Archive for the ‘Inflammation’ Category
The Biologically Plausible Model Of Perturbed Microglial Colonization And Developmental Trajectories Participating In (Some Cases Of) Autism And The Ribs Hypothesis Of Maternal Inflammation And Causation, Or, Venturing Into Uncharted Realms Of Guessing
Posted February 3, 2013on:
- In: Autism | Beautiful Complexity | Bilbo | Developmentall Programming | Early Life Immune Activation | Glial Priming | Humbling Complexity | Immunology | Inflammation | LPS | Microglia | microglia colonization | microglial proliferation | Mr. Rat | Phenotypes | Primed Phenotype | Proliferation | Pruning | Synapse | Uncategorized
- 9 Comments
Hello Friends – There are (at least) two big
classifications of microglia findings in autism, an altered
morphology (i.e., shape and function, or ‘activated’ versus
‘quiescent’), and an increased number (i.e., more), with both
parameters varying with each other and spatially. In other
words, disparate parts of the brain have different numbers of
microglia in them, and the functional profile of those microglia
also varies from one area to another.
[Note: There is ongoing
discussion regarding the appropriate definition of
‘activation’ of microglia, with evidence of (at least) four states
of microglial morphology.] Recently I saw a discussion on the SFARI
site about the fancy in
vivo study of microglia numbers in high functioning males with
autism. (I believe I am growing
increasingly jaded, as it occurs to me that radio tracing against
show microglial activation is a fancy trick, but one leaving us
open to detecting other stuff too.) In any case, the findings
are not especially unexpected by now, well not to me anyways, but a
comment at the SFARI site really got me thinking about the chain of
events that could lead to different spatial and
morphological characteristics of microglia. Perhaps we could
gain insight into the question of what the microglia are doing by
trying to understanding how they got there. Do we have any
biologically plausible models that might educate us on how a
different morphology and distribution of microglia could be
achieved? A while ago I got a copy of a few
articles that don’t have autism in them per se, but they kept
coming to the forefront of my mind when I thought about that
question. The first is Distribution
of microglia in the postnatal murine nigrostriatal
system, which had a disease focus on
Parkinson’s, but what really grabbed my attention is what they
learned about the developmental pathway microglia took to populate,
and then depopulate the substantia nigra (SN), a little wedge of
brain involved with motor skills, reward seeking, and addiction.
Interestingly, the SN
has been shown to contain more microglia than
adjacent structures. We have analysed
changes in microglia numbers and in microglial morphology in the
postnatal murine nigrostriatal system at various stages ranging
from postnatal day 0 (P0) up to 24 months of age. We
clearly show that the microglia numbers in the SN and in the
striatum dramatically increase from P0 to P15 and
significantly decrease in both areas in 18-month-old and
[Note: There seems to be
some variance in the appropriate ‘rat-to-human-age’ approximations;
especially when trying to do something as
expeditionary as comparing brain development. We should
extrapolate only with caution.] The part that makes me grin is that
it illustrates our nascent understanding of the process of
microglial colonization into the CNS, the hows,
whens, wheres, and whys are still
shrouded in mystery. The broadest outlines tell us that microglial
penetration into the brain is a long running, dynamic process; the
microglia are slow infiltrators, gaining access into parts of the
brain in concert with a swath of proliferating and inhibitory
factors, all at a time of once in a lifetime neurodevelopmental
of postnatal forebrain amoeboid microglial cell proliferation and
development by the transcription factor Runx1 paints a
beautiful portrait of functionality. Runx1 is a chemical
messenger that participates in phenotyopic determination of blood
cell progenitors into mature cells. The researchers observed
spatial, time dependent expression of Runx1 in the developing
forebrain, and differential levels following injury.
Here we show that the mouse
transcription factor Runx1, a key regulator of myeloid
cell proliferation and differentiation, is expressed in forebrain
amoeboid microglia during the first two postnatal weeks.
Runx1 expression is then downregulated in ramified microglia. Runx1
inhibits mouse amoeboid microglia proliferation and promotes
progression to the ramified state. We show further that
Runx1 expression is upregulated in microglia following nerve injury
in the adult mouse nervous system. These findings provide
insight into the regulation of postnatal microglia activation and
maturation to the ramified state and have implications for
microglia biology in the developing and injured brain.
It doesn’t really tell us much about a
persistent change in microglia per se, but it does render a picture
of proliferation and differentiation as an easily
disrupted symphony. When we think about the
developing brain, I won’t pretend to have more than a lightyear
close guess at what microglia might be doing
differently between amoeboid and ramified
morphologies in this locale, at this time, but I
very highly doubt there isn’t
a functional impact on microenvironment neurodevelopment; our
developing brains are using opportunities like the Indians used the
buffalo, no waste, no excess, and because balance is important,
everything is important. Moving back to the
question of the plausibility of a pathway to the autism state,
luckily (or unluckily?) the literature is veritably littered with
insults that perturb microglial development, leading to
persistent changes to microglial morphology, ultimately
percolating up to behavioral changes. Prenatal stress is a bad, bad
thing, and here is a study that finds that extreme mice stress can
persistently alter the mice activation profile of mice microglia.
stress increases the expression of proinflammatory cytokines and
exacerbates the inflammatory response to LPS in the hippocampal
formation of adult male mice, was just published, and
comes wrapped up with a double hit, and
different resting and stimulated neuroimmune environments.
Under basal conditions,
prenatally stressed animals showed increased expression of
interleukin 1ß and tumor necrosis factor-a (TNF-a) in the
hippocampus and an increased percentage of microglia cells with
reactive morphology in CA1 compared to non-stressed males.
Furthermore, prenatally stressed mice showed increased TNF-a
immunoreactivity in CA1 and increased number of Iba-1
immunoreactive microglia and GFAP-immunoreactive astrocytes in the
dentate gyrus after LPS administration. In contrast, LPS did not
induce such changes in non-stressed animals. These
findings indicate that prenatal stress induces a basal
proinflammatory status in the hippocampal formation during
adulthood that results in an enhanced activation of microglia and
astrocytes in response to a proinflammatory
Note: I have not read this
paper so I do not know if a qualitative number of microglia, or
just more immune-targeted microglia were found, but likely the
latter. A similar, full free paper, Prenatal
stress causes alterations in the morphology of microglia and the
inflammatory response of the hippocampus of adult female
mice, found broadly similar results; perturbed resting
and stimulated states in the treatment group.
Prenatal stress, per se,
increased IL1ß mRNA levels in the hippocampus, increased the total
number of Iba1-immunoreactive microglial cells and increased the
proportion of microglial cells with large somas and retracted
cellular processes. In addition, prenatally stressed and
non-stressed animals showed different responses to peripheral
inflammation induced by systemic administration of LPS.
LPS induced a significant increase in mRNA levels of IL-6,
TNF-a and IP10 in the hippocampus of prenatally stressed mice but
not of non-stressed animals.
Going back to my
ramblings on glial priming, it seems that here we have an
example of a type of cross system priming (sweet!), where
disturbing the stress response system changed the immune system;
such is the way of the polyamorous chemical families interacting in
our brain. It also occurs to me that given the
delicate nature of the developing brain, and the
tasks going on in there, we might want to think very
carefully before we ‘induced a significant increase in
mRNA levels of IL-6, TNF-a and IP10 in the hippocampus‘
on subgroups who might be environmentally predisposed to react with
exaggerated vigor. But what do I know? Of course, the
prenatal immune challenge arena holds a ton of studies on
persistent microglial function, and ‘consequences’. There are
way too many to list, but a quick overview of some very recent ones
would include: Enduring
consequences of early-life infection on glial and neural cell
genesis within cognitive regions of the brain, an early
life real infection model with e coli that
concludes, “Taken together, we have provided evidence that
systemic infection with E. coli early in life has significant,
enduring consequences for brain development and subsequent adult
function.” (Staci Bilbo!) This paper was sort
of a quinella, as it showed both changes in immune responsiveness
into adulthood; it also demonstrated the ability
of an immune insult to alter
the developmental trajectory of the
microglia, i.e., E. coli increased the number of newborn
microglia within the hippocampus and PAR compared to controls. The
total number of microglia was also significantly increased in E.
coli-treated pups, with a concomitant decrease in total
lipopolysaccharide exposure induces long-lasting learning
impairment, less anxiety-like response and hippocampal injury in
adult rats very directly blasted rats with some LPS
immune activation action, and includes, ”Neonatal LPS
exposure also resulted in sustained inflammatory responses in the
P71 rat hippocampus, as indicated by an increased number of
activated microglia and elevation of interleukin-1ß content in the
rat hippocampus.” (Sound familiar?) Interleukin-1
receptor antagonist ameliorates neonatal lipopolysaccharide-induced
long-lasting hyperalgesia in the adult rats took the
extra step of adding a set of animals that got inhibited
inflammatory responses. Results are increasingly
administration of an IL-1 receptor antagonist (0.1mg/kg)
significantly attenuated long-lasting hyperalgesia induced by LPS
and reduced the number of activated microglia in the adult rat
brain. These data reveal that neonatal intracerebral LPS
exposure results in long-lasting hyperalgesia and an elevated
number of activated microglia in later life. This effect is similar
to that induced by IL-1ß and can be prevented by an IL-1 receptor
I love how (once again) we
can see how interrupting the immune response can have an effect.
Environmental impacts outside of the immune
activation realm may also find a place within the ‘big tent’ of
microglial agitation with consequent developmental impacts.
The people who made the first big neuroimmune / autism splash at
Johns Hopkins later came out with Neuroinflammation
and behavioral abnormalities after neonatal terbutaline treatment
in rats: implications for autism, which found that an
agent used to prevent labor in some situations could
“produced a robust increase in microglial activation on PN
30 in the cerebral cortex” in treatment animals.
The drug in question, terbutaline, has been weakly associated with
increased incidence of autism, i.e., Prenatal
exposure to ß2-adrenergic receptor agonists and risk of autism
spectrum disorders, and beta2-adrenergic
receptor activation and genetic polymorphisms in autism: data from
dizygotic twins. And now, in 2013, Beta-adrenergic
receptor activation primes microglia cytokine production,
displays another example of cross system
if ß-AR stimulation is sufficient to prime microglia, rats were
intra-cerebroventricularly administered isoproterenol (ß-AR
agonist) or vehicle and 24h later hippocampal microglia were placed
in culture with media or LPS. Prior isoproterenol treatment
significantly enhanced IL-1ß and IL-6, but not TNF-a production
following LPS stimulation. These data suggest that central
ß-AR stimulation is sufficient to prime microglia cytokine
In other words, they gave
the rats a drug in the class of terbutline, and subsequently
observed an increased microglia responsiveness in cultured
cells. What a crazy coincidence. Detecting total
populations of microglia in adulthood, either regionally
or in the brain as a whole is a little more difficult, the little
buggers are a lot easier to detect when we light them up with neon
green tracers that stick to proteins expressed at ‘activation’
time, and it just doesn’t look like the question has been asked too
many times. I did, however, find something that has a sort of
chip shot on this analysis, Prenatal
stress alters microglial development and distribution in postnatal
rat brain, which looked at regional microglia populations
and phenotypes at two time periods following prenatal stress
stress consisting of 20 min of forced swimming occurred on
embryonic days 10–20. On postnatal days 1 and 10, stressed and
control pups were killed. Microglia were identified using Griffonia
simplicifolia lectin and quantified in the whole encephalon.
In addition, plasma corticosterone was measured in dams at
embryonic day 20, and in pups on postnatal days 1 and 10.
At postnatal day 1, there was an increase in number of
ramified microglia in the parietal, entorhinal and frontal
cortices, septum, basal ganglia, thalamus, medulla oblongata and
internal capsule in the stressed pups as compared to controls, but
also there was a reduction of amoeboid microglia and the total
number of microglia in the corpus callosum. By postnatal
day 10, there were no differences in the morphologic type or the
distribution of microglia between the prenatal stress and control
groups, except in the corpus callosum; where prenatal stress
decreased the number of ramified microglia. The stress procedure
was effective in producing plasma rise in corticosterone levels of
pregnant rats at embryonic day 20 when compared to same age
controls. Prenatal stress reduced the number of immature
microglia and promoted an accelerated microglial differentiation
into a ramified form.
They did a lot
of clever stuff at analysis time, taking samples from several
locations after birth and ten days later, and
also did some fine grained classification of the
shape of the microglia. They include spatial and temporal
mappings of four microglial developmental profiles. It looks
as if prental stress was able to alter the developmental speed of
microglia from one morphology to another in different parts of the
brain. There was as small section in the discussion that
speculated on what such changes might mean for neurodevelopment.
