar to D3 (an extra hydroxyl group at position 1) and can be
converted to 1,25(OH)
2D by adding another hydroxyl group at position
25. As far as I know, it is an artificial substance not found
in-vivo.
Generally the article (and this is a preprint) uses the term "VitD" to
refer to one of the three compounds, usually 1,25(OH)
2D, but sometimes 25(OH)D, which I think is confusing. It would be better to refer to
the specific compounds, in all cases.
1,25(OH)
2D, when in circulation in the blood, acts as a hormone: endocrine
cell-to-cell signaling between cells anywhere, or in many distant
places in the body, with the compound being circulating in many parts
of the body via the bloodstream.
This does not mean that 1,25(OH)
2D
is
a hormone. It can act as a hormone when in circulation. Nor
does it make any sense to state that "vitamin D" is a hormone, with
this meaning either just D3 or collectively D3, 25(OH)D and 1,25(OH)
2D.
However, this "vitamin D is a hormone" statement and this confused and
confusing use of terminology is industry standard practice, since
it is so common in vitamin D research articles, including a recent
article co-written by the acknowledge leader of the field, Michael Holick:
Immunologic Effects of Vitamin D on Human Health and Disease
Nipith Charoenngam, Michael F. Holick 2020-07-15
Nutrients 2020, 12(7), 2097 https://doi.org/10.3390/nu12072097
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which I think is a great article, apart from these terminological problems.
(Page 5 to 6.) The researchers prepared activated
*
Th1 cells and treated them with 1,25(OH)
2D to determine which genes were
upregulated (296) and downregulated (157) by this robust VDR
activation. (The precise details of these transcriptional - DNA to
mRNA - changes would depend on the exact state of the cells, since
there all sorts of subtleties in the direct mechanisms of
transcription, and, for instance, acetylation of histones in ways which
also affect the rate at which particular genes are transcribed.) Among
these transcriptional changes were:
- Reduced transcription ("repressed") of the IFNG (interferon gamma) gene which, when
copied to mRNA, causes the protein making machinery (ribosomes) to
create IFNγ. So this change reduces the amount of IFNγ produced.
- Likewise, repression of the gene for the pro-inflammatory cytokine IL-17 [WP].
- Increased transcription ("induced") of the genes for IL-10, IL-6 (generally, but not always, a pro-inflammatory cytokine [WP]) and several transcription factor genes including JUN (for c-Jun [WP]), BACH2 (for the "broad complex-tramtrack-bric a brac and Cap'n'collar homology 2" protein [WP]) and STAT3 (Signal transducer and activator of transcription 3 [WP]). I discuss these further below. (It is 1AM and I can feel myself, very slowly, becoming a cell biologist - though I would not want to be examined on that BACH2 thang.)
The induction of IL-6 was a surprise to the researchers, since this is
a frequently encountered and typically pro-inflammatory cytokine.
However, as I best understand it, it seems that in these circumstances
IL-6 is being produced for purposes of internal signaling (intracrine, I
guess) rather than to stimulate high levels of inflammation in the
vicinity of this cell.
* "Activation" in
this context means that the T cells had C3b bound to their CD46
receptors which initiated the transcription of the genes for VDR
(vitamin D receptor) and the CYP27B1 1-hydroxylase enzyme, both which
were made as a result. I assume that the medium in which
these cells were tested had little or no 25(OH)D, since this would have
been converted to 1,25(OH)
2D, which would have bound to the VDR and so
caused the repressed and induced patterns of mRNAs described above and
below.
The researchers repeated these tests and found the same patterns of
gene transcription when, instead of directly adding 1,25(OH)
2D, they added 25(OH)D (calcifediol). This shows that the activated cells, when
they had enough 25(OH)D for the activation-created CYP27B1 1-hydroxylase
enzyme molecules in their cytosol to convert to 1,25(OH)
2D, which then
binds to the VDR molecules there (also created by the
activation). Then
the bound complex of 1,25(OH)
2D and VDR "migrates" (or does it simply
diffuse?) to the nucleus, complexes with some other molecules (the retinoid X "receptor") and the
resulting 3 molecule ensemble changes gene expression as
expected so the cells transition successfully from their initial
pro-inflammatory state
B, through their pro- and anti-inflammatory state
C, to their shutdown mode: anti-inflammatory state
D.
