T Rex - for what turned
out to be rather a monster post
If the title
of this post already makes sense, you probably do not need to read it.
It is about regulatory
T cells (Tregs), which are an interesting way to treat what I have termed the over-activated
immune system in autism. The same ideas
can be extended to other conditions related to mast cells, and also potentially Multiple
Sclerosis (MS), Irritable bowel Disease (IBD) and even obesity.
Take Home Summary
For those
more interested in what can be done, rather than why, here is the conclusion
from this post:-
There are at
least four possible ways to increase the number of regulatory T cells (Tregs),
which should reduce pro-inflammatory cytokines (particularly IL-6) and increase
anti-inflammatory cytokines (like IL-10).
It should also reduce obesity, protect against diabetes and protect
against organ damage in those already diabetic.
The simplest
method is to increase production of small-chain fatty acids, which are the
main metabolic products of bacteria fermentation that occurs naturally in the
intestines. You either eat more fibre or
eat the specific bacteria, that causes the fermentation.
1. Increase
specific gut microbiota, namely B. fragilis and Clostridia
4. Have a bone marrow transplant (not
recommended)
For regular readers you may recall that B. fragilis appeared in an
earlier post:-
Why this post? - Bumetanide has stopped working
I recently
received a comment from a lady who has tried Bumetanide in her child with autism. After the expected two week delay, she
noticed lots of positive behavioral changes, but sadly latter on the Bumetanide
“stopped working”.
In the past
I received comments about “NAC has stopped working”.
Since I also
experienced the same effect of “everything stops working” in the summer, I know
how these people feel.
In reality,
as I eventually discovered, it is not that Bumetanide/NAC has stopped working,
but rather something else has started working.
I wrote once about autism being a Dynamic Encephalopathy, which to be fair was Martha Herbert’s idea and
not mine. This is one reason that a new type of doctor will be needed if autism is ever to be treated. It is a moving target.
In some
types of autism it seems that the immune system can switch to an over-activated
state and when in this state all my clever autism drugs appear to stop
working.
In some
people the problem is driven by so-called mast cells. Mast cells play a key role in the
inflammatory process. When activated they release granules and various hormonal mediators. Histamine and the pro-inflammatory cytokine
IL-6 are produced and this wreaks havoc in the brain, undoing all the good done
by Bumetanide, NAC etc.
Regulatory T cells (Tregs)
In earlier
posts I think I have exhaustively covered mast cells and to how to stabilize
them. However, I decided to look further
back up the chain in the immune system at what may modulate the mast cells. Regulatory T cells caught my attention.
The regulatory T cells (Tregs),
formerly known as suppressor T cells,
are a subpopulation of T cells which modulate the immune system,
maintain tolerance to self-antigens, and abrogate
autoimmune disease. These cells generally suppress or downregulate induction
and proliferation of effector T cells.
T regulatory cells are a
component of the immune system that suppress immune responses of other cells.
This is an important "self-check" built into the immune system to
prevent excessive reactions.
The immune
system must be able to discriminate between self and non-self.
When self/non-self discrimination fails, the immune system destroys cells and
tissues of the body and as a result causes autoimmune diseases. Regulatory T
cells actively suppress activation of the immune system and prevent
pathological self-reactivity, i.e. autoimmune disease
The immunosuppressive cytokines TGF-beta and Interleukin
10 (IL-10) have also been implicated in regulatory T cell function.
Recent evidence suggests that mast
cells may be important mediators of Treg-dependent peripheral
tolerance.
Regulatory T cells come in many
forms with the most well-understood being those that express CD4, CD25, and
Foxp3 (CD4+CD25+ regulatory T cells).
Abstract
Mast
cell degranulation is a hallmark of allergic reactions, but mast cells can also
produce many cytokines that modulate immunity. Recently, CD25(+) regulatory T
cells (Tregs) have been shown to inhibit mast cell degranulation and
anaphylaxis, but their influence on cytokine production remained unknown. In
this study, we show that, rather than inhibit, Tregs actually enhance mast cell
production of IL-6. We demonstrate that, whereas inhibition of degranulation
was OX40/OX40 ligand dependent, enhancement of IL-6 was due to TGF-β.
