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From: Kofi on 9 Apr 2007 05:12
Imagine my surprise when a recent search of the regulatory T-cell
literature turned up a vein of research on histones shortly after I
started taking sodium butyrate. The vein is about a year old. I could
kick myself for missing it.

Butyrate is an HDAC inhibitor formed from the digestion of fiber by
friendly microflora in the gut. In addition to supplying healthy fuel
for intestinal cells, butyrate also inhibits the removal of acetyl
groups from histones by blocking the enzyme which removes them - histone
deacetylase (HDAC). This potently inhibits the formation of colon
cancer. When histones are properly acetylated, the FoxP3 gene works. A
working FoxP3 gene turns on regulatory T-cells which then block the
body's autoimmune reactions - theoretically preventing everything from
allergy to multiple sclerosis. This means HDAC inhibitors promote
immunotolerance by increasing the number/function of regulatory T-cells
(Tregs). Sodium butyrate, then, may have broad application against
everything from autoimmunity to the rejection of transplanted organs. A
proper dose in prior studies was on the order of 4g daily [PMID
16225487] but might require carnitine with it.

Butyrate also promotes prostaglandin E2 (PGE2) production in certain
cells [PMID 9733608]. PGE2 is an essential activation signal for Tregs.
This prostaglandin also mediates stem cell repair/growth in hair
follicles, cartilage and bone. Blocking COX-2 function not only leads
to autoimmunity but also joint erosion (two things never told to
arthritis patients taking Vioxx, Celebrex or other NSAIDs which block
COX-2).

Butyrate is more than a mere immunosuppressant. Along with vitamin D3,
butyrate appears to prompt the production of antimicrobial innate
defenses [PMID 16895558]. A loss of butyrate might account for the
chronic intestinal infections seen in Crohn's patients. Without
butyrate, innate intestinal defenses protecting the gut lining from
infection could collapse.

For those of you familiar with the helminth hypothesis of autoimmunity
(see
<http://www.discover.com/issues/sep-93/features/ofparasitesandpo264/>,
<http://www.cosmosmagazine.com/node/695> and <http://www.ovamed.de/>),
butyrate may be synergistic with helminth therapy or may be one means by
which helminths calm the immune system and activate Tregs. The
literature mentions at least one helminth infection which alters gut
microflora and increases butyrate production. Whether intestinal
parasites do this generally or not I don't know. If generally true,
this would directly connect helminths, butyrate and Tregs to each other.

I can't yet find a direct connection with butyrate, IgG4 and mast cell
inhibition. Helminths crank up production of IgG4 which then blocks
mast cell-degranulation, thus preventing allergic symptoms. It does
appear to be the case that butyrate generally inhibits mast cell
inflammatory responses, thus 86'ing allergy symptoms [PMID 16949031].
However, it also seems to enhance certain aspects of the remaining mast
cell response [PMID 8026597]. It may make mast cells less likely to go
off but more potent when they do. The literature is thin on the matter
and I'm speculating with what little is available.

Clearly butyrate is an important regulator of the body's inflammatory
response and important to the treatment of autoimmune diseases.
Carnitine, in turn, is central to butyrate metabolism. Carnitine is
needed for butyrate uptake in the gut. When cells can't get enough
carnitine, they can't make use of butyrate. In the type of inflammatory
bowel injury commonly seen in Crohn's and I.B.D., carnitine transporters
OCTN2 and Atb0+ can be disabled [PMID 17065219]. Knock out carnitine
transport in the gut with an inflammatory toxin (like mercury, in my
case) or perhaps through a genetic defect and you lose butyrate uptake,
HDAC inhibition, FoxP3 and then, ultimately, immunotolerance (along with
innate defenses and mast cell sanity). Although this hypothesis has yet
to be tested in a human biopsy, the metabolic chain of events is clear.

Mercury poisoning, known to cause autoimmunity, knocks out this exact
same carnitine transporter (reported in the literature as OCT-2) in
other types of cells like nerves. Mercury goes on to deplete betaine,
molybdenum and histidine over time by overtaxing production of the
natural chelator, metallothionein. (This leads into the mu
opioid/protein kinase C/cannabinoid networks and a host of other things
I've discussed before.) There is an OCT-2 receptor on B-cells, although
I'm unaware of its exact function. I suspect it might play an
antiproliferative role similar to one of the cannabinoid receptors. (If
true, then carnitine might have an anticancer effect against certain
leukemias. Is anyone aware of a connection?)

