Archive for the ‘M.I.N.D’ Category
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 February 22, 2010on:
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.
Neat looking study: Plasma cytokine profiles in Fragile X subjects: Is there a role for cytokines in the pathogenesis?
Posted January 29, 2010on:
I have yet to get my hands on a copy of Plasma cytokine profiles in Fragile X subjects: Is there a role for cytokines in the pathogenesis?, but plan on doing so relatively soon. Even still, the abstract looks pretty good:
BACKGROUND: Fragile X syndrome (FXS) is a single-gene disorder with a broad spectrum of involvement and a strong association with autism. Altered immune responses have been described in autism and there is potential that in children with FXS and autism, an abnormal immune response may play a role. OBJECTIVES: To delineate specific patterns of cytokine/chemokine profiles in individuals with FXS with and without autism and to compare them with typical developing controls. METHODS: Age matched male subjects were recruited through the M.I.N.D. Institute and included: 19 typically developing controls, 64 subjects with FXS without autism and 40 subjects with FXS and autism. Autism diagnosis was confirmed with ADOS, ADI-R and DSM IV criteria. Plasma was isolated and cytokine and chemokine production was assessed by Luminex multiplex analysis. RESULTS: Preliminary observations indicate significant differences in plasma protein levels of a number of cytokines, including IL-1alpha, and the chemokines; RANTES and IP-10, between the FXS group and the typical developing controls (p<0.01). In addition, significant differences were observed between the FXS group with autism and the FXS without autism for IL-6, Eotaxin, MCP-1 (p<0.04). CONCLUSIONS: In this study, the first of its kind, we report a significantly altered cytokine profile in FXS. The characterization of an immunological profile in FXS with and without autism may help to elucidate if an abnormal immune response may play a role and help to identify mechanisms important in the etiology of autism both with and without FXS.
I love the MIND guys. Anyways, the quick glance gives me some ideas:
- The big one here would seem to be that we seem to have additional evidence that immune dysregulation has very specific ties to autism; a pattern not just of immune dysregulation, but indeed, the beginings of an immunological signature, one so precise that given nothing but blood samples, we could beat Vegas odds in selecting which child has Fragile-X, and which child has Fragile-X and autism. This is also more evidence that an abberant immunological response might be playing a causative role in autistic behavior genesis; just having Fragile-X isn’t enough for you to have this profile, you also have to have autism.
- Some of the players there we know about already; IL-6 was found in increased levels in the CNS by Vargas, and Li , and was found to be generated at higher levels in several studies including Ashwood, Enstrom , Jyonouchi, and Jyonouchi. It also has great deal of support for a place in seizure generation. MCP-1 was also found in Vargas, Eotaxin is also chemotaxic, though I haven’t read anything about it yet.
That’s the abstract view, the whole paper should get here soon.