Interestingly, our data demonstrate that the Treg-derived TGF-β was
surface-bound, because the interaction was contact dependent, and no TGF-β was
detectable in the supernatant. Soluble TGF-β1 alone was sufficient to enhance
mast cell IL-6 production, and these supernatants were sufficient to promote
Th17 skewing, but those from Treg-mast cell cultures were not, supporting this
being surface-bound TGF-β from the Tregs. Interestingly, the augmentation of
IL-6 production occurred basally or in response to innate stimuli (LPS or
peptidoglycan), adaptive stimuli (IgE cross-linking by specific Ag), and
cytokine activation (IL-33). We demonstrate that TGF-β led to enhanced
transcription and de novo synthesis of IL-6 upon activation without affecting IL-6
storage or mRNA stability. In vivo, the adoptive transfer of Tregs inhibited
mast cell-dependent anaphylaxis in a model of food allergy but promoted
intestinal IL-6 and IL-17 production. Consequently, our findings establish that
Tregs can exert divergent influences upon mast cells, inhibiting degranulation
via OX40/OX40 ligand interactions while promoting IL-6 via TGF-β.
Treg cells are reduced in people with Autism
The following study showed that 73%
of subjects with autism had reduced levels of Tregs and in particular those
with allergies of a familial history of autoimmune disease.
Those in the 73% with allergies are
the ones who fit my over activated immune system category.
Abstract
Autoimmunity may have a role in autism,
although the origins of autoimmunity in autism are unknown. CD4( +)CD25(high)
regulatory T cells play an important role in the establishment of immunological
self-tolerance, thereby preventing autoimmunity. The authors are the first to
study the frequency of CD4(+)CD25( high) regulatory T cells in the blood of 30
autistic and 30 age- and sex-matched healthy children. Patients with autism had significantly lower
frequency of CD4(+)CD25(high) regulatory T cells than healthy children (P <
.001). These cells were deficient in 73.3% of children with autism.
Autistic patients with allergic manifestations (40%) and those with a family
history of autoimmunity (53.3%) had a significantly lower frequency of
CD4(+)CD25(high) regulatory T cells than those without (P < .01 and P <
.001, respectively). In
conclusion, CD4(+)CD25( high) regulatory T cells are deficient in many children
with autism. Deficiency
of these cells may contribute to autoimmunity in a subgroup of children with
autism. Consequently, CD4(+)CD25(high) regulatory T cells could be new
potential therapeutic targets in these patients.
Her group are not researching
autism, they are researching inflammation, particularly in the colon.
But inflammation can occur
anywhere.
Their recent work and some relating
to it is covered in the following
excellent article is from the Multiple Sclerosis Discovery Forum. It is very readable.
Common compounds made by gut
microbes that break down dietary fiber appear to boost the number and function
of regulatory T cells (Tregs) in the colons of mice, a new
study found. The findings expand the known ways that intestinal
bacteria can influence Tregs, which can dial down an immune response and may be
malfunctioning in autoimmune and inflammatory disorders, including multiple
sclerosis (MS) and inflammatory bowel disease (IBD).
The microbial metabolites, known as short-chain fatty acids (SCFAs),
restored the depleted Tregs of germ-free mice, the researchers reported. In
mice with normal intestinal bacteria, supplemental SCFAs expanded the existing
Treg population and activity. In a mouse model of colitis, SCFAs in drinking
water reduced intestinal inflammation by enhancing Treg function.
"It's a terrific
paper," said Sarkis Mazmanian, Ph.D., a microbiologist at the California
Institute of Technology in Pasadena, in an interview with MSDF. Mazmanian first
reported that PSA on the surface of B. fragilis converts CD4+
T cells into Foxp3+ Treg cells that produce IL-10 in the colon (Round
and Mazmanian, 2010). "We have been working with a specific
organism that makes a molecule unique to B. fragilis that induces
Tregs and suppresses inflammation, and Wendy has discovered a more general
metabolite produced by multiple bacterial groups that does something
similar."
The study builds on discoveries (Nagano et al., 2012)
showing that Tregs are dependent upon gut microbiota, specifically B.
fragilis and Clostridia, Garrett told MSDF in an email. "We
all may not have B. fragilis," she wrote. "In addition,
human and mice both have many different strains of Clostridia.