OCTN1 is another carnitine transporter. Besides carnitine, it ferries
the potent antioxidant ergothioneine, which is also the precursor for
metallothionein [PMID 15795384]. It's not quite clear why yet but OCTN1
is abnormally expressed in the joints of rheumatoid arthritis patients
[PMID 17142562] and certain mutations in OCTN1 and OCTN2 increase
genetic susceptibility to Crohn's disease [PMID 16961737] and other
autoimmune conditions. I have suspected before that carnitine played
some direct role in preventing autoimmunity since men have naturally
higher carnitine levels than women and are less susceptible to
autoimmunity. The recent research on butyrate and FoxP3 clarifies why
this should be the case. Carnitine is the key that lets butyrate into
cells for beta oxidation. These mutations in carnitine transporters
could result in less butyrate uptake, less HDAC inhibition and lower
FoxP3 expression.

Notice the close chemical relationships among

+ betaine (trimethylglycine)
+ carnitine (gamma-trimethyl-beta-hydroxybutyrobetaine aka
4-N-trimethylammonium-3-hydroxybutyric acid)
+ ergothioneine (2-thiol-L-histidine-betaine)
+ metallothionein (ergothioneine plus a molybdenum-containing
prosthetic group)
+ methionine (2-amino-4-(methylthio)butyric acid)

Methionine is used to synthesize SAMe (s-adenosylmethionine) and
homocysteine. It's a principle figure in the methyl cycle, something
mercury is known to disturb. On the positive side, methionine
restriction tends to slow the aging process in lower organisms.
Methylation and histone acetylation are two of the main epigenetic
regulators. By putting methyl groups on sections of DNA or adding
acetyl groups to histones in the DNA, the body changes how active or
inactive genes become. Methylating an oncogene, for instance, muffles
it and inhibits the development of cancer. Demethylating it frees it to
promote cancer. While it's a lot more complicated than I can explain in
a paragraph, you can see how mercury or any defect in the
carnitine/butyrate dance would throw havoc into the entire epigenetic
cycle.

As an aside, the non-intuitive ability of HDAC inhibitors to promote
Tregs might limit their usefulness as solitary anticancer agents.
Cancer cells often employ Treg umbrellas to evade immune system attack
by secreting PGE2 [PMID 15958566]. While HDAC inhibitors do have
anticancer effects of their own, blocking Treg function through another
pathway with something like, say, a COX-2 inhibitor might enhance the
chemotherapeutic properties of sodium butyrate and trichostatin A.

Notes:

