Aop: 464


A descriptive phrase which references both the Molecular Initiating Event and Adverse Outcome.It should take the form “MIE leading to AO”. For example, “Aromatase inhibition leading to reproductive dysfunction” where Aromatase inhibition is the MIE and reproductive dysfunction the AO. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE. More help

Toll-like receptor 4 (TLR4) activation leads to neurodegeneration via increase in neuroinflammation

Short name
A name that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
TLR4 activation leads to neurodegeneration

Graphical Representation

A graphical representation of the AOP.This graphic should list all KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs. More help
Click to download graphical representation template Explore AOP in a Third Party Tool


The names and affiliations of the individual(s)/organisation(s) that created/developed the AOP. More help

Natalie Prowse, MSc. (

Point of Contact

The user responsible for managing the AOP entry in the AOP-KB and controlling write access to the page by defining the contributors as described in the next section.   More help
Arthur Author   (email point of contact)


Users with write access to the AOP page.  Entries in this field are controlled by the Point of Contact. More help
  • Arthur Author


Provides users with information concerning how actively the AOP page is being developed, what type of use or input the authors feel comfortable with given the current level of development, and whether it is part of the OECD AOP Development Workplan and has been reviewed and/or endorsed. OECD Status - Tracks the level of review/endorsement the AOP has been subjected to. OECD Project Number - Project number is designated and updated by the OECD. SAAOP Status - Status managed and updated by SAAOP curators. More help
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 Do not cite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

Page Revision Date/Time
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


A concise and informative summation of the AOP under development that can stand-alone from the AOP page. The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance. More help

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


Used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development.The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. More help

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.


Provides a description of the approaches to the identification, screening and quality assessment of the data relevant to identification of the key events and key event relationships included in the AOP or AOP network.This information is important as a basis to support the objective/envisaged application of the AOP by the regulatory community and to facilitate the reuse of its components.  Suggested content includes a rationale for and description of the scope and focus of the data search and identification strategy/ies including the nature of preliminary scoping and/or expert input, the overall literature screening strategy and more focused literature surveys to identify additional information (including e.g., key search terms, databases and time period searched, any tools used). More help

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

This section is for information that describes the overall AOP. The information described in section 1 is entered on the upper portion of an AOP page within the AOP-Wiki. This is where some background information may be provided, the structure of the AOP is described, and the KEs and KERs are listed. More help


Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a prototypical stressor and the biological system) of an AOP. More help
Key Events (KE)
A measurable event within a specific biological level of organisation. More help
Adverse Outcomes (AO)
An AO is a specialized KE that represents the end (an adverse outcome of regulatory significance) of an AOP. More help
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
KE 188 Neuroinflammation Neuroinflammation
AO 2039 N/A, Neurodegeneration (updated) N/A, Neurodegeneration (updated)

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarizes all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP.Each table entry acts as a link to the individual KER description page. More help

Network View

This network graphic is automatically generated based on the information provided in the MIE(s), KEs, AO(s), KERs and Weight of Evidence (WoE) summary tables. The width of the edges representing the KERs is determined by its WoE confidence level, with thicker lines representing higher degrees of confidence. This network view also shows which KEs are shared with other AOPs. More help

Prototypical Stressors

A structured data field that can be used to identify one or more “prototypical” stressors that act through this AOP. Prototypical stressors are stressors for which responses at multiple key events have been well documented. More help

Life Stage Applicability

The life stage for which the AOP is known to be applicable. More help
Life stage Evidence
Adults Moderate
Old Age High

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available. More help
Term Scientific Term Evidence Link
human Homo sapiens High NCBI
human and other cells in culture human and other cells in culture High NCBI
mouse Mus musculus High NCBI
Canis domesticus Canis lupus familiaris Low NCBI
rat Rattus norvegicus High NCBI

Sex Applicability

The sex for which the AOP is known to be applicable. More help
Sex Evidence
Mixed High

Overall Assessment of the AOP

Addressess the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and Weight of Evidence (WoE) for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). More help
Attached file: Aop 464 evidence tables

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

Addressess the relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context. More help

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

The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently, evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence. The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs. More help

Essentiality of Key events is captured in the attached document. 

Evidence Assessment

Addressess the biological plausibility, empirical support, and quantitative understanding from each KER in an AOP. More help

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

Modulating factors (MFs) may alter the shape of the response-response function that describes the quantitative relationship between two KES, thus having an impact on the progression of the pathway or the severity of the AO.The evidence supporting the influence of various modulating factors is assembled within the individual KERs. More help

Quantitative Understanding

Optional field to provide quantitative weight of evidence descriptors.  More help

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)

Addressess potential applications of an AOP to support regulatory decision-making.This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. More help


List of the literature that was cited for this AOP. More help

Allan SM, Rothwell NJ (2001) Cytokines and acute neurodegeneration. Nat Rev Neurosci 2001 210 2:734–744 Available at: [Accessed August 15, 2022].

