Archive for the ‘Toll Like Receptors’ Category
The Interconnectedness of the Brain, Behavior, and Immunology and the Difficult to Overstate Flaccidity of The Correlation Is Not Causation Argument
Posted on: May 12, 2011
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.
-pD
Intriguing Findings – Maternal Obesity, Inflammation, and Consequent Priming of Microglia, Immune Alterations, and Spatial Processing in Offspring (!)
Posted on: May 4, 2010
Hello friends –
I’ve been forced to modify my pubmed alerts so that I don’t miss abstracts like this:
Enduring consequences of maternal obesity for brain inflammation and behavior of offspring
Obesity is well characterized as a systemic inflammatory condition, and is also associated with cognitive disruption, suggesting a link between the two. We assessed whether peripheral inflammation in maternal obesity may be transferred to the offspring brain, in particular, the hippocampus, and thereby result in cognitive dysfunction. Rat dams were fed a high-saturated-fat diet (SFD), a high-trans-fat diet (TFD), or a low-fat diet (LFD) for 4 wk prior to mating, and remained on the diet throughout pregnancy and lactation. SFD/TFD exposure significantly increased body weight in both dams and pups compared to controls. Microglial activation markers were increased in the hippocampus of SFD/TFD pups at birth. At weaning and in adulthood, proinflammatory cytokine expression was strikingly increased in the periphery and hippocampus following a bacterial challenge [lipopolysaccharide (LPS)] in the SFD/TFD groups compared to controls. Microglial activation within the hippocampus was also increased basally in SFD rats, suggesting a chronic priming of the cells. Finally, there were marked changes in anxiety and spatial learning in SFD/TFD groups. These effects were all observed in adulthood, even after the pups were placed on standard chow at weaning, suggesting these outcomes were programmed early in life.
WOW. [All emphasis is mine]
You may note that there isn’t any mention of autism per se here, but we do seem to hit a lot of sweet spots that immediately grabbed my attention for a couple of reasons. While my primary persona as Some Jerk On The Internet is a self appointed autism investigator, somewhere along the line in real life I’ve been trying some relatively strange (for the US) dietary practices; a ‘veganesque’ ingredient selection and the move to a diet based on whole foods, organic when possible. What I’ve noticed during this timeframe is just how fat so many Americans are. The obesity epidemic is real, folks, and doesn’t have the fuzzy nature of ‘increased awareness’ to allow us to (pretend) hope that there isn’t something real happening; we have been getting fatter and fatter for the past few decades. And here we have evidence that obesity can create physiological and behavioral changes in offspring through our mediator de jour, inflammation.
So, why am I blogging about this paper on an autism blog? Creepily enough, a lot of the differences listed here (well, all of them, actually), have similarities to findings in the autism realm.
At weaning and in adulthood, proinflammatory cytokine expression was strikingly increased in the periphery and hippocampus following a bacterial challenge [lipopolysaccharide (LPS)] in the SFD/TFD groups compared to controls.
With human subjects, it is a bit problematic to determine if there are ‘striking increases’ in proinflammatory cytokine expression in the hippocampus following bacterial challenge, but in vitro, we have scads of evidence that the autism population creates an exaggerated innate immune response when compared to ‘normal’. The most recent example of this is, Differential monocyte responses to TLR ligands in children with autism spectrum disorders, by Enstrom, which I also blogged about. We also have Ashwood, and several by Jyonouchi showing similar findings; increased production of proinflammatory cytokines TNF-alpha, IL-6, and IL1-B to some TLR agonists, including TLR4.
Microglial activation within the hippocampus was also increased basally in SFD rats, suggesting a chronic priming of the cells.
Of course, the seminal paper in this regard was Vargas, Neuroglial Activation and Neuroinflammation in the Brain of Patients with Autism, which found increased levels of microglial activation in an autism cohort; their focus seemed to be areas other than the hippocampus. Similar findings of an ongoing immune response within the CNS in autism population can be found in Elevated immune response in the brain of autistic patients, and Immune transcriptome alterations in the temporal cortex of subjects with autism. The concept of ‘primed microglia’ is touched on in another paper by Bilbo, Early-life programming of later-life brain and behavior: a critical role for the immune system, which I’d like to get to eventually, but haven’t had the time for yet, but essentially suggests that there are time critical periods during which the microglia are vulnerable to persistent immunological modification, changing their resting state and response to future immune challenges.
Finally, there were marked changes in anxiety and spatial learning in SFD/TFD groups.
Oh yeah. Everyone has heard the story about the kid with autism who can paint New York City after a helicopter ride, or seeing colors in sound, or whatever, but there does also seem to be a lot of applied research involving specific kinds of visual tests that people with autism seem to do better at than people without. Curiously, we even have a knock out model in rodents that show superior spatial processing skills. Anyone who knows a couple of kids with autism knows one that has anxiety problems; there are some evaluations of this, but honestly, this type of thing suffers a bit from my mind because in order to ask the right question, ‘Are you anxious?’, you’ve eliminated a chunk of the autism population. If we break down to the chemical level and start looking at known biomarkers for the stress response, the HPA-Axis, we’ve got tons of evidence that something is amiss.