FOXP3 interactions with histone acetyltransferase and class II histone
deacetylases are required for repression; FOXP3 acts as a repressor of
transcription and is both an essential and sufficient regulator of the
development and function of regulatory T cells; transcriptional
repression by FOXP3 involves a histone acetyltransferase-deacetylase
complex that includes histone acetyltransferase TIP60 (Tat-interactive
protein, 60 kDa) and class II histone deacetylases HDAC7 and HDAC9; the
N-terminal 106-190 aa of FOXP3 are required for TIP60-FOXP3, HDAC7-FOXP3
association, as well as for the transcriptional repression of FOXP3 via
its forkhead domain; FOXP3 can be acetylated in primary human regulatory
T cells and TIP60 promotes FOXP3 acetylation in vivo; overexpression of
TIP60 but not its histone acetyltransferase-deficient mutant promotes,
whereas knockdown of endogenous TIP60 relieved, FOXP3-mediated
transcriptional repression; a minimum FOXP3 ensemble containing native
TIP60 and HDAC7 is necessary for IL-2 production regulation in T cells;
FOXP3 association with HDAC9 is antagonized by T cell stimulation and
can be restored by the protein deacetylation inhibitor trichostatin A
[PMID 17360565]; transcriptional control of Foxp3 expression itself
contributes to development of a stable Treg lineage; an evolutionarily
conserved region within the foxp3 locus upstream of exon-1 possesses
transcriptional activity; complete demethylation of CpG motifs as well
as histone modifications within the conserved region in ex vivo isolated
Foxp3+CD25+CD4+ Tregs takes place, but not in naive CD25-CD4+ T cells;
partial DNA demethylation is already found in developing Foxp3+
thymocytes; however, Tregs induced by TGF-beta in vitro display only
incomplete demethylation despite high Foxp3 expression; in contrast to
natural Tregs, TGF-beta-induced Foxp3+ Tregs lose both Foxp3 expression
and suppressive activity upon restimulation in the absence of TGF-beta;
this suggests expression of Foxp3 must be stabilized by epigenetic
modification to allow the development of a permanent suppressor cell
lineage [PMID 17298177]; ectopic expression of Foxp3 in non-Tregs leads
to repression of the IL-2 and IFN-gamma genes, gain of suppressor
function and induction of genes like CD25, GITR, and CTLA-4; Foxp3 binds
to the endogenous IL-2 and IFNgamma loci in T cells but only after T
cell receptor (TCR) stimulation; the activation-induced Foxp3 binding
was abrogated by cyclosporin A (a calcineurin inhibitor) suggesting a
role for the phosphatase calcineurin in Foxp3 function; binding of Foxp3
to the IL-2 and IFNgamma genes induces active deacetylation of histone
H3, a process that inhibits chromatin remodeling and opposes gene
transcription; binding of Foxp3 to the GITR, CD25, and CTLA-4 genes
results in increased histone acetylation; Foxp3 may regulate
transcription through direct chromatin remodeling and Foxp3 function is
influenced by signals from the TCR [PMID 17028180]; foxp3 (forkhead box
protein 3) binds to the endogenous IL-2 locus and promotes histone
deacetylation in an activation-dependent manner [PMID 16974603]; histone
deacetylase inhibition promotes foxp3 acetylation and the generation and
suppressive functions of regulatory T cells, leading - in conjunction
with a brief sub-therapeutic course of rapamycin - to robust,
donor-specific tolerance [PMID 16974602]; FOXP3 acts in vivo as a
transcriptional regulator by assembling a multisubunit protein complex
involved in histone modification as well as chromatin remodeling�[PMID
16903909]

schistosomiasis infection in mice reduces levels of the tricarboxylic
acid cycle intermediates including citrate, succinate, and
2-oxoglutarate in urine while increasing levels of pyruvate, suggesting
stimulated glycolysis; taurine, 2-oxoisocaproate and 2-oxoisovalerate
are depleted and and tryptophan is elevated in the urine; various
microbial-related metabolites like trimethylamine, phenylacetylglycine,
acetate, p-cresol glucuronide, butyrate, propionate and hippurate were
also coupled with an S. mansoni infection, indicating disturbances in
the gut microbiota [PMID 15314235] (this may indicate that intestinal
helminths increase or enhance the gut�s production of butyrate, perhaps
as a survival strategy; the overall effect would promote certain
bacteria and enhance Treg effectiveness);

asthma patients have a large number of NK cells and show stronger NK
activity, indicating NK cell activity may be related to total IgE level
in healthy serum; sodium butyrate (NaBu) enhances IL-4-induced IgE
production in LPS-stimulated murine splenocytes in vitro and inductive
rat IgE production in vivo and enhanced the NK cell activity ex vivo;
IgE production may be involved in butyrate-enhanced NK cell activity in
vivo; mice intraperitoneally treated/immunized with NaBu or/and Ascaris
suum extract (ASC) and the spleen NK cell activity were evaluated;
spleen NK cell activity and IL-2- or IFN-gamma-induced spleen NK cell
activity of mice treated/immunized with NaBu or/and ASC were stronger
than those of untreated/unimmunized mice; although IL-4 blocked IL-2
(100 U/ml)- or IFN-gamma (100 U/ml)-induced increase in NK cell
activity, these NK cell activities in mice treated/immunized with
NaBu/ASC were not inhibited; IgE production tended to rise in
NaBu-treated mice serum and a synergistic effect was observed with
treatment of NaBu and ASC; the NAS (serum) significantly increased IL-2-
or IFN-gamma-induced NK cell activity; its effect was inhibited by
anti-mouse IgE mAb; IgE is important for NAS-enhanced
IL-2/IFN-gamma-induced NK cell activity and IL-4 does not inhibit IgE
and IL-2/IFN-gamma-induced NK cell activity in mice [PMID 12901494]