Given that during the
early postnatal period occur numerous brain developmental processes
(e.g. neurogenesis, myelination, synaptogenesis, astrogliogenesis,
neuronal cell death and blood–brain barrier maturation) [6, 19, 22,
25, 36, 52] it is possible that altered microglial
development induced by in utero stress may affect other
developmental processes either changing microenvironment molecular
constitution or triggering earlier inflammatory changes secondary
to the blood–brain barrier opening induced by prenatal
stress . Although punctual, the altered microglial
development might alter extensively the other
neurodevelopmental processes ensuing perdurable
structural changes; for example it is possible that the
change in the distribution pattern of microglia in the prenatal
stress group may render vulnerable some neuroanatomic
regions due to the reduction of neurotrophic factors,
such as the corpus callosum where there is a continuous axonal
No kidding! [There is also some very
interesting notes regarding microglial participation in purkinje
cell death that deserves and entire post. . .] This should be the
point that any rational observer must accept that we several lines
of evidence that early life experiences can persistently alter
microglial function with plausible mechanisms that could affect
neurodevelopment. Our data concerning total population
numbers in adulthood is a lot more difficult to come by, but I
think this will probably be getting looked at soon enough. Of
course, in any particular individual it is difficult (or
impossible?) to know how they may have arrived at a state of
increased microglial activation, but at the same time, it is not as
if we have no clue on possible pathways to this destination; our
short list of environmental factors includes immune insult, stress,
and chemical agents. If the question is, ‘what are the microglia
doing in the autism population?’, one plausible answer is ‘their
phenotype was persistently altered by an early life event through a
developmental programming model’. As I was mulling all of this
over, two things happened. First, a maternal CRP
study came out, and found a pretty strong relationship
between direct measurements of mommy inflammation with increased
risk of baby autism. The nice part is that they had a
gigantic data set (1.2M births!) to work with thanks to a few
decades of single payer medicine. (Very
For maternal CRP
levels in the highest quintile, compared with the lowest quintile,
there was a significant, 43% elevated
risk. This finding suggests that
maternal inflammation may have a significant role in autism, with
possible implications for identifying preventive strategies and
pathogenic mechanisms in autism and other neurodevelopmental
Just after that paper came out, I
made some Fred Flintstone style beef ribs. I ‘primed’ the
meat with a Moroccan inspired spice rub overnight, then
slow, slow, slow cooked them with a
low, low, low heat all day
long, and blasted away with a date glaze under the
broiler just before go time and they were caveman style
primal fucking awesome. The key to arriving there
was the slow cooking. The rib preparation
process got me thinking about our population wide experiment
in replacing infection with inflammation where we have
traded in death by pathogens or other once fatal ailments in
exchange for a longer life frequently plagued by conditions
associated with higher inflammation. Our analysis on long
term alterations to microglial proliferation and morphology is
largely comprised of studying acute insults
(sound familiar?), i.e., injection of purified bacterial cell
components known to trigger a robust immune response, ten sessions
of mouse based pregnant forced swimming, or exposure to chemicals
with rare and particular exposure routes in humans. Mostly I
think this is due to the black swan nature of the developmental programming
model alongside the very new idea that microglia are
doing jobs other than responding to infections; our models are
crude because of our relative ignorance. What will we find
when our filters are appropriately powered to detect for chronic,
but subtle insults? It occurs to me that there may be a ribs model
of altered microglial colonization of the fetal brain; it seems
clear that proliferation and differentiation of microglia can
clearly be changed by powerful inputs, but the
chemical messengers that impact that change are closely related (or
the same) as the measurement points in the maternal CRP study.
Could a slow cooking of slightly higher but not acutely
increased maternal inflammation be participating in the
genesis of autism (in some children) through altering the migration
and proliferation of microglia into the neonatal brain? Could
the same chemical messengers of inflammation be subtly
priming the microglia to respond with increased vigor to
insults later in life? Has our replacement of infection with
inflammation included an unanticipated effect that alters the
developmental pathway of the very cells that help shape our
children’s brains? I don’t think we are (quite) clever enough to
answer these types of questions yet, but we are at least starting
to generate the right kind of data to inform us on how to get
started. I don’t know what we will find, but the initial data
doesn’t look very good. In the meantime, I am recommending
you go get some ribs and let them cook all day long. –
A Brief Overview Of Glial Priming, How It (Probably) Applies To (Some Cases Of) Autism, And Worrisome Speculation On A Model Of A Low Penetrant Effect
Posted October 26, 2012on:
Hello friends –
The concept of glial priming (and implicit double multi hits) is the nexus of developmental programming, low penetrant effects, and an altered microglial responsiveness, a blueprint for a change in function in the tightly entangled neuroimmune environment; sort of an all time greats theory mashup for this blog. The basic idea is that microglia can become sensitized to insults and subsequently respond to similar insults with greater robustness and/or for increased timespans later in life. Here is a snippet from Microglia in the developing brain: A potential target with lifetime effects on the primed glial phenotype:
There is a significant amount of evidence regarding what is often termed ‘‘priming’’ and ‘‘preconditioning’’ events that serve to either exacerbate or provide neuroprotection from a secondary insult, respectively. In these states, the constitutive level of proinflammatory mediators would not be altered; however, upon subsequent challenge, an exaggerated response would be induced. The phenomena of priming represent a phenotypic shift of the cells toward a more sensitized state. Thus, primed microglia will respond to a secondary ‘‘triggering’’ stimulus more rapidly and to a greater degree than would be expected if non-primed.
Glial priming may be the fulcrum on which much of the underlying early immune activation research balances, the machinery that drives environmental influences during development leading to irregular neuroimmune functionality through the lifespan. Even though this type of finding is not really unexpected when considered within the prism of programming effects in other systems and the perturbed immune milieu in many (all?) neurological disorders, it is still pretty cool.
The first paper that I read that specifically mentioned glial priming was Glial activation links early-life seizures and long-term neurologic dysfunction: evidence using a small molecule inhibitor of proinflammatory cytokine upregulation, (Somera-Molina KC , 2007) which totally kicked ass. They brought a lot of heat at design time of the study; (very powerful) seizures were induced /saline given in animals at postnatal day 15 and 45; at day 55 animals were analyzed and showed distinct increases in microglial activation, neurologic injury, and future susceptibility to seizures in the ‘two hit’ group (i.e., animals that got seizure inducing kainic acid instead of saline on both day 15 and 45). Even better, it was shown that a CNS available inhibitor of inflammatory cytokine production rescued the effect of the seizure. In other words, it didn’t matter if the animals had a seizure, what mattered was the presence or absence of an unmitigated inflammatory response associated with the seizure.
Treatment with Minozac, a small molecule inhibitor of proinflammatory cytokine upregulation, following early-life seizures prevented both the long-term increase in activated glia and the associated behavioral impairment.
That is an important step in understanding the participation of inflammation in seizure pathology. There were also observable effects (worse) in animals that got seizures just once, if they got induced on day 15 versus 45, and even worse symptoms for the “double hit” animals. That was pretty fancy stuff in 2007. The similarity in terms of seizure susceptibility really reminded me of another paper, Postnatal Inflammation Increases Seizure Susceptibility in Adult Rats, which also showed altered susceptibility to seizures in animals subjected to seizures in early life, with the effect mediated through inflammation related cytokines. Here, however, the same effect observed, but with the addition of clinical evidence of chronically perturbed microglia phenotype in the treatment group. Nice!
The same group followed up with Enhanced microglial activation and proinflammatory cytokine upregulation are linked to increased susceptibility to seizures and neurologic injury in a ‘two-hit’ seizure model (full version), with more of the same. Here is part of the Discussion:
First, in response to a second KA ‘hit’ in adulthood, there is an enhancement of both the upregulation of proinflammatory cytokines, microglial activation, and expression of the chemokine CCL2 in adult animals who had previously experienced early-life seizures. Consistent with the exaggerated proinflammatory cytokine and microglial activation responses after the second hit, these animals also show greater susceptibility to seizures and greater neuronal injury. Second, administration of Mzc to suppress of the upregulation of proinflammatory cytokines produced by early-life seizures prevents the exaggerated cytokine and microglial responses to the second KA hit in adulthood. Importantly, regulating the cytokine response to early-life seizures also prevents the enhanced neuronal injury, behavioral impairment, and increased susceptibility to seizures associated with the second KA insult. These results implicate microglial activation in the mechanisms by which early-life seizures lead to increased susceptibility to seizures and enhanced neurologic injury with a second hit in adulthood.
Not only that, but the authors speculated on the possibility of a rescue effect through neuroimmune modulation!
Our data support a role for activated glia responses in the mechanisms by which early-life seizures produce greater susceptibility to a second neurologic insult. The improved outcomes with Mzc administration in multiple acute or chronic injury models where proinflammatory cytokine upregulation contributes to neurologic injury (Hu et al., 2007; Somera-Molina et al., 2007; Karpus et al., 2008; Lloyd et al., 2008) suggest that disease-specific interventions may be more effective if combined with therapies that modulate glial responses. These results are additional evidence that glial activation may be a common pathophysiologic mechanism and therapeutic target in diverse forms of neurologic injury (Akiyama et al., 2000; Craft et al., 2005; Emsley et al., 2005; Hu et al., 2005; Perry et al., 2007). Therapies, which selectively target glial activation following acute brain injury in childhood, may serve to prevent neurologic disorders in adulthood. These findings raise the possibility that interventions after early-life seizures with therapies that modulate the acute microglial activation and proinflammatory cytokine response may reduce the long-term neurologic sequelae and increased vulnerability to seizures in adulthood.
(Please note, the agent used in the above studies, kainic acid, is powerful stuff, and the seizures induced were status epileptcus, a big deal and a lot different than febrile seizures. That doesn’t mean that febrile seizures are without effect, I don’t think we are nearly clever enough to understand that question with the level of detail that is needed, but they are qualitatively different and not to be confused.)
The idea of modulating glial function as a preventative measure seems especially salient to the autism community alongside the recent (totally great) bone marrow studies observing benefits to a Rett model and an early life immune activation model of neurodevelopment.
A lot of kids with autism go on to develop epilepsy in adolescence, with some studies finding prevalence in the range of 30%, which terrifies the shit out of me. Is a primed microglial phenotype, a sensitization and increased susceptibility to seizures one of the mechanisms that drive this finding?
After Somera-Molina, I started noticing a growing mention of glial priming as a possible explanation for altered neuroimmune mechanics in a lot of places. Much of the early life immune literature has sections on glial priming, Early-Life Programming of Later-Life Brain and Behavior: A Critical Role for the Immune System (full / highly recommended / Staci Bilbo!) is a nice review of 2010 data that includes this:
However, there is increasing support for the concept of “glial priming”, in which cells can become sensitized by an insult, challenge, or injury, such that subsequent responses to a challenge are exaggerated (Perry et al., 2003). For instance, a systemic inflammatory challenge in an animal with a chronic neurodegenerative disease leads to exaggerated brain inflammation compared to a control animal (Combrinck et al., 2002). The morphology of primed glial cells is similar to that of “activated” cells (e.g. amoeboid, phagocytic), but primed glial cells do not chronically produce cytokines and other pro-inflammatory mediators typical of cells in an activated state. Upon challenge, however, such as infection or injury in the periphery, these primed cells will over-produce cytokines within the brain compared to cells that were not previously primed or sensitized (Perry et al., 2002). This overproduction may then lead to cognitive and/or other impairments (Cunningham et al., 2005; Frank et al., 2006; Godbout et al., 2005).
Other studies included increased effects of pesticide exposure following immune challenge, Inflammatory priming of the substantia nigra influences the impact of later paraquat exposure: Neuroimmune sensitization of neurodegeneration, which includes, “These data suggest that inflammatory priming may influence DA neuronal sensitivity to subsequent environmental toxins by modulating the state of glial and immune factors, and these findings may be important for neurodegenerative conditions, such as Parkinson’s disease (PD).” Stress was also found to serve as a priming agent in Glucocorticoids mediate stress-induced priming of microglial pro-inflammatory responses, which studied the effect of stress mediated chemicals on inflammatory challenges; the authors get bonus points for using glucocorticoid receptor agonists and surgical procedures to eliminate glucocorticoid creation to observe a priming effect of stress on neuroimmune response.
Here is a terrifying but increasingly unsurprising study on how neonatal experience modifies the physical experience of pain in adulthood, recently published in Brain, Priming of adult pain responses by neonatal pain experience: maintenance by central neuroimmune activity
Adult brain connectivity is shaped by the balance of sensory inputs in early life. In the case of pain pathways, it is less clear whether nociceptive inputs in infancy can have a lasting influence upon central pain processing and adult pain sensitivity. Here, we show that adult pain responses in the rat are ‘primed’ by tissue injury in the neonatal period. Rats that experience hind-paw incision injury at 3 days of age, display an increased magnitude and duration of hyperalgesia following incision in adulthood when compared with those with no early life pain experience. This priming of spinal reflex sensitivity was measured by both reductions in behavioural withdrawal thresholds and increased flexor muscle electromyographic responses to graded suprathreshold hind-paw stimuli in the 4 weeks following adult incision. Prior neonatal injury also ‘primed’ the spinal microglial response to adult injury, resulting in an increased intensity, spatial distribution and duration of ionized calcium-binding adaptor molecule-1-positive microglial reactivity in the dorsal horn. Intrathecal minocycline at the time of adult injury selectively prevented both the hyperalgesia and early microglial reactivity associated with prior neonatal injury. The enhanced neuroimmune response seen in neonatally primed animals could also be demonstrated in the absence of peripheral tissue injury by direct electrical stimulation of tibial nerve fibres, confirming that centrally mediated mechanisms contribute to these long-term effects. These data suggest that early life injury may predispose individuals to enhanced sensitivity to painful events.
One of the primal drivers of behavior in any animal, pain, can be persistently modified at a molecular level! Have you ever known someone that seemed to have a higher pain tolerance than you? Maybe they did, and the training of their microglia (or yours) in early life might be why. The most basic physiologic responses can be organized through the crucible of early life events sensitizing microglia to the future environment. Multi hit wow!
The effect that befalls us all, getting older, has a ton of studies on the effect of aging on glial priming, with greatest, err, ‘hits’ including Immune and behavioral consequences of microglial reactivity in the aged brain, Aging, microglial cell priming, and the discordant central inflammatory response to signals from the peripheral immune system (full),Immune and behavioral consequences of microglial reactivity in the aged brain (full), and the autism implication heavy Microglia of the Aged Brain: Primed to be Activated and Resistant to Regulation, and others. Broadly, these studies spoke of the same pattern, a primed neuroimmune response, except in this instance, the “hits” that predisposed towards altered microglial reactivity weren’t a vigorous insult during development, but just the hum drum activity of growing older. It wasn’t a hit so much, more like a then gentle force of a relentless tide, but the functional effect on microglia response was largely similar, responses to stimuli were changed and programming was observed. I do not believe that the underlying instrument of change in age related priming is understood, but the thought occurs to me that it could simply be an exhaustion effect; a lifetime of exposure to inflammatory cytokines gradually changes the microglial phenotype.
