Posts Tagged ‘Immune’
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.
A Sense Of Relief After (Some Of) Your Phantoms Are Observed By Others, A Distillation of Humbling Complex Early Life Neuroimmune Literature: “Microglia in the developing brain: a potential target with lifetime effects”, and The Need For Dispassionate Analysis
Posted June 15, 2012on:
Hello friends –
I have a confession to make. The fact that a lot of very smart people have ignored or flat out laughed at my arguments bothers me sometimes. I have applied non-trivial, not to be rebated time and effort to put forth what I considered to be logical views, scientifically defendable and important ideas; and yet people I knew were otherwise rational, and in some cases, very intelligent, just hadn’t seemed to get what I was saying. Often this was within the context of a discussion argument of vaccination, but my larger concern, that of a non-imaginary, non-trivial increase in children with autism in the past decades, also usually falls on deaf ears. If “environmental changes” incorporate the chemical milieu of our mother’s wombs, the microbial world our infants are born into, or the ocean of synthetic chemicals we all swim through every day, we have no rational conclusion but that our environment has changed a lot in the past few decades. Considered within the context of the reality based model where the events of early life can be disproportionally amplified through the lifetime of an organism, clinging to the idea that there has been a stable incidence of autism seems dangerously naïve, at most charitable.
And yet, for the most part, many or most of the people who are alarmed are crackpots. There were times I questioned myself. Am I missing something? Am I chasing phantoms? Why aren’t any of these other smart people as worried as I am?
A while ago I got a copy of Microglia in the developing brain: A potential target with lifetime effects (Harry et all), a paper that tells me that if nothing else, I have some good company in pondering the potential for disturbances in early life to uniquely affect developmental outcome, in this instance through alterations to the neuroimmune system. If I am incorrect about the validity of a developmental programming model with lifetime effects, lots of prolific researchers are wrong about the same thing in the same way. Harry is a very thorough (and terrifying) review of the relevant literature. Here is the abstract:
Microglia are a heterogenous group of monocyte-derived cells serving multiple roles within the brain, many of which are associated with immune and macrophage like properties. These cells are known to serve a critical role during brain injury and to maintain homeostasis; yet, their defined roles during development have yet to be elucidated. Microglial actions appear to influence events associated with neuronal proliferation and differentiation during development, as well as, contribute to processes associated with the removal of dying neurons or cellular debris and management of synaptic connections. These long-lived cells display changes during injury and with aging that are critical to the maintenance of the neuronal environment over the lifespan of the organism. These processes may be altered by changes in the colonization of the brain or by inflammatory events during development. This review addresses the role of microglia during brain development, both structurally and functionally, as well as the inherent vulnerability of the developing nervous system. A framework is presented considering microglia as a critical nervous system-specific cell that can influence multiple aspects of brain development (e.g., vascularization, synaptogenesis, and myelination) and have a long term impact on the functional vulnerability of the nervous system to a subsequent insult, whether environmental, physical, age-related, or disease-related.
The body of Microglia in the developing brain: A potential target with lifetime effects has tons of great stuff. From the Introduction
The evidence of microglia activation in the developing brain of patients with neurodevelopmental disorders(e.g., autism) and linkage to human disease processes that have a developmental basis (schizophrenia) have raised questions as to whether developmental neuroinflammation actively contributes to the disease process. While much of the available data represent associative rather than causative factors, it raises interesting questions regarding the role of these ‘‘immune-type’’ cells during normal brain development and changes that may occur with developmental disorders. Within the area of developmental neurotoxicology, the potential for environmental factors or pharmacological agents to directly alter microglia function presents a new set of questions regarding the impact on brain development.
There is a short section on what is known about the colonization of the brain by microglia, it is a busy, busy environment, and while we are just scratching the surface, microglia seem to be involved in scads of uber-critical operations, many of which pop up in the autism literature. It is just being confirmed that microglia constitute a distinct developmental path that diverges as an embryo, two papers from 2007 and 2010 are referenced as reasons we now believe microglia are a population of cells that migrate into the CNS before birth and are not replaced from the periphery in adulthood. From there, the beautiful complexity is in full effect; as the microglia develop and populate the brain there are specific spatial and morphological conditions, microglia are first evident at thirteen weeks after conception, and do not reach a stable pattern until after birth. In fact, it appears that microglia aren’t done finishing their distribution in the CNS until the postnatal period, “With birth, and during the first few postpartum weeks, microglia disseminate throughout all parts of the brain, occupying defined spatial territories without significant overlap (Rezaie and Male, 2003) suggesting a defined area of surveillance for each cell.”
