Very Neat Paper – Differential monocyte responses to TLR ligands in children with autism
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.