So what about autism?
First and foremost, it provides us a line of insight into the likelyhood of a causal relationship between an altered neuroimmune milieu and autism (or nearly any other neurological disorder); that is, the question of whether or not our continued and repeated findings of altered neuroimmune parameters in the autism population represent a participating force in autism, as opposed to an artifact, a function of something else, which is also causing autism, or perhaps a result of having autism. While these are still possible explanations, the findings of glial priming provide additional detail on available mechanisms to affect brain activity and behavior through neuroimmune modifications alone.
If nothing else, we now know that we need not rely on models with no underlying substrate except the lamentations of ‘correlation does not equal causation’ and the brash faith of another, as of yet undefined, explanation. These models tell us that immune mediated pathologies can be created (and removed!) in very well established animal models of behavioral disturbances with corollaries to autism findings.
For more direct links to autism, we can look at the autism immune biomarker data set and find evidence of primed peripheral (i.e., outside the CNS) programming, literal examples where the autism population responds with a different pattern than the control group including an increased response to some pathogen type agonists, increased immune response following exposure to pollutants, of even dietary proteins.
The pattern we see of an altered microglial phenotype in the autism population, a state of chronic activity, is certainly consistent with disturbed developmental programming; it does not seem unlikely to me that a priming effect is also present, the initial prime seems to be responsible for the programming. As far as I know, there are no studies that have directly attempted to evaluate for a primed phenotype in the microglia of the autism population; I’d be happy to be corrected on this point.
Thinking about the possibility of increased microglial responsiveness and possible cognitive effects of a sustained neuroimmune toggling got me wondering if this is one of the mechanisms of a change in behavior following sickness? Or, alternatively, for some of us, “Is This Why My Child Goes Goddamn Insane And Stims Like Crazy For A Week After He Gets Sick?”
If we look to a lot of the studies that have shown a priming effect, they share a common causative pathway as some cases of autism, an early life immune insult. For some examples, the interested reader could check out Neonatal programming of the rat neuroimmune response: stimulus specifc changes elicited by bacterial and viral mimetics (full paper), Modulation of immune cell function by an early life experience, or the often mentioned Postnatal Inflammation Increases Seizure Susceptibility in Adult Rats (full paper). If there are some cases of autism that have an early life immune insult as a participating input, it is very likely a primed microglia phenotype is also present.
The studies on aging are bothering me, not only am I getting older, but the findings suggest that a priming need not necessarily mandate a distinct ‘hit’, it can be more like a persistent nudge. Our fetuses and infants develop in an environment with an unprecedented number of different nudges in the past few decades as we have replaced infection with inflammation. Acknowledging this reality, however, raises the troubling thought that our embrace of lifestyles associated with increased inflammation has reached a tipping point that we are literally training the microglia of our children to act and react differently; we aren’t waiting a lifetime to expose our fetuses and infants to environments of increased inflammation, we are getting started from the get go.
Even with all of that, however, there is a genuinely microscopic Google footprint if you search for “autism ‘glial priming’”. So, either I’m seeing phantoms (very possible), or the rest of the autism research community hasn’t caught on yet, at least in such a way that Google is notified.
Even if I am chasing phantoms, there is evidence of a widespread lack of understanding of the depth of the neuroimmune/behavioral crosstalk literature, even by the people who should be paying the most attention. This was brought to my attention by a post at Paul Patterson’s blog, where Tom Insel was quoted as finding the recent Patterson and Derecki findings ‘unexpected’.
A bone marrow transplant, which replaces the immune system, corrected both the immune response and the behavior. This finding, which was unexpected, is surprisingly similar to another recent paper reporting disappearance of the symptoms of Rett syndrome in mice following a bone marrow transplant.
Keep in mind, this is from the guy who is the head of the IACC! I can tell you one thing; while the studies were impressive, I don’t think that the findings were especially unexpected. The researchers took the time to give mice bone marrow transplants, and in Wild-type microglia arrest pathology in a mouse model of Rett syndrome, the authors utilized a variety of knockout mice and even partial body irradiation to illuminate the question of neuroimmune participation in disorder. This work was not initiated in a vacuum, they did not throw a dart at a barn door sized diagram of study methodologies and land on ‘bone marrow transplants with subsequent analysis of microglia population properties and behaviors, accounting for different exposure timeframes, radiation techniques, and genotypes’. These were efforts that had a lot of supporting literature in place to justify the expense and researcher time. [I really want to find time to blog both of those papers in detail, but for the record, I did feel the rescue effects are particularly nice touches.]
So given that the head of the IAAC was surprised to find that immune system replacement having an effect on behavior was ‘surprising’, I’m not all together shocked at the relative lack of links on ‘glial priming’ and autism, but I don’t think it will stay that way for too much longer. As more experiments demonstrating a primed phenotype start stacking up, we are going to have to find a way to understand if generation autism exhibits a primed glial phenotype. I don’t think we are going to like the answer to that question very much, and the questions that come afterwards are going to get very, very inconvenient.
Spelling it out a bit more, with bonus speculation, we should remember our recent findings of the critical role microglia are playing in shaping the neural network; our microglia are supposed to be helping form the physical contours of the brain, a once in a lifetime optimization of synaptic structures that has heavy investment from fetushood to toddlerhood. Unfortunately, it appears that microglia perform this maintenance while in a resting state, i.e., not when they have been alerted of an immune response and taken on a morphology consistent with an ‘activated state’. An altered microglia morphology can be instigated during infection, or perceived infection and consequent immune response. For examples of peripheral immune challenges changing microglial morphology, the neuroimmune environment and behavior some examples include: Peripheral innate immune challenge exaggerated microglia activation, increased the number of inflammatory CNS macrophages, and prolonged social withdrawal in socially defeated mice, Exaggerated neuroinflammation and sickness behavior in aged mice following activation of the peripheral innate immune system, or Long-term changes of spine dynamics and microglia after transient peripheral immune response triggered by LPS in vivo.
But what if we have a susceptible population, a population sensitized such that the effects of an immune challenge would result in an exaggerated and extended microglial response, effectively increasing the length of time the microglia would be ‘not resting’. What might be the changes in this population in response to a series of ‘hits’?
It does not seem to be a large logical leap to assume that if some of the altered brain physiology in autism is due to abnormal microglia function during the period of robust synaptic pruning, triggering the microglia to leave their resting state for an extended period in response could be a reasonable participant. Think of it as an exaggerated loss of opportunity effect, essentially a longer timeframe during which the microglia are not performing synaptic upkeep when compared to the microglia in an individual that is not sensitized. While our brains do show a lot of ability to ‘heal’, that does not mean that all things or times are created equally; there are some very distinct examples of time and spatially dependent neurochemical environments during early synapse development, environments that change as time goes on; i.e., Dynamic gene and protein expression patterns of the autism-associated met receptor tyrosine kinase in the developing mouse forebrain (full paper), or A new synaptic player leading to autism risk: Met receptor tyrosine kinase. In other words, recovering from a delay in microglial participation in synaptic pruning during development may not be as simple as ‘catching up’; if the right chemical environment isn’t available when the microglia get done responding, you might not be able to restart like a game of solitaire. The Met levels might be different, the neurexin levels might be different, a thousand other chemical rally points could be set that much of a nudge differently; in a system dependent on so many moving variables being just so, an opportunity missed is an opportunity lost. For good.
While the effects of a series of challenges and consequent obstructions of synaptic maintenance might not be acutely clear, I am becoming less and less convinced of the ‘safety’ of an observed lack of immediately obvious effects. I think that an intellectually honest evaluation of our recent ‘discoveries’ in many areas of early life disturbances (i.e., antibiotics and IDB risk, C-section and obesity risk, birth weight and cardiovascular risk) tell us that subtle changes are still changes, and many rise to the level of a low penetrant, environmentally induced effect once we get clever enough to ask the right question. And boy are we a bunch of dummies.
Taking all of this into consideration, all I can think is thank goodness we haven’t been artificially triggering the immune system of our infants for the past two decades while we were blissfully unaware of the realities of microglial maintenance of the brain and glial priming! What a relief that we did not rely on an assumption of lack of effect as a primary reason not to study the effect of an immune challenge. If we had done those things, we might start kicking ourselves when we realized out that our actions could be affecting susceptible subsets of children who were predisposed to reacting in difficult to measure but real ways that could literally affect the physical structure of their brains.
Considering the Scaffolds of Interconnectedness, “Environmental enrichment alters glial antigen expression and neuroimmune function in the adult rat hippocampus” and How Seeing The Obvious Can Still Be A Pleasant Surprise
Posted March 9, 2012on:
I’ve been thinking a lot lately about the beauty and trials of the tightly coupled systems, the interconnected pathways that keep popping up when pubmed tells me something that might be of interest on journey autism. One theme bubbling to the top of my thoughts is that there is a large set of inputs capable of tweaking the areas we see altered in autism; broken isn’t necessarily appropriate, but the research increasingly tells us that a delicately balanced set of connected processes is readily changed, and the way that the physics work out, there is no way to change just one thing when you have a polygamous marriage of chemical systems.
Imagine a orchestra where all of the musicians were physically bound to one or more of their counterparts, a system of wires, pulleys, springs and levers such that the musicians are actually participating in the playing of each other, not soccer players doing synchronized flips so much as a set of violin-em-cello-em robots, connected to play their instruments in unison, wind them up and create a symphony. Different orchestras might have a tighter wire from one member to another, or an older spring, but when they worked together, you could tell what composition they were playing. In this analogy, you cannot have the drummers start beating harder and faster without also changing how hard the French horn players blow. The situation only gets more complicated if some of our musicians were connected to several other musicians simultaneously. There would still be music if the cellist couldn’t keep a steady rhythm, but it would be different music, not just a different cello.
The communication between a lot of our “systems”, immune, endocrine, stress response and central nervous systems are a lot like musicians in the orchestra, interdependent and intimately connected.
The funny thing is, this same message is being blared to me, and to you, all the time, damn near every time you turn on the TV, but it is hidden in plain sight by legislatively mandated doublespeak. Consider how many advertisements each of us have seen for pharmaceutical drugs where the number of complications and contra-indicated conditions far, far exceed the number of desired effects?
Here is a list of common side effects of Viagra:
Diarrhea, dizziness, flushing, headache, heartburn, stuffy nose, upset stomach
So right off the bat, besides what we are looking for, we can see it is common to expect Viagra to also affect your GI system, immune system, and/ or brain function. These are the types of things that are “common”. (One wonders how Viagra would sell if it always caused headaches and diarrhea, and sometimes transiently ameliorated erectile dysfunction? ) A list of ‘severe’ side effects includes memory loss and a sudden decrease in hearing or vision. Even after decades of work by a lot of exceptionally smart people and hundreds of billions of dollars, the interlocked complexity of our bodies are continuing to prove very difficult to adjust in only the way we’d like, and seemingly minor perturbations in one area can pop up in very unpredictable fashion in other functions.
Trying to put my mind around the implications of this in regards to autism often leaves me with a sense of being profoundly humbled and woefully underprepared, not unlike a lot of my experiences with autism in the real world. Secondarily, again with great similarity to personal experience, I (eventually) come to the (re-)realization that we should rejoice in opportunities to be challenged and learning more about something makes us richer in ways more important than dollars.
A superb example of all of this and more landed in my inbox the other day, Environmental enrichment alters glial antigen expression and neuroimmune function in the adult rat hippocampus (Williamson et all). [Also on this paper, blog favorite, Staci Bilbo]
Williamson reported that animals given a so called ‘enriched environment’ exhibited significantly decreased immune responses in certain portions of the brain following immune challenge, with reduced levels of several chemokines and cytokines in the hippocampus in the treatment group. (A previous discussion about environmental enrichment on this blog can be found here) In this instance, the treatment group got to spend twelve hours a day in a different area, a housing unit with “a running wheel, a PVC tube and various small objects and toys”, while the control group of animals stayed in their drab, Soviet era proletariat cages all day and all night long. Here is the abstract:
Neurogenesis is a well-characterized phenomenon within the dentate gyrus (DG) of the adult hippocampus. Environmental enrichment (EE) in rodents increases neurogenesis, enhances cognition, and promotes recovery from injury. However, little is known about the effects of EE on glia (astrocytes and microglia). Given their importance in neural repair, we predicted that EE would modulate glial phenotype and/or function within the hippocampus. Adult male rats were housed either 12h/day in an enriched environment or in a standard home cage. Rats were injected with BrdU at 1week, and after 7weeks, half of the rats from each housing group were injected with lipopolysaccharide (LPS), and cytokine and chemokine expression was assessed within the periphery, hippocampus and cortex. Enriched rats had a markedly blunted pro-inflammatory response to LPS within the hippocampus. Specifically, expression of the chemokines Ccl2, Ccl3 and Cxcl2, several members of the tumor necrosis factor (TNF) family, and the pro-inflammatory cytokine IL-1ß were all significantly decreased following LPS administration in EE rats compared to controls. EE did not impact the inflammatory response to LPS in the cortex. Moreover, EE significantly increased both astrocyte (GFAP+) and microglia (Iba1+) antigen expression within the DG, but not in the CA1, CA3, or cortex. Measures of neurogenesis were not impacted by EE (BrdU and DCX staining), although hippocampal BDNF mRNA was significantly increased by EE. This study demonstrates the importance of environmental factors on the function of the immune system specifically within the brain, which can have profound effects on neural function.