However, all healthy humans have regulatory T cells. Since SCFA are such
abundant microbial metabolites, we hypothesized that SCFA may regulate Tregs in
the colon."
"SCFA exert so many different effects on Tregs by
altering molecules that affect the structure of DNA, making some areas of the
DNA more open and available for transcription," Garrett wrote in an email.
"In this way, SCFA can affect several different Treg functions."
For Garrett and
others, the findings advance the therapeutic potential of dietary-based
interventions using the SCFA mix and perhaps other molecules that boost
signaling through GPR43 to improve Treg function in patients with inflammatory
bowel disease and other autoimmune diseases. The concept was also advanced in
another new study from Kenya Honda, M.D., Ph.D., of the RIKEN Center for
Integrative Medical Sciences in Yokohama, Japan, in a recent Nature
paper. A mixture of 17 strains of human-derived Clostridia designed to
expand and differentiate Tregs relieved symptoms of colitis and allergic
diarrhea in mouse models (Atarashi et al., 2013).
The full paper is here:-
So much for the colon, what about the
effect of increasing Treg in autism?
We already know that in the MIA (maternal immune activation) mouse model of autism, treating mice pups with B. fragilis reduces their autistic behaviours.
'Friendly' bacteria treat autism-like symptoms in mice
That is a pretty good start, since we know that B. fragilis causes more SCFAs to be produced in the intestines.
The most
effective way to reset an immune system would be a bone marrow transplant. The following article from SFARI looks about
what happens in mice.
An altered
immune system can cause autism-like behaviors, suggests a study published 31
July in the Proceedings of the National Academy of Sciences1. The researchers found that a
bone marrow transplant, which restores the animals’ immune system, alleviates
some of their symptoms, including anxiety and repetitive behavior.
Such
transplants are too dangerous for treating people with autism, but the findings
suggest other treatments targeting immune cells, the researchers say.
When
confronted with foreign cells — for example, when infected with a virus — the
body typically activates immune cells called T cells to release signaling
molecules called cytokines. A different set of T cells, called regulatory T
cells, then keep that immune response in check by suppressing the activated T
cells.
In the
study, researchers injected pregnant mice with a mock flu virus that sets off
their immune response. The
offspring carry overly responsive T cells and have too few regulatory T cells
throughout their lifetime, the study found. These two things together point to
an immune system that's overly reactive.
The findings are provocative because several lines of evidence,
including studies of postmortem brain tissue, cerebrospinal fluid and blood, suggest that some individuals with autism
have an overactive immune system that leads to high levels of inflammation. One
study has also reported that some children with the disorder have a dearth of
regulatory T cells in their blood2
Studies on the effect of Small Chain
Fatty Acids (SCFAs) on Humans
The good
news is that numerous studies show that Wendy Garrett’s findings seem to apply
far beyond the colon.
The reason
is that SCFAs are able to cross the Intestinal Epithelium (i.e. cross from the
gut to the bloodstream)
CONCLUSIONS Data suggest a potential therapeutic value
of Tregs to improve insulin resistance and end organ damage in type 2 diabetes
by limiting the proinflammatory milieu.
Abstract
Short-chain fatty acids (SCFAs) are the main products of dietary fiber
fermentation and are believed to drive the fiber-related prevention of the
metabolic syndrome. Here
we show that dietary SCFAs induce a peroxisome proliferator-activated receptor
(PPAR) γ-dependent switch from lipid synthesis to utilization. Dietary SCFA supplementation
prevented and reversed high-fat diet-induced metabolic abnormalities in mice by
decreasing PPARγ expression and activity. This increased the expression
of mitochondrial uncoupling protein 2 and raised the AMP/ATP ratio, thereby
stimulating oxidative metabolism in liver and adipose tissue via AMP-activated
protein kinase. The SCFA-induced reduction in body weight and stimulation of insulin
sensitivity were absent in mice with adipose-specific disruption of PPARγ.