Here are some of the juicier parts of the paper. From the introduction:
Obesity and insulin resistance are also strongly linked to cognitive dysfunction, including Alzheimer’s disease (13, 14). Neuroinflammation is independently linked to cognitive disruption (15); brain IL-1 expression, in particular, is implicated in Alzheimer’s disease pathogenesis (16). However, a direct mechanism linking these diverse factors is lacking; that is, whether peripheral inflammation in obesity contributes directly to inflammation/ cytokine production in cognitive regions of the brain, and thus cognitive disruption, remains unclear. Moreover, whether maternal obesity may program inflammation within the brains of offspring long term, particularly in regions important for cognition, such as the hippocampus, is virtually unknown. Tozuka et al. (17) recently reported long-term impairments in neurogenesis within the dentate gyrus as a consequence of being born to obese mouse dams, although the researchers did not explore a potential role for inflammation. Notably, White et al. (18) reported increased glial activation and oxidative stress in the cortex of high-fat-diet-fed rats that were also born to high-fat dams. Glia are the primary immunocompetent cells of brain; thus, long-term changes in their activity as a consequence of diet could be critical in long-term programming of neural function.
The whole ‘long term programming of neural function’ theory is beautiful and terrifying. From the discussion section:
A central question in this study was to identify whether systemic inflammation is transferred to cognitive regions of the brain. The answer to that question is clearly yes, especially in the SFD rats. These animals exhibited increased peripheral cytokines (in liver, fat,and serum) and hippocampal IL-1 responses to an LPS challenge. At P20 and in adults, rats from SFD dams exhibited a very large increase in hippocampal IL-1 following a moderate dose of LPS (Fig. 4). Males exhibited a larger response than females in adulthood, although the diet effect was significant in both sexes. The exaggerated response in adults was particularly striking, given that these animals had been fed a low-fat diet since weaning, a period of time longer in duration (9 wk) than the total time they were exposed to the high-fat diets during development (6 wk). Notable as well was the increase in basal levels of IL-1 in high-fat diet groups in adulthood. These data indicate a basal shift in the expression of this cytokine.
It is almost as if being male provides risks every time they bother to look. Oh well. This would seem to be an illustration of ‘long term programming’, what happened during development was more important than what happened afterwards. There were some differences found in the transfat group compared to the saturated fat group, primarily observed in differences in neural and peripheral response to LPS; the authors theorize that this is related to the deposition locations of the different kinds of fats; i.e., saturated fats make it into the brain more easily than trans fats, which are stored in the liver (where they measured perfipheral inflammation).
We explored the influence of a trans-fat-rich vs. saturated-fat-rich diet independently in this study. This comparison yielded surprising findings as well, as the SFD appeared to be much more deleterious for body weight, leptin, and IL-1 compared to the TFD, especially in males. Conversely, CRP expression in the liver, a reliable risk factor for heart disease in humans, was significantly increased in the TFD groups following LPS compared to the SFD and LFD groups. It should be noted, however, that the dams consumed less of the TFD than of the SFD, suggesting it may have been less palatable, and therefore, induced fewer changes. Furthermore, the role of CRP as an acute-phase protein in rodents is controversial (52); support for this idea is the lack of increase in response to LPS in every diet group. Thus, the role of CRP in this experimental model, if any, remains to be further explored.
Remarkably, the authors found that low fat diet rats performed better in some tasks, and the authors speculate that a high fat diet may produce some cognitive gains.
A very intriguing possibility as well is that increased basal IL-1 in the high-fat-diet groups facilitated cognition. A growing body of evidence suggests a role for IL-1 in normal, nonpathological, synaptic plasticity mechanisms within the brain, including memory (44). IL-1 is critical for long-term potentiation (LTP) maintenance during learning (45, 46). However, exaggerated IL-1 within the brain is also strongly associated with memory impairment, providing support for an inverted-U function for optimal IL-1 and cognition(46, 47).
The inveterd U function is, I believe, similar to the concept of hormesis, wherein exposure to an agent and physiological response does not necessarily follow a straight linear response. A good example of this that may be the Pessah studies, which found that for some types of PCBs, low level exposure caused more problems than high levels of exposure. [Note: Beware of anyone who wants to use the ‘poison is in the dose’ cannard, which might be meaningful if the measurement endpoints are mortality, but increasingly less worthwhile if you want to measure subtle effects.]
Here is the closing paragraph:
In closing, it is clear that maternal high-fat diet has a profound influence on the innate immune response of the offspring, in both the periphery and the brain, and that this has enduring consequences for cognition and affect in both males and females. Future studies are needed to assess whether peripheral signals such as leptin vs. central targets such as microglia may be driving the responses in brain, and whether immune targeting (e.g., TLR4 signaling) may be sufficient to prevent exaggerated CNS inflammation in high-fatdiet-exposed pups.
Great idea at the end! I’d love to see a paper where they replicated these groups, but one group of rats also got fish oil or other anti-inflammatories to see if the effect of the inflammation was attenuated. TLR4 knockout mice might also be neat to see. This is a cool study that tells us just how much we still have to learn about how our choices can have very difficult to predict effects.
