Ligands and Inhibitors of TLR Reporter Cell Lines

tlrs and ligands

The earliest phase of response to an infection is dependent on the innate immune system. As well as relying on anatomical barriers such as skin, tears and saliva, and humoral barriers such as the complement system, innate immunity involves the recognition of conserved features of pathogens. Whilst adaptive immunity builds up a memory, resulting in the rapid destruction of pathogens upon a subsequent exposure, innate immunity is our first line of defence against infection.

Initial recognition of micro-organisms by the innate immune system relies on germline-encoded Pattern Recognition Receptors (PRRs). PRRs are expressed by antigen processing and presentation cells such as macrophages and dendritic cells. These receptors are activated upon detection of Pathogen-Associated Molecular Patterns (PAMPs) or Damage-Associated Molecular Patterns (DAMPs). PAMPs are molecular sequences shared by large groups of pathogens, and are essential for microbial survival and/or pathogenicity. They are distinct from self-antigens and include cell wall constituents such as lipopolysaccharide (LPS), peptidoglycan, lipoteichoic acid or lipoarabinomannan, as well as single or double stranded RNA and unmethylated CpG DNA. DAMPs are endogenous molecules that are released from damaged cells, and vary widely depending on cell type.

PRRs include transmembrane Toll-Like Receptors (TLR) and C-type Lectin Receptors (CLR), as well as cytoplasmic Nod-Like Receptors (NLR) and Retinoic-acid Inducible gene-like Receptors (RIG). TLRs are amongst the most comprehensively studied PRRs due to their central role in host defences and their involvement in a number of diverse pathological processes. As well as triggering the innate immune responses which are so crucial to preventing the early spread of pathogens during an infection, TLRs also activate antigen presenting cells (APC), leading to the induction of adaptive immunity (1). The Toll receptor is part of an evolutionarily conserved recognition and signalling system, and was originally discovered in Drosophila in 1988 during a screen for embryonic polarity genes. Toll, known as TLR (Toll-Like Receptor) in mammals, is highly conserved across species. Most mammals have between ten and fifteen types of TLRs. TLRs are Type I transmembrane proteins consisting of an extracellular leucine-rich repeat domain which is involved in ligand recognition, a transmembrane domain, and an intracellular Toll-Interleukin 1 receptor (TIR) domain which is involved in signalling. The TIR domain is so called because of its homology to the cytoplasmic domain of the mammalian Interleukin-1 receptor (IL-1R) (2).

TLRs are typically localised to the plasma membrane (TLR1, 2, 4, 5, 6, 10) or to endolysosomes (TLR3, 7, 8, 9, 11, 12, 13), and each TLR recognises distinct PAMPs. Following PAMP recognition, adaptor molecules are recruited to the intracellular TIR domain of TLRs. This leads to the activation of signalling cascades and the expression of genes involved in immune responses. The adaptor molecules themselves also contain a TIR domain, and several such molecules have been identified including MyD88, TIR domain-containing adaptor inducing IFN-β (TRIF/TICAM-1), Toll/IL-1R domain containing adaptor protein/MyD88 adapter-like (TIRAP/Mal) and TRIF-related adaptor molecule (TRAM) (3). MyD88 is essential for signalling by all TLR family members, with the exception of TLR3 (4). Many TLR signalling pathways are still being elucidated, however activated pathways are known to include NF-κB, p38 MAPK, JNK and Interferon, and key signalling proteins include IL-1 receptor associated kinases (IRAKs), transforming growth factor kinase (TAK-1), IκB kinases (IKKs) and TNF receptor associated factors (TRAFs).
Novus Biologicals sells a range of SEAPorterTM reporter cell lines, which can be used to screen for TLR agonists and antagonists. SEAPorterTM are HEK293T cells that have been stably co-transfected with TLR and SEAP (Secreted Alkaline Phosphatase) genes. The SEAP reporter gene is under the transcriptional control of an NF-κB response element. This gene enables kinetic measurements to be performed through sequential sampling of the growth media, and then SEAP activity is readily measured using the Secreted Alkaline Phosphatase Reporter Assay Kit (NBP2-25285). Novus Biologicals also sells an NF-κB SEAP reporter cell line without an added TLR gene (NBP2-26260). This is a useful control that can be used alongside a TLR specific cell line.

Novus Biologicals also sells a number of LUCPorterTM reporter cell lines. These HEK293T cells have been stably transfected with a vector encoding the Renilla luciferase reporter gene, which is under the transcriptional control of various promoters or response elements. For TLR research these include HEK293T cell lines containing the TLR3 gene and luciferase reporter which is either under the control of an IFNβ promoter (NBP2-26262) or an interferon sensitive response element (NBP2-26292), as well as a HeLa cell line with genes for TLR4, MD-2 and CD14 where the luciferase reporter is under the control of an IL8 promoter (NBP2-26263). Measurement of luciferase activity is readily accomplished by using the LightSwitch Luciferase Reporter Assay Reagent (NBP2-25287) following cell lysis. This assay system is optimized to pair with all of the LUCPorterTM cell lines.

In addition to the reporter cell lines, Novus Biologicals also sells a range of TLR ligands and inhibitors.