Similarly, SCFA-induced reduction of hepatic steatosis was absent in mice
lacking hepatic PPARγ. These results demonstrate that adipose and hepatic PPARγ
are critical mediators of the beneficial effects of SCFA on the metabolic
syndrome, with clearly distinct and complementary roles. Our findings indicate
that SCFAs may be used therapeutically as cheap and selective PPARγ modulators.
Recall that from earlier posts, I am already on the look out for selective PPARγ modulators (like Tangeretin)
Increased intake of dietary carbohydrate that is fermented in the colon by the microbiota has been reported to decrease body weight, although the mechanism remains unclear. Here we use in vivo11C-acetate and PET-CT scanning to show that colonic crosses the blood–brain barrier and is taken up by the brain. Intraperitoneal results in appetite suppression and hypothalamic neuronal activation patterning. We also show that administration is associated with activation of acetyl-CoA carboxylase and changes in the expression profiles of regulatory neuropeptides that favour appetite suppression.
Tregs and Allergies
Fortunately some
researchers have indeed looked at Tregs and allergies, but they did not seem to
know about SCFAs.
Abstract
Dysregulated immune response results in
inflammatory symptoms in the respiratory mucosa leading to asthma and allergy
in susceptible individuals. The T helper type 2 (Th2) subsets are primarily
involved in this disease process. Nevertheless, there is growing evidence in
support of T cells with regulatory potential that operates in non-allergic
individuals. These regulatory T cells occur naturally are called natural T
regulatory cells (nTregs) and express the transcription factor Foxp3. They are
selected in the thymus and move to the periphery. The CD4 Th cells in the
periphery can be induced to become regulatory T cells and hence called induced
or adaptive T regulatory cells. These cells can make IL-10 or TGF-b or both, by which they attain most
of their suppressive activity. This review gives an overview of the
regulatory T cells, their role in allergic diseases and explores possible
interventionist approaches to manipulate Tregs for achieving therapeutic goals.
Regulation of Inflammation by Short
Chain Fatty Acids
Here is a
very good paper from Brazil, for those who need more convincing.
Short chain fatty acids (SCFAs), which are the major metabolic
products of anaerobic bacteria fermentation, have been suggested to be the link
between microbiota and host tissues. The concentration of these fatty acids in
the GI tract and blood may predispose to or prevents pathological conditions
such as IBD, cancer and diabetes. Modifications in the concentrations or the
ability of host
tissues to use SCFAs have
been described in these conditions.
Mode of action of SCFAs
If anyone is interested in how SCFAs
work their tricks, this is what they say in Brazil:
The main mechanism
described for these effects is the attenuation of HDAC activity. Among the SCFAs, butyrate is the
most potent, whereas acetate is the least potent inhibitor of HDAC.
This enzyme, together
with the histone acetyltransferases (HAT), controls the degree of protein acetylation. By
inhibiting the HDAC activity, SCFAs increase the acetylation of histone and non histone proteins
including NFκB, MyoD, p53 and N-FAT [57] and, consequently, modulate gene
expression.
The production of
prostaglandin E2 (PGE2) is also modified by SCFAs. These fatty acids stimulated
the in vitro production of this eicosanoid by human monocytes
[58]. In accordance with this result, induction of PGE2 production was observed
three hours after intraplantar injection of SCFAs and LPS in rat paws [34]. PGE2 has
been considered an anti-inflammatory prostanoid due to its ability to attenuate
the production of IL-1β and TNF-α by macrophages and Th1 differentiation.
However, there is now evidence in favor of a pro-inflammatory action of this
molecule [59]. PGE2, through activation of its receptor EP4,
facilitates Th1 differentiation and Th17 expansion, two subsets of T helper involved
in immune inflammation [59,60]. Considering these findings, SCFAs may also
affect T cell differentiation.
In addition to the
classical eicosanoids, such as PGE2, other lipid mediators are also generated
from polyunsaturated fatty acids including lipoxins, resolvins, protectins and
maresins [61]. Despite their relevance to the resolution of the inflammatory
process [61], at the moment, no study has been conducted in order to
investigate the effect of SCFAs on the production of these lipid mediators.
Anti-inflammatory
actions of SCFAs have been also observed in neutrophils. Acetate, propionate and
butyrate at 30 mM reduce TNF-α production by LPS-stimulated human neutrophils
[62].