One of the authors of this paper, Staci Bilbo, has been on a bit of a tear lately regarding the effect of early life immune challenges and subsequent immune and behavioral differences in the treatment animals, including recent hits like Early-life infection is a vulnerability factor for aging-related glial alterations and cognitive decline, Enduring consequences of early-life infection on glial and neural cell genesis within cognitive regions of the brain, and Early-life programming of later-life brain and behavior: a critical role for the immune system, all of which may have implications for everyone’s favorite environmental agent. I’ll be tackling those papers, and several others that have come out with similar methodologies soon enough, but the entire Frontline debacle has left me a little exhausted on the issue.
– pD
The Antigen Gambit Part 1 – Or – Can We Understand Immunology Through Addition?
Posted on: April 14, 2010
I hate to write another vaccination related post, but I keep on running into the same, tired argument, and thought it might be nice to have a single place to list and link the reasons that one of the most commonly used defenses of why we don’t need to study the vaccination schedule can be dismantled. The scary part, the really fucking scary part, is how easy it is to deconstruct the metrics being provided by experts as to why questioning the process of vaccination need not be thoroughly evaluated, and how people that ought to know better keep regurgitating the antigen gambit despite its obvious shortcomings when held to the most primitive logical tests.
For some background, lets start with basic immunology and the hows and whys of how vaccines actually work. But even before that, lets be clear: Vaccines work. I have absolutely no doubt that the purpose of vaccines, providing protection against microbial invaders is successful, and saves millions of lives every year. What I’m not so sure of, is whether or not this is the only thing our increasingly aggressive vaccination schedule has been accomplishing.
The functional success of vaccination is that we have crafted a technique that allows us to train our immune system to recognize some very nasty, dangerous, and deadly bacterial and viral pathogens. How is this done? Well, it turns out that at a very detailed molecular level, many bacteria and viruses have very specific patterns on their exterior, for our purposes, an immunological fingerprint that identifies, for example, the tetanus bacteria from the diphtheria bacteria. These fingerprints are known as antigens, and our immune systems use them to store a memory of particular pathogens we have been exposed to, so the next time such a pattern is encountered, a robust immune response can be mounted rapidly, before the pathogen gets a chance to reproduce and get us sick. The memorization of these molecular patterns, the fingerprints of specific bacteria and viruses, is the foundational premise of vaccination; by presenting these antigens to our immune system in a hopefully(?) harmless way, we train our immune system to respond to these invaders without actually having to endure the virulence of the actual bacteria or virus. Making things a bit more complicated, some pathogens have more than one molecular face to present, and as such, more than one fingerprint is necessary for our immune system to recognize. Some others, such as flu, regularly shift their molecular fingerprint; this is why there are seasonal flu shots, each year scientists must make educated guesses as to which particular influenza fingerprints will be most prevalent; when they guess correctly, the vaccine mostly works, because we have trained our immune system to see that particular antigen pattern. Other pathogens, like HIV, undergo such rapid transformation of their outward facing molecular structure that tailoring a molecular portrait of them has proven exceedingly difficult.
So, again at a very high level, vaccines work because they present antigens, immune fingerprints, from viruses or bacteria to our bodies, without the associated virulence of the organisms. The hows of creating the antigens without the problems of actual infection aren’t necessary for this discussion; lets just assume that for our purposes, you can have bacterial or viral fingerprints introduced in a vaccine without having to worry about the traditional ramifications of the actual bacteria or virus they came from. Great!
Given that, lets imagine you are a skeptic and are a bit bothered by the fact that our existing vaccine and autism research seems to be wholly comprised of studies involving either thimerosal, or the MMR. It seems a bit confusing that these two types of studies are sufficient for us to have certainty that the act of vaccination itself, or other vaccines administered at very different ages might be contributing to our apparent observations of increases in autism (or other behavioral or autoimmune disorders). If you raise a question involving this glaring blind spot in our research, a lot of the time you’ll see a response like some of these:
The only thing that makes biological sense in the discussion really is antigens and excipients and if you look at that, today’s kids get FAR fewer than say, my generation.
What is relevant is the number of antigens, and not the number of vaccines, that matters. Antigens are the active part of the vaccine which stimulates the immune response.
Another point directed to those who think that multiple vaccines overload the immune system. In actual fact, even though we are vaccinating against more diseases than in the past, we are actually using fewer antigens (the part of the vaccine which stimulates the immune response) in these vaccines than was previously the case.
You get the picture; the only measurement of interest is the number of antigens in vaccines. To be completely fair to some people that use the antigen gambit, it is in response to its equally simplistic counterpart, the ‘Vaccines Overload The Immune System’ gambit. That’s no excuse, at the end of the day, the people using crank arguments are supposed to be the cranks. What worries me is the people using the antigen gambit, are in many cases, the experts, and in the rest of the cases, folks that have listened to the experts, and parrot something that sounds sciency. It is a frightening day when you realize that if infectious disease experts had a reason, a real reason, we shouldn’t study the entire vaccination schedule, they’d provide one better than the antigen gambit.