It occurred to me to wonder if there are differences in microglia settlement patterns in males and females in human infants, as has been observed in other models? Could a spatially or temporally different number of micoglia, or different developmental profiles of microglia based on sex be a participant in the most consistent finding in the autism world, a rigid 4:1 male/female ratio?
Speaking towards the extremely low replacement rates for microglia in adulthood, the authors wonder aloud on the possible effects of perturbations of the process of microglial colonization.
The slow turn-over rate for mature microglia raises an issue related to changes that may occur in this critical neural cell population. While this has not been a primary issue of investigation there is limited data suggesting that microglia maintain a history of previous events. Thus, if this history alters the appropriate functioning of microglia then the effects could be long lasting. Additionally, a simple change in the number of microglia colonizing the brain during development, either too many or too few, could have a significant impact not on only the establishment of the nervous system network but also on critical cell specific processes later in life.
Perhaps coincidentally (*cough*), we have abundant evidence of an altered microglial state and population in the autism population; while we do not know that these findings are the result of a disturbance during development, it is an increasingly biologically plausible mechanism, and thus far, I’ve yet to see other mechanisms given much thought, excepting the chance of an ongoing, undetected infection.
There is a brief section concerning the changes found in adult microglial populations in terms of density, form, and gene expression in different areas of the brain, “With further investigation into the heterogeneity of microglia one would assume that a significant number of factors, both cell membrane and secreted, will be found to be differentially expressed across the various subpopulations.” Nice.
There is a section of the paper on microglial phenotypes, there are a lot of unknowns and the transformation microglia undergo between functional states is even more nebulously understood during brain development. “It is now becoming evident that in the developing brain, many of the standards for microglia morphology/activation may require readdressing.” We haven’t even figured out what they’re doing in the adult brain!
There is a really cool reference for a study that shows altered microglial function dependent on the age of the organism.
In the adult rodent, ischemia can induce microglia to display either a more ramified and bushy appearance or an amoeboid morphology depending on the level of damage and distance from the infarct site(s). In the immature rodent, ischemia-induced changes in capillary flow or, presumably, altered CNS vascularization can retain the microglia in an amoeboid phenotype for longer and delay the normal ramification process (Masuda et al., 2011).
One way of looking at this would be to say that we should exercise extreme caution in trying to translate our nascent understanding of how mature microglia react when speculating on how immature microglia will act. To follow up on just how little we know, there is a long discussion about the shortcomings of a the term ‘activated’ microglia with some details on chemical profiles of broadly generalized ‘classically inflammatory, ‘alternatively activated’, ‘anti-inflammatory’, and ‘tissue repair’ phenotypes.
Next up is a dizzyingly list of brain development functions that microglia are known, or suspected to participate in. Without getting too deep in the weeds, of particular interest to the autism realm, that list includes neurogenesis and differentiation in the cortex [related: Courchesne, me], cell maturation via cytokine generation, axon survival and proliferation [related: Wolff, me], programmed cell death of Purkinje cells, clearance of ‘early postnatal hippocampul neurons’, and the ‘significant contribution to synaptic stripping or remodeling events’, i.e., pruning (Paolicelli / fractaltine), and even experience dependent microglia / neuron interactions. Taking all of this (and more) into consideration, the authors conclude “Thus, one can propose that alterations in microglia functioning during synapse formation and maturation of the brain can have significant long-term effects on the final established neural circuitry. “ Ouch.
Next up is a summary of many of the animal studies on microglial participation in brain formation, there is a lot there. Interestingly (and particularly inconvenient) is the finding that a lot of the functional actions of microglia during development appear to operate after birth. “Overall, the data suggest that microglial actions may be most critical during postnatal brain maturation rather than during embryonic stages of development.” Doh!
Early life STRESS gets some attention, and for once there is some good news if you look at it the right way. There is something about a very cool study from Schwarz (et all / Staci Bilbo!) involving drug challenge that peered deep into the underlying mechanisms of an environmental enrichment model; animals given a preferential handling treatment were found by two metrics to have differential microglia response in adulthood with (biologically plausible) observations, increased mRNA levels for IL-10 production, and decreased DNA methylation; i.e., less restriction on the gene that produces IL-10, and more messenger RNA around to pass off the production orders [totally beautiful!]. There is more including thyroid disruption (though in a way that I found surprising), and the observations of time dependent effects on immue disturbances. (super inconvenient)
There is so much data that keeps piling on that the authors end up with “Overall, the existing data suggest a critical regulatory role for microglia in brain development that is much expanded from initial considerations of microglia in the context of their standard, immune mediated responses.”
A terrifying concept that I haven’t found time to dedicate a post towards is microglia priming, which gets some attention in Harry.