Total interconnectedness kick ass!
Considering the wide ranging and predominantly ‘rather-not-have-than-have’ properties of ‘extra’ TNF-alpha and IL-1beta in the CNS, this is a pretty interesting finding. Not only that, animals ‘protected’ through environmental enrichment also showed increased levels of growth factors known to be altered in autism, again in the hippocampus. In a very real and measurable sense, it was possible to shuffle the neuroimmune cocktail of the brain by changing things like the availability of quality leisure time. As we have seen in other areas, altering the chemical milieu of immunomodulatory factors in the brain isn’t trivial, and is increasingly associated with a variety of conditions classically diagnosed through the study of behaviors.
It should be noted that there were unexpected, and generally negative findings from this study, namely, a relative lack of biomarkers indicative of increased neurogenesis in the environmental enrichment group; something that I think took the authors by a bit of surprise.
There is a short discussion on the possibilities on why the findings of differential neuroimmune responses were found only in the hippocampus, with reference being made to previous studies indicating that this area of the brain has been found to be more susceptible to a variety of insults.
There were some other findings that struck me as particularly intriguing; something that has been hinted at previously in other studies (or transcripts), but not yet well described, likely due to the fact that the area is still largely unknown to us. Specifically, the authors reported a state of glial activation, somewhat the opposite of what they expected.
The data instead suggest that EE changes the phenotype of glia, altering their activation and attenuating their pro-inflammatory response to peripheral LPS, although this remains to be directly tested. Interestingly, the blunted neuroinflammatory response within the DG of EE rats occurring concomitant with the increase in classical glial ‘‘activation’’ markers runs counter to our initial prediction. However, we believe these data simply highlight the fact that little is known about the function of these markers. Moreover, there is a growing literature that distinguishes classical versus alternative activation states in microglia, the latter of which is associated more strongly with repair (Colton, 2009; Colton and Wilcock, 2010).
Thus, it is possible that EE shifts microglia into an alternatively activated phenotype, an intriguing possibility that we are currently exploring.
The authors discuss the fact that their findings were highly spatially specific within the brain, involved a subset of cytokines and chemokines, and environmental enrichment did not seem to affect immune response in the periphery.
The immune response within the hippocampi of EE rats was markedly attenuated for a subset of cytokines and chemokines measured in our study. Importantly, not all measured immune molecules were blunted in the hippocampi of EE rats. Furthermore, the immune response was similar for each housing group in the parietal cortex as well as in the periphery. Within the hippocampus, however, EE rats had an attenuated response of interleukin-1b (IL-1b), the TNF family of genes, and several chemokines involved in the recruitment of leukocytes and monocytes. These families of genes indicate an altered hippocampal milieu in EE rats that may be less pro-inflammatory, more neuroprotective and less permeable to peripheral infiltrating immune cells.
There is a short discussion on the existing knowledge concerning IL-B and TNF-alpha in normal and pathological conditions, and how these findings are consistent with other findings involving environmental enrichment and cognition.
Tumor necrosis factor alpha (TNFa) is well characterized for its roles in inflammation and host defense, sepsis and, most intriguing for this study, apoptosis cascades (for review, see Hehlgans and Pfeffer, 2005). The observed attenuation after an immune challenge of TNFa and several associated genes in EE rats compared to HC controls indicates a potential enduring change in the hippocampal microenvironment of enriched rats, such that one mechanism by which EE may increase neuroprotection following insults to the CNS (Briones et al., 2011; Goldberg et al., 2011; Young et al., 1999) is via altered TNF tone and function, increasing the likelihood of cell survival by reducing apoptotic signaling. In addition to attenuated IL-1b and TNF responses, EE rats showed blunted responses for several chemokines known to influence the recruitment of circulating monocytes and leukocytes to the CNS.
Finally, the authors conclude how their findings add to the literature on environmental enrichment and brain function.
In summary, environmental enrichment is a relatively simple manipulation that results in robust beneficial outcomes for the brain. While previous studies have shown a role in post-insult rehabilitation for EE, our study provides evidence that enrichment need not follow the insult in order to be beneficial. Indeed, neuroinflammatory disease states might be attenuated or delayed in their onset in the face of ongoing EE. The translational reach of this manipulation remains to be explored, but in animal models of neuroinflammation, EE may provide a simple preventative measure for negative outcomes.
The bottom line is that a fuller rat life experience resulted in different neuroimmune profiles, findings with some consistency with previous observations that an enriched rat house resulted in improved behavioral manifestations of cognitive performance. The qualities of these different neuroimmune profiles are also consistent with chemical profiles associated with positive outcomes in several conditions.
There is a deceivingly startling realization hidden in these finding, startling because it reveals the malleable nature of the seemingly different, but basic systems interacting and deceptive because it is so obvious. How many of us have known someone who deteriorated upon entering a nursing home, or even retiring from working? How many of us have kept their children inside for a week due to weather and watched their children go crazy after the already inferior indoor entertainment options are long exhausted? Those changes in emotion, in behaviors and function, just like the findings from this study, are founded by chemistry.
But seeing evidence that relatively simple environmental modifications can rejigger the molecular atmosphere of the brain is still more than a little awe inspiring. Knowing there is machinery underneath the hood is a little different than observing the cogs of cognition swell , shrink, or slow down; nothing less than a deeper understanding of the chemical basis of thought. And that is pretty cool.
The Interconnectedness of the Brain, Behavior, and Immunology and the Difficult to Overstate Flaccidity of The Correlation Is Not Causation Argument
Posted May 12, 2011on:
Hello friends –
I’ve gotten into a lot of discussions online about the vaccines and autism; generally with very poor, if not nonexistent, evidence of having changed any opinions, but relatively strong evidence ( p > .001) that persisting in making my arguments can get you called ‘an antivaccine loon’, ‘idiot’, someone who engages in ‘Gish Gallop’, or the worst insult I’ve received so far, ‘anti-science’. While I am really torn on the vaccine issue, I am certain that both peripheries of this debate are at least somewhat wrong in the conclusions that they’ve drawn from the available evidence. I do believe that lots of parents have witnessed a very real change in their children post vaccination, and I also don’t believe for a single second that vaccines are the cause of an epidemic of autism. It’s a mess and I’ve been poking around the Internet almost five years into journey autism and from my eyes, it hasn’t improved any in the past half decade. This is very sad.
That being said, while I do think we need to have a rational and dispassionate discussion about what our existing vaccine studies can and cannot tell us about autism, I’m really concerned about the fact that the vaccine wars seem to have inoculated otherwise intelligent people from any semblance of intellectual curiosity regarding the immunological findings in the autism realm. That’s a problem, because there are lots of things other than vaccines that can modify the immune response, various environmental agents and cultural changes that are relatively new, and ignoring immunological findings in autism because they happen to intersect with the function of vaccination is a huge, massive, supernova sized disservice to what history will view us poorly on, refusing to perform honest evaluation due to fear and the comfort of willful ignorance.
Here, in this post, I will make the case that this lack of curiosity on immunological findings in autism is either born of a lack of understanding on how much we know about the ties between the immune system and the brain, or alternatively, originates from a deep seated desire to avoid honest interactions. This isn’t to make the case that vaccines can cause autism, or even that the immunological disturbances observed in autism are causative, but rather that an obstinate refusal to consider these as possibilities is the sign of someone who cannot, or will not accept, the biological plausibility of immunologically driven behaviors despite a constellation of evidence.
One of the things that jumps out to me why the autism population might be a subgroup of the population susceptible to changes as a result of immune dysfunction (and thus, potentially adversely affected as a result of vaccination), is the sheer volume of evidence we now have available to us indicating an altered immune response, and indeed, an ongoing state of inflammation within the brain in the autism population, and most strikingly, repeated observations of a correlation between the degree of immune dysregulation as a propensity of an inflammatory state, and the severity of autism behaviors. Again and again we’ve seen that as markers indicative of an inflammatory state increase, so too, do severity of autism behaviors. Not only that, but there are instances wherein the decrease of components known to regulate the immune response decrease, autistic behaviors are more severe. Subtle shifts in either the start or the resolution of the immune response seems to affect autistic behavior severity in the same way. I know coincidences happen all the time, but that doesn’t mean that everything is a coincidence.
We also have a large number of studies that tell us that in vitro, similar levels of stimulation with a variety of agents cause exaggerated or dysregulated production of immune markers in the autism population.
A large percentage of the time that I mention these findings, usually within discussions with an origin in vaccination, someone decides to educate me on one of the most rudimentary scientific axioms:
Correlation does not equal causation.
It must be stated, the above statement is absolutely true. Unfortunately for the people for whom this accurate, but simplistic catchphrase comprises the entirety of their argument, it completely ignores a wealth of research that tells us in very unambiguous terms that there is incontrovertible evidence that crosstalk between the immune system and central nervous system can modify behavior. The research indicating a relationship between immune dysregulation and autism does not exist in a vacuum, but rather, is only a tiny fragment of evidence, mostly accumulated within the last few years, that tells us that the paradigm of the past decades, that of the brain as a immune privileged organ without communication to the immune system, is as antiquated as refrigerator moms and a one in ten thousand prevalence.
From a common sense, why didn’t I think of that standpoint, the best example of the interaction between the brain and the immune response is the old standard, just plain old getting sick. You live in the dirty world, you pick up a pathogen, you get sick, and suddenly you get lethargic and you start to run a fever. But is it the pathogen itself that is actually making you feel like staying in bed all day?
What is being learned is that it is not necessarily the microbial invader that is causing you to get tired and feel sore, but rather, that your decreased energy levels are centrally mediated through your brain, and the triggers for your brain to start a fever include molecules our bodies use for a wide range of communications, including immune based messaging, cytokines. Some of the most common cytokines in the research to follow include IL-6, IL-1B, and TNF-Alpha; so called ‘pro-inflammatory’ cytokines. Researchers have been plugging away at just how the immune response is capable of modifying behaviors, i.e., inducing, sickness behavior for a while now, at least in terms of autism research. From 1998, we have Molecular basis of sickness behavior:
Peripheral and central injections of lipopolysaccharide (LPS), a cytokine inducer, and recombinant proinflammatory cytokines such as interleukin-1 beta (IL-1 beta) induce sickness behavior in the form of reduced food intake and decreased social activities. Mechanisms of the behavioral effects of cytokines have been the subject of much investigation during the last 3 years. At the behavioral level, the profound depressing effects of cytokines on behavior are the expression of a highly organized motivational state. At the molecular level, sickness behavior is mediated by an inducible brain cytokine compartment that is activated by peripheral cytokines via neural afferent pathways. Centrally produced cytokines act on brain cytokine receptors that are similar to those characterized on peripheral immune and nonimmune cells, as demonstrated by pharmacologic experiments using cytokine receptor antagonists, neutralizing antibodies to specific subtypes of cytokine receptors, and gene targeting techniques. Evidence exists that different components of sickness behavior are mediated by different cytokines and that the relative importance of these cytokines is not the same in the peripheral and central cytokine compartments.
The first sentence in this abstract references a practice that is extremely common in studying the immune system, intentionally invoking a robust immune response by exposing either animals, or cells in vitro, to the components that comprise the cell wall of certain types of bacteria; lipopolysaccharide, or LPS. LPS could be considered a sort of bacterial fingerprint, a pattern that our immune systems, and the immune system of almost everything, has evolved to recognize, and correspondingly initiates an immune response.
Because this is a conversation that frequently has an origin in vaccination, essentially the act of faking an infection, it is salient to remember that the animals or cell cultures aren’t really getting sick when exposed to LPS; there is no pathology associated with whatever type of bacteria might be housed within a cell membrane containing LPS. Usually, when the body is exposed to a gram negative bacteria, and the consequent LPS exposure, there are also effects of the bacteria that interact with the organism, but by only incorporating the alert signal for a bacterial invader, we can gain insight into the effect of the immune response itself; there isn’t anything else to cause any changes. This means that similarly to LPS administration, straight administration of these pro-inflammatory cytokines are similar to the result of getting sick with a pathogen, at least as far as the immune response is concerned.
In the above instance, administration of LPS, or simply cytokines, had been shown to be capable of causing reduced food intake and ‘decreased social activities’.
Later in 1998, Central administration of rat IL-6 induces HPA activation and fever but not sickness behavior in rats (full version), was published wherein the authors report that central administration (i.e., directly into the CNS), of cytokines in isolation (IL-6) or in combination (IL-6 + IL-1B) were capable of inducing altered HPA activation, fevers, and sickness behaviors. Effects of peripheral administration of recombinant human interleukin-1 beta on feeding behavior of the rat was published a few years later, and observed that peripheral administration (i.e., not the CNS) of IL-1B could affect how much a rat ate, with sucrose ingestion being consistently altered during periods of sickness.
Jumping ahead a few years, a review paper Expression and regulation of interleukin-1 receptors in the brain. Role in cytokines-induced sickness behavior reviewed how cytokines participate in sickness behavior, Interleukin-6 and leptin mediate lipopolysaccharide-induced fever and sickness behavior examined the interactions of IL-6 and leptin in sickness behavior, and Behavioral and physiological effects of a single injection of rat interferon-alpha on male Sprague-Dawley rats: a long-term evaluation reported “these data suggest that a single IFN-alpha exposure may elicit long-term behavioral disruptions”.