We see from this that
if all the cell's internal mechanisms are functioning normally, that successful transition from
B to
D would involve these
external factors and these
internal processes:
- High levels of complement
- perhaps created in part by the population of Th1 cells in the same
vicinity (millimetres, I guess) which are in the same initial B
state, (or is some or all of this complement, C3 protein at lest, made inside the Th1 cell??) lead to (by some
processes I am not trying to include in this summary) to the C3b part
of these complement compounds binding to the Th1 cell's CD46 receptors,
with their binding site on the outside of the cell's plasma
membrane.
- The activated CD46 receptor
alters (by means I am not trying to summarize) gene expression in many
ways, including by inducing (increasing the number of mRNA copies of)
the genes for the CYP27B1 1-hydroxylase enzyme and VDR.
These two of the many other gene expression altering changes caused by the activation of the CD46 receptors initiates this cell's vitamin D based autocrine (and potentially paracrine) signaling process.
This process is initiated in a response to this particular cell's
circumstances - it is not some kind of homeostasis-maintaining feedback
loop. Each cell type which uses 25-hydroxyvitamin D based intracrine signaling has similar
principles to steps 2 to 4, but the details of the initiating
process and of which genes are induced and repressed are completely
different between the cell types.
I think of this as the initiation
step of this cell's vitamin D based intracrine signaling system.
(I am leaving out various mRNA editing steps, if such steps occur with
these genes - such as splicing removing introns - see
post-transcriptional modification [WP].)
- These mRNAs leave the nucleus, get into the cytoplasm and there
direct ribosomes to make both the CYP27B1 1-hydroxylase enzyme and VDR
proteins. This is translation [WP],
but just of these two proteins (always the same for every cell which
uses 25-hydroxyvitamin D based intracrine / paracrine signaling).
(Meanwhile, other gene transcription changes caused directly by CD46
activation also result in more or less of other proteins being made,
but this is not part of the 25-hydroxyvitamin D based intracrine signaling process we
are focusing on at present.)
- Externally supplied 25-hydroxyvitamin D (AKA calcifediol and 25(OH)D) is necessary to the next step.
The cell cannot (generally <- added 2023-06-29) make its own
25(OH)D. Even if vitamin D3 is present in the interstitial fluid [WP]
(or perhaps the plasma, if the cell is in the bloodstream) it lacks the
25-hydroxylase enzyme to convert it to 25(OH)D. (2023-06-29 note,
this is not necessarily absolutely the case, since I recall that there
is evidence that some cells, other than those in the liver, can
hydroxylate vitamin D3 to 25(OH)D. For simplicity, here I assume
this is not the case to any significant degree for these Th1
lymphocytes.)
Generally we
assume that UV-B produced or ingested D3 is converted, over days
to a week or so, in the liver by this enzyme into 25(OH)D, where it goes
into circulation in the blood plasma (more on binding proteins
below).
As
best I understand it, circulating 25(OH)D diffuses from the plasma
into the interstitial fluid, without any particular active transporters
or energy expenditure. From there, it diffuses - again without
transmembrane transporters, energy expenditure or any other directional
processes - from outside the cell's plasma membrane and into its
cytosol. As far as I know, the 25(OH)D molecules are largely, but not
entirely hydrophobic - only two hydroxyl groups and all the other
outside parts of the molecule being oliophilic [WP]
hydrogen atoms and small. I assume the molecule makes its own way
across the plasma membrane. Can anyone
provide more details or confirm this? (2023-06-29 note: I recall
that kidney cells have an active transport arrangement to get 25(OH)D
into the cells by carrying it attached to vitamin D binding protein,
which is what the great majority of circulating 25(OH)D molecules are
bound to.)
Inside the cell, 25(OH)D is subject to degradation by the 24-hydroxylase
membrane, which is encoded by the CYP24A1 gene, the name of which is
often applied to the enzyme itself. [WP].
(This is all basic vitamin D stuff - not specific to cells with vitamin
D based intracrine / paracrine signaling systems.)
