Toll like receptor 4 Activation

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).

TLR4 EXPRESSION

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).

TLR4 ACTIVATION

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.
  • HEK-Blue(TM)-hTLR4 cell line (human) produces SEAP when TLR4 induction activates NF-κB and AP-1.(Hardy et al., 2012; Sharma et al., 2019)
  • TLR4/MD-2/NF-κB/SEAPorter(TM) HEK 293 produces SEAP when TLR4 and MD-2 are transcribed by NF-κB 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).

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).

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