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Key Event Title
Toll like receptor 4 Activation
|Level of Biological Organization|
Key Event Components
|toll-like receptor 4 signaling pathway||Toll-like receptor 4||increased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|TLR4 activation in microglia leads to neurodegeneration||MolecularInitiatingEvent||Arthur Author (send email)||Under development: Not open for comment. Do not cite|
|Rattus norvegicus||Rattus norvegicus||High||NCBI|
|Macaca mulatta||Macaca mulatta||Moderate||NCBI|
|Canis lupus familiaris||Canis lupus familiaris||Moderate||NCBI|
|Troglodytes troglodytes||Troglodytes troglodytes||Moderate||NCBI|
|All life stages||High|
Key Event Description
Part of the innate immune system response, Toll-like receptor 4 (TLR4) is a type-1 trans-membrane pattern recognition receptor (PRR) that is part of the toll-like receptor family.
Initially thought to be primarily a receptor for sensing and responding to bacterial infection, the most studied and canonical TLR4 ligand is lipopolysaccharide (LPS), the endotoxin coating of gram-negative bacteria. Extensive research on TLR4 mechanisms has identified many other agonists which can act as pattern associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPS) such as high mobility group box 1 (HMGB1) (Yang et al., 2010), palmitic acid (Nicholas et al., 2017), Amyloid beta proteins (Liu et al., 2020), alpha-synuclein (Fellner et al., 2013), ethanol (Fernandez-Lizarbe et al., 2009), “sterile inflammation”-promoting substances such as ozone, atmospheric particulate matter, and nanoparticles (Lucas and Maes, 2013), and a host of other molecules released from injured or dying cells (Gaikwad et al., 2017).
Prototypical TLR4 activation in differentiated cells of myeloid lineage is dependent on complexing a ligand with the co-receptor molecule cluster of differentiation 14 (CD-14). CD-14 is a phospholipid transporter that transfers LPS to a specific recognition motif on myeloid differentiation factor 2 (MD-2), which connects with a specific binding pocket on the TLR-4 receptor. The binding of the CD-14/ligand to MD-2 causes a conformational change in TLR4, initiating signal transduction (Jerala, 2007; Kim et al., 2013).
TLR4 activation in myeloid cells can directly induce signaling via 2 pathways, one of which is dependent on myeloid differentiation primary response protein 88 (MyD88), and the other of which is dependent on toll-interleukin-1 receptor domain-containing adaptor inducing interferon-β (TRIF). Activation of the MyD88-dependent pathway results in a specific set of signaling patterns leading to activation of the transcription factors activator protein 1 (AP-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). NF-κB and AP-1 are primarily responsible for upregulation of inflammatory cytokines, inducible nitric oxide synthase (iNOS), and key components that support assembly of the NLR family pyrin domain containing 3 (NLRP3) inflammasome (Guha and Mackman, 2001; Qin et al., 2005; Lu et al., 2008; Bauernfeind et al., 2009).
TLR4 signaling via MyD88 is followed by CD-14-mediated endocytosis, which initiates the TRIF-dependent pathway. TRIF-dependent signaling results in upregulation of type-1 interferons, anti-inflammatory mediators, and termination of inflammatory signaling (Ciesielska et al., 2021).
Image from (Ciesielska et al., 2021), Cellular and molecular Life Sciences
Release of inflammatory cytokines and reactive oxygen species (ROS) into the extracellular milleu as a result of TLR4 activation has been implicated in a number of human diseases including, but not limited to cardiovascular diseases, autoimmune diseases, diabetes mellitus, macular degeneration, and pulmonary diseases (Kim and Sears, 2010; Lucas and Maes, 2013; Yang et al., 2016).
Evidence for Perturbation by Stressor
TLR4 has been extensively studied over the last 3 decades facilitating an enormous body of evidence and numerous comprehensive reviews. Activation by the prototypical stressor, LPS, in numerous cell types has been unequivocably established (Guha and Mackman, 2001; Lu et al., 2008; Sepulcre et al., 2009a). More recently, many other TLR4 stressors have been identified including small molecules (Peri et al., 2010), high mobility group box 1 (HMGB1) (Kim et al., 2013), fatty acids (Shi et al., 2006; Rocha et al., 2016; Nicholas et al., 2017), and nanoscale particulate matter in air pollution (Woodward et al., 2017). In addition, synthetic compounds and small-molecule activators have been developed and tested to specifically activate TLR4 for immunotherapies and as vaccine adjuvants (Romerio and Peri, 2020).