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. . . Exactly how long this primed state will last has not been determined; however, data from microglia suggest that it can extend over an expanded period of time. Preconditioning can also represent changes that would occur not only over the short term but may be long lasting.”
I happen to think that microglia priming is going to be a very important cog in the machinery for this journey when all is said and done; the evidence to support a preconditioning system is strong, and in parallel, the things we see different in autism (and elsewhere) is consistent with a different set of operations of microglia, AND we also have evidence the disturbances that would invoke microglial change are subtle but real risk factors for autism.
What comes next is a type of greatest hits mashup of very cool papers on developmental programming in the CNS.
Galic et al.(2008) examined age related vulnerabilities to LPS in rats to determine critical age periods. Postnatal injection of LPS did not induce permanent changes in microglia or hippocampal levels of IL-1b or TNFa; however, when LPS was given during the critical postnatal periods, PND 7 and 14, an increased sensitivity to drug induced seizures was observed in 8-week-old rats. This was accompanied by elevated cytokine release and enhanced neuronal degeneration within the hippocampus after limbic seizures. This persistent increase in seizure susceptibility occurred only with LPS injection at postnatal day 7 or 14 and not with injections during the first day of life or at PND 20. Similar long-lasting effects were observed for pentylenetetrazol-induced seizures when PND 11 or 16 rat pups were subjected to LPS and hyperthermic seizures (Auvin et al., 2009). These results again highlight this early postnatal period as a ‘‘critical window’’ of development vulnerable to long-lasting modification of microglia function by specific stimuli. Work by Bilbo and co-workers demonstrated LPS-induced deficits in fear conditioning and a water maze task following infection of PND 4 rats with Escherichia coli. In the young adult, an injection of LPS induced an exaggerated IL-1b response and memory deficits in rats neonatally exposed to infection (Bilbo et al., 2005). Consistent with the earlier work by Galic et al. (2008), an age dependency for vulnerability was detected with E. coli-induced infection at PND 30 not showing an increased sensitivity to LPS in later life (Bilbo et al., 2006).
In particular, Galic 2008, or Postnatal Inflammation Increases Seizure Susceptibility in Adult Rats (full paper) was a very formative paper for me; it was elegant in design and showed alarming differences in outcome from a single immune challenge experience, if it occurred during a critical developmental timeframe. If you haven’t read it, you should.
This paper has a nice way of distilling the complexity of the literature in a readable way.
One hypothesis for developmental sensitivity is the heterogeneous roles for inflammatory factors and pro-inflammatory cytokines during development, including their timing-, region and situation-specific neurotrophic properties. Many of the proinflammatory cytokines are lower at birth with a subsequent rapid elevation occurring during the first few weeks of life. In an examination of the developing mouse cortex between PND 5 and 11, mRNA levels for TNFa, IL-1b, and TNFp75 receptor remained relatively constant while a significant increase in mRNA levels of CR3, macrophage-1 antigen (MAC-1), IL-1a, IL-1 receptor 1 (IL- )R1, TNFp55 receptor (TNFp55R), IL-6, and gp130 occurred (Fig. 2). This data suggests that an upregulation of interleukins and cytokine receptors may contribute to enhanced cytokine signaling during normal cortical development.
One hypothesis put forward using a model reliant on postnatal exposure to LPS suggests that these types of exposure may ‘‘reprogram’’ neuroimmune responses such that adult stress results in hyperactivation of the hypothalamic pituitary adrenal (HPA) axis (Mouihate et al., 2010) and corticosterone changes (Bilbo and Schwarz, 2009).While limited, the available data suggest that events occurring during development, especially postnatal development, have the potential to cause long term alterations in the phenotype of microglia and that this can be done in a region specific manner.
In what could, conceivably, be a coincidence, our available information on the autism brain also shows region specific changes in microglia populations, microglial activation profiles, and oxidative stress. I do not believe the findings reviewed in Microglia in the developing brain: A potential target with lifetime effects will be meaningless artifacts; the likelihood that our observations of an altered neuroimmune state in autism are not, at least, participatory has become vanishingly small.
Can these findings inform us on the incidence question? I was lurking on a thread on Respectful Insolence a while ago, and someone gave what I thought was a very succinct way of thinking about the changes that our species has encountered the past few decades; it went something like “we have replaced infection with inflammation”. That’s a pretty neat way of looking at how things have gotten different for humanity, at least lots of us, and especially those of us in the first world. We used to get sick and die early; now we live longer, but oftentimes alongside chronic disorders that share a common underlying biological tether point, inflammation.