Much more recently, Sickness-related odor communication signals as determinants of social behavior in rat: a role for inflammatory processes more elegantly found that behavior was modified by LPS exposure, and that this effect was neutralized by concurrent administration of the anti-inflammatory cytokine, IL-10. Similarly, Inhibition of peripheral TNF can block the malaise associated with CNS inflammatory diseases observed another distinct means by which interfering with the immune response could attenuate the effect of faux sickness, in part, concluding, “Thus behavioral changes induced by CNS lesions may result from peripheral expression of cytokines that can be targeted with drugs which do not need to cross the blood-brain barrier to be efficacious.” In other words, what is happening in the periphery, outside of the protective boundaries of the blood brain barrier, can none the less manipulate behaviors that are controlled by the brain.
There are tons, tons more studies like this, but the point should be clear by now; it is accepted that you can achieve some of the same behaviors the come alongside illness, such as fever and lethargy, without the presence of an actual bacteria or virus; all you need is for your brain to think that you are sick.
While it must be acknowledged that the behavioral disturbances observed in autism are a lot different than feeling the need to watch TV all day, these types of studies were among the first clues that the traditional view of the CNS as a separate entity within the gated community of the blood brain barrier needed revision.
Measuring how much sugar water a rat drank is great stuff, but the reality is that we have conservatively a gazillion studies telling us that disorders that manifest behaviorally have strong, strong ties to the immune system; and once we begin to understand the vast scope of these findings, the utter frailty of “correlation does not equal causation” becomes painfully clear to the intellectually honest observer.
The big problem I found myself with in crafting this posting was that the sheer volume of studies available really makes a complete illustration of the literature impossible; I started looking and pubmed nearly puked trying to return to me a listing of all of the things I wanted to summarize. So here is some of the best of the best; to keep things interesting, I thought I’d only include findings from 2007 or later as a mechanism to show just how nascent our understanding of the connections between the brain and the immune system really are.
Initially, we can start with a condition that nearly everyone agrees is diagnosed based on behavior, depression. It turns out, the number of findings establishing a link between immune system markers and depression is wide and deep.
Here’s a great one, Elevated macrophage migration inhibitory factor (MIF) is associated with depressive symptoms, blunted cortisol reactivity to acute stress, and lowered morning cortisol, which reports, that MIF can modify HPA axis function and is tied to depression; a particularly compelling finding considering well documented alterations in HPA axis metabolites in autism, and the fact that increased MIF has also been found in the autism population, and as levels increased, so too did autism severity.
Here is part of the abstract for Inflammation and Its Discontents: The Role of Cytokines in the Pathophysiology of Major Depression (full paper)
Patients with major depression have been found to exhibit increased peripheral blood inflammatory biomarkers, including inflammatory cytokines, which have been shown to access the brain and interact with virtually every pathophysiologic domain known to be involved in depression, including neurotransmitter metabolism, neuroendocrine function, and neural plasticity. Indeed, activation of inflammatory pathways within the brain is believed to contribute to a confluence of decreased neurotrophic support and altered glutamate release/reuptake, as well as oxidative stress, leading to excitotoxicity and loss of glial elements, consistent with neuropathologic findings that characterize depressive disorders.
Somewhere along the way, researchers discovered that some anti-depressants can exert anti-inflammatory effects, for examples of these findings we could look to Fluoxetine and citalopram exhibit potent antiinflammatory activity in human and murine models of rheumatoid arthritis and inhibit toll-like receptors, or Plasma cytokine profiles in depressed patients who fail to respond to selective serotonin reuptake inhibitor therapy, which concludes in part, “Suppression of proinflammatory cytokines does not occur in depressed patients who fail to respond to SSRIs and is necessary for clinical recovery”.
In Investigating the inflammatory phenotype of major depression: focus on cytokines and polyunsaturated fatty acids, the authors report that, “The findings of this study provide further support for the view that major depression is associated with a pro-inflammatory phenotype which at least partially persists when patients become normothymic.” A nice review of the evidence of immunological participation in depression can be found in The concept of depression as a dysfunction of the immune system (full paper).
Moving forward, we can look to schizophrenia, we have similar findings, including Serum levels of IL-6, IL-10 and TNF-a in patients with bipolar disorder and schizophrenia: differences in pro- and anti-inflammatory balance, which observed an imbalanced baseline cytokine profile in the schizophrenic group; findings very similar in form with An activated set point of T-cell and monocyte inflammatory networks in recent-onset schizophrenia patients involves both pro- and anti-inflammatory forces. Similarly, the findings from Dysregulation of chemo-cytokine production in schizophrenic patients versus healthy controls, (full paper) which states, in part:
Growing evidence suggests that specific cytokines and chemokines play a role in signalling the brain to produce neurochemical, neuroendocrine, neuroimmune and behavioural changes. A relationship between inflammation and schizophrenia was supported by abnormal cytokines production, abnormal concentrations of cytokines and cytokine receptors in the blood and cerebrospinal fluid in schizophrenia
Their findings include differentially increased and decreased production of chemokines and cytokines as a result of LPS stimulations in the case group. Of particular note, a similarly dysregulated immune profile of cytokine and chemokine generation has been found in the autism population in several studies.
We also have several trials of immunomodulatory drugs in the schizophrenic arena that further implicate the immune system in pathology, including Adjuvant aspirin therapy reduces symptoms of schizophrenia spectrum disorders: results from a randomized, double-blind, placebo-controlled trial, a ‘gold standard’ trial which found that, “Aspirin given as adjuvant therapy to regular antipsychotic treatment reduces the symptoms of schizophrenia spectrum disorders. The reduction is more pronounced in those with the more altered immune function. Inflammation may constitute a potential new target for antipsychotic drug development”. A similar clinical trial, Celecoxib as adjunctive therapy in schizophrenia: a double-blind, randomized and placebo-controlled trial , another gold standard trial, which also had findings in the same vein, “Although both protocols significantly decreased the score of the positive, negative and general psychopathological symptoms over the trial period, the combination of risperidone and celecoxib showed a significant superiority over risperidone alone in the treatment of positive symptoms, general psychopathology symptoms as well as PANSS total scores.” [Celecoxib is a cox-2 inhibitor; i.e., anti-inflammatory, i.e., immunomodulatory]
What about bi-polar disorder? More of the same, including, The activation of monocyte and T cell networks in patients with bipolar disorder, or Elevation of cerebrospinal fluid interleukin-1ß in bipolar disorder, which reports, in part, “Our findings show an altered brain cytokine profile associated with the manifestation of recent manic/hypomanic episodes in patients with bipolar disorder. Although the causality remains to be established, these findings may suggest a pathophysiological role for IL-1ß in bipolar disorder.”. These studies were published in April and March, 2011, respectively.
Brain tissue from persons with bi-polar disorder also showed increased levels of excitotoxicity and neuroinflammation in Increased excitotoxicity and neuroinflammatory markers in postmortem frontal cortex from bipolar disorder patients (full version), and authors report differential cytokine profiles depending on state of mania, depression, or remission in Comparison of cytokine levels in depressed, manic and euthymic patients with bipolar disorder.
Another disorder based solely around behavior, Tourette syndrome, has increasingly unsurprising findings. Polymorphisms of interleukin 1 gene IL1RN are associated with Tourette syndrome reports “The odds ratio for developing Tourette syndrome in individuals with the IL1RN( *)1 allele, compared with IL1RN( *)2, was 7.65.” (!!!) , and Elevated expression of MCP-1, IL-2 and PTPR-N in basal ganglia of Tourette syndrome cases is yet another example of observations of CNS based immune participation in a disorder that is diagnosed by behavior.
There are also some reviews that perform a cross talk of sorts between disorders; i.e., The mononuclear phagocyte system and its cytokine inflammatory networks in schizophrenia and bipolar disorder, or Immune system to brain signaling: Neuropsychopharmacological implications, published in May 2011, which has this abstract:
There has been an explosion in our knowledge of the pathways and mechanisms by which the immune system can influence the brain and behavior. In the context of inflammation, pro-inflammatory cytokines can access the central nervous system and interact with a cytokine network in the brain to influence virtually every aspect of brain function relevant to behavior including neurotransmitter metabolism, neuroendocrine function, synaptic plasticity, and neurocircuits that regulate mood, motor activity, motivation, anxiety and alarm. Behavioral consequences of these effects of the immune system on the brain include depression, anxiety, fatigue, psychomotor slowing, anorexia, cognitive dysfunction and sleep impairment; symptoms that overlap with those which characterize neuropsychiatric disorders, especially depression. Pathways that appear to be especially important in immune system effects on the brain include the cytokine signaling molecules, p38 mitogen-activated protein kinase and nuclear factor kappa B; indoleamine 2,3 dioxygenase and its downstream metabolites, kynurenine, quinolinic acid and kynurenic acid; the neurotransmitters, serotonin, dopamine and glutamate; and neurocircuits involving the basal ganglia and anterior cingulate cortex. A series of vulnerability factors including aging and obesity as well as chronic stress also appears to interact with immune to brain signaling to exacerbate immunologic contributions to neuropsychiatric disease. The elucidation of the mechanisms by which the immune system influences behavior yields a host of targets for potential therapeutic development as well as informing strategies for the prevention of neuropsychiatric disease in at risk populations.
All of the conditions above, depression, schizophrenia, bi-polar, and tourettes are diagnosed behaviorally; it is only in the last few years that the medical dimension of these disorders were even understood to exist. None of the studies that I referenced above are more than five years old; the idea that behavioral disorders were so closely entangled with the immune system is very, very new. It should be noted that I intentionally left out disorders that also have reams of evidence of immune participation, but which are more degenerative in nature; i.e., Alzheimer’s, ALS, Parkinson’s. When discussing autism, I also left out studies involving aberrant presence of auto-antibodies, of which there are many.
One of the things that I have learned in trying to refine my thought processes during my time on the Internet is that rarely does a single study tell us much about a condition; but the converse also holds true, if we have many studies with different methodologies or measurement end points, but they all reach similar conclusions, then the likely-hood that the findings are accurate is much, much greater. All of the studies I have listed above tell us something similar; that the immune system is clearly, unmistakably playing a part in a lot of conditions classically considered neurological and diagnosed behaviorally. It isn’t enough to nitpick flaws in a single one of the studies in order for ‘correlation does not equal causation’ to make meaningful headway into the implications of these studies; instead, all of the studies above, and lots more, have to be wrong in the same way if we would like to return to a place where we can keep our heads in the sand, hoping for coincidences and bleating out catchphrases in the face of clinical findings. That isn’t going to happen. Given this reality, we should not and cannot ignore the growing evidence of immune abnormalities in the autism population, no matter how inconvenient following that trail of evidence might become.
Increasingly Unsurprising Findings – Microglial Activation and Increased Microglial Density Observed in the Dorsolateral Prefrontal Cortex in Autism – With Bonus Theoretical Pontifications
Posted August 22, 2010on:
Hello friends –
A new paper looking for evidence of an ongoing immune reaction in the brain of people with autism landed the other day, Microglial Activation and Increased Microglial Density Observed in the Dorsolateral Prefrontal Cortex in Autism
BACKGROUND: In the neurodevelopmental disorder autism, several neuroimmune abnormalities have been reported. However, it is unknown whether microglial somal volume or density are altered in the cortex and whether any alteration is associated with age or other potential covariates. METHODS: Microglia in sections from the dorsolateral prefrontal cortex of nonmacrencephalic male cases with autism (n = 13) and control cases (n = 9) were visualized via ionized calcium binding adapter molecule 1 immunohistochemistry. In addition to a neuropathological assessment, microglial cell density was stereologically estimated via optical fractionator and average somal volume was quantified via isotropic nucleator. RESULTS: Microglia appeared markedly activated in 5 of 13 cases with autism, including 2 of 3 under age 6, and marginally activated in an additional 4 of 13 cases. Morphological alterations included somal enlargement, process retraction and thickening, and extension of filopodia from processes. Average microglial somal volume was significantly increased in white matter (p = .013), with a trend in gray matter (p = .098). Microglial cell density was increased in gray matter (p = .002). Seizure history did not influence any activation measure. CONCLUSIONS: The activation profile described represents a neuropathological alteration in a sizeable fraction of cases with autism. Given its early presence, microglial activation may play a central role in the pathogenesis of autism in a substantial proportion of patients. Alternatively, activation may represent a response of the innate neuroimmune system to synaptic, neuronal, or neuronal network disturbances, or reflect genetic and/or environmental abnormalities impacting multiple cellular populations.
This is a neat paper, to my eye not as comprehensive as the landmark paper on microglial activation, Neuroglial Activation and Neuroinflammation in the Brain of Patients with Autism Neuroglial Activation andNeuroinflammation in the Brain of Patientswith Autism, but still a very interesting read. Here are the some areas that caught my eye. From the introduction:
These results provide evidence for microglial activation in autism but stop short of demonstrating quantifiable microglial abnormalities in the cortex, as well as determining the nature of these abnormalities. Somal volume increases are often observed during microglial activation, reflecting a shift toward an amoeboid morphology that is accompanied by retraction and thickening of processes (13). Microglial density may also increase, reflecting either proliferation of resident microglia or increased trafficking of macrophages across a blood-brain barrier opened in response to signaling by cytokines, chemokines, and other immune mediators (13–16). These results provide evidence for microglial activation in autism but stop short of demonstrating quantifiable microglial abnormalitiesin the cortex, as well as determining the nature of these abnormalities. Somal volume increases are often observed during microglial activation, reflecting a shift toward an amoeboid morphology that is accompanied by retraction and thickening ofprocesses (13). Microglial density may also increase, reflecting either proliferation of resident microglia or increased trafficking of macrophages across a blood-brain barrier opened in response to signaling by cytokines, chemokines, and other immune mediators(13–16).