How It Is Measured or Detected
Commercially available antibodies exist for the detection of TLR4 and its accessory proteins CD14 and MD-2. Western blot assays can be used for semi-quantitative measures of expression; immunohistochemistry using 3,3’ diaminobenzidine tetrahydrochloride (DAB) is considered qualitative, whereas immunofluorescent stains can be quantified using a variety of techniques including relative intensity (Ungaro et al., 2009).
Fluorescent in Situ Hybridization (FISH) can be used to detect TLR4 expression in paraffin-embedded tissues, using specific probes for TLR4 mRNA (Neal et al., 2012). Similarly, the receptor can be detected in specific tissues and cell types using quantitative real-time polymerase chain reaction assays (qRT-PCR) and commercially available enzyme-linked immunosorbent assays (ELISA).
It is difficult to perform direct measurement of TLR4 activation, however, quantitative measurement of TLR4 endocytosis can be made using commercially available TLR4 antibodies conjugated to fluorophores with flow cytometry to detect the loss of TLR4 and CD14 at the cell surface after stimulation with LPS (Schappe and Desai, 2018).
In general, most assays rely on indirect methods of detection by identifying molecular changes down-stream of the TLR4 pathway of interest. With respect to the MyD88 pathway, indirect measurement of activation can be made by stimulating cells with LPS and then using commercially available ELISA kits to measure increases in cytokine release. Similarly, semi-quantitative indirect measurements of activation can be made by leveraging commercial antibodies to phosphorylated subunits of NF-κB, primarily through western blotting.
Transgenic Mouse models
Activation can also be verified by comparing experimental results of downstream effectors from WT and either constitutive or conditional TLR4-KO mouse strains. Several transgenic mouse models are available including constitutive TLR4-KO, (B6(Cg)-Tlr4tm1.2Karp/J and B6.B10ScN-Tlr4lps-del/JthJ), conditional TLR4-KO (B6.129-Tlr4tm1.1Jke/J), a conditional TLR4-knock-in (TLR4 is effectively knocked out by a blocking cassette until exposed to Cre recombinase) (B6.129-Tlr4tm2Jke/J), and 2 reporter strains (B6(FVB)-Tlr4tm1.1Gbrt/J, C57BL/6-Gt(ROSA)26Sortm3(Tlr4,-GFP)Gbrt/J. A mouse reporter mouse strain was also produced to specifically identify where TLR4 is selectively knocked out and vs still present (Neal et al., 2012).
Cell reporter lines:
- As an indirect measure of TLR4 activation, there are commercially available human cell lines that produce secreted embryonic alkaline phosphatase (SEAP) on activation of TLR4, or when TLR4 is transcribed. Commercially available chemiluminescent and fluorescent SEAP Assays can then be used determine the amount of alkaline phosphatase secreted into cell supernatant, which can be used to quantify the degree of TLR4 activation.
- TLR4, MD2 and CD14 can be transduced into transcriptional NF-κB::eGFP reporter cells based on the human T cell line Jurkat JE6-1, with quantitative activation assessed by flow cytometry at very low levels of LPS (Radakovics et al., 2022).
Domain of Applicability
Mammalian TLR4 is a homolog of the drosophila “Toll” receptor (Lemaitre et al., 1996), and is present on a variety of myeloid cell types including monocytes, macrophages, dendritic cells, B cells, and granulocytes, and peripheral blood leukocytes, as well as placental tissue and endothelial cells in pulmonary, heart and spleen tissue (Zarember and Godowski, 2002; Zeuke et al., 2002; Taylor et al., 2004; Vaure and Liu, 2014). In the brain, there is extensive evidence of TLR4 on microglia and to a lesser extent, on astrocytes (Fernandez-Lizarbe et al., 2009; Gorina et al., 2011; Rodríguez-Gómez et al., 2020), with some evidence this receptor may also be expressed in low levels on neurons (Acioglu et al., 2022).
TLR4 and its accessory proteins are evolutionarily conserved in amniotes (Loes et al., 2018), however most fish and amphibians do not have MD-2 and CD14, and few fish express TLR4 (Sepulcre et al., 2009).