The tour de force take down of the Vaccines Overload the Immune System gambit is “Addressing Parents’ Concerns: Do Multiple Vaccines Overwhelm or Weaken the Infant’s Immune System?“, by Paul Offit and others. It’s my guess that this document, published in the highly read Pediatrics journal, plays a big part in people believing that the only important thing about the vaccine schedule is the number of antigens involved. Here is the abstract:
Recent surveys found that an increasing number of parents are concerned that infants receive too many vaccines. Implicit in this concern is that the infant’s immune system is inadequately developed to handle vaccines safely or that multiple vaccines may overwhelm the immune system. In this review, we will examine the following: 1) the ontogeny of the active immune response and the ability of neonates and young infants to respond to vaccines; 2) the theoretic capacity of an infant’s immune system; 3) data that demonstrate that mild or moderate illness does not interfere with an infant’s ability to generate protective immune responses to vaccines; 4) how infants respond to vaccines given in combination compared with the same vaccines given separately; 5) data showing that vaccinated children are not more likely to develop infections with other pathogens than unvaccinated children; and 6) the fact that infants actually encounter fewer antigens in vaccines today than they did 40 or 100 years ago.
The biggest problem here is that the acknowledged, ‘implicit’ concern is that multiple vaccines may overwhelm the immune system. The concern we should be more concerned with is, can vaccines modify the immune system in ways that we cannot predict? This is a question that is not addressed here, but if your premise starts with the wrong question, or in this case, a bad question your conclusions shouldn’t be worth much.
All of the bullet points provided suffer from one or more maladies, including a foundational structure of gross over simplifications, insulting the intelligence of the reader, or in one case, wildly optimistic claims of a study conclusions; the same kind of thing what would get you a special article by the Chicago Tribune if you recommended children with autism try not to eat wheat for a few weeks and see what happens.
For this post, we’ll just focus on the last bullet point, and the text that supports it:
6) the fact that infants actually encounter fewer antigens in vaccines today than they did 40 or 100 years ago
This is the lead in for this question:
Parents who are worried about the increasing number of recommended vaccines may take comfort in knowing that children are exposed to fewer antigens (proteins and polysaccharides) in vaccines today than in the past.
To prove this comforting point, the authors provide this fancy table:
(Bigger view on the link to full paper – they don’t have this table exploded as its own supplement link). The good news is in green here, as noted in the text, the only reduction count in the vaccine schedule after 1960 was the change from DTP to DTAP.
The bad news is that, if counting antigens were a meaningful metric, of well, anything, the chicken pox vaccine, Varicella, now contains more antigens than the rest of the shot schedule combined.
This puts us in somewhat of a conundrum. If the ‘number of antigens’ in vaccines is what is relevant, does this mean that the Varicella vaccine puts nine times more stress on the immune system than the Pneumococcus vaccine? Does the Varicella vaccine initiate an immune response sixty nine times more strenuous than the diphtheria component of the DTAP vaccine? [Good luck finding a study to measure the innate immune response to any of those vaccines in a pediatric population.]
The DTAP was licensed in the 1980s, but Varicella didn’t get licensed until 1990; so this means that children who got DTAP, but didn’t get Varicella, got far fewer antigens, half as many, than children born just a few years later. Is this meaningful?
Here is an interesting way to view the question. Imagine the CDC was addressing a set of parents whose children was born in 1985 who were concerned about those vaccinations overloading the immune system of their children, and this was the response:
Parents who are worried about the increasing number of recommended vaccines may take comfort in knowing that your children were exposed to fewer antigens (proteins and polysaccharides) than in vaccines today.
Does this sound like a good argument?
We might also take a look at how frequently children experience mild side effects from vaccination, according to the CDC web site. Fever is an indicator of innate immune activation, though you will occasionally see arguments made that it is insufficiently characterized to draw conclusions from, but if we are trying to understand if addition of antigens is a useful measurement or not, it would seem the rates of side effects are valid goalposts. Here are some quotes; there isn’t a fancy table of this information yet.
- Varicella: Fever (1 person out of 10, or less) [69 antigens]
- Pneumococcal: Up to about 1 out of 3 had a fever of over 100.4 degrees Fahrenheit, and up to about 1 in 50 had a higher fever (over 102.2 degrees Fahrenheit). [8 antigens]
- MMR Fever (up to 1 person out of 6) [24 antigens]
- DTAP: Fever (up to about 1 child in 4) [4 – 7 antigens]
Now that is curious. According to the CDC, the vaccine with the most antigens causes fever far less frequently than vaccines with many times fewer antigens in them. If we can use addition to gain comfort from the fact that the current vaccine schedule includes fewer antigens than it used to, how do we incorporate in this information?
But if we can’t use addition for our purposes? What if, in fact, the system we are interacting with is much, much too complicated to be usefully outlined with simple addition? What if antigens aren’t the only relevant measuring point in evaluating vaccine impact on the immune system? In this case, why use the reduction in antigens in vaccines as an argument to ‘address parents concerns’? Why has such a gross over simplification achieved ubiquity in the blogosphere and indeed, why was it promulgated by the most frequently interviewed physician when the subject is autism and vaccination?