while mast cells contribute to type I allergic conditions they have only
recently been associated with chronic relapsing/remitting autoimmune
diseases like celiac disease and ulcerative colitis; the short chain
fatty acid n-butyrate downregulates TNF-alpha transcription in mast
cells; this correlates with an impaired activation of the Jun
NH(2)-terminal kinase (JNK) but not other MAP kinases like ERK and p38
that are mostly unaffected by n-butyrate; as a consequence, there was a
decreased nuclear activity of AP-1 and NF-AT transcription factors;
butyrate inhibits critical inflammatory mediators in mast cells by
relatively selectively targeting the JNK signalling [PMID 16949031]

mast-cell-derived mediators have mitogenic activities on
mouse-transformed epidermal cell line Pam 212 cells; these activities
were partially blocked by antihistamines or anticytokine antibodies,
including anti-IL1 alpha, -IL1 beta or IL6 antibodies; pretreatment of
mast cell lines with sodium butyrate enhanced the production of these
factors; calcium ionophore or Concanavalin A (ConA) stimulate mast cells
to generate factor production; this suggests mast-cell-derived mediators
might play a role in the epidermal hyperplasia seen in lichenified
lesions in atopic dermatitis [PMID 1422267]

mast cells derived from the bone marrow of BALB/mice (BMMC) were
cultured and their growth ceased with sodium butyrate; the sodium
butyrate treatment (1mM, 4 days) caused maturation of the granules, an
increased histamine content from approx. 1 pg/cell to 4 pg/cell;
maturation of the granules was accompanied by the increase in relative
weight percent of sodium, phosphorus and sulphur, with a decrease in
chloride; the sulphur-to-potassium ratio increased three-fold in
butyrate-treated mast cells [PMID 8026597]

portal venous blood transfusions in organ transplantation is
immunosuppressive and may be mediated by increased Kupffer cell
production of the immunosuppressive arachidonic acid metabolite
prostaglandin E2 (PGE2); butyrate is known to enhance gene transcription
and enhances Kupffer cell PGE2 production by altering cyclooxygenase and
phospholipase A2 (PLA2) activity and augmenting the immunosuppressive
effect of portal venous transfusion in Lewis rats; Kupffer cells from
portally transfused animals produced significantly more PGE2 than
saline-transfused controls; adding butyrate to the culture medium
further increased PGE2 production as much as sevenfold in Kupffer cells
of portally transfused animals; short-chain fatty acids propionate and
hexanoate did not increase PGE2 production; butyrate added to Kupffer
cells from transfused animals slightly upregulated inducible
cyclooxygenase (COX-2) mRNA levels and increased PLA2 activity fivefold;
Kupffer cell immune function was affected by in vitro butyrate treatment
causing a significant drop in production of TNFalpha; butyrate may be a
useful immunoregulatory agent in organ transplantation protocols which
seek to enhance transcription of immunosuppressive molecules [PMID
9733608]

antimicrobial peptides like cathelicidin and beta-defensin are produced
in various cell types; production is independent and specific to the
cell type and stimulus; vitamin D3 induced cathelicidin expression in
keratinocytes and monocytes but not in colonic epithelial cells;
butyrate induced cathelicidin in colonic epithelia but not in
keratinocytes or monocytes; in all cell types with a functional vitamin
D responsive element, vitamin D3 activated the cathelicidin promoter; in
colonic epithelia butyrate induced cathelicidin expression without
increasing promoter activity and vitamin D3 activated the cathelicidin
promoter without a subsequent increase in transcript accumulation;
cathelicidin transcript induction correlated with increased processed
mature peptide and enhanced antimicrobial activity against
Staphylococcus aureus; on the other hand, induction of beta-defensin-2
expression did not alter the innate antimicrobial capacity of cultured
cells; this suggests antimicrobial peptide expression is regulated in a
tissue-specific manner at transcriptional, post-transcriptional and
post-translational levels; innate antimicrobial activity can be
triggered independently of the release of other pro-inflammatory
molecules, suggesting strategies for boosting innate immune defence
without increasing inflammation [PMID 16895558]
From: myancov on 1 May 2007 13:26
Kofi, I appreciate your posts... please keep it coming

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