CD14 is expressed in mouse bone marrow-derived macrophages, dendritc cells (DCs) and embryonic fibroblasts, but not in BALB/c-derived A20 cell line, with evidence that mature DCs express more CD14, enhancing endocytotic signaling (Maeshima and Fernandez, 2013).
There is a high degree of homology between human and murine TLR4. However, while promoter regions are homologous, differences at human exon II and mouse exon III can lead to splice variants that are not shared between species (Rehli, 2002). Importantly, while the intracellular signaling domain is highly conserved, there are species-specific differences and intraspecies polymorphisms in the extracellular domain that are tied to ligand recognition (Vaure and Liu, 2014). This variation may partially explain why Lipid A variants from different pathogenic bacteria, differentially activate human and murine TLR4 (Maeshima and Fernandez, 2013).
Despite high TLR4 homology between humans and primates, baboons, unlike humans and chimpanzees are highly resistant to LPS, and require >100-fold higher doses to exhibit the same responses (Haudek et al., 2003)
Compared to mice, humans are highly sensitive to LPS, with the severe illness induced by just 2-4ng/kg (Sauter and Wolfensberger, 1980). By comparison in C57BL/6 and BALBc mice, the typical LD50 of LPS is 10mg/kg. Differences in toxicity may be linked to how rapidly each species upregulates of TNF-α and IFN-γ (Dinges and Schlievert, 2001; Taylor et al., 2022). Age also plays a role in LPS-induced toxicity, with an LD50 of 25.6mg/kg for young mice and 1.6 mg/kg for aged mice, suggesting that inflammatory tone increases with age (Tateda et al., 1996). Similarly, inflammatory cytokine levels were significantly higher in aged mice, relative to young mice 72 hours after intratracheal LPS challenge (Ito et al., 2007).
There are fundamental differences in TLR4 activation in murine and human DCs, including triggering of apoptotic pathways in mature murine DCs. Activation of TLR4 in other types of immune cells in both mice and humans can produce cell-type specific responses due to differences in expression of TLR4/MD-2 and CD14 (Ciesielska et al., 2021).
Human neonates are also more susceptible to LPS, possibly due to reduced cell-surface expression of CD-14, which mediates the termination of TLR4 inflammatory signaling (Levy, 2005). Both the human placenta and fetal membranes express TLR4 and respond to bacterial pathogens by upregulating inflammatory cytokines (Firmal et al., 2020). TLR4 activation alters between pro-inflammatory to anti-inflammatory during different phases of pregnancy, and LPS-mediated TLR4 signaling is significantly associated with pre-term birth (Firmal et al., 2020).
Acioglu C, Heary RF, Elkabes S (2022) Roles of neuronal toll-like receptors in neuropathic pain and central nervous system injuries and diseases. BRAIN Behav Immun 102:163–178.
Bauernfeind FG, Horvath G, Stutz A, Alnemri ES, MacDonald K, Speert D, Fernandes-Alnemri T, Wu J, Monks BG, Fitzgerald KA, Hornung V, Latz E (2009) NF-kB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J Immunol 183:787 Available at: /pmc/articles/PMC2824855/ [Accessed August 5, 2022].
Ciesielska A, Matyjek M, Kwiatkowska K (2021) TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling. Cell Mol LIFE Sci 78:1233–1261.
da Silva Correia J, Soldau K, Christen U, Tobias PS, Ulevitch RJ (2001) Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex. transfer from CD14 to TLR4 and MD-2. J Biol Chem 276:21129–21135.
Dinges MM, Schlievert PM (2001) Comparative Analysis of Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Activity in Serum and Lethality in Mice and Rabbits Pretreated with the Staphylococcal Superantigen Toxic Shock Syndrome Toxin 1. Infect Immun 69:7169 Available at: /pmc/articles/PMC100117/ [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.
Firmal P, Shah VK, Chattopadhyay S (2020) Insight Into TLR4-Mediated Immunomodulation in Normal Pregnancy and Related Disorders. Front Immunol 11:807 Available at: /pmc/articles/PMC7248557/ [Accessed August 8, 2022].