Any dispassionate analysis of the available data can tell us that we have, indeed, replaced infection with inflammation; we suffer from less death and misery from infection, but more metabolic disorder, more diabetes, more hypertension, more asthma and autoimmune conditions than previous generations. We have largely replaced good fatty acids with poor ones in our diet. All of these conditions are characterized by altered immune biomarkers, including an increase in proinflammatory cytokines. Those are the facts that no one can deny; we have replaced infection with inflammation.
But when we look to the findings of Microglia in the developing brain: A potential target with lifetime effects, it becomes clear that our newfound knowledge of microglial function and crosstalk with the immune system raises some very troubling possibilities.
Lately it has been quite in vogue among a lot of the online posting about autism to at least mention environmental factors which could participate in developmental trajectory leading to autism; that’s a big step, an important and long overdue acknowledgement. If you pay close attention, you will notice that 99% of these admissions are handcuffed to the word “prenatal”. This is likely an attempt to deflect precise questions about the robustness of our evaluation of the vaccine schedule, but the big question, the incidence question, still hinges on fulcrum of the genetic versus environmental ratio ; that is a problem for the purveyors of the fairytale because the prenatal environment of our fetuses, the chemical milieu of their development, is qualitatively different compared to generations past. That chemical soup is their environment; and that environment has unquestionably changed in the past decades as we have replaced infection with inflammation.
Our previous analysis tells us that invoking inflammation outside the brain modifies microglial function inside the wall of the blood brain barrier; good or bad, no honest evaluation of the literature can argue against a lack of effect. What happens outside the brain affects what happens inside the brain. If, however, microglia are active participants in brain formation, as a swath of recent research indicates, can this fact give us insight into the incidence question?
Is a state of increased inflammation the pathway between maternal asthma, depression, stress, and obesity being associated with increased risk of autistic offspring? Have we replaced infection with inflammation plus?
What could be more lethal to the fairytale of a static tale of autism than a positive relationship between a lifestyle characterized by increased inflammation and the chances of having a baby with autism?
Are we totally fucked?
We cannot know the answers unless we have the courage to ask the difficult questions with methods powerful enough to provide good data, and it won’t be easy. The static rate of autism fairytale is a comforting notion; it expunges responsibility for the coronal mass ejection sized change to our fetuses developing environment, and while hiding behind the utterly frail findings of social soft scientists, we can happily place tin foil hats and accusations of scientific illiteracy on anyone who might be worried that our abilities have outstripped our wisdom. That is a terrible, cowardly way to approach the incidence question, what we should be doing is exactly the opposite, ridiculing the epidemic sized error bars in prevalence studies and demanding more answers from the hard scientists. Eventually we will get there and it will be a critical mass of information from studies like Harry that will propel decision makers to abandon the fairytale for a course regulated by dispassionate analysis.
Additional Findings of an Altered NeuroImmune Environment In Autism with Intriguing Questions Raised – Microglia in the Cerebral Cortex in Autism
Posted April 17, 2012on:
Hello friends –
A study with a beautifully terse title, Microglia in the Cerebral Cortex in Autism landed in my inbox the other day. It adds to the growing literature showing perturbations in neuroimmune system in the autism population, this time by measuring the number of microglia in different parts of the brain. Here is the abstract:
We immunocytochemically identified microglia in fronto-insular (FI) and visual cortex (VC) in autopsy brains of well-phenotyped subjects with autism and matched controls, and stereologically quantified the microglial densities. Densities were determined blind to phenotype using an optical fractionator probe. In FI, individuals with autism had significantly more microglia compared to controls (p = 0.02). One such subject had a microglial density in FI within the control range and was also an outlier behaviorally with respect to other subjects with autism. In VC, microglial densities were also significantly greater in individuals with autism versus controls (p = 0.0002). Since we observed increased densities of microglia in two functionally and anatomically disparate cortical areas, we suggest that these immune cells are probably denser throughout cerebral cortex in brains of people with autism.
[Note: You don’t see p-values of .0002 too often!] This paper is at a high level largely similar to another recent paper, Microglial Activation and Increased Microglial Density Observed in the Dorsolateral Prefrontal Cortex in Autism (discussed on this blog, here). The authors were clever here, they intentionally used two very anatomically different, and spatially separated parts of the brain to evaluate for microglia population differences, a sort of bonus slice to learn more about the population of microglia in the brain.
The specific measurement technique in use, staining for specific antibodies, does not give us information regarding the activated/non activated state of the microglia, a determination which must be made with evaluations of morphology, though several other studies have measured this directly, and many more provide indirect evidence of a chronic state of activation of microglia. Not only did the author s report an increase in population density in the autism group, the number of microglia was also positively correlated between sites; i.e., a patient with more microglia in the visual cortex was also more likely to have more microglia in the fronto-insular.