Propionate and butyrate
inhibit the expression of pro-inflammatory mediators (TNF-α, CINC-2αβ and NO)
in rat neutrophils, an effect that seems to involve attenuation of NF-κB
activation [21].
Microglial cells are
resident immune cells of the central nervous system (CNS). Activation of these cells
leads to production of several inflammatory mediators (e.g., cytokines and NO)
that participate in the defense reaction of the CNS against insults including
microorganisms and damaged cells [63].
Chronic or excessive
activation of these cells has detrimental effects on the CNS and seems to be involved
in the initiation and progression of neurodegenerative diseases including
Alzheimer and Parkinson’s disease. In spite of some controversy about the
effect of SCFAs on microglial production of inflammatory mediators [52,53], most of the studies indicate
that these fatty acids attenuate microglial activation, an effect that seems to
involve HDAC inhibition [53,54]. These observations and the data obtained in vivo [64] support the proposition that SCFAs
and other inhibitors of HDAC may be useful in preventing inflammation in the CNS.
Indeed, Kim et al. [64] have shown that butyrate, valproic acid and
trichostatin A (all inhibitors of HDAC activity) present antineuroinflammatory
and neuroprotective effects in the ischemic brain of rats.
Effectors Mechanisms of Phagocytes
Once in the inflammatory
site, neutrophils and macrophages internalize, kill and digest bacteria and fungi
through mechanisms including production of reactive oxygen species (ROS) and
release of granule enzymes. SCFAs affect the production of ROS and the phagocytic
capacity of phagocytes.
This effect is important
in the course of anaerobic bacteria infection. Both inhibition [65,66,68] and stimulation
[4,68] of neutrophil phagocytosis by SCFAs have been described. In macrophages,
butyrate reduce the phagocytic activity, an effect that probably arises from
its inhibitory action on cell differentiation and maturation [69].
The effects of SCFAs on
ROS production by neutrophils remain controversial. Some groups have found that
SCFAs induce ROS production [4,70,71], whereas others have shown inhibition
[65,67,72–74].
The discrepancy in the
results obtained may be explained by differences in the protocols used such as the
concentrations of SCFAs, measurement of ROS by using different methodologies (e.g., lucigenin-amplified
chemiluminescence or reduction of cytochrome c), stimuli (e.g., PMA or fMLP),
solution pH, source and state of neutrophil activation (e.g., neutrophils
isolated from human blood or elicited rat neutrophils).
3.3. Lymphocyte Activation and Response
Lymphocytes are involved
in the adaptive immune response. These cells display membrane receptors that recognize
a broad range of non-self antigens and allow them to generate specific responses to liminate
invading pathogens and infected or tumoral cells. SCFAs modify lymphocytes function
as follows:
T-cell
proliferation: butyrate inhibits lymphocyte proliferation in response to
several stimuli including concanavalin-A
and immobilized anti-CD3 monoclonal antibody [41,75].
Production
of cytokines: incubation of lymphocytes with butyrate reduces the production of interleukin-2; this
cytokine stimulates growth, differentiation and survival of antigen-selected
T-lymphocytes, and
interferon-γ (IFN-γ) after stimulation with concanavalin-A or anti-CD3 and anti-CD8 [76,77]. This
latter cytokine is particularly important in response to viral infection, tumor cells and in
auto-immune conditions. On the other hand, butyrate presents an opposite effect on the production
of IL-10 by lymphocytes [75].
Production of regulatory T (Treg)
cells: this subpopulation of T cells actively suppresses immune function and is
considered an attractive target for the treatment of immunological and inflammatory pathologies. HDAC inhibitors enhance the
production and suppressive function of regulatory T cells [77]. Considering that SCFAs, as
previously described, also suppress the activity of HDAC, we hypothesize that these fatty acids
may also exert their effects on inflammation
and immune responses through regulation of this subset of T cells.
Conclusion
Within
reasonable limits, short chain fatty acids (SCFAs) are good for you.
Particularly
if you have an inflammatory condition or need to lose some weight.
You already
produce them and some people would benefit from some more.