Tragically, my ongoing google based degree in neurology has yet to cover the chapters on specific brain geography, so the finer points, such as the difference between the middle frontal gyri and the neocortex are lost on me. None the less, several things jump out at me from what I have managed to understand so far. The shift to an ‘ameboid morphology’ is one that I’ve run into previously, notably in Early-life programming of later-life brain and behavior: a critical role for the immune system, which is a paper I really need to dedicate an entire post towards, but as applicable here, the general idea is that the microglia undergo structural and functional changes during times of immune response; the ‘ameboid’ morphology is associated with an active immune response. Regarding increased trafficking of macrophages across the BBB, Vargas 2005 noted chemokines (MCP-1) increases in the CNS, so we do have reason to believe such signalling molecules are present.
The authors went on to look for structural changes in microglia, differences in concentration of microglia, and evaluated for markers indicative of an acute inflammatory response. Measurements such as grey and white matter volumes and relationships to microglia structural differences, and correlations with seizure activity were also performed. There were three specimens from children under the age of six that were analyzed as a subgroup to determine if immune activation was present at early ages. From the discussion section:
Moderate to strong alterations in Iba-1 positive microglial morphology indicative of activation (13,29) are present in 5 of 13 postmortem cases with autism, and mild alterations are present in an additional 4 of 13 cases. These alterations are reflected in a significant increase in average microglial somal volume in white matter and microglial density in gray matter, as well as a trend in microglial somal volume in gray matter. These observations appear to reflect a relatively frequent occurrence of cortical microglial activation in autism.
Of particular interest are the alterations present in two thirds of our youngest cases, during a period of early brain overgrowth in the disorder. Indeed, neither microglial somal volume nor density showed significant correlation with age in autism, suggesting long running alteration that is in striking contrast with neuronal features examined in the same cases (Morgan et al., unpublished data, 2009). The early presence of microglial activation indicates it may play a central pathogenic role in some patients with autism.
The authors evaluated for IL-1R1 receptor presence, essentially a marker for an inflammatory response, and found that the values did not differ between the autism population and controls, and that in fact the controls trended towards expressing more IL-1R1 than the autism group. I think this was the opposite of what the authors expected to find.
While Iba-1 staining intensity increases modestly in activated microglia (30), strong staining and fine detail were apparent in Iba-1 positive resting microglia in our samples. Second, there is no increase in microglial colocalization with a receptor, IL-1R1, typically upregulated in acute inflammatory reactions (28). The trend toward an increase in colocalization in control cases may also hint at downregulation of inflammatory signal receptors in a chronically activated system.
I don’t think I’ve seen this type of detail in qualitative measures of the neuroimmune response in autism measured previously, so I definitely appreciate the detail. Furthermore, from a more speculative standpoint, we may have some thoughts on why we might see this in the autism population specifically that I’ll go into detail below a little bit.
The authors failed to find a relationship between seizure activity and microglial activation, which came as a surprise to me, to tell the truth. Also discussed was the large degree of heterogeneity in the findings in so far as the type and severity of microglial morphological differences observed. The potential confounds in the study included an inability to control for medication history, and the cause of death, eight of which were drowning in the autism cases. There was some discussion of potential causes, including, of course, gene-environment interactions, maternal immune activation, neural antibodies, and the idea that “chronic innate immune system activation might gradually produce autoimmune antibodies via the occasional presentation of brain proteins as antigens” (!) There was also this snipet:
Microglial activation might also represent an aberrant event during embryonic monocyte infiltration that may or may not also be reflected in astroglial and neuronal populations (17), given the largely or entirely separate developmental lineage of microglia (13). Alternatively, alterations might reflect an innate neuroimmune response to events in the brain such as excessive early neuron generation or aberrant development of neuronal connectivity.
There is a short discussion of the possible effects of an ongoing microglial immune response, including damage to neural cells, reductions in cells such as Purkinjes, and increases in neurotrophic factors such as BDNF.
This is another illustration of an ongoing immune response in the CNS of the autism population, though in this instance, only some of the treatment group appeared to be affected. It would have been nice to see if there were correlations between behavioral severity and/or specific behavior types, but it would seem that this information is was not available in sufficient quality for this type of analysis, which is likely going to be an ongoing problem with post mortem studies for some time to come. I believe that an effort to develop an autism tissue bank is underway, perhaps eventually some of these logistical problems will be easier to address. The fact that some of the samples were from very young children provides evidence that when present, the neuroinflammatory response is chronic, and indeed, likely lifelong.
Stepping away from the paper proper, I had some thoughts about some of these findings that are difficult to defend with more than a skeletal framework, but have been rattling around my head for a little while. Before we move forward, let’s be clear on a couple of things:
1) The jump from rodent to human is fraught with complications, most of which I doubt we even understand.
2) We can’t be positive that an activated neuroimmune system is the cause of autistic behaviors, as opposed to a result of having autism. I still think a very strong argument can be made that an ongoing immune response is ultimately detrimental, even if it cannot be proven to be completely responsible for the behavioral manifestation of autism.
3) At the end of the day, I’m just Some Jerk On The Internet.
Those caveats made, Morgan et all spend a little time on the potential cause of a persistent neuroinflammatory state as referenced above. One of the ideas, “an aberrant event during embyronic monocyte infiltration that may or may not also be reflected in astroglial and neuronal populations given the largely or entirely separate developmental lineage of microglia” struck me as particularly salient when considered alongside the multitude of data we have concerning the difficult to predict findings regarding an immune insult during critical developmental timeframes.
We now have several papers that dig deeper into the mechanism by which immune interaction during development seem to have physiological effects with some parallels to autism; specifically, Enduring consequences of early-life infection on glial and neural cell genesis within cognitive regions of the brain (Bland et all), and Early-Life Programming of Later-Life Brain and Behavior: A Critical Role for the Immune System (Bilbo et all) ; both of which share Staci Bilbo as an author and I think she is seriously onto something. Here is the abstract for Bland et all:
Systemic infection with Escherichia coli on postnatal day (P) 4 in rats results in significantly altered brain cytokine responses and behavioral changes in adulthood, but only in response to a subsequent immune challenge with lipopolysaccharide [LPS]. The basis for these changes may be long-term changes in glial cell function. We assessed glial and neural cell genesis in the hippocampus, parietal cortex (PAR), and pre-frontal cortex (PFC), in neonates just after the infection, as well as in adulthood in response to LPS. E. coli increased the number of newborn microglia within the hippocampus and PAR compared to controls. The total number of microglia was also significantly increased in E. coli-treated pups, with a concomitant decrease in total proliferation. On P33, there were large decreases in numbers of cells coexpressing BrdU and NeuN in all brain regions of E. coli rats compared to controls. In adulthood, basal neurogenesis within the dentate gyrus (DG) did not differ between groups; however, in response to LPS, there was a decrease in neurogenesis in early-infected rats, but an increase in controls to the same challenge. There were also significantly more microglia in the adult DG of early-infected rats, although microglial proliferation in response to LPS was increased in controls. Taken together, we have provided evidence that systemic infection with E. coli early in life has significant, enduring consequences for brain development and subsequent adult function. These changes include marked alterations in glia, as well as influences on neurogenesis in brain regions important for cognition.
Bland et all went on to theorize on the mechanism by which an infection in early life can have such long lasting effects.
We have hypothesized that the basis for this vulnerability may be long-term changes in glial cell function. Microglia are the primary cytokine producers within the brain, and are an excellent candidate for long-term changes, because they are long-lived and can become and remain activated chronically (Town et al., 2005). There is increasing support for the concept of ‘‘glial priming”, in which cells can become sensitized by an insult, challenge, or injury, such that subsequent responses to a challenge are exaggerated (Perry et al., 2003).
The authors infected some rodents with e-coli on postnatal day four, and then evaluated for microglial function in adulthood.
We have hypothesized that the basis for early-life infection-induced vulnerability to altered cytokine expression and cognitive deficits in adulthood may be due to long-term changes in glial cell function and/or influences on subsequent neural development. E. coli infection on P4 markedly increased microglial proliferation in the CA regions of the hippocampus and PAR of newborn pups, compared to a PBS injection (Figs. 3 and 4). The total number of microglia, and specifically microglia with an ‘‘active” morphology (amoeboid, with thick processes), were also increased as a consence of infection. There was a concomitant decrease in non microglial newborn cells (BrdU + only) in the early-infected rats, in the same regions.
Check that shit out! Rodents infected with E-coli during the neonatal period had an increased number of active microglia when compared to rodents that got saline as neonates. Keep in mind that the backbone of these studies, and studies from other groups indicate that this persistence of effects are not specific to an e-coli infection, but rather, can be triggered by any immune response during critical timeframes. In fact, at least two studies have employed anti-inflammatory agents, and observed an attenuation of effect regarding seizure susceptibility.
A final snipet from Bland et all Discussion section:
Although the mechanisms remain largely unknown, the ‘‘glial cell priming” hypothesis posits that these cells have the capacity to become chronically sensitized by an inflammatory event within the brain (Perry et al., 2003). We assessed whether glial priming may be a likely factor in the current study by measuring the volume of each counted microglial cell within our stereological analysis. The morphology of primed glial cells is similar to that of ‘‘activated” cells (e.g., amoeboid, phagocytic), but primed glial cells do not chronically produce cytokines and other pro-inflammatory mediators typical of cells in an activated state. There was a striking increase in cell volume within the CA1 region of adult rats infected as neonates (Figs. 2 and 8), the same region in which a marked increase in newborn glia was observed at P6. These data are consistent with the hypothesis that an inflammatory environment early in life may prime the surviving cells long-term, such that they over-respond to a second challenge, which we have demonstrated at the mRNA level in previous studies (Bilbo et al., 2005a, 2007; Bilbo and Schwarz, in press).
The concept of glial priming, close friends with the ‘two hit’ hypothesis (or soon to be, the multi-hit hypothesis?), has some other very neat studies behind it, the coolest ones I’ve found so far are from a group at Northwestern, and include “hits” such as Glial activation links early-life seizures and long-term neurologic dysfunction: evidence using a small molecule inhibitor of proinflammatory cytokine upregulation, Enhanced microglial activation and proinflammatory cytokine upregulation are linked to increased susceptibility to seizures and neurologic injury in a ‘two-hit’ seizure model and Minozac treatment prevents increased seizure susceptibility in a mouse “two-hit” model of closed skull traumatic brain injury and electroconvulsive shock-induced seizures. Also the tragically, hilariously titled, Neonatal lipopolysaccharide and adult stress exposure predisposes rats to anxiety-like behaviour and blunted corticosterone responses: implications for the double-hit hypothesis. (!) These are potentially very inconvenient findings, the details for which I’ll save for another post.
Moving on to Bilbo et all, though a pure review paper than an experiment, it provides additional detailed theories on the mechanisms behind persistent effects of early life immune challenge. Here’s the abstract:
The immune system is well characterized for its critical role in host defense. Far beyond this limited role however, there is mounting evidence for the vital role the immune system plays within the brain, in both normal, “homeostatic” processes (e.g., sleep, metabolism, memory), as well as in pathology, when the dysregulation of immune molecules may occur. This recognition is especially critical in the area of brain development. Microglia and astrocytes, the primary immunocompetent cells of the CNS, are involved in every major aspect of brain development and function, including synaptogenesis, apoptosis, and angiogenesis. Cytokines such as tumor necrosis factor (TNF)α, interleukin [IL]-1β, and IL-6 are produced by glia within the CNS, and are implicated in synaptic formation and scaling, long-term potentiation, and neurogenesis. Importantly, cytokines are involved in both injury and repair, and the conditions underlying these distinct outcomes are under intense investigation and debate. Evidence from both animal and human studies implicates the immune system in a number of disorders with known or suspected developmental origins, including schizophrenia, anxiety/depression, and cognitive dysfunction. We review the evidence that infection during the perinatal period of life acts as a vulnerability factor for later-life alterations in cytokine production, and marked changes in cognitive and affective behaviors throughout the remainder of the lifespan. We also discuss the hypothesis that long-term changes in brain glial cell function underlie this vulnerability.
Bilbo et all go on to discuss the potential for time sensitive insults that could result in an altered microglial function. Anyone that has been paying attention should know that the concept of time dependent effects is, to my mind, the biggest blind spot in our existing research concerning autism and everyones favorite environmental agent.
Is there a sensitive period? Does an immune challenge early in life influence brain and behavior in a way that depends on developmental processes? Since 2000 alone, there have been numerous reports in the animal literature of perinatal immune challenges ranging from early gestation to the juvenile period, and their consequences for adult offspring phenotypes (see Table 1). It is clear that the timing of a challenge is likely a critical factor for later outcomes, impacting the distinct developmental time courses of different brain regions and their underlying mechanisms (e.g., neurotransmitter system development, synapse formation, glial and neural cell genesis, etc; Herlenius and Lagercrantz, 2004; Stead et al., 2006). However, the original question of whether these changes depend on development has been surprisingly little addressed. We have demonstrated that infection on P30 does not result in memory impairments later in life (Bilbo et al., 2006), nor does it induce the long-term changes in glial activation and cytokine expression observed with a P4 infection (Bilbo et al., unpublished data). The factors defining this “sensitive period” are undoubtedly many, as suggested above. However, our working hypothesis is that one primary reason the early postnatal period in rats is a sensitive or critical period for later-life vulnerabilities to immune stimuli, is because the glia themselves are functionally different at this time. Several studies have demonstrated that amoeboid, “macrophage-like”, microglia first appear in the rat brain no earlier than E14, and steadily increase in density until about P7. By P15 they have largely transitioned to a ramified, adult morphology. Thus, the peak in density and amoeboid morphology (and function) occurs within the first postnatal week, with slight variability depending on brain region (Giulian et al., 1988; Wu et al., 1992). [emphasis theirs]
[Note: The authors go on to state that this time period is likely developmentally equivalent to the late second, to early third trimester of human fetal development.]