Gaikwad S, Patel D, Agrawal-Rajput R (2017) CD40 Negatively Regulates ATP-TLR4-Activated Inflammasome in Microglia. Cell Mol Neurobiol 37:351–359 Available at: https://pubmed.ncbi.nlm.nih.gov/26961545/ [Accessed January 7, 2021].
Gorina R, Font-Nieves M, Márquez-Kisinousky L, Santalucia T, Planas AM (2011) Astrocyte TLR4 activation induces a proinflammatory environment through the interplay between MyD88-dependent NFκB signaling, MAPK, and Jak1/Stat1 pathways. Glia 59:242–255 Available at: https://onlinelibrary-wiley-com.proxy.library.carleton.ca/doi/full/10.1002/glia.21094 [Accessed August 1, 2022].
Guha M, Mackman N (2001) LPS induction of gene expression in human monocytes. Cell Signal 13:85–94.
Hardy E, Kamphuis T, Japaridze A, Wilschut JC, Winterhalter M (2012) Nanoaggregates of micropurified lipopolysaccharide identified using dynamic light scattering, zeta potential measurement, and TLR4 signaling activity. Anal Biochem 430:203–213 Available at: https://www.sciencedirect.com/science/article/pii/S0003269712004435.
Haudek SB, Natmessnig BE, Fürst W, Bahrami S, Schlag G, Redl H (2003) Lipopolysaccharide dose response in baboons. Shock 20:431–436 Available at: https://pubmed.ncbi.nlm.nih.gov/14560107/ [Accessed August 8, 2022].
Ito Y, Betsuyaku T, Nasuhara Y, Nishimura M (2007) Lipopolysaccaride-induced neutrophilic inflammation in the lungs differs with age. Exp Lung Res 33:375–384.
Jerala R (2007) Structural biology of the LPS recognition. Int J Med Microbiol 297:353–363.
Kim JJ, Sears DD (2010) TLR4 and Insulin Resistance. Gastroenterol Res Pract 2010.
Kim S, Kim SY, Pribis JP, Lotze M, Mollen KP, Shapiro R, Loughran P, Scott MJ, Billiar TR (2013) Signaling of high mobility group box 1 (HMGB1) through toll-like receptor 4 in macrophages requires CD14. Mol Med 19:88–98.
Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA (1996) The dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86:973–983.
Levy O (2005) Innate immunity of the human newborn: distinct cytokine responses to LPS and other Toll-like receptor agonists. J Endotoxin Res 11:113–116 Available at: https://pubmed.ncbi.nlm.nih.gov/15949138/ [Accessed August 8, 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.
Loes AN, Bridgham JT, Harms MJ (2018) Coevolution of the toll-like receptor 4 complex with calgranulins and lipopolysaccharide. Front Immunol 9:304.
Lu Y-C, Yeh W-C, Ohashi PS (2008) LPS/TLR4 signal transduction pathway. Cytokine 42:145–151.
Lucas K, Maes M (2013) Role of the Toll Like Receptor (TLR) Radical Cycle in Chronic Inflammation: Possible Treatments Targeting the TLR4 Pathway. Mol Neurobiol 48:190–204.
Maeshima N, Fernandez R (2013) Recognition of lipid A variants by the TLR4-MD-2 receptor complex . Front Cell Infect Microbiol 3 Available at: https://www.frontiersin.org/articles/10.3389/fcimb.2013.00003.
Neal MD, Sodhi CP, Jia H, Dyer M, Egan CE, Yazji I, Good M, Afrazi A, Marino R, Slagle D, Ma C, Branca MF, Prindle TJ, Grant Z, Ozolek J, Hackam DJ (2012) Toll-like receptor 4 is expressed on intestinal stem cells and regulates their proliferation and apoptosis via the p53 up-regulated modulator of apoptosis. J Biol Chem 287:37296–37308.
Nicholas DA, Zhang K, Hung C, Glasgow S, Aruni AW, Unternaehrer J, Payne KJ, Langridge WHR, De Leon M (2017) Palmitic acid is a toll-like receptor 4 ligand that induces human dendritic cell secretion of IL-1β. PLoS One 12 Available at: /pmc/articles/PMC5413048/ [Accessed August 1, 2022].
Qin L, Li G, Qian X, Liu Y, Wu X, Liu B, Hong JS, Block ML (2005) Interactive role of the toll-like receptor 4 and reactive oxygen species in LPS-induced microglia activation. Glia 52:78–84.
