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Toll-like receptor 4 (TLR4) activation leads to neurodegeneration via increase in neuroinflammation
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|Author status||OECD status||OECD project||SAAOP status|
|Under development: Not open for comment. Do not cite|
This AOP was last modified on August 15, 2022 23:40
Revision dates for related pages
|Toll like receptor 4 Activation||August 09, 2022 13:30|
|Induction, Nuclear Transcription Factor kappa B (NFkB)||August 16, 2022 00:07|
|NLRP3 inflammasome activity, increased||June 25, 2021 08:29|
|Pro-inflammatory cytokines, increased||August 15, 2022 15:28|
|N/A, Neurodegeneration (updated)||August 09, 2022 13:04|
|Neuroinflammation||November 17, 2022 04:44|
|TLR-4 Activation leads to Induction, NFkB||August 09, 2022 00:59|
|TLR-4 Activation leads to Pro-inflammatory cytokines, increased||August 15, 2022 23:50|
|Pro-inflammatory cytokines, increased leads to N/A, Neurodegeneration (updated)||August 09, 2022 12:39|
|Induction, NFkB leads to Pro-inflammatory cytokines, increased||August 09, 2022 01:00|
|Induction, NFkB leads to inflammasome activity, increased||August 09, 2022 01:00|
|Pro-inflammatory cytokines, increased leads to Neuroinflammation||August 09, 2022 01:13|
|inflammasome activity, increased leads to Neuroinflammation||August 15, 2022 17:41|
|Neuroinflammation leads to N/A, Neurodegeneration (updated)||August 09, 2022 01:14|
|Lipopolysaccharride||May 29, 2018 07:05|
|aminoalkyl glucosaminide phopsphates||August 01, 2022 11:47|
|Pathogen Associated Molecular Patterns (PAMPs)||March 26, 2021 04:14|
|Danger Associated Molecular Patters (DAMPs)||March 26, 2021 04:13|
|Amyloid-beta protein 25-35||August 01, 2022 11:49|
|alpha-synuclein||August 01, 2022 11:49|
|palmitic acid||August 01, 2022 13:21|
|high mobility group box 1||August 07, 2022 15:50|
Inflammation-based neurodegeneration is a critical mediator in diseases of the central nervous system (CNS), that result in the degradation and loss of neurons and glia, ultimately leading to cognitive decline and death. Innate immune-mediated inflammation plays a role in CNS damage both acutely, such as in stroke or traumatic brain injury, and in a host of chronic neurodegenerative diseases such as Alzheimer’s, Parkinson’s, multiple sclerosis, and amyotrophic lateral sclerosis (Glass et al., 2010)
As the primary immune cells of the brain, microglia are key mediators in the CNS inflammatory response (Block and Hong, 2005), primarily through pattern recognition receptors (PRRs) that have evolved to identify and target invading viruses and pathogens collectively known as pathogen-associated molecular patterns (PAMPS) and danger-associated molecular patterns (DAMPS) (Kofler and Wiley, 2011). Microglia also act as macrophages, responding to and cleaning up debris based on signals sent from dead and dying cells (Wolf et al., 2017).
A key activator of the inflammatory response in microglia is Toll-like receptor 4 (TLR4), which evolved in tetrapods primarily respond to bacterial invaders (Sepulcre et al., 2009; Schroder et al., 2012). While the canonical activator of TLR4 (and the most studied), is a component of the gram-negative bacterial cell wall, lipopolysaccharide (LPS) (Jerala, 2007; Park and Lee, 2013), recent research has identified many other molecules and environmental toxins that can activate TLR4, including misfolded protein aggregates such as amyloid-beta (Aβ) and alpha-synuclein, produced in Alzheimer’s and Parkinson’s disease, respectively (Fernandez-Lizarbe et al., 2009; Wong et al., 2009; Fellner et al., 2013; Liu et al., 2020; Romerio and Peri, 2020). Termed “sterile inflammation”, there is mounting evidence that endogenous release of DAMPS from damaged and dying neurons results in chronic activation of TLR4 which can both cause and exacerbate neurodegeneration, both directly and through damage to blood-brain-barrier integrity (Graeber et al., 2011; Kuperberg and Wadgaonkar, 2017; Wilkins et al., 2017). In addition to disease-produced DAMPS, an increasing number of environmental toxins such as nanoparticles, have been found to produce or exacerbate TLR4-mediated inflammation (Bianchi et al., 2015).
AOP Development Strategy
Toll-like receptor 4 (TLR4) is present on a large number of cell types in the periphery of mammalian systems including those of the immune system, macrophages and epithelial cells. In the central nervous system, TLR4 is present on microglia and astrocytes, and to a lesser extent, neurons. Its primary role is to identify and defend the body against bacterial pathogens, however, overactivation can trigger signaling cascades that lead to run-away inflammation resulting in degeneration of cells and tissue. TLR4 agonists have been developed as vaccine adjuvants (Mata-Haro et al., 2007; Casella and Mitchell, 2008) and are under development for potential cancer treatments (Toroghian et al., 2022). Off-target effects of TLR-4 activation could have deleterious effects on brain and immune function and must be considered in any application. Importantly, TLR4 can be chronically activated by endogenous danger-associated molecular patterns (DAMPS) and pattern-associated molecular patterns (PAMPS) which can lead to "sterile inflammation" (Andersson and Tracey, 2011), while environmental toxins such as lead and titanium dioxide particles can augment and enhance TLR4 signaling (Luna et al., 2012; Bianchi et al., 2015). Finally, TLR4 signaling, alone or complexed with cluster of differentiation 36 (CD36) and TLR2, has been extensively studied in the context of neurodegeneration, particularly focussing on the role of chronically activated microglia in the aged and injured brain (Wendimu and Hooks, 2022).