These findings demonstrate that, at the time of death, there were significantly higher microglial densities in the subjects with autism compared to the control subjects, and that this change in microglial density is widespread throughout the cerebral cortex in autism. The microglial densities in FI and VC in the same subject were significantly correlated (both measures were available in 10 controls and 8 autistic subjects for a total of 18 subjects) with Pearson’s r2 = 0.4285, p = 0.0024 (Fig. 6). This indicates that the elevation in density is consistent between these areas, and probably throughout the cortex, in both subjects with autism and controls.
Also of interest, in the control group microglia densities tended to decrease with age, but this change was not seen in the autism population.
There is some discussion about a big problem in the autism research world, a very real and meaningful dearth of available tissue samples, this study shared five patients with Morgan, and one from Vargas. [Note: Sign up to help. Morbid but necessary.]
The authors went on to ask the exact same question I had, “How and when does the increased density of autistic microglial arrays arise, and how is it maintained?” Unfortunately, while there aren’t any good answers, I was still a little disappointed with the analysis. There is a quick rundown of a variety of neuroimmune and peripheral immune findings in autism, and some thoughts on ‘sickness behavior’ with the implicit interconnectedness of the immune state and behaviors, and some discussion on some of the many animal models of maternal immune activation in autism.
In an stroke of amazing serendipity, the authors wonder aloud towards the possibility of a type of distracted worker effect of microglia on neural networks, sort of a bank shot on the autism paradox I struggled with in my previous post when I said,
Are increased neuron number and altered white matter tracts the result of microglia not performing the expected maintenance of the brain? Are the findings from Courchesne and Wolff the opportunity costs of having a microglia activated during decisive developmental timeframes?
The authors of Microglia in the Cerebral Cortex in Autism state
In contrast, microglia can also phagocytize synapses and whole neurons, thus disrupting neural circuits. For example,when the axons of motor neurons are cut, the microglia strip them of their synapses (Blinzinger and Kreutzberg 1968; Cullheim and Thams 2007; Graeber et al. 1993). Another example of the disruption of circuitry arises from the direct phagocytosis of neurons. Neurons communicate with microglia by emitting fractalkine*, which appears to inhibit their phagocytosis by microglia. Deleting the gene for the microglial fractalkine receptor (Cx3cr1) in a mouse model of Alzheimer’s disease has the effect of preventing the microglial destruction and phagocytosis of layer 3 neurons that was observed in these mice in vivo with 2-photon microscopy (Furhmann* et al. 2010). In particular, Cx3cr1 knockout mice have greater numbers of dendritic spines in CA1 neurons, have decreased frequency sEPSCs and had seizure patterns which indicate that deficient fractalkine signaling* reduces microglia-mediated synaptic pruning, leading to abnormal brain development, immature connectivity, and a delay in brain circuitry in the hippocampus (Paolicelli* et al. 2011). In summary, the increased density of microglia in people with autism could be protective against other aspects of this condition, and that a possible side-effect of this protective response might involve alterations in neuronal circuitry.
Oh hell yeah. (* concepts and papers discussed on this blog, here)
Going back to the big dollar question, How and when does the increased density of autistic microglial arrays arise, and how is it maintained?”, the possibility of an ongoing infection was raised as a one option, “The increase of microglial densities in individuals with autism could be a function of neuroprotection in response to harmful microorganisms.” Vargas had a dedicated section towards a failure to find agents of the peripheral immune system that are consistent with infiltration from the peripheral immune system commonly observed during acute infection, I do not think other papers have looked for that per se, but will cede to someone with better data. (?) There was a very weird paper from Italy that pointed to a possible polyomavirus transmission from the father in the autism group, though this study was not referenced in Microglia in the Cerebral Cortex in Autism. [Note: I showed my wife this paper, and she told me, “Good job with the autism gametes.” Nice.] Could a virus cause autism, is a nice discussion on this that includes blog and personal favorites, Fatemi, Patterson, and Persico discussing the possibilities and limitations of the study. Great stuff!
While I must admit the possibility that the chronically activated microglia in autism are working on purpose, the irony gods mandate that I wonder aloud if certain segments of the autism Some-Jerk-On-The-Internet population will cling to the possibility that autism is caused by a disease in order to disavow a causative role for neuroinflammation? Those are some tough choices.
There is a discussion on the myriad of ways that microglia could directly participate in autism pathogenesis, starting the discussion off right to the point, “By contrast, there are diseases that arise from intrinsic defects in the microglia themselves which can cause stereotypic behavioral dysfunctions.” There is a short discussion of Nasu-Hakola disease, something I’d never heard of, which has evidence of an increase in cytokines as a result of genetically driven microglial deficiencies, and shows striking behavioral manifestations.