The 24-hydroxylase enzyme is inside the mitochondria [WP]
and its molecular numbers, or at least its overall activity in the
body, is upregulated by high 25(OH)D levels. (The diagram https://www.wikipathways.org/index.php/Pathway:WP1531
shows this enzyme degrading 25(OH)D, here referred to as calcidiol, and
being upregulated only by parathyroid hormone and 1,25(OH)2D
calcitriol,
whether circulating or perhaps locally produced, rather than
25(OH)D. However, my potentially faulty understanding is that its
activity is also driven by 25OHD itself, in a self-regulatory system
which causes the curved D3 or 25(OH)D input to 25(OH)D level response
in
the Ekwaru et al. 2014 diagram: https://vitamindstopscovid.info/01-supp/a-ratios/ .)
This converts some 25(OH)D to 24,25(OH)D which is an irreversible operation, leading to the
latter being metabolised and its components excreted. This is the
primary, or perhaps sole, self-regulatory mechanism which tends to
limit 25(OH)D levels (in the bloodstream and/or in individual cells) if there is a large input of vitamin D3 (and/or its UV-B
creation) or (artificially) 25(OH)D into the body.
Secondly, this 24-hydroxylase enzyme also degrades 1,25(OH)2D, which has a much shorter half-life than 25(OH)D. (I
guess this shorter half-life is due primarily to the greater affinity
of 1,25(OH)2D for this 24-hydroxylase enzyme, but this is the limit of my knowledge and I
don't have time now to dive into another rabbit hole to find out for
sure.)
This externally supplied 25(OH)D (or that which remains
subject to 24-hydroxylase activity AND it being already consumed as
described next) finds its way to the active site of the 1 hydroxylase
enzyme. The concentration of 25(OH)D in the cytosol is very low -
so there is probably only one such molecule every 320 nanometres cubed
(my rough calculations at https://vitamindstopscovid.info/02-intracrine/#03-minlev from which the next illustration is drawn) when the molecule, in red below, is only 0.2 nanometres long.
The 25(OH)D molecule also relies on thermal vibrations and some local
electrostatic attraction to rotate it into the correct orientation and
so
slot into the active site correctly. I mention this to highlight
that this reaction proceeds at a rate limited by the concentration of
25(OH)D, which is very low, and the likely still lower concentration of
the 1-hydroxylase enzyme. (Cofactors are necessary for the
1-hydroxylation
reaction, some of which are consumed in the process.)
The newly created 1,25(OH)2D molecule leaves the active site and the
enzyme is ready for another 25(OH)D molecule to float into position.
The rate at which 1,25(OH)2D production proceeds is directly dependent on
the 25(OH)D level in the cytosol, and this is reduced for every 25(OH)D
molecule hydroxylated to 1,25(OH)2D, or lost to 24-hydroxylase.
For the intracrine signaling system to operate rapidly and fully, there
needs to be a substantial rate of 25(OH)D to 1,25(OH)2D conversion, because
the latter is short-lived. This means there needs to be a
continual supply of 25(OH)D to the cell, by diffusion from the
interstitial fluid (or perhaps directly from the plasma).
So the presence of adequate, externally supplied, 25(OH)D in the cytosol (which
should be the case at all times - this presence not created by the CD46
initiation of this intracrine / paracrine signaling
system) together with the newly created (by the initiation of the intracrine / paracrine signaling process) 1-hydroxylase enzyme leads directly to the outcome of this step: in-cytosol production of 1,25(OH)2D.
Some of this 1,25(OH)2D is degraded by 24-hydroxylase - over time, most of
it
which remains in the cell will presumably be degraded in this way. Some of this 1,25(OH)2D diffuses from the cell and may act on nearby cells as a
paracrine agent.
Some of this 1,25(OH)2D drives the next step.
- The newly created 1,25(OH)2D molecules in the cytosol soon find themselves bound to the VDR, for which they have a very high affinity. There are a bunch of details regarding how the complex of the two "migrates"*
to the nucleus, binds with other things and (by various mechanisms I am
far from understanding - and I am not sure if anyone fully understands
them all) the presence of large numbers of bound VDR complexes alters
the transcription (AKA expression) of numerous genes. The exact
details of which genes are induced or suppressed, and to what degree,
depends entirely on the cell type.
*
"Migration" is the usual term, but as far as I know there is no active,
powered, directional mechanism for this. Can anyone improve my
knowledge? I wrote "migrate" to be compatible with all I have
read, but for now I assume the bound complex diffuses around the
place randomly, with some of them getting into the nucleus.