Ponder the above at your own risk.
– pD
Neat Study: “Increased serum levels of high mobility group box 1 protein in patients with autistic disorder”
Posted on: March 27, 2010
- In: Autism | Immunology | Inflammation | Intriguing | Seizures | Toll Like Receptors | Uncategorized
- 1 Comment
Hello friends – The other day a pretty neat abstract hit my inbox: Increased serum levels of high mobility group box 1 protein in patients with autistic disorder
BACKGROUND: High mobility group box 1 (HMGB1) is a highly conserved, ubiquitous protein that functions as an activator for inducing the immune response and can be released from neurons after glutamate excitotoxicity. The objective of the present study was to measure serum levels of HMGB1 in patients with autistic disorder and to study their relationship with clinical characteristics. METHODS: We enrolled 22 adult patients with autistic disorder (mean age: 28.1+/-7.7years) and 28 age- and gender-matched healthy controls (mean age: 28.7+/-8.1years). Serum levels of HMGB1 were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with healthy subjects, serum levels of HMGB1 were significantly higher in patients with autistic disorder (10.8+/-2.6ng/mL versus 5.6+/-2.5ng/mL, respectively, P<0.001). After adjustment for potential confounders, serum HMGB1 levels were independently associated with their domain A scores in the Autism Diagnostic Interview-Revised, which reflects their impairments in social interaction. CONCLUSIONS: These results suggest that HMGB1 levels may be affected in autistic disorder. Increased HMGB1 may be a biological correlate of the impaired reciprocal social interactions in this neurodevelopmental disorder.
I had not heard of “high mobility group box 1” before, but as being described as an ‘activator for inducing the immune response’, my interest was definitely piqued! The author, Emanuele Enzo, was extremely gracious in providing me a copy of his manuscript. Below are the juicy parts: From the introduction:
In recent years, many different mechanisms have been suggested to play a role in the pathophysiology of ASD, including impaired neurotransmission, genetic mutations, viral infections, gastrointestinal factors, and excitotoxicity (Levy et al., 2009; Rapin and Tuchman, 2008). Growing evidence has also suggested that inflammation (Cohly and Panja, 2005), neuroinflammation (Pardo et al., 2005), and oxidative stress (McGinnis, 2004) may be involved in the pathogenesis of ASD. High mobility group box 1 (HMGB1) is a highly conserved, ubiquitous protein released from inflammatory cells that functions as a signal for inducing inflammation and as an activator for inducing the immune response (Klune et al., 2008; Bianchi and Manfredi, 2007). The action of extracellular HMGB1 appears to be dependent on interaction with several cell surface receptors, including toll-like receptors 2/4 (TLRs-2/4) (Yu et al., 2006) and the receptor for advanced glycation endproducts (RAGE) (Rauvala and Rouhiainen, 2007). RAGE is a member of the immunoglobulin superfamily of cell surface receptors that is activated by HMGB1 but also by advanced glycation end products and S100 proteins (Yan et al., 2009), all of which have been shown to be altered in autism (Boso et al., 2006; Junaid et al., 2004). In addition, HMGB1 seems to be released from neurons after glutamate excitotoxicity (Kim et al., 2006; Kim et al., 2008). [emphasis and links are mine]
Some familiar players here , namely, neuroinflammation [Vargas, Li, Garbett], and TLR2 and TLR4 [Engstrom, Jyounouci]. I read Klune this afternoon and it is a very good review paper regarding HMGB1, which essentially illuminates on its description as ‘a signal for inducing inflammation’ involved with TLR2 and TLR4. In it, HMGB1 is termed an ‘alarmin’, an endogenous immune adjuvant, or more plainly, a homegrown danger signal. There is mention of synergistic effects in promoting an inflammatory response in conjunction with tnf-alpha, a presence in autoimmunity, cancer and other nasty conditions, as well as potential restorative effects. Anyone who has been paying attention to the ‘abnormal immune response’ findings in autism is going to see a lot of crosstalk here. [Interested by the semantics, I would encourage readers to take a look at paperwork on resolvins as potential mediators of inflammation]. The RAGE stuff is another paper when Enzo is an author that I haven’t read yet, but mean to. From the results section:
After allowance for age, BMI, and Raven’s Progressive Matrices scores, we found a positive independent association between HMGB1 levels and the ADI-R Social Scores (HMGB1, the worse the social interaction β=0.39, t=2.81, P < 0.01, Fig. 2); the higher the serum level of HMBG1, the worse the social interaction.
Now, this is pretty interesting, because it is another instance where we observe a correlation between immunomodulators and autism severity; Grigorenko found that MIF, a known upregulator of the innate immune response, was positively associated with autism severity, and Ashwood found an inverse relationship between the immune regulating cytokine, TGF-Beta1 and autism severity. It would seem that by several measurements, a propensity towards an inflammatory state seems to be able to affect the degree of impairment.