Radakovics K, Battin C, Leitner J, Geiselhart S, Paster W, Stöckl J, Hoffmann-Sommergruber K, Steinberger P (2022) A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands . Front Immunol 12 Available at: https://www.frontiersin.org/articles/10.3389/fimmu.2021.817604.
Rehli M (2002) Of mice and men: Species variations of Toll-like receptor expression. Trends Immunol 23:375–378 Available at: https://pubmed.ncbi.nlm.nih.gov/12133792/ [Accessed January 11, 2021].
Rodríguez-Gómez JA, Kavanagh E, Engskog-Vlachos P, Engskog MKR, Herrera AJ, Espinosa-Oliva AM, Joseph B, Hajji N, Venero JL, Burguillos MA (2020) Microglia: Agents of the CNS Pro-Inflammatory Response. Cells 9 Available at: /pmc/articles/PMC7407646/?report=abstract [Accessed December 16, 2020].
Sauter C, Wolfensberger C (1980) Interferon in human serum after injection of endotoxin. Lancet (London, England) 2:852–853 Available at: https://pubmed.ncbi.nlm.nih.gov/6159510/ [Accessed August 8, 2022].
Schappe MS, Desai BN (2018) Measurement of TLR4 and CD14 Receptor Endocytosis Using Flow Cytometry. Bio-protocol 8.
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].
Sharma J, Boyd T, Alvarado C, Gunn E, Adams J, Ness T, Dunwoody R, Lamb J, House B, Knapp J, Garner R (2019) Reporter Cell Assessment of TLR4-Induced NF-κB Responses to Cell-Free Hemoglobin and the Influence of Biliverdin. Biomedicines 7.
Tateda K, Matsumoto T, Miyazaki S, Yamaguchi K (1996) Lipopolysaccharide-induced lethality and cytokine production in aged mice. Infect Immun 64:769–774 Available at: https://pubmed.ncbi.nlm.nih.gov/8641780/ [Accessed August 8, 2022].
Taylor KR, Trowbridge JM, Rudisill JA, Termeer CC, Simon JC, Gallo RL (2004) Hyaluronan Fragments Stimulate Endothelial Recognition of Injury through TLR4. J Biol Chem 279:17079–17084.
Taylor MD, Fernandes TD, Yaipen O, Higgins CE, Capone CA, Leisman DE, Nedeljkovic-Kurepa A, Abraham MN, Brewer MR, Deutschman CS (2022) T cell activation and IFN gamma modulate organ dysfunction in LPS-mediated inflammation. J Leukoc Biol 112:221–232.
Ungaro R, Abreu MT, Fukata M (2009) Practical techniques for detection of Toll-like receptor-4 in the human intestine. Methods Mol Biol 517:345–361.
Vaure C, Liu Y (2014) A comparative review of toll-like receptor 4 expression and functionality in different animal species. Front Immunol 5:316.
Yang H, Hreggvidsdottir HS, Palmblad K, Wang H, Ochani M, Li J, Lu B, Chavan S, Rosas-Ballina M, Al-Abed Y, Akira S, Bierhaus A, Erlandsson-Harris H, Andersson U, Tracey KJ (2010) A critical cysteine is required for HMGB1 binding to Toll-like receptor 4 and activation of macrophage cytokine release. Proc Natl Acad Sci U S A 107:11942–11947.
Yang Y, Lv J, Jiang S, Ma Z, Wang D, Hu W, Deng C, Fan C, Di S, Sun Y, Yi W (2016) The emerging role of Toll-like receptor 4 in myocardial inflammation. Cell Death Dis 7:e2234–e2234 Available at: https://doi.org/10.1038/cddis.2016.140.
Zarember KA, Godowski PJ (2002) Tissue Expression of Human Toll-Like Receptors and Differential Regulation of Toll-Like Receptor mRNAs in Leukocytes in Response to Microbes, Their Products, and Cytokines. J Immunol 168:554–561 Available at: https://www.jimmunol.org/content/168/2/554 [Accessed August 8, 2022].
Zeuke S, Ulmer AJ, Kusumoto S, Katus HA, Heine H (2002) TLR4-mediated inflammatory activation of human coronary artery endothelial cells by LPS. Cardiovasc Res 56:126–134.