The purpose of this AOP is to document the biology of this receptor signaling and implications of adverse outcomes, particularly through microglial activation, for potential future hazard identification that could lead to or exacerbate neurodegeneration.
This AOP was developed based on prior research into the activation of TLR4, supported by a large and well-documented breadth of research including systematic reviews on stressor-specific activation and identification of key events in the pathway.
Key events such as "(188) Neuroinflammation" and "(352) N/A, Neurodegeneraton" have been reused, however, extensive changes were made to (352) and in the interests of not overwriting the existing without the author's permission, a new event "( 2039 ) N/A, Neurodegeneration (updated)" was created with additions to the prior event in red. Ideally, these changes would be merged in the non-training wiki, with the original author's permission.
Given the TLR4 activation pathways apply to a number of immune and epithelial cells, key events and relationships created specifically for this AOP were designed to be broadly applicable to a variety of cell types and initiating events. A primary goal in developing this AOP is broad resusability of the KEs and KERs. This AOP includes key events and key event relationships within the cellular pathway at a high level of detail as each of these events are possible targets for drug development.
Evidence was collected via comprehensive search using Web of Science and Pubmed to identify key research and domain applicablity pertaining to this AOP. Due to the overwhelming breadth and depth of research published with respect to TLR4 signaling, citations focused on high-quality reviews with high citation indexes, balanced with content pertaining to the most recent research available.
Summary of the AOP
Molecular Initiating Events (MIE)
Key Events (KE)
Adverse Outcomes (AO)
|Type||Event ID||Title||Short name|
|MIE||2033||Toll like receptor 4 Activation||TLR-4 Activation|
|KE||2034||Induction, Nuclear Transcription Factor kappa B (NFkB)||Induction, NFkB|
|KE||2036||Pro-inflammatory cytokines, increased||Pro-inflammatory cytokines, increased|
|KE||1895||NLRP3 inflammasome activity, increased||inflammasome activity, increased|
|AO||2039||N/A, Neurodegeneration (updated)||N/A, Neurodegeneration (updated)|
Relationships Between Two Key Events (Including MIEs and AOs)
|TLR-4 Activation leads to Induction, NFkB||adjacent||Low||Moderate|
|Induction, NFkB leads to Pro-inflammatory cytokines, increased||adjacent||Low||Moderate|
|Induction, NFkB leads to inflammasome activity, increased||adjacent||Low||Low|
|Pro-inflammatory cytokines, increased leads to Neuroinflammation||adjacent||Low||Moderate|
|inflammasome activity, increased leads to Neuroinflammation||adjacent||Low||Low|
|Neuroinflammation leads to N/A, Neurodegeneration (updated)||adjacent||Low||Low|
|TLR-4 Activation leads to Pro-inflammatory cytokines, increased||non-adjacent||Low||Moderate|
|Pro-inflammatory cytokines, increased leads to N/A, Neurodegeneration (updated)||non-adjacent||High||Moderate|
Life Stage Applicability
Overall Assessment of the AOP
The attached document includes:
- Support for biological plausibility of KERs
- Support for essentiality of KEs
- Empirical support for KERs
- Support for Quantitative Understanding of KERs
- Known Modulating Factors (because the in-line section in this AOP is not working)
Biological plausibility is considered high for KERs leading up to neuroinflammation, increased, as the induction of nuclear factor kappa B (NF-κB) by TLR4 activation have been extensively studied in a variety of cell types using both knockout and inhibitor models for over 20 years and multiple reviews have been written on this subject. Similarly, NF-κB induction has also been tied to increased release of pro-inflammatory cytokines has also been extensively studied and thus also has high biological plausibility. There is increasing evidence that inflammatory cytokine release induces neuroinflammation, though the cause-effect is not definitively established, thus biological plausibility is moderate. Biological plausibility for NLRP3 involvement in induction of NLR family pyrin domain containing 3 (NLRP3) is as it may rely on other co-factors. Similarly, the induction of cytokines downstream of TLR4 activation or NF-κB induction that can be directly tied to increases in inflammatory cytokines. Thus, empirical support for the AO can only be considered low as the bulk of evidence tying TLR4 to the AO is correlational and has been focused on activation in the context of existing neurodegenerative disease, with quantitative measurements largely focused on the activating ligand, not the activation of the receptor itself. Strong biological plausibility for molecular events exists in this AOP, however definitive links to neurodegeneration are weak. Thus the overall evidence supporting this AOP should be considered Low.