TLR Ligands

  • Unmethylated deoxycytosine-deoxyguanosine (CpG) motifs are abundant in bacterial genomes, but occur much less frequently in mammalian DNA (1). These motifs consist of a cytosine joined to a guanine by a phosphodiester link, and are recognised by TLR9. Synthetic oligodeoxynucleotides (ODNs) containing these motifs can be used to stimulate TLR9. Novus Biologicals offers several formats of TLR9 ODNs, as listed in Figure 1.

Figure 1. TLR9 ODNs.

  • Flagellin (NBP2-25289) is a key component of bacterial flagellae, which are appendages used primarily for locomotion. It is highly conserved amongst both Gram positive and Gram negative bacteria, and is recognised by TLR5 (5).
  • Histones (NBP2-26236) are found in eukaryotic cell nuclei where they play a key role in packaging the genomic DNA in to nucleosomes. Extracellular histones are major mediators of cell death and sepsis, which occurs as a result of interaction with TLR2 and TLR4 (6).
  • Imidazoquinoline Resiquimod/R848 (NBP2-26231) is a synthetic imidazoquinoline, and has been demonstrated to activate TLR7 and TLR8 (7).
  • Imiquimod/R837 (NBP2-26228) is a synthetic imidazoquinoline analogue of guanosine, and has been shown to activate TLR7 and/or TLR8 (8).
  • LPS-EB (NBP2-25295, NBP2-31066) is a standard lipopolysaccharide preparation. LPS is a major component of the cell wall of Gram-negative bacteria. Recognition of LPS requires TLR4 in addition to CD14, and is enhanced by MD-2 (lymphocyte antigen 96), a secreted glycoprotein which forms a receptor complex with TLR4 (9).
  • Macrophage-Activating Lipopeptide-2, or MALP-2, (NBP2-26219) is a lipopeptide that was originally isolated from Mycoplasma fermentans, and it has subsequently been synthesised chemically and used in multiple studies. MALP-2 is recognised by a heterodimer which is formed between TLR2 and TLR6 (10).
  • PAM3CSK4 (NBP2-25297) is a synthetic lipopeptide that mimics the acylated amino terminus of bacterial lipoproteins; it is recognised by a heterodimer which is formed between TLR1 and TLR2 (11).
  • Polyinosinic-polycytidylic acid (NBP2-25288) is a synthetic analogue of double-stranded RNA, produced during the replication of many viruses; it binds directly to TLR3 (12).
  • Zymosan (NBP2-26233) is a cell wall preparation of Saccharomyces cerevisiae and is composed primarily of β-glucans, mannans, mannoproteins and chitin. It activates TLR2 in synergistic collaboration with Dectin-1, a C-type lectin that is expressed on monocytes, macrophages, and dendritic cells as a phagocytic receptor for β-glucan containing particles (13).

The TLR ligands that have just been discussed are listed in Figure 2:

SEAPorterTM cell line
LUCPorterTM cell line

Figure 2. The SEAPorterTM cell lines are HEK293 cells stably transfected to express the reporter gene for SEAP under the transcriptional control of an NF-κB response element. The target gene is human for TLR1–10, and murine for TLR11-13. The LUCPorterTM cell lines express the luciferase reporter gene under the transcriptional control of various promoters or response elements (the HEK293 cell line NBP2-26262 is controlled by the IFNβ promoter, whilst NBP2-26292, also a HEK293 line, is controlled by the interferon-sensitive response element). The target genes for NBP2-26263 (a HeLa cell line) are TLR4, MD-2 and CD14, and these are controlled by the IL-8 promoter.

TLR inhibitors

  • Chloroquine (NBP2-29386) is an anti-malarial, and is also used to treat a number of autoimmune conditions such as rheumatoid arthritis and systemic lupus erythematosus (SLE). It partitions in to acidic vesicles such as lysosomes and endosomes, resulting in the inhibition of lysosomal enzyme activity and endosomal acidification. The acidic pH of endosomes is required for the activation of endosomal TLRs. Researchers have shown that the therapeutic activity of Chloroquine results from the inhibition of TLR9 (14).
  • The MyD88 inhibitor peptide set (NBP2-29328) consists of an inhibitor peptide and a control peptide. MyD88 is an adaptor molecule that links TLRs and IL-1Rs with downstream signaling molecules. A key step in this signaling process is the homo-dimerisation of MyD88 molecules, which is prevented by binding of the inhibitor peptide to MyD88 (15).
  • Quinacrine (NBP2-29385) is an analogue of Chloroquine, and exerts its effects in a similar manner (14).
  • The adaptor molecule Toll/IL-1R domain containing adaptor protein/MyD88 adapter-like (TIRAP/Mal) facilitates the recruitment of MyD88 to TLR2/TLR4 complexes. The TIRAP inhibitor peptide sets (NBP2-26245 and NBP2-29331) each contain a peptide which is derived from TIRAP/Mal, consisting of a protein transduction sequence followed by an inhibitor sequence; this peptide interferes with assembly of the MyD88/TLR2/TLR4 signalling complex (17). Control peptide is also included in the sets, consisting only of the protein transduction sequence.
  • Viral Inhibitory Peptide of TLR4 (VIPER) consists of an 11 amino acid inhibitory sequence derived from the A46 Vaccinia virus protein, linked to a 9R homopolymer delivery sequence (18). VIPER specifically inhibits TLR4 signalling, although it’s mechanism of action has yet to be fully elucidated. The TLR4 inhibitor peptide set (NBP2-26244) consists of the VIPER peptide, plus a control peptide consisting of an inert sequence also linked to a 9R homopolymer.
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