The possibility of some areas of the brain being more susceptible to alterations than others is there too, “Thus, while changes in microglial density appear to be widespread in brains of autistic individuals, some areas may be more vulnerable than others to its effects.” Considering this idea alongside the extremely heterogeneous set of symptoms assigned to autism, a curious question to ponder becomes; if neuroinflammation is a participatory process in the behavioral manifestation of autism, could some of the variability in autistic behaviors be explained by spatially specific gradients of microglial activity? Going further, considering the still largely mysterious migration of microglia into the brain during development, could the temporal origin of microglial activation in autism be a determinant in the eventual behavioral manifestations? These are tricky questions, and I don’t think that our current methodological capacities are sufficient to start thinking about forming a model for analysis.
One concept I was surprised to not receive attention was a developmental programming model, where animal studies tell us that if something happens during critical developmental timeframes, the effect can propagate into adulthood. In fact, one study, Enduring consequences of early-life infection on glial and neural cell genesis within cognitive regions of the brain (Bland et all) exposed four day old animals to e-coli, which found, among other things, “significantly more microglia in the adult DG of early-infected rats”, something seemingly of considerable salience to the current findings, especially considering the known risk factors of early infections as autism risk factors. In Bland, no external agent other than an infection during early life was necessary; this is the essence of the developmental programming model, even after the infection was long since cleared, patterns of physiology were imprinted, the animals recovered from e-coli but were changed from the experience. This my biggest issue with the possibility of an as of yet undefined, and continued evidence free pathogen or process that is causing the immune abnormalities we see in autism, it mandates we ignore existing biologically plausible models that fit well within known risk factors for autism. Why?
Another area this paper was curiously silent on is the data regarding differences in males and females in the timeframes of microglial migration into the brain, something I’d like to learn much more about soon. As an example, Sex differences in microglial colonization of the developing rat brain [yet another by blog favorite, Staci Bilbo] reported “the number and morphology of microglia throughout development is dependent upon the sex and age of the individual, as well as the brain region of interest” among other findings broadly consistent with a beautiful complexity. This is interesting fodder for a discussion concerning possibly the most persistent finding in autism, a very high male to female ratio that has a series of possible explanations [somewhat discussed on this blog, here].
So we know more, but still have only increased our knowledge incrementally. It is increasingly likely that an increased number of microglia in many areas of the brain is characteristic of autism, but the whys, hows, whens, wheres, and whoms still hold many mysteries. The more things change, the more they stay the same.
The Increasingly Multifaceted Resume Of Microglia, Speculations On What It Might Mean For An Autism Paradox and The Swan Song Of Another Autism Canard
Posted March 26, 2012on:
Hello friends –
I’ve had a couple of interesting papers land in my pubmed feed the past few weeks that seem to be tangentially touching on something that has been at the back of my mind for a long time; namely, the repeated findings of a state of an ongoing immune response in the CNS of the autism population, coupled with a behavioral state that is either static, or in many cases, showing gradual improvement over time. [Discussions of ongoing immune response in the brain in autism, here, here, or here]. This is exactly the opposite of what I expected. Most of the conditions I had generally associated with a state of neuroinflammation, i.e., Alzheimer’s or Parkinson’s show a behavioral profile opposite to autism over time, i.e., a deterioration of skills and cognitive abilities. The diagnosis for these conditions is never a straight line or a gradual curve upwards, but a dispassionately reliable trajectory of a downward spiral.
This is something that has been really bugging me a lot as a riddle, I’ve mentioned it here in comments, and other places on the Internet. While outright signs of neuroinflammation are clearly associated with conditions you would rather not have, as opposed to have, we must admit that the available evidence tells us that we cannot just wave our hands, say ‘neuroinflammation!’, and know much more than the broad strokes. [Note: In my early days of my AutismNet life, my view was somewhat less nuanced.] I think that part of what was bothering me is the result of an oversimplified model in my mind’s eye, but I’d formed that model on top of a set of measurements that had empirical precision but underpowered understandings, alongside a more fundamental lack of knowledge.
We know a little more now.
The first paper that really got me thinking along these lines was Synaptic pruning by microglia is necessary for normal brain development, (discussed on this blog, here), which provided evidence of microglial involvement in the ‘pruning’ of synapses, an important step in brain development thought to streamline neural communication by optimizing neuron structure. This was the first paper I’d read that hinted at microglia participation in ‘normal’ brain function; it was only very recently that microglia were considered to have any role in non pathological states. Another paper, Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer’s disease, also implicated microglia in synaptic pruning.
Then I got myself a copy of The role of microglia at synapses in the healthy CNS: novel insights from recent imaging studies. It is a review of several recent studies on the non-excited life of microglia.