This is the transcription phase of the intracrine signaling system.
There is some kind of degradation process for bound VDR complexes in
the nucleus, so they don't accumulate and alter gene translation
forever. I don't know the details, but I guess the VDR itself is
retained and is free to diffuse back into the cytosol. However,
there is surely a degradation process for the VDR as well, otherwise
they would accumulate and this part of the intracrine signaling system
would not be turned off when the initiating condition for the system is
no longer active. The same goes for the 1-hydroxylase enzyme, which I assume is degraded in some way at a modest rate.
- The changed mRNA numbers for the various genes, by altering the protein produced by ribosomes, alters the function of the cell.
This is the final output of this entire vitamin D driven intracrine
signaling process for this particular type of cell.
mRNAs are rapidly degraded, and proteins (which may be exported or
transformed in various ways) don't last forever. So for the
autocrine signaling system to continue to function in its activated
state, the original initiating stimulus (in the Th1 cell type, C3b
binding to the CD46 receptors bound to, and sticking out of, the cell's plasma membrane) must
continue to be present, 25(OH)D needs to be continually supplied, by
diffusion, so 1,25(OH)2D production can continue. So there needs to
be a continuing supply of 25(OH)D, by diffusion from the bloodstream into
the cytosol of each cell.
The exact details by which IFNγ and IL-10 are produced by these Th1
lymphocytes is somewhat more complex than getting their mRNAs into the
cytosol. More details of this were discovered by the researchers
and are summarized below. However, these details do not alter the
central role of 25-hydroxyvitamin D based intracrine signaling controlling the
Th1 cells' production of these two crucially important cytokines other
than that they also rely to some extent on the hydroxyvitamin D based
intracrine signaling system also upregulating the transcription of the gene which produces
IL-6.
This has been a rather detailed excursion into the molecular details of
the central (common to all cell types which use it) mechanisms of 25-hydroxyvitamin D based intracrine signaling.
We need to think about this because at present (March 2021) the world
is going to hell in a handbasket, in large part due to most humans
having insufficient 25(OH)D in the cytoplasm of their immune cells to
initially fight the SARS-CoV-2 infection, and in particular, in the
context of humans' genetic predisposition to overly inflammatory immune
responses due to lack of helminths:
(2023-06-29 update: on helminths, see: https://vitamindstopscovid.info/06-adv/ .
If everyone had about
50ng/ml
or more - twice is just fine (2023-06-29 note: for most people, there
may be some people who have bad reactions to 100 ng/mL 250 nmol/L, but
I have not yet been able to find all the best research) - 25(OH)D in
their bloodstream, all these immune cell types, Th1 lymphocytes
included, and all
other cells in the body which use 25-hydroxyvitamin D based intracrine
(and perhaps
paracrine) signaling, would be working pretty well. Omega-3 and
other nutrients such as
magnesium, boron (
#08-boron) and vitamin C are also important, but for
now we focus on 25(OH)D, since this is the most important nutritional
deficiency which clobbers all aspects of the immune system.
This McGregor et al. preprint concerns low 25(OH)D greatly increasing the (already
genetically driven - see helminth material must mentioned) overly-inflammatory pattern of immune responses -
and this response (perhaps just from these Th1 cells, but likely
involving weak and dysregulated responses from all types of immune
cell) is the primary driving reason why some COVID-19 sufferers
progress to severe symptoms in their lungs, with high levels of endothelial damage,
which causes the hypercoagulative blood which does the real damage with
microembolisms and larger clots all through the body.
Inquiring minds now want to know how the (ideal, as far as we know
~50ng/ml 125nmol/L
or more 25(OH)D blood levels relate to the levels of 25(OH)D in the cytoplasm of
the Th1 cells (and all other cell types which also use vitamin D for
autocrine / paracrine signaling). See my pages at the current
site and at
https://vitamindstopscovid.info
for research which shows we need such levels, at least for intracrine
signaling to work properly. (Some people, with auto-immune diseases,
need two or three times these levels to significantly reduce or abolish
their symptoms - here I am discussing most people, not these. See
the McCullough et al. article mentioned above for examples.)