P.S. for the Diehards
Proprionic Acid (PPA) in Rats
There is
also research indicating that injecting large amounts of one particular SCFA,
Propionic acid into the brains of rats does them no good at all. In fact the opposite of all the good things
notes by the Brazilians and others.
Having
read an awful lot of autism research, I have to point out that sometimes a
little of what does you harm, can actually do you some good. For example the Valproate mouse model of
autism is based on feeding Valproic Acid to the female mouse to make her pup be
born with autistic features. Yet the
same drug Valproic Acid, in lower doses, is an effective treatment for autism
with seizures in humans.
In pregnant
humans the risk of Valproate is slightly different. According to Harvard:-
Valproate. It’s best to
avoid taking valproate (Depakote) during pregnancy, especially during the first
trimester, as this drug increases the risk of neural tube defects such as spina
bifida. Risk increases with dose. In absolute terms, researchers estimate that
one to six babies out of every 100 exposed to valproate in the first trimester
of fetal development are born with some type of neural tube defect.
Abstract
Clinical observations suggest that certain gut and dietary factors
may transiently worsen symptoms in autism spectrum disorders (ASD), epilepsy
and some inheritable metabolic disorders. Propionic acid (PPA) is a short chain
fatty acid and an important intermediate of cellular metabolism. PPA is also a
by-product of a subpopulation of human gut enterobacteria and is a common food
preservative. We examined the behavioural, electrophysiological,
neuropathological, and biochemical effects of treatment with PPA and related
compounds in adult rats.
Intraventricular infusions of PPA produced reversible repetitive
dystonic behaviours, hyperactivity, turning behaviour, retropulsion, caudate
spiking,
and the progressive development of limbic kindled seizures,
suggesting that this compound has central effects. Biochemical analyses of
brain homogenates from PPAtreated rats showed an increase in oxidative stress
markers (e.g., lipid peroxidation and protein carbonylation) and glutathione S-transferase activity
coupled with a decrease in glutathione and glutathione peroxidase activity.
Neurohistological examinations of hippocampus and adjacent white matter
(external capsule) of PPA treated rats revealed increased reactive astrogliosis
(GFAP immunoreactivity) and activated microglia (CD68 immunoreactivity)
suggestive of a neuroinflammatory process. This was coupled with a lack of
cytotoxicity (cell counts, cleaved caspase 3_ immunoreactivity),
and an increase in phosphorylated CREB immunoreactivity. We propose that some
types of autism may be partial forms of genetically inherited or acquired
disorders involving altered PPA metabolism. Thus, intraventricular
administration of PPA in rats may provide a means to model some aspects of
human ASD in rats.
The short chain fatty acids (SCFAs) acetate
(C2), propionate (C3) and butyrate (C4) are the main metabolic products of
anaerobic bacterial fermentation in the intestine. In addition to their
important role as fuel for intestinal epithelial cells, SCFAs modulate
different processes in the gastrointestinal (GI) tract such as electrolyte and
water absorption. These fatty acids have been recognized as potential mediators
of the effects of the gut microbiota on intestinal immune function and
gut-brain axis interaction [4]. Recently it was reported that
the three types of SCFAs (acetate, propionate, and butyrate) reduce the
production of proinflammatory factors, including TNF-α, IL-1β, IL-6, and NO.
Additionally, SCFAs
enhance the production of the anti-inflammatory cytokine IL-10 in low concentrations
(1–1,200 μmol/L) [5].
In
spite of the protective effects of SCFAs, propionic acid (PPA) neurotoxicity was
recently demonstrated via intraventricular direct infusion into rat brains [6],
passage from the gut to the brain in the case of acute PPA orally administered
to rat pups [7]
or Chronic administration on postnatal days 5–28 [8] and,
most recently, subcutaneous injection once a day (500 mg/kg) in pregnant
rats on gestation days G12–16 [9].
I am very much minded
to go with Wendy, the Brazilians and the Egyptians (who found Trep low in
autism).
I think the
Saudis, with their PPA-neurointoxicated rats, are barking up the wrong
tree.
In fact, the
Saudis say that PPA is low in humans with autism.
Low SCFAs,
like PPA, help produce low Trep, which helps produces high IL-6 and low IL-10,
just as I expect to find in autism.