From the very lightweight discussion section:
HMGB1 has been shown to function as a proinflammatory cytokine-like involved in both excitotoxicity (Kim et al., 2006) and glial activation (Pedrazzi et al., 2007) . Of note, growing evidence has suggested a pathophysiological role for excitotoxicity (Blaylock and Strunecka, 2009) and glutamatergic dysregulation (Blaylock, 09; Shinohe et al., 2006) in ASD. In addition, neuroinflammation may be an important feature in some patients with autistic disorder (Pardo et al., 2005; Vargas et al., 2005). Recently, Pedrazzi et al. (2007) have shown that HMGB1 promotes a specific proinflammatory program in primary astrocytes. Increased oxidative stress and immune dysregulation are other important feature in ASD (McGinnis, 2004; Cohly and Panja, 2005; Blaylock, 2009), and HMGB1 protein plays important roles in both processes (Bianchi and Manfredi, 2007; Klune et al., 2008). Interestingly, HMGB1 may induce a prooxidative state through interaction with its cell-surface receptor RAGE (Rauvala and Rouhiainen, 2007), a molecule previously implicated in the pathogenesis of ASD (Boso et al., 2006). An interesting observation in this study is that raised HMGB1 levels in our patient group were correlated with disturbances in social function as assessed with ADI–R, suggesting that this molecule may be a biological correlate of the impaired reciprocal social interactions in this neurodevelopmental disorder. This finding is intriguing, but needs to be confirmed with further studies.
You don’t see Blaylock get referenced too often, I need to read those papers, he does seem to have an online credibility problem that I can’t figure out. Anyways, the statement that HMGB1 and astrocytes is particularly interesting, because we can see from the seminal Vargas paper, Neuroglial Activation and Neuroinflammation in the Brain of Patients with Autism that astrocytes were the primary producer of the increased cytokine IL-6 and chemokine MCP-1 in the brains of autistics. A link to increased oxidative stress doesn’t surprise me too much, though again, I haven’t read anything about RAGE, so seeing another pathway towards increased oxidative stress is a nice touch. There is a section on the weaknesses of the study including smaller study sizes and uncertainty towards the source of HMGB1. As always, there is a call for additional study.
Brain inflammation is a major factor in epilepsy, but the impact of specific inflammatory mediators on neuronal excitability is incompletely understood. Using models of acute and chronic seizures in C57BL/6 mice, we discovered a proconvulsant pathway involving high-mobility group box-1 (HMGB1) release from neurons and glia and its interaction with Toll-like receptor 4 (TLR4), a key receptor of innate immunity. Antagonists of HMGB1 and TLR4 retard seizure precipitation and decrease acute and chronic seizure recurrence. TLR4-defective C3H/HeJ mice are resistant to kainate-induced seizures. The proconvulsant effects of HMGB1, like those of interleukin-1b (IL-1b), are partly mediated by ifenprodil-sensitive N-methyl-d-aspartate (NMDA) receptors. Increased expression of HMGB1 and TLR4 in human epileptogenic tissue, like that observed in the mouse model of chronic seizures, suggests a role for the HMGB1-TLR4 axis in human epilepsy.
Very Neat Paper – Differential monocyte responses to TLR ligands in children with autism
Posted on: February 22, 2010
- In: Autism | IL-6 | Immunology | LPS | M.I.N.D | Phenotypes | Some Jerk On The Internet | Tnf-Alpha | Toll Like Receptors | You Can Call Me Curebie
- 2 Comments
Hello friends –
One of the most frequent omissions in the pre-eminent autism debate is the very, very different immune response that our population of interest seems to have in comparison with children without a diagnosis of autism. A 2009 paper from the MIND institute is a good example of this type of finding, “Differential monocyte responses to TLR ligands in children with autism“.
Some background is critical here to understand this paper. The very first step in the initation of an immune response is the identification of an invading pathogen as foreign to the body, an intruder, and subsequently marshalling other immune system cells to launch a counterattack on the foreign attacker. The components of the immune system that are responsible for this are the Toll Like Receptors, or TLRS. [The Wiki link, at left, has a very nice table of the known TLRs and the triggering molecular structure, immune system cells that express the TLR, and signaling mechanisms. ] At a very detailed molecular level, these proteins have developed the ability to discriminate different classifications of microbial pathogens; in other words, some TLRs can identify cell structures common to bacteria, some TLRs identify signatures associated with viruses, and so on. It is the TLRs that launch the first phase of the immune response, the innate immune response, and there is increasing evidence that TLRs also play a role coordinating the adaptive immune response. For our purposes, it is sufficient to understand that Toll Like Receptors are the critical starting point of the generation of innate immune cytokines that we see abnormal in so many studies in autism.