Looking at the experimenter's graphs for the IFNγ and IL-10 responses
to 1,25(OH)
2D (Fig 2C) and 25(OH)D (Fig S5d in the Supplementary data PDF),
we see that most of the change occurs between concentrations of 0 and:
1,25(OH)2D: 1nM, with about half the action at 0.1nM. (However, I chose 10nM for the levels which I think reflect the complete transition to anti-inflammatory state D.)
25(OH)D: (My guess, based on interpolating between the 0, 10 and 50nM levels.) 25nM with about half the action around 7nM.
nM == nano Mols == nmol/L = a billionth of a mole of molecules per litre.
1 nanogram per millilitre of D3, 25(OH)D or 1,25(OH)
2D
is the same concentration as 2.5nmol/L of these compounds. (This
is ignoring the slightly heavier mass of the latter two.)
A Mole [
WP] is 6.022 x
1023 molecules. 1 nanomol is 6 x
1014 molecules, so 1nM = 1nmol/L = 0.4ng/L means that for each molecule, there is 1.66 x
10-15
litres of water. This volume is a cube with sides 1.185
micrometres = 1185 nanometres, which is big for a molecule which is
only 0.2 nanometres long. This is something like one 1mm
long object in a home swimming pool.
The nM concentrations reported in these graphs are for the solution in
which the researchers bathed the cells, and for now we assume reasonably
free diffusion into the much smaller cell bodies - so these figures
roughly represent the concentrations in the cytosol of these Th1
lymphocytes.
So it seems that to get the intracrine signaling system of these cells
to respond fully, we need about 25nmol/L 25(OH)D in the cytosol, which is
about
10ng/ml.
I think the discrepancy between this very approximate figure and the abovementioned
~50ng/mL for circulating 25(OH)D in the bloodstream can be accounted for roughly as follows:
- Most of the 25(OH)D in the blood plasma is bound tightly to vitamin D binding protein (DBP) molecules [WP],
which with 458 amino acids, dwarf the 25(OH)D molecule which is not much
bigger than a single amino acid. There is is considerable
individual and racial variation in the form of these DBP molecules and in
the concentration in which they are present in the plasma.
Although it is possible to measure the level of 25(OH)D which is not
bound to DBP, this is not a commonly available from pathology labs,
although such measurements might be more clinically relevant regarding the immune system.
DBP also binds D3 and the low level of circulating (hormonal) 1,25(OH)2D.
- Most of the 25(OH)D which is not bound to DBP molecules is bound - much less strongly, I recall - to albumin proteins [WP].
So the fraction of 25(OH)D available to diffuse into the interstitial
fluid, and then into the cytosol of immune cells such as these Th1
lymphocytes, is the unbound fraction plus some part of the albumin-bound
fraction.
- In any intracellular parts of the 25(OH)D molecules paths of diffusion from
the plasma to the cytosol of the Th1 lymphocytes (such as diffusing
through endothelial and other cells of capillaries), there will be some
losses due to the actions of the 24-hydroxylase enzyme.
In March 2021 we know, from numerous research articles in the last
year - many of which are analysed by some apparently expert, but
anonymous, souls at:
https://vdmeta.com , some of which are listed at
https://aminotheory.com/cv19/#lr
- that people who succumb to severe COVID-19 have generally lower, on
average, 25(OH)D levels than those who don't. This becomes
particularly clear when we consider the dramatic improvements due to
giving hospitalised COVID-19 patients in Cordoba, Spain, just 0.532mg
of oral 25(OH)D calcifediol:
https://aminotheory.com/cv19/#2020-Castillo , raising their 25(OH)D levels from a likely
4 to
20ng/ml range to around
60ng/ml in a few hours.
So lets think about D3 intakes and these low 25(OH)D levels for a
moment. For 70kg adults, 0.125mg 5000IU D3 a day maintains about
50ng/ml. This is
1 gram every 22 years. These patients
lower 25(OH)D levels of
4 to
20ng/ml probably result from much lower total quantities of D3
from UV-B, food and few, if any, supplements. Let's say their D3
input was 0.01mg 400IU a day, with some patients, in recent years at
least, with even less, due to being aged, being indoors, having dark
skin, covering their bodies almost entirely when outside etc. The
UK government recommends 0.01mg 400IU D3 a day for adults - in winter
only.
