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

Natalie Prowse, MSc. (natalie.prowse@carleton.ca)

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).

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.

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.

Allan SM, Rothwell NJ (2001) Cytokines and acute neurodegeneration. Nat Rev Neurosci 2001 210 2:734–744 Available at: https://www-nature-com.proxy.library.carleton.ca/articles/35094583 [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: 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].

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: http://www.jimmunol.org/content/183/7/4733.abstract.

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: https://pubmed.ncbi.nlm.nih.gov/21078923/ [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: https://www-nature-com.proxy.library.carleton.ca/articles/emm201397 [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: www.pnas.org/cgi/doi/10.1073/pnas.1110156109 [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: https://www.jimmunol.org/content/182/4/1836 [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.