From the abstract:
Autism spectrum disorders (ASD) are characterized by impairment in social interactions, communication deficits, and restricted repetitive interests and behaviors. Recent evidence has suggested that impairments of innate immunity may play an important role in ASD. To test this hypothesis, we isolated peripheral blood monocytes from 17 children with ASD and 16 age-matched typically developing (TD) controls and stimulated these cell cultures in vitro with distinct toll-like receptors (TLR) ligands: TLR 2 (lipoteichoic acid; LTA), TLR 3 (poly I:C), TLR 4 (lipopolysaccharide; LPS), TLR 5 (flagellin), and TLR 9 (CpG-B). Supernatants were harvested from the cell cultures and pro-inflammatory cytokine responses for IL-1b, IL-6, IL-8, TNFa, MCP-1, and GM-CSF were determined by multiplex Luminex analysis. After in vitro challenge with TLR ligands, differential cytokine responses were observed in monocyte cultures from children with ASD compared with TD control children. In particular, there was a marked increase in pro-inflammatory IL-1b, IL-6, and TNFa responses following TLR 2, and IL-1b response following TLR stimulation in monocyte cultures from children with ASD (p < 0.04). Conversely, following TLR 9 stimulation there was a decrease in IL-1b, IL-6, GM-CSF, and TNFa responses in monocyte cell cultures from children with ASD compared with controls (p < 0.05). These data indicate that, monocyte cultures from children with ASD are more responsive to signaling via select TLRs. As monocytes are key regulators of the immune response, dysfunction in the response of these cells could result in long-term immune alterations in children with ASD that may lead to the development of adverse neuroimmune interactions and could play a role in the pathophysiology observed in ASD.
So, at a high level we can see that in the test tube, blood from children with autism generates a different immune response than blood from children without autism, and further, that this differentiation seems to be TLR specific. In a surpizingly common finding, we observe an increase in pro-inflammatory cytokines such as IL-1B, IL-6, and TNF-Alpha, all of which have many other findings in autism, seizures, and other neurological conditions. More curious, to my mind, is the decreased response to TLR9, another TLR responsible for orchestrating the immune response to some types of bacterial invaders.
From the discussions section:
Our results indicate notable differences in cytokine production following TLR stimulation in monocyte cell cultures from ASD children including increased pro-inflammatory cytokine production following exposure to the TLR 2 ligand, LTA with increased production of IL-1b, IL-6, and TNFa (3.3-, 3.1-, and 2.9-fold increases, respectively) relative to TD controls. In addition, there was an almost twofold increase in IL-1b responses following TLR 4 stimulation with its ligand LPS. Our current findings are consistent with previous reports of enhanced innate immune activity in ASD (Croonenberghs et al., 2002; Jyonouchi et al., 2001), and further indicates that a dysfunctional innate immune response may occur in a number of individuals with ASD.
TLR2 and TLR4 are both involved with sensing and responding to bacteria; I’m not up to speed currently to give a good description of the specific bacterial populations; for example, TLR4 is responsible for sensing gram negative bacteria, which refers to a specific protein structure on some types of bacteria. The paper then goes on to describe some of the other known findings involving TLRs or their outputs for autism or other neurological conditions.
Pro-inflammatory cytokines, IL-1b, IL-6, and TNFa, which are predominantly derived from cells of the monocyte lineage, are of special interest in the study of neuroimmunological contributions to psychiatric disorders. These cytokines can act both locally and centrally to increase neuroinflammatory responses and/or to affect brain function such as the induction of serotonin from the hypothalamus; changes that may affect behavioral responses (Dunn, 2006). Of the TLR ligands analyzed in this study, those specific to induce TLR 2 signaling, elicited the most profound pro-inflammatory response in monocyte cell cultures derived from children with ASD. TLR 2 is constitutively expressed on the surface of microglial cells (Bsibsi et al., 2002; Kielian et al., 2005; Olson and Miller, 2004) and deficiencies in TLR 2 but not TLR 4, reduce T cell recruitment, microglial proliferation, and cytokine/chemokine expression in a neonatal murine model (Babcock et al., 2006). Previous animal studies have demonstrated that TLR 2 stimulation, leading to pro- inflammatory cytokine production, is sufficient to induce neuroinflammation and the neuronal degeneration that is characteristic of bacterial meningitis, and that TLR 2 deficient animals are protected from such changes (Hoffmann et al., 2007). In a murine EAE model of multiple sclerosis, the clinical disease course and severity of the condition correlated with increased brain expression of CD14 and TLR 2 transcripts, suggesting that there is an increase in or upregulation of microglial cells and monocytes in this model, and that TLR signaling may be actively involved in neuroinflammation and autoimmune development (Zekki et al., 2002). The induction of an inflammatory cytokine storm, initiated by monocyte activation, could produce downstream effects leading to the generation of neuroinflammatory and/or autoimmune responses. An autoimmune sequelae such as the generation of anti-neuronal antibodies to a wide variety of targets have been described in individuals with ASD and may be a consequence of responses originally started by inappropriate innate immune activity (Cabanlit et al., 2007; Connolly et al., 2006, 1999; Croen et al., 2008; Kozlovskaia et al., 2000; Silva et al., 2004; Singh and Rivas, 2004b; Singh et al., 1997a,b; Wills et al., 2009).