Andersson U, Tracey KJ (2011) HMGB1 Is a Therapeutic Target for Sterile Inflammation and Infection. In: ANNUAL REVIEW OF IMMUNOLOGY, VOL 29 (Paul WE, Littman DR, Yokoyama WM, eds), pp 139–162. Karolinska Univ Hosp, Karolinska Inst, Dept Womens & Childrens Hlth, S-17176 Stockholm, Sweden.

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: [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: [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: [Accessed August 1, 2022].

Fernandez-Lizarbe S, Pascual M, Guerri C (2009) Critical Role of TLR4 Response in the Activation of Microglia Induced by Ethanol. J Immunol 183:4733 LP – 4744 Available at:

Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH (2010) Mechanisms Underlying Inflammation in Neurodegeneration. Cell 140:918–934.

Jerala R (2007) Structural biology of the LPS recognition. Int J Med Microbiol 297:353–363.

Kodama L, Gan L (2019) Do Microglial Sex Differences Contribute to Sex Differences in Neurodegenerative Diseases? Trends Mol Med 25:741–749.

Kofler J, Wiley CA (2011) Microglia: key innate immune cells of the brain. Toxicol Pathol 39:103–114 Available at: [Accessed August 1, 2022].

Liu Y, Dai Y, Li Q, Chen C, Chen H, Song Y, Hua F, Zhang Z (2020) Beta-amyloid activates NLRP3 inflammasome via TLR4 in mouse microglia. Neurosci Lett 736:135279.

Luna AL, Acosta-Saavedra LC, Martínez M, Torres-Avilés N, Gómez R, Calderón-Aranda ES (2012) TLR4 is a target of environmentally relevant concentration of lead. Toxicol Lett 214:301–306.

Lynch MA (2022) Exploring Sex-Related Differences in Microglia May Be a Game-Changer in Precision Medicine. Front Aging Neurosci 14.

Mata-Haro V, Cekic C, Martin M, Chilton PM, Casella CR, Mitchell TC (2007) The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4. Science (80- ) 316:1628–1632.

Olmos G, Llado J (2014) Tumor Necrosis Factor Alpha: A Link between Neuroinflammation and Excitotoxicity. Mediators Inflamm 2014.

Park BS, Lee JO (2013) Recognition of lipopolysaccharide pattern by TLR4 complexes. Exp Mol Med 2013 4512 45:e66–e66 Available at: [Accessed August 1, 2022].

Romerio A, Peri F (2020) Increasing the Chemical Variety of Small-Molecule-Based TLR4 Modulators: An Overview. Front Immunol 11:1210 Available at: /pmc/articles/PMC7381287/ [Accessed July 27, 2022].

Schroder K et al. (2012) Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages. Proc Natl Acad Sci U S A 109:E944–E953 Available at: [Accessed January 11, 2021].

Scuderi C, Golini L (2021) Successful and Unsuccessful Brain Aging in Pets: Pathophysiological Mechanisms behind Clinical Signs and Potential Benefits from Palmitoylethanolamide Nutritional Intervention. Anim  an Open Access J from MDPI 11:2584 Available at: /pmc/articles/PMC8470385/ [Accessed August 8, 2022].

Sepulcre MP, Alcaraz-Pérez F, López-Muñoz A, Roca FJ, Meseguer J, Cayuela ML, Mulero V (2009) Evolution of Lipopolysaccharide (LPS) Recognition and Signaling: Fish TLR4 Does Not Recognize LPS and Negatively Regulates NF-κB Activation. J Immunol 182:1836–1845 Available at: [Accessed August 1, 2022].

Shah K, McCormack CE, Bradbury NA (2014) Do you know the sex of your cells? Am J Physiol - Cell Physiol 306:C3 Available at: /pmc/articles/PMC3919971/ [Accessed August 10, 2022].

Toroghian Y, Khayyami R, Hassanian SM, Nassiri M, Ferns GA, Khazaei M, Avan A (2022) The therapeutic potential of targeting Toll like receptor pathway in breast  cancer. Curr Pharm Des.

Vaure C, Liu Y (2014) A comparative review of toll-like receptor 4 expression and functionality in different animal species. Front Immunol 5:316.

Wendimu MY, Hooks SB (2022) Microglia Phenotypes in Aging and Neurodegenerative Diseases. Cells  11.

Wolf SA, Boddeke HWGM, Kettenmann H (2017) Microglia in Physiology and Disease. Annu Rev Physiol 79:619–643.

Wong SW, Kwon MJ, Choi AMK, Kim HP, Nakahira K, Hwang DH (2009) Fatty acids modulate toll-like receptor 4 activation through regulation of receptor dimerization and recruitment into lipid rafts in a reactive oxygen species-dependent manner. J Biol Chem 284:27384–27392.