In the healthy brain, quiescent microglia continuously remodel their shape by extending and retracting highly motile processes. Despite a seemingly random sampling of their environment, microglial processes specifically interact with subsets of synaptic structures, as shown by recent imaging studies leading to proposed reciprocal interactions between microglia and synapses under non-pathological conditions. These studies revealed that various modalities of microglial dynamic behavior including their interactions with synaptic elements are regulated by manipulations of neurotransmission, neuronal activity and sensory experience. Conversely, these observations implied an unexpected role for quiescent microglia in the elimination of synaptic structures by specialized mechanisms that include the phagocytosis of axon terminals and dendritic spines. In light of these recent discoveries, microglia are now emerging as important effectors of neuronal circuit reorganization.
This review by Tremblay was published in 2012, evidence of the nascent nature of our available data on microglial involvement in the normal brain environment; Tremblay states that part of the reason this type of finding is so recent is the relative difficulty of measuring microglia in non excited states. They were the electrons of brain measurements; our previous attempts to measure them were capable of causing them to change morphology.
The roles of ‘resting’ or immunologically quiescent microglia have remained relatively unknown (also see Tremblay et al., 2011). This is largely due to the difficulties of studying microglia in their non-activated state. Microglia respond promptly to any changes occurring in their environment, and therefore experimental ex vivo and in vitro preparations inevitably result in transformation of their normally prevailing behavior.
Anyway, some new whizbang technologies (i.e., in vivo two-photon laser scanning microscopy)[?] are allowing researchers to peer into the ho-hum everyday activities of ‘non activated’ microglia, and what they are finding is that the term ‘activated microglia’ might be a bit of a misnomer, microglia have been participating in brain function all along, it is just that our filters were insignificantly powered to detect some of their actions until very recently. Several studies have shown that so called ‘resting’ microglia are constantly evaluating their environment with protusions that seemed to operate rather quickly in relationship to other types of neurons.
This unexpected behavior suggested that resting or surveillant microglia may continuously survey the brain parenchyma as part of their immune function, which would justify the substantial expenditure of energy required to continuously maintain microglial dynamics in the normal brain, without excluding the possibility of an additional, distinct contribution to normal brain physiology
Several papers are reviewed that utilized a couple of highly technical methods, including double roll your own transgenic mouse models to visualize the interactions of microglia in a non excited state and synapses. Specific areas of the brain were measured in different studies, microglia were observed transiently engaging with neurons and seemed to target some dendrites for removal. The authors speculate that this could be a mechanism by which neuronal network maintenance, plasticity, could be affected.
In the mature healthy CNS, neuronal networks are continuously remodeled through the formation, modification and elimination of synaptic structures (see Fortin et al. (2011) for molecular mechanisms of structural plasticity) in relation with behavioral and sensory experience.
To determine a possible role of surveillant microglia in the structural remodeling of synaptic structures under normal physiological conditions, Tremblay et al. (2010b) also examined the size changes of spines and terminals before, during and after microglial contacts. Spines contacted by microglial processes during imaging (30–120 min sessions) were found to be smaller initially than those which remained non-contacted. Spines, but not terminals, also underwent transient increases in size during microglial contact, with smaller spines showing the most pronounced changes. Surprisingly, chronic imaging over 2 days further revealed a statistically significant difference in the elimination rate of microglia-contacted spines: spines contacted by microglia were more frequently eliminated than non-contacted spines (24 versus 7%; P 0.05), and in all cases, only the small spines were seen to disappear. These observations suggest that despite an apparently random sampling of the parenchyma, microglial processes specifically target a subset of small, structurally dynamic and transient dendritic spines.
There is also some description of studies that seemed to indicate that the microglial/synapse interactions could be modified through environmental stimulus, two experiments were described involving sensory deprivation and consequent changes in microglia activity. Other experiments described changes in microglial surveillance as a result of induced changes in neuronal excitability by chemical agonists or antagonists of glutamate receptors. [Perhaps this is the basis of the curious findings in Neuroprotective function for ramified microglia in hippocampal excitotoxicity?]
In their concluding statements, Tremblay provides a good description of just how little we know, and in a style that I love, poses open questions for the newer rounds of literature to address.