These people would generally not be patients in hospital with
COVID-19 if they had been supplementing 0.125mg D3 a day (or more
according to bodyweight, with more for people suffering from obesity:
https://vitamindstopscovid.info/01-supp/
). 2023-06:29: see the chart of vitamin D3 supplemental intake
quantities, as ratios of body weight, at:
https://brownstone.org/articles/vitamin-d-everything-you-need-to-know/
.
So this severe ill-health, and numerous other aspects of ill health, has
been in large part caused by these patients having disastrously low D3
intakes (though generally about enough to ensure they don't get rickets
or unusual levels of osteoporosis), of about
1 gram every 100,000 days
= 274 years. Pharma grade D3 costs about USD$2.50 a gram ex
factory in 1kg quantities. (That said, UK intracrine signaling pioneer Martin Hewison states in a 2021-03-03
video
that the UK is the vitamin D deficiency centre of the world, and that
rickets is not uncommon in certain communities - presumably of people
with dark skin and/or who avoid direct sun exposure of their skin.)
This global epidemic of vitamin D deficiency has been running for
decades and centuries. (2023-06-23 update: since humans started
living in Europe and especially northern Europe, eventually migrating
to various other locations far from the equator.) Without it,
there would be little or no
need for the disastrous lockdowns, social distancing, economic and
travel shutdowns etc. which governments have imposed to combat
COVID-19, and surely will continue to impose in the years as vaccine
manufacturers (and populations of entire countries) play cat-and-mouse,
or whack-a-mole, with
increasingly efficient viral variants which
are also under strong selection pressure to evolve avoidance of
existing infection- and vaccine-induced immune responses.
Back to the research article!
The researchers demonstrate further details of the Th1 immune shutdown
program, which I discuss below. This research shows that
the shutdown is dependent on adequate 25(OH)D levels in the cytosol of
the cell, since, in-vitro (as we surmise, in-vivo, from high 25(OH)D
levels apparently causing a reduction in this hyper-inflammatory immune
dysregulation) the cells do not respond
to high levels of complement by transitioning from their
pro-inflammatory program to their anti-inflammatory shutdown program,
unless sufficient 25(OH)D is supplied to them.
This concludes the section of my summary and discussion which is most
relevant to 25-hydroxyvitamin D based intracrine signaling in Th1 lymphocytes.
What follows is an attempt to summarize some of the other molecular
details of exactly how IFNγ, IL-10 and IL-6 production is regulated in
these cells, within the context of these Th1 cells' 25-hydroxyvitamin D based intracrine
/ paracrine signaling system.
Again, please remember this is the best effort of an electronic
technician to understand and describe complex cell-biology
experimentation.
The researchers noticed that IL-10 production was proportional to IL-6
production. They added IL-6 to cells which had their CD46
receptors stimulated, and so the 25-hydroxyvitamin D based intracrine signaling system had been
initiated, but with the cells remained stuck in their pro-inflammatory
B
state due to there being insufficient 25(OH)D in the cytosol. (With sufficient 25(OH)D
they would have converted some of it to 1,25(OH)
2D, completed the
autocrine signaling pathway and transitioned through
C to the anti-inflammatory
D
state.) This caused the cells to produce pro-inflammatory IL-17,
which had been reported by other researchers. This seems
clinically relevant to me, since this is an additional mechanism for
pro-inflammatory dysregulation in patients with low 25(OH)D levels, IL-6
being at high levels in all these COVID-19 lung infections.
Both c-JUN and STAT3 transcription factors were produced by the full
operation of the 25-hydroxyvitamin D based intracrine signaling system (leading to
state
D). c-JUN phosphorylation [
WP]
was also driven directly by this. STAT3 phosphorylation was
dependent not on this completion of the intracrine signaling
pathway, but on the presence of IL-6, the production of which was
directly caused by the completion of the intracrine signaling system. The researchers confirmed
that the IL-6 receptor was necessary for this process by introducing an
IL-6 receptor blocker Tocilizumab.
(Imagine the dreams of the people whose job it is to come up with these names for pharmaceuticals.)
Since this IL-6 was, at least in part, produced inside the cell,
and activates receptors in the same cell, this is a separate IL-6 based
intracrine pathway with these IL-6 molecules acting as an intracrine
agent. To the extent that some of the IL-6 which activates these
receptors was produced in nearby cells (Th1 or other types), those IL-6
molecules are acting as paracrine agents.