Of particular interest here is the discussion that TLR seems to play a very important role in the immune response in the CNS, and in fact, animals bred without TLR2 expression fail to develop a neuroinflammatory state when induced in normal rodents. Given what we know from Vargas, Li, Chez, and Garbett, we seem to be observing an ongoing immune response in the CNS in autism, the fact that TLR2 seems to respond more robustly in the autism population would seem to be a piece of the puzzle as to why this might be occurring. In a very real way, for reasons still unclear, people with autism are predisposed to respond more robustly using mechanisms already associated with neuroinflammatory conditions.
Following is a section focusing on a variety of research involving prenatal immune challenges and subsequent behavioral outcomes in the offspring. Then there is a section that has a lot of very cautiously placed ‘ifs’, ‘maybe’s, and ‘possibles’ that still raises a lot of intriguing possibilities.
In this study, we demonstrated that there is differential signaling in monocytes through different TLRs in children with ASD compared to TD controls. For instance, while LTA induced an increased pro-inflammatory IL-1b, IL-6, and TNFa response and LPS induced increased IL-1b in ASD compared to TD, exposure to poly I:C or flagellin produced similar responses between cases and controls, and CpG produced a significantly lower monocyte response in ASD compared to TD. This may mean that signals generated through different TLR by the recognition of distinct PAMPS expressed by specific bacteria or viruses may lead to differential innate immune activity in ASD. For example, in the current study, signaling through TLR 9 by CpG stimulation was notable for resulting in significantly lower IL-1b, TNFa, MCP-1, and GM-CSF release in ASD compared with TD. Typically, TLR 9 ligand recognition induces downstream anti-viral responses, mainly through interferon a/b production (Kawai and Akira, 2007). The clinical significance of this is unknown but may suggest that children with ASD respond poorly to TLR 9 stimulation that may lead to an ineffective anti-viral interferon response and may to inappropriate responses which could lead to infection, chronic inflammation and tissue destruction and could hence expose the individual to increased levels of autoantigens.
In contrast, signaling through TLR 2 and TLR 4 leads to the marked release of pro-inflammatory cytokines. The pronounced increase in the production of these cytokines in response to LTA and LPS ligation warrants further investigation to elucidate the signaling cascade generated from TLR 2 and TLR 4. A previous report indicated that in the first month of life, children that later develop ASD have more infections than their counterparts (Rosen et al., 2007). These previous findings documenting the presence of increased bacterial and viral infections in conjunction with our observations that children with ASD are hyper-responsive to LTA and LPS stimulation could suggest that aberrant signaling through TLR 2 and TLR 4 may participate in this disorder. Inappropriate stimulation of innate immune responses during critical neurodevelopmental junctures, such as early childhood, could contribute to alterations in neurodevelopment and potentially lead to changes characteristic of ASD (Rosen et al., 2007).
I haven’t read Rosen 2007 yet, but it is on my list. [Does anyone have a copy?]
This altered innate immune response may have widespread effects on the activation and response of other immune cells and may also impact on neuronal activity given the extent of cytokine receptors present on neuronal and glial cells (Gladkevich et al., 2004). Furthermore, altered innate responses may ultimately play a role in the initiation and perpetuation of autoimmune responses that are present in some individuals with ASD. Our observations might also reflect genetic alterations in TLR signaling pathways, or pathways that control
monocyte function, such as the MET pathway, and ultimately lead to monocyte activation and cytokine production. MET is a pleiotropic receptor tyrosine kinase and is a key negative regulator of immune responses (Beilmann et al., 1997, 2000; Ido et al., 2005; Okunishi et al., 2005) that exerts its effects through engagement of its ligand, hepatocyte growth factor (HGF). Notably, MET signaling induces a tolerogenic phenotype in innate immune cells without affecting their antigen presenting capabilities (Okunishi et al., 2005; Rutella et al., 2006). Interestingly, the gene encoding MET carries a common polymorphism, the rs1858830 ‘C’ allele, which is functional and increases the relative risk for autism approximately 2.25-fold (Campbell et al., 2006). Thus, the MET ‘C’ variant may predispose to the absence of down-regulation of innate immune cell activation in ASD, and that the combination of a MET polymorphism and increased response to TLR ligands could combine to increase susceptibility to loss of self-tolerance and increased immune responsiveness.
The MET stuff is very cool and isn’t going away; I need to do some more reading on it, but having a particular downregulating allele for MET increases your risk of autism in a subtle, but real fashion. The resultant molecule from MET, HGF, serves a lot of different functions, including neuron formation, gastrointestinal repair, and, as noted above, as an immunoregulator. The allele is still relatively common, close to one half of everyone has it, but it is, nonetheless, over represented in the autism population. It would seem that you need something else, (probably a lot of something elses) at a genetic level to really start increasing your risk of autism; and above the authors speculate that an inherited downregulatory immune control in conjunction with an upregulated immune response could be a example of multiple low penetrance genes interacting to more greatly increase risk of developing autism.
There are more papers on TLR responsiveness in autism, and other neurological conditions that I’d like to get too eventually, but this one is the most recent, and as a result benifits greatly from a larger base of knowledge from a variety of related areas. I’d like to read a lot of the papers listed as references here; they are all pieces of the puzzle, its just tough to see how they fit in.
– pD
passionless Droning about autism 