Since the recent studies have barely scratched the surface (of the brain in this case), the modalities of microglial interactions with excitatory and inhibitory synapses throughout the CNS, much as their functional significance and particular cellular and molecular mechanisms still remain undetermined. For example, in which contexts do quiescent microglia directly phagocytose axon terminals and dendritic spines, use other mechanisms such as proteolytic remodeling of the extracellular space, or refrain from intervening? How do surveillant microglia recognize and respond to the various molecular signals in their environment, including dynamic changes in neurotransmission and neuronal activity at individual synapses? How do these immune cells cooperate with other glial cells, as well as peripheral myeloid cells, in maintaining or shaping neuronal architecture and activity? And, as in the case of microglial memory of past immune challenges (see Bilbo et al., 2012), do surveillant microglia somehow remember their previous behavioral states, the flux of information processing in the brain, or the structural changes of synaptic elements in recent and not so recent windows of intervention?
The last sentence there, I think, is especially salient considered within a context of developmental programming.
So what we’ve learned is that decades after the discovery of microglia cells as the immune regulators in the CNS, they appear to also be participating in more fundamental maintenance of the neural structure of our brains; there is increasing evidence of direct relationships in synaptic and axonal removal as well as roles in neurotransmission and the regulation of excitability. Is more on the horizon?
But what about autism and our apparent autism paradox of a static or improving behavioral state alongside conditions of immune activation within the CNS?
Well, I have also been thinking about two brain scanning studies that have come out not too long ago, Neuron Number in Children With Autism (Courchesne et all) , which found increased numbers of neurons in the autism cohort, and Differences in White Matter Fiber Tract Development Present From 6 to 24 Months in Infants With Autism (Wolff et all) which found that the autism group showed denser bundled of white matter, so called wiring, between different parts of the brain. In both of these studies mention is made of the fact that it was possible that their findings, increased cell numbers could be the result of inappropriate removal of excess neurons during development.
Apoptotic mechanisms during the third trimester and early postnatal life normally remove subplate neurons, which comprise about half the neurons produced in the second trimester. A failure of that key early developmental process could also create a pathological excess of cortical neurons.
For example, differences in structural organization prior to a period of experience-dependent development related to social cognition (52–54) may decrease neural plasticity through limitations on environmental input, preventing typical neural specialization (52). These alterations could have a ripple effect through decreasing environmental responsiveness and escalating invariance*, thus canalizing a specific neural trajectory that results in the behavioral phenotype that defines ASDs. In typical development, the selective refinement of neural connections through axonal pruning (55) along with constructive processes such as myelination (56) combine to yield efficient signal transmission among brain regions. One or both of these mechanisms may underlie the widespread differences in white matter fiber pathways observed in the current study.
So, we have growing evidence of microglial participation of neural maintenance alongside growing evidence of impaired maintenance in the autism cohort.
Can our autism paradox be explained by microglia converging in the center of these related lines of thought? Is the answer to our riddle that the ongoing immune response in the brain is not sufficiently powered, or targeted, to cause increasing loss of abilities, but instead, was enough to keep critical, once in a lifetime chances for brain organization from occurring? Are increased neuron number and altered white matter tracts the result of microglia not performing the expected maintenance of the brain? Are the findings from Courchesne and Wolff the opportunity costs of having a microglia activated during decisive developmental timeframes?
That is a pretty neat idea to consider.
Even without the Courchesne and Wolff, the findings that specifically mention impaired network maintenance as possible culprits, the findings of active participation of ‘non-active’ microglia in brain optimization and normal processes is a very problematic finding for another autism canard, the idea that findings of neuroinflammation may not be pathological. The intellectually honest observer will admit that the crux of this defense lay in vaccine count trial testimony presented by John Hopkin’s researchers after their seminal neuroinflammation paper was published. Unfortunately, the vigor with which this testimony is trotted out online does not match the frequency with which such ideas actually percolate into the literature.
But with the data from Tremblay, Paolicelli, and others, such an idea becomes even more difficult to defend, we must now speculate on a mechanism by which either microglia could be in an excited state and continue to perform streamlining of the neural structure, or insist that it is possible that microglia were not excited during development, and something else happened to interfere with neuron numbers, and then, subsequently the microglia became chronically activated.
This is unlikely, and unlikelier still when we consider that anyone proposing such a model must do so with enough robustness to overcome a biologically plausible pathway supported by a variety of studies. And that is only if there was anything underneath the vapor! Make no mistake, if you ever press someone to actually defend, with literature, the mechanisms by which a state of chronic neuroinflammation might be beneficial in autism, or even the result of something else that also causes autism, no further elucidation of that mechanism is ever forthcoming. There isn’t anything there.
At some point, it becomes incumbent of people wishing to make an argument that they propose a biologically plausible mechanism if they wish to continue to be taken seriously. If they cannot, if the literature cannot be probed to make such a case with more empirical support than it might be, the notion so add odds with available evidence should be summarily discarded, unless and until a transcendent set of findings is presented. There should always be room for more findings in our worldview, but precious limited space for faith in the face of contradictory findings.
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.
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.