We seem to have a growing abundance of evidence that immune stimulation in utero can have neurological impacts on the fetus that include schizophrenia, and autism. In some instances, we have specific viral triggers; i.e., the flu or rubella, but I’d further posit that we have increasing reason to believe that any immune response can have a similar effect. The Patterson studies involving IL-6 in a rodent model of maternal activation seem to make this point with particular grace, as the use of IL-6 knockout mice attenuated the effect, as did IL-6 antibodies; and direct injection of IL-6 in the absence of actual infection produced similar outcomes. In animal models designed to study a variety of effects, we have a veritable spectrum of studies that tell us that immune insults during critical developmental timeframes can have lifelong effects on neuroimmune activity, HPA-axis reactions, seizure susceptibility, and ultimately, altered behaviors. I believe that we are rapidly approaching a point where there will be little question as towards if a robust immune response during development can lead to a developmental trajectory that includes autism, and will instead be faced with attempting to detangle the more subtle, and inconvenient, mechanisms of action, temporal windows of vulnerability, and indeed if there are subgroups of individuals that are predisposed to be more likely to suffer from such an insult.
Another thing that struck me about Morgan was the speculation that an increased presence of IL-1R in controls may have been suggestive of an attempt to muzzle the immune response in the case group; repeated from Morgan “The trend toward an increase in colocalization in control cases may also hint at downregulation of inflammatory signal receptors in a chronically activated system.” In other words, for controls it wasn’t a big deal to be expressing IL-1R in a ‘normal’ fashion, because the immune system is in a state of balance. Another way of looking at our observations would be to ask the question as towards what has caused the normally self regulating immune system to fail to return to a state of homeostasis? Ramping up an immune response to fight off pathogens and ratcheting back down to avoid unnecessary problems is something most peoples immune systems do with regularity. Is the immune system in autism trying to shut down unsuccessfully?
There are clues that the homeostatic mechanisms are trying to restore a balanced system. For example, in Immune transcriptome alterations in the temporal cortex of subjects with autism, researchers reported that the genetic pathway analysis reveals a pattern that could be consistent with “an inability to attenuate a cytokine activation signal.” Another paper that I need to spend some read in full, Involvement of the PRKCB1 gene in autistic disorder: significant genetic association and reduced neocortical gene expression describes a genetic and expression based study that concludes, in part, that downregulation of PRKCB1 “could represent a compensatory adjustment aimed at limiting an ongoing dysreactive immune process“.
If we look to clinical evidence for a decreased capacity to regulate an immune response, one paper that might help is Decreased transforming growth factor beta1 in autism: a potential link between immune dysregulation and impairment in clinical behavioral outcomes, the authors report an inverse dose relationship between peripheral levels of an important immune regulator, TGF-Beta1, and autism severity; i.e., the less TGF-Beta1 in a subject, the worse the autism behaviors [the autism group also, as a whole, had less TGF-Beta1 than the controls].
And then, in between the time that Morgan came out, and I completed this posting, another paper hit my inbox that might provide some clues,a title that is filled to the brim with autism soundbytes, “Effects of mitochondrial dysfunction on the immunological properties of microglia“. The whole Hannah Poling thing seemed so contrived to me, basically two sets of people trying to argue past each other to reach a predetermined conclusions, and as a result, I’ve largely shied away from digging too deeply into the mitochondrial angle. This may not be a luxury I have anymore after reading Ferger et all. For our purposes, lets forget about classically diagnosed and acute mitochonrdrial disease, as Hannah Poling supposedly has, and just acknowledge that we have several studies that show that children with autism seem to have signs of mitochondrial dysfunction, as I understand it, sort of a halfway between normal mitochondrial processing and full blown mitochondrial disorder. Given that, what does Ferger tell us? Essentially an in vitro study, the group took microglial cells from mice, exposed some of them to toxins known to interferre with the electron transport chain, and exposed the same cells to either LPS or IL-4 to measure the subsequent immunological response. What they observed was that the response to LPS was unchanged, but the response to IL-4, was blunted; and pertinently for our case, the IL-4 response is a so called ‘alternative’ immune response, that participates in shutting down the immune response. From the conclusion of Ferger:
In summary, we have shown that mitochondrial dysfunction in mouse microglial cells inhibit some aspects of alternative activation, whereas classic activation seems to remain unchanged. If, in neurological diseases, microglial cells are also affected by mitochondrial dysfunction, they might not be able to induce a full anti-inflammatory alternative response and thereby exacerbate neuroinflammation. This would be associated with detrimental effects for the CNS since wound healing and attenuation of inflammation would be impaired.
If our model of interest is autism, our findings can begin to fit together with remarkable elegance. And we haven’t even gone over our numerous studies that show the flip side of the immunological coin; that children with autism have been shown time and time again to have a tendency towards an exaggerated immune response, and increased baseline pro-inflammatory cytokines when compared with their non diagnosed peers!
Anyways, those are my bonus theoretical pontifications regarding Morgan.
Implications for Autism Or Just Interesting? “Epigenetic and immune function profiles associated with posttraumatic stress disorder”
Posted June 25, 2010on:
Hello friends –
One of my tangential pubmed alerts notified me of this study the other day: Epigenetic and immune function profiles associated with posttraumatic stress disorder
The biologic underpinnings of posttraumatic stress disorder (PTSD) have not been fully elucidated. Previous work suggests that alterations in the immune system are characteristic of the disorder. Identifying the biologic mechanisms by which such alterations occur could provide fundamental insights into the etiology and treatment of PTSD. Here we identify specific epigenetic profiles underlying immune system changes associated with PTSD. Using blood samples (n = 100) obtained from an ongoing, prospective epidemiologic study in Detroit, the Detroit Neighborhood Health Study, we applied methylation microarrays to assay CpG sites from more than 14,000 genes among 23 PTSD-affected and 77 PTSD-unaffected individuals. We show that immune system functions are significantly overrepresented among the annotations associated with genes uniquely unmethylated among those with PTSD. We further demonstrate that genes whose methylation levels are significantly and negatively correlated with traumatic burden show a similar strong signal of immune function among the PTSD affected. The observed epigenetic variability in immune function by PTSD is corroborated using an independent biologic marker of immune response to infection, CMV—a typically latent herpesvirus whose activity was significantly higher among those with PTSD. This report of peripheral epigenomic and CMV profiles associated with mental illness suggests a biologic model of PTSD etiology in which an externally experienced traumatic event induces downstream alterations in immune function by reducing methylation levels of immune-related genes.
Essentially the authors took a bunch of people that are more likely to experience stressful situations and PTSD, urban Detroit residents, who amazingly report PTSD symptoms at twice the level that previous studies have found in analysis of larger areas. [Apparently, getting physically attacked is more common there, which gives rise to PTSD even more than ‘other traumatic event types’, and was reported by 50% of the participants from a larger study which formed the population pool of this study. (!!)] With this population base, blood was drawn and methylation profiles were analyzed between participants who reported PTSD symptoms (n=23) and those who ‘only’ had ‘potentially traumatic events’ (PTE). PTSD and ‘controls’ where matched by race, age, sex, and blood profiles.
Once methylation levels were identified, a functional annotation clustering analysis was performed, which I believe is similar a pathway analysis; essentially a bioinformatic tool to gain insight into which biological functions were being manipulated as a result of differential methylation of the genome. This is a powerful new tool in discerning what is happening in autism and elsewhere, and I expect it will provide some surprising answers in the future. Here is their text on what they found:
Consistent with previous findings from gene expression (4, 5) and psychoeneuroimmunologic studies (3), each of the top three FACs determined from uniquely unmethylated genes among PTSD-affected individuals shows a strong signature of immune system involvement. This signature includes genes from the innate immune system (e.g.,TLR1 andTLR3), as well as from genes that regulate innate and adaptive immune system processes (e.g., IL8, LTA, and KLRG-1). In contrast, pathways and processes relevant to organismal development in general—and neurogenesis in particular—figure prominently among the genes uniquely unmethylated in the PTSD-unaffected group (e.g., CNTN2 and TUBB2B; Fig. S2). Notably, similar clusters were obtained using an alternative approach based on genes differentially methylated between the two groups at P < 0.01, with annotations in the top five FACs that include signal, cell proliferation, developmental process, neurologic system process, and inflammatory response
Keeping in mind that reduced methylation results in increased gene expression, if we take a look at Table 1, some of the parallels to autism jump out a little more robustly:
In the ‘Uniquely Unmethylated’ (i.e., higher expression), area, we find that participants affected by PTSD had showed greater enrichment in genes related to the immune response, and specifically the inflammatory response and innate immune response. Our evidence for similar immunological profiles in the autism realm is deep, and includes multiple observations of an active immune response in the CNS, highly significant over expression of genes related to immune function in the CNS, several observations of known upregulators of the innate immune response that are associated with inflammatory conditions, and multiple studies finding an exaggerated innate immune response in vitro when compared to controls. The correlations with developmental process and neuron creation are pretty straightforward.
In the ‘Uniquely Methylated’ area (i.e., lower expression), the sensory perception differences hit close to home, and xenobiotic metabolism has been implicated by several studies.
Going further, the researchers attempted to evaluate for correlations between the number of potentially traumatic experiences and the methylation profile, and somewhat unsurprisingly found that as the number of experiences increased, the methylation differentials showed wider variation.
Here again we see a distinct signature of immune-related methylation profiles among the PTSD-affected group only. More specifically, we see methylation profiles that are suggestive of immune activation among persons with more PTE exposure in the genes that are significantly negatively correlated with increasing number of PTEs—a pattern reflective of that observed for the uniquely unmethylated genes in this same group (Table 1).
From the discussion section:
Among the many analyses performed in this work, the immune related functions identified in the PTSD-affected group were consistently identified only among gene sets with relatively lower levels of methylation (Tables 1 and 2). Demethylation has previously been shown to correlate with increased expression in several immune system–related genes (reviewed in ref. 22), including some identified here [e.g., IL8 (23)]. In contrast, methylation profiles among the PTSD-unaffected are distinguished by neurogenesis-related functional annotations. Neural progenitor cells have previously been identified in the adult human hippocampus (24); however, stress can inhibit cell proliferation and neurogenesis in this brain region (reviewed in ref. 25), and recent work suggests that adult neurogenesis may be regulated by components of the immune system (reviewed in ref. 26). Thus, immune dysfunction among persons with PTSD may be influenced by epigenetic profiles that are suggestive of immune activation or enhancement and also by an absence of epigenetic profiles that would be consistent with the development of normal neural-immune interactions (27).
Among the genes uniquely methylated in the PTSD-affected group, it is striking that the second most enriched cluster—sensory perception of sound—directly reflects one of the three major symptom clusters that define the disorder (Fig. 3B). Genes in this FAC thatmay be particularly salient to this symptom domain include otospiralin (OTOS),which shows decreased expression in guinea pigs after acoustic stress (28) and otoferlin (OTOF), mutations in which have been linked to nonsyndromic hearing loss in humans (29). Exaggerated acoustic startle responses, often measured via heart rate or skin conductance after exposure to a sudden, loud tone, have been well documented among the PTSD affected (30) and are indicative of a hyperarousal state that characterizes this symptom domain. Notably, prospective studies have demonstrated that an elevated startle response is a consequence of having PTSD, because the response was not present immediately after exposure to trauma but developed with time among trauma survivors who developed the disorder (30, 31).
My son had some very severe auditory related problems earlier in his life, and still occasionally struggles with either sudden loud noises, or some very specific noises, such as some dog barks, or the sound of an infant crying. Previously the only physiology based attempt at an explanation I’d heard of for this type of response involved fine grained brain architecture and consequent filtering and/or overexcitation problems. The idea that sound sensitivities in particular can be obtained environmentally is of particular interest to the autism community.
From the common sense angle, I find this completely fascinating; we’ve known for a long time that living with consistent stress is bad for you with a variety of nasty endpoints, but this type of finding narrows down the means by which this happens. In the far off future, perhaps targeted methyl affecting drugs could be considered for people who experience extremely stressful events, as sort of a ‘PTSD vaccine’ [hehe] could be developed.
From an ASD perspective, increased feeling of anxiety, or just generally being ‘stressed out’ is a consistent finding both in research and from what I’ve read of readings from people with autism on the Internet. I’ve seen several explanations, with sensory based problems being mentioned several times. From a biological standpoint we seem to have a growing body of evidence of an abnormally regulated stress response in the autism cohort. An internet friend of mine, Loftmatt, has written extensively on his thoughts concerning the increase in stress in modern society and the mechanisms by which this could be contributing to our apparent observations of an increase in autism. This study would seem to provide insight towards a possible mechanism by which a frequent state of stress could lead to some of our immunological findings in the autism realm; a possibility I hadn’t considered previously when trying to detangle a means by which our observations of immune activation were not participating in autistic behavior. The thought of a feedback loop also strikes me looking at this, something causes a feeling of extreme stress, which leads to abnormal methylation levels and genetic expression, which leads to increased physiological (and behavioral?) alterations, and even more stress.
I may try to poke through the supplementary materials to see if any specific genes or pathways found to be differentially regulated have parallels in some of the other studies we’ve seen recently such as Garbett or Hu, although this may be somewhat of a crapshoot unless I could figure out how to actually submit gene lists to GSEA and read the responses.
And we may need to consider the possibility that these types of effects can be trans-generational. One of the most fascinating studies I’ve seen on epigentics involved exactly that, a multi-generational effect of famine in Holland, wherein the grandchildren of women who were pregnant during a time of famine bore striking differences in a variety of endpoints compared to children whose grandmothers were not pregnant during that time.