With further evidence from the cells of two patients with a rare STAT3 mutation (!) the researchers concluded:
VitD [they
mean completion of the 25-hydroxyvitamin D based intracrine signaling response to high levels of
complement, which is only possible with sufficient 25(OH)D] induces STAT3 and IL-6, and IL-6 phosphorylates STAT3 to promote production of [anti-inflammatory] IL-10.
|
Page 7 to 8 describes the researchers' investigation of the molecular
details of how VDR activation (which will transition these cells from
the
B state, through
C to
D) works, such as by the VDR, complexed to other molecules, affecting the degree to which histones [
WP] are acetylated [
WP]
in particular places. The 46 chromosomes add up to 1.8 metres of
DNA which is wound around much smaller histone molecules. This
typically tight winding impedes access to most of the DNA by the
enzymes which can copy its information into messenger RNA molecules
(mRNAs). Acetylation in particular parts of the histone enables
looser DNA winding and therefore greater access of these enzymes to
particular parts of the DNA - including the genes which, in this
particular cell type, VDR activation increases the transcription
of. I don't clearly understand their observations regarding
c-Jun, BACH2 and STAT3 transcription factors, their phosphorylation and
super enhancer structures arising from histone acetylation.
BACH2 - a transcription factor whose production is increased "by
vitamin D" (meaning the successful completion of the 25-hydroxyvitamin D based intracrine process leading to state
D)
- is crucial to the increased transcription of other genes by this
process, including the gene for the IL-6 receptor which, as as noted
previously, is an essential link in the chain of IL-6 based intracrine /
paracrine signaling which leads to the production of anti-inflammatory
IL-10.
Expression
of VitD-repressed genes . . . was significantly higher in
bronchoalveolar fluid Th cells of patients with COVID-19 than
healthy controls.
|
So the Th1 cells from hospitalised COVID-19 patients did not have their (high levels
of complement driven) CD46 activated 25-hydroxyvitamin D based intracrine signaling function run to completion (from state
B through
C to
D),
presumably due to lack of 25(OH)D, leading to little or no production of
intracrine agent 1,25(OH)
2D, and so little or no activation of VDRs which,
if activated, would have repressed the transcription of these genes.
Here endeth the lesson. There will be a test on Friday!
This is a review of observations and mechanisms. I haven't had a chance to read it yet.
Further to the discussion above on
intracrine and paracrine signaling of 1,25OHD, here is a pertinent
article, from 2008. The lead author is a critical care
pulmonologist in Iowa.
Also of interest is this analysis of
COVID-19 damage to lung tissue differs from that resulting from
H1N1 influenza in 2009:
Here is an excellent paper, from
researchers in Singapore and Liverpool regarding the role of
inappropriate, over-inflammatory, immune responses in severe symptoms
with COVID-19.
There's no mention of vitamin D - though deficiency in this clearly
drives weakened and dysregulated immune responses which allow disease
progression and which, as this article documents, drive a lot of the
damage which occurs. Nor is there any mention of coagulation or
vasoconstriction - both key elements in the material above, which was
developed after this late April article would have been finalised.
Here's another article concerning
endothelial failure in the lungs, with the researchers not knowing why
this happens to some people.
These would be mainly the patients with
the weakest and most dysregulated immune systems - the primary,
currently known (I suspect boron deficiency as well) preventable cause
of which is vitamin D deficiency. Yet vitamin D is not mentioned
in this article.
We're not done yet . . .
Yes, but why? This is where
anyone who has read the research I have read - and which you will read
if you follow my links - thinks of the low 25OHD levels of these
patients, and how this will reduce the ability for numerous types of
immune cell (and it seems we should count some or all endothelial cells
as immune cells too) to act strongly and successfully against the
virus, while regulating the destructive responses which cause almost
all the harm described in this article.
There are no doubt other articles with detailed accounts of COVID-19
pathology in the epithelium of the lungs. I don't have time to
find or read them all, but here are some of potential interest:
I haven't read this yet. Would
vitamin K supplementation reduce the most destructive aspect of severe
COVID-19: the hypercoagulative state?