Domain of Applicability
Life Stage Applicability: TLR4 activation in microglia and astrocytes, through sepsis or traumatic brain injury, can lead to encephalitis and neurodegeneration at any age. Thus, the overarching domain of applicability for this AOP is all life stages, though susceptibility to inflammation and neurodegeneration increases with age, particularly as it relates to degenerative diseases.
Taxonomic Applicability: Data used to support the molecular aspects of this AOP came from studies on mouse, rat and human cell lines, as well as in vivo studies on mice and rats. TLR4 homology is highly conserved across species, however mice are much more resilient than humans to LPS, requiring 250-500 times higher dose to induce a similar cytokine response (Copeland et al., 2005). Phenotypic differences in responsiveness are likely routed in species-specific TLR4-regulated gene expression (Copeland et al., 2005; Schroder et al., 2012; Vaure and Liu, 2014), so rodent-derived data should be treated with caution with respect to potential impact on humans. Of equal importance, mice do not naturally suffer from neurodegenerative diseases that afflict humans, yet much of the study of the neurodegenerative impacts of TLR4 signaling has been performed in transgenic mouse models which do not completely replicate human conditions. Age-related neurodegeneration that has been linked to increased neuroinflammation has been identified canine, feline and non-human primates (Emborg, 2017; Scuderi and Golini, 2021), though few studies have been conducted with respect to inflammatory mediators.
Sex Applicability: Studies on molecular aspects of this AOP have largely been conducted in human and mouse cell lines. In 2014 an analysis of papers that used cell lines determined that just 5% reported the sex as female, with 20% reported as male, and 75% not reporting sex (Shah et al., 2014). To date, the vast majority of in vivo studies use male rodents. Rodent microglia, the primary immune cells of the brain, have demonstrated sex differences in gene expression, though studies don’t all agree on which has a higher inflammatory expression profile (Kodama and Gan, 2019; Lynch, 2022). Despite these limitations, inflammation associated with neurodegeneration has been documented with respect to both sexes (Lynch, 2022), and as such this AOP is applicable to both males and females.
Essentiality of the Key Events
Essentiality of Key events is captured in the attached document.
See attached document. Overall, the empirical support for the KERs in the AOP is LOW since quantifiable measurements of key events upstream such as TLR4 activation and Induction of NF-κB are not readily measureable leading to a lack of evicence for incidence, dose and temporal concordance.
Known Modulating Factors
See attached document. Overall, the Quantitative Understanding of this the AOP is low. It is well-accepted that the application of LPS induces TLR4 activation, and using LPS dose as a proxy, studies have derived semi-quantitative dose and time-response data for induction of pro-inflammatory cytokine release in both human and mouse plasma, and in cell lines. There is indirect evidence that pro-inflammatory cytokine increases lead to neurodegeneration both chronically and acutely, however release of multiple cytokines and inflammatory factors can have synergistic effects on cell-death and a true quantitative understanding has yet to be developed (Allan and Rothwell, 2001). TNF-α application in vitro has demonstrated neurotoxicity (Olmos and Llado, 2014), but a quantitative understanding in vivo is not well established.
Considerations for Potential Applications of the AOP (optional)
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Bianchi MG, Allegri M, Costa AL, Blosi M, Gardini D, Del Pivo C, Prina-Mello A, Di Cristo L, Bussolati O, Bergamaschi E (2015) Titanium dioxide nanoparticles enhance macrophage activation by LPS through a TLR4-dependent intracellular pathway. Toxicol Res (Camb) 4:385–398 Available at: https://pubs.rsc.org/en/content/articlehtml/2015/tx/c4tx00193a [Accessed August 4, 2022].
Block ML, Hong JS (2005) Microglia and inflammation-mediated neurodegeneration: Multiple triggers with a common mechanism. Prog Neurobiol 76:77–98.
Casella CR, Mitchell TC (2008) Putting endotoxin to work for us: monophosphoryl lipid A as a safe and effective vaccine adjuvant. Cell Mol Life Sci 65:3231 Available at: /pmc/articles/PMC2647720/ [Accessed July 27, 2022].
Copeland S, Warren HS, Lowry SF, Calvano SE, Remick D (2005) Acute inflammatory response to endotoxin in mice and humans. Clin Diagn Lab Immunol 12:60–67.
Emborg ME (2017) Nonhuman Primate Models of Neurodegenerative Disorders. ILAR J 58:190–201 Available at: https://academic.oup.com/ilarjournal/article/58/2/190/4080221 [Accessed August 8, 2022].
Fellner L, Irschick R, Schanda K, Reindl M, Klimaschewski L, Poewe W, Wenning GK, Stefanova N (2013) Toll-like receptor 4 is required for α-synuclein dependent activation of microglia and astroglia. Glia 61:349–360 Available at: https://onlinelibrary-wiley-com.proxy.library.carleton.ca/doi/full/10.1002/glia.22437 [Accessed August 1, 2022].
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Jerala R (2007) Structural biology of the LPS recognition. Int J Med Microbiol 297:353–363.
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Lynch MA (2022) Exploring Sex-Related Differences in Microglia May Be a Game-Changer in Precision Medicine. Front Aging Neurosci 14.
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