The more we learn, the more complicated the world becomes.
Posted June 7, 2010on:
Hello friends –
I’ve been referencing this paper in some discussions online for a while; I’ve read it, and in fact, while working on another project, got the opportunity to speak with one of the authors of the paper. It’s a very cool paper with a lot of information in it, some of which, could be considered inconvenient findings. Here is the abstract:
Autism is a severe disorder that involves both genetic and environmental factors. Expression profiling of the superior temporal gyrus of six autistic subjects and matched controls revealed increased transcript levels of many immune system related genes. We also noticed changes in transcripts related to cell communication, differentiation, cell cycle regulation and chaperone systems. Critical expression changes were confirmed by qPCR (BCL6, CHI3L1, CYR61, IFI16, IFITM3, MAP2K3, PTDSR, RFX4, SPP1, RELN, NOTCH2, RIT1, SFN, GADD45B, HSPA6, HSPB8and SERPINH1). Overall, these expression patterns appear to be more associated with the late recovery phase of autoimmune brain disorders, than with the innate immune response characteristic of neurodegenerative diseases. Moreover, a variance-based analysis revealed much greater transcript variability in brains from autistic subjects compared to the control group, suggesting that these genes may represent autism susceptibility genes and should be assessed in follow-up genetic studies.
(emphasis is mine) [Full paper freely available from that link]
I am particularly intrigued by the second bolded sentence regarding the “these expression patterns appear to be more associated with the later recovery phase of autoimmune brain disorders, than the innate immune response characteristic of neurodegenerative diseases”. I’ve had it put to me previously that we should not necessarily implicate neuroinflammation in autism, the argument being that even though we had evidence of chronically activated microglia, what we do not seem to have evidence for is actual damage to the brain, and ergo, the neuroinflammation may actually be a byproduct of having autism, as opposed to playing a causative role, or that in fact, the neuroinflammation might even be beneficial. There have been some other places where the claim has been made that because our profile of neuroinflammation doesn’t match more classically recognized neurodegenerative disorders (i.e., MS/Alzheimer’s/Parkinson’s), that therefore, certain environmental agents need not be fully investigated as a potential contributor to autism. This is the first time that I am aware that someone has attempted to classify the neuroinflammatory pattern observed in autism not only as distinctly different from classical neurodegenerative diseases, but to also go so far as to provide a more refined example.
From the Introduction:
In order to better understand the molecular changes associated with ASD, we assessed the transcriptome of the temporal cortex of postmortem brains from autistic subjects and compared it to matched healthy controls. This assessment was performed using oligonucleotide DNA microarrays on six autistic-control pairs. While the sample size is limited by the availability of high-quality RNA from postmortem subjects with ASD, this sample size is sufficient to uncover robust and relatively uniform changes that may be characteristic of the majority of subjects. Our study revealed a dramatic increase in the expression of immune system-related genes. Furthermore, transcripts of genes involved in cell communication, differentiation, cell cycle regulation and cell death were also profoundly affected. Many of the genes altered in the temporal cortex of autistic subjects are part of the cytokine signaling/regulatory pathway, suggesting that a dysreactive immune process is a critical driver of the observed ASD-related transcriptome profile.
I was initially very skeptical about this, with a sample set so small, wasn’t it difficult to ascertain if their findings were by chance or not? It turns out, the answer depends on the type of datapoint you are evaluating against. A powerful tool in use by the researchers is a recent addition to the genetic analysis research suite, not only the ability to scan for thousands of gene activity levels simultaneously, but the use of known gene networks to identify if among those thousands of results, related genes are being expressed differentially. This is important for some amazingly robust findings presented later in the paper, so lets sidetrack a little bit. Here is a nice overview of the process being used:
Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation. A common approach involves focusing on a handful of genes at the top and bottom of L (i.e., those showing the largest difference) to discern telltale biological clues. This approach has a few major limitations.
(i) After correcting for multiple hypotheses testing, no individual gene may meet the threshold for statistical significance, because the relevant biological differences are modest relative to the noise inherent to the microarray technology.
(ii) Alternatively, one may be left with a long list of statistically significant genes without any unifying biological theme. Interpretation can be daunting and ad hoc, being dependent on a biologist’s area of expertise.
(iii) Single-gene analysis may miss important effects on pathways. Cellular processes often affect sets of genes acting in concert. An increase of 20% in all genes encoding members of a metabolic pathway may dramatically alter the flux through the pathway and may be more important than a 20-fold increase in a single gene.
(iv) When different groups study the same biological system, the list of statistically significant genes from the two studies may show distressingly little overlap (3).
So, back to Garbett, not only did the authors find a great number of genes overexpressed in the autism group (and a smaller number, underexpressed), when they threw their thousands of results of individual genes into the GSEA, what came back was that several genetic pathways were very significantly altered, many of them immune mediated. This is a big step in understanding in my opinion. I believe we have likely come full circle on our understanding of very high penetrance genes that might be driving towards a developmental trajectory of autism; i.e., Rhett, Fragile-X. But using this technique we can determine if entire biological pathways are altered by measuring the output of genes. Specifically the point made in bullet (iii) stands out to me; having a twenty fold increase in a single gene might not be too big a deal if the other participants in the proteins function cannot be altered by twenty fold as a result due to other rate limiting constraints; but if we can see related sets of genes with similar expression profiles, we can get a much better picture of the biological results of different expression.
The methods get dense pretty quickly, but are worth a shot to show how thorough the researchers were to insure that their findings were likely to be signficant. Essentially they performed three different statistical tests against their results of differentially expressed genes and broke their results down into genes that passed all three tests, two of three test, or one of three tests. Furthermore, a selected twenty genes were targeted with qPCR validation, which in all cases showed the expected directionality; i.e., if the expression was increased in the transcriptome analysis, qPCR analysis confirmed the increased expression.To provide another benchmark, they tested for other genes known to be associated with autism, REELN, and GFAP and found results consistent with other papers.
Having determined a large number of differentially expressed genes, the authors then went to try to analyze the known function of these genes.
These classifications were performed on a selected gene set that is differentially expressed between AUT and CONT subjects; based on the success of our qPCR validation, we decided to perform this analysis using transcripts that both reported an |ALR>1| and that reached p<0.05 in at least 2/3 statistical significance comparisons. Of 221 such transcripts, 186 had increased expression in AUT compared to CONT, while only 35 genes showed reduced expression in the AUT samples. We subjected these transcripts to an extensive literature search and observed that 72 out of 193 (37.3%) annotated and differentially expressed transcripts were either immune system related or cytokine responsive transcripts (Supplemental Material 2). Following this first classification, we were able to more precisely sub-classify these 72 annotated genes into three major functional subcategories, which overlap to a different degree; 1) cell communication and motility, 2) cell fate and differentiation, and 3) chaperones (Figure 3). The deregulation of these gene pathways might indicate that the profound molecular differences observed in the temporal cortex of autistic subjects possibly originate from an inability to attenuate a cytokine activation signal.
That last sentence packs a lot of punch for a couple of reasons. It would seem to be consistent with their statements regarding a “late recovery phase” of an autoimmune disorder; i.e., an immune response was initiated at some point in the past, but has yet to be completely silenced. This also isn’t the first time that the idea of problems regulating an immune response (i.e., the inability to attenuate a cytokine activation signal) has been suggested from clinical findings, for example, in Decreased transforming Growth Factor Beta1 in Autism: A Potential Link Between Immune Dysregulation and Impairment in Clinical Behavioral Outcomes, the authors found an inverse correlation between TGF-Beta1 and autism behavioral severity:
Given that a major role of TGFβ1 is to control inflammation, the negative correlations observed for TGFβ1 and behaviors may suggest that there is increased inflammation and/or ongoing inflammatory processes in subjects that exhibit higher (worse) behavioral scores.
As such, TGFβ has often been considered as one of the crucial regulators within the immune system and a key mediator in the development of autoimmune and systemic inflammation.
In summary, this study demonstrates that there is a significant reduction in TGFβ1 levels in the plasma of young children who have ASD compared with typically developing children and with non-ASD developmentally delayed controls who were frequency-matched on age. Such immune dysregulation may predispose to the development of autoimmunity and/or adverse neuroimmune interactions that could occur during critical windows in development.
[full paper from the link]
The theme of a critical window of development and enduring consequences of insults during that window is one that is getting more and more attention recently; this is an area that is going to get more and more attention as time goes by, and eventually, as the clinical data continues to pile up, meaningless taglines aren’t going to be enough to keep us from dispassionately evaluating our actions.
The Discussion section is particularly nice, I’ll try not to just quote the entire thing. Here are the really juicy parts.
The results of our study suggest that 1) in autism, transcript induction events greatly outnumbers transcript repression processes; 2) the neocortical transcriptome of autistic individuals is characterized by a strong immune response; 3) the transcription of genes related to cell communication, differentiation and cell cycle regulation is altered, putatively in an immune system-dependent manner, and 4) transcriptome variability is increased among autistic subjects, as compared to matched controls. Furthermore, our study also provides additional support for previously reported involvement of MET, GAD1, GFAP, RELN and other genes in the pathophysiology of autism. While the findings were obtained on a limited sample size, the statistical power, together with the previously reported postmortem data by other investigators suggest that the observed gene expression changes are likely to be critically related to the pathophysiology seen in the brain of the majority of ASD patients.
There is some description of studies using gene expression testing in the autism realm where the authors ultimately conclude that technical and methodological differences between the studies make them difficult to tie together coherently. There is another small section re-iterating the findings that were similar to single gene studies; i.e., REELN, MET, and GAD genes.
The most prominent expression changes in our dataset are clearly related to neuroimmune disturbances in the cortical tissue of autistic subjects. The idea of brain inflammatory changes in autism is not novel; epidemiological, (DeLong et al., 1981; Yamashita et al., 2003; Libbey et al., 2005) serological studies (Vargas et al., 2005; Ashwood et al., 2006) and postmortem studies (Pardo et al., 2005; Vargas et al., 2005; Korkmaz et al., 2006) over the last 10 years have provided compelling evidence that immune system response is an essential contributor to the pathophysiology of this disorder (Ashwood et al. 2006). Finally, converging post-mortem assessments and measurements of cytokines in the CSF of autistic children (Vargas et al., 2005), may indicating an ongoing immunological process involving multiple brain regions
Nothing really new here to anyone that is paying attention, but good information for the extremely common, gross oversimplification that ‘immune abnormalities’ have been found in autism, but we don’t have any good reason to think they may be part of the problem. Of course, this is an argument you’ll see a lot of the time regarding everyone’s favorite environmental agent.
Altered immune system genes are often observed across various brain disorders, albeit there are notable differences between the observed transcriptome patterns. The majority of neuroimmune genes found activated in the autistic brains overlap with mouse genes that are activated during the late recovery or “repair” phase in experimental autoimmune encephalomyelitis (Baranzini et al., 2005). This suggests a presence of an innate immune response in autism. However, the altered IL2RB, TH1TH2, and FAS pathways suggest a simultaneously occurring, T cell-mediated acquired immune response. Based on these combined findings we propose that the expression pattern in the autistic brains resembles a late stage autoimmune event rather than an acute autoimmune response or a non-specific immune activation seen in neurodegenerative diseases. Furthermore, the presence of an acquired immune component could conceivably point toward a potential viral trigger for an early-onset chronic autoimmune process leading to altered neurodevelopment and to persistent immune activation in the brain. Interestingly, recently obtained gene expression signatures of subjects with schizophrenia (Arion et al., 2007) show a partial, but important overlap with the altered neuroimmune genes found here in autism. These commonly observed immune changes may represent a long-lasting consequence of a shared, early life immune challenge, perhaps occurring at different developmental stages and thus affecting different brain regions, or yielding distinct clinical phenotypes due to different underlying premorbid genetic backgrounds.
The last sentence, regarding ‘long-lasting’ consequences of early life immune challenges is one that has a large, and growing body of evidence in the literature that report physiological and behavioral similarities to autism. We also have recent evidence that hospitilization for viral or bacterial infection during childhood is associated with an autism diagnosis. There is, of course, a liberal sprinkling of ‘mays’, ‘propose’, and ‘conceivably’ caveats in place here.
Earlier I mentioned that the authors studied gene networks in addition to single gene expressions., and that some of those findings were very significant. The results of this are found in Table 2. In one discussion, I had it pointed out to me by ScienceMom that it appeared that some of the networks were not found to be statistically significant (and ergo we should not necessarily assume that immune dysfunction was a participant in autism). [If you look at Table 2, some networks like a p value of 0000]. I decided to use the data in this paper for another project that isn’t ready yet, but in that process I was able to speak directly with one of the authors of this paper. I asked him about this, and he told me that this was a function of space limitations; all of the gene networks described were found to be statistically signficant, but in some instances there wasn’t enough space to typeset the p value. In fact, some networks were found to be differentially expressed with a p-value of .000000000000001. (!!!!!!!!) That isn’t a value that you see very often.
I recently got a copy of Mitochondrial dysfunction in Autism Spectrum Disorders: cause or effect, which shares an author with this paper, Persico. In that paper, they reference Immune Transcriptome Alterations In the Temporal Cortex of Subjects With Autism, invoking a potential cascade effect of prenatal immune challenge, inherited calcium transport deficiencies, and resultant mitochondrial dysfunction that could lead to autism. I’ve generally stayed away from the mitochondria stuff in the discussion realm; even though I think it is probably somewhat important to some children, and critically important to a select few children, I’ve mostly found that the discussion of mitochondrial issues is comprised of two sets of people talking past one another so as to prove something, or disprove something about everyones favorite environmental agent; but this is a neat paper that I’d like to get to eventually.