The innate immune system represents the first line of host defense against microbial pathogens and relies on germline-encoded receptors known as pattern-recognition receptors (PRRs) [1,2,3]. These receptors recognize molecular signatures expressed on microbes, known as pathogen-associated molecular patterns (PAMPs), as well as molecular motifs expressed on damaged host cells, known as damage-associated molecules patterns (DAMPs) [1,2,3]. The interaction of PRRs with PAMPs and DAMPs can result in expression of pro-inflammatory cytokines and other immune mediators (Figure 1) [1,2,3]. PRRs are primarily expressed by antigen-presenting cells (APCs), including dendritic cells and macrophages, but they have also been found to be expressed on other immune and non-immune cells [2,4].
Among the several different classes of PRRs in mammals, the Toll-like receptors (TLRs) have been the most extensively studied [2,4,5]. Other classes of PRRs include RIG-I–like receptors (RLRs), Nod-like receptors (NLRs), AIM2-like receptors (ALRs), and C-type lectin receptors (CLRs), as well as cGas and other intracellular DNA sensors (Figure 1) [6,7,8]. This overview will focus on the TLR and RLR classes of PRRs.
Figure 1. Various pathogens are recognized by innate immune cells (such as dendritic cells and macrophages) [2,3,4]. These immune cells contain pattern recognition receptors (PRRs) that identify pathogen-induced infections through certain protein and other molecular motifs known as pathogen- and damage-associated molecular patterns (PAMPs and DAMPs), found on pathogens and damaged host cells, respectively [2,3]. The PPRs include Toll-Like receptors (TLRs), nucleotide-binding oligomerization domain-like receptors (Nod-like receptors, NLRs), and C-type lectin receptors (CLRs) [6,8].
TLRs play an important role in the innate immune system by recognizing PAMPs [1,2,4,5]. TLRs are expressed by dendritic cells (DCs), macrophages, and non-immune cells such as fibroblasts. TLRs can be classified based on their cell localization [2,3,4]. In humans, the TLR family consists of 10 members (TLR1–TLR10), whereas mice have 12 members (TLR1–TLR9, TLR11–TLR13). The cell-surface TLRs include TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10 and contain extracellular domains [2,3,4]. The intracellular TLRs include TLR 3, TLR7, TLR8, TLR9, TLR11, TLR12, and TLR 13 and are found in the endoplasmic reticulum (ER), endosomes, and lysosomes [2,6,7]. Cell-surface TLRs recognize bacterial membrane components such as lipids, lipoproteins, and proteins, while intracellular TLRs predominantly recognize nucleic acids [8,9]. For a nonexhaustive list of PAMPs recognized by TLRs, see Table 1.
TLRs are considered to be type-I transmembrane proteins consisting of an N-terminal ligand recognition domain, a single transmembrane region, and a C-terminal signaling domain . The extracellular domain of TLRs contains tandem copies of a motif known as the leucine-rich repeat structure (LRR), which is responsible for recognizing PAMPs . The intracellular signaling domain of TLRs is homologous to the IL-1 receptor and is known as Toll/intereukin-1 receptor (TIR) domain . This signaling domain is necessary to initiate downstream signaling pathways . The two major signaling pathways involved in TLR activation include the MyD88-dependent pathway and the TRIF-dependent pathway (Figure 2) .
Figure 2. Signaling pathways triggered in APCs by pathogens through TLRs. Cell-surface TLRs such as TLR1 contain extracellular domains, whereas intracellular TLRs like TLR3 localize to endosomal compartments [2,6,10]. Formation of homo- or heterodimers induces downstream signaling through either the MyD88- or TRIF-dependent pathways . Downstream engagements of TLR leads to expression of proinflammatory cytokines, including interleukin-6 (IL-6), IL-8, IL-12, and tumor necrosis factor alpha (TNFα) .
|TLR type (Structure)||Adapters (Structure)||PAMPs/Activators||Species|
|TLR1-TLR2 (LRR-TIR)||MyD88 (TIR-DD), TIRAP (TIR)||Triacyl lipopeptides||Bacteria|
|TLR2–TLR6 (LRR–TIR)||MyD88 TIRAP||Diacyl lipopeptides|
|TLR2 (LRR–TIR)||MyD88, TIRAP||PGN Lipoarabinomannan|
Virus (measles virus)
|TLR3 (LRR–TIR)||TRIF (TIR)||dsRNA||Virus|
|TLR4 (LRR–TIR)||MyD88, TIRAP, TRIF, TRAM (TIR)||LPS|
Virus (RSV, MMTV)
|TLR7 (LRR–TIR)||MyD88||ssRNA||RNA virus|
|hTLR8 (LRR–TIR)||MyD88||ssRNA||RNA virus|
|TLR9 (LRR–TIR)||MyD88||CpG DNA|
|mTLR11 (LRR–TIR)||MyD88||Not determined|
|Bacteria (uropathogenic bacteria)|
Parasites (Toxoplasma gondii)
Abbreviations: dsRNA, double-stranded ribonucleic acid; HA, hemagglutinin; LPS, lipopolysaccharides; LRR, leucine rich repeat; LTA, lipoteichoic acid; MMTV, mouse mammary tumor virus; MyD88, myeloid differentiation primary response 88; PGN, peptidoglycan; RSV, respiratory syncytial virus; ssRNA, single-stranded ribonucleic acid; tGPI-mucin (glycosylphosphatidylinositol-anchored mucin-like glycoproteins); TIR, Toll/interleukin-1 receptor; TIRAP, TIR domain–containing adaptor protein; TLR, Toll-like receptor; TRIF, TIR domain–containing adapter-inducing interferon-β; TRAM, TRIF-related adaptor molecule.
Whereas the endosomal RNA–detecting TLRs contribute to antiviral immunity, RLRs are essential for immune recognition of and response to most RNA viruses [11,12]. RLRs primarily recognize single- and double-stranded RNA (ssRNA and dsRNA). Members of the RLR family of receptors can be found in the cytoplasm of all cell types and consist of RIG-I (retinoic acid–inducible gene I), MDA5 (melanoma differentiation–associated gene 5) and LGP2 (laboratory of genetics and physiology 2) [13,14]. RIG-I and MDA5 induce type I interferon (IFN) and chemokines upon activation by viral infection or the presence of bacterial RNA.
RLRs are generally composed of two N-terminal caspase recruitment domains (CARDs), a central DEAD box helicase/ATPase domain, and a C-terminal regulatory domain [12,13,14]. LGP2 does not contain a CARD domain and therefore its effect on downstream antiviral signaling is likely due to interaction with dsRNA viral ligand or with other RLRs (RIG-I and MDA5). RLRs are localized in the cytoplasm and recognize the genomic RNA of dsRNA viruses and dsRNA generated as the replication intermediate of ssRNA viruses [15,16]. The expression of RLRs is greatly enhanced in response to type I IFN stimulation or virus infection [13,14,15]. Stimulation of RIG-I or MDA5 by viral RNA releases the associated CARD domains, which aggregate with K63 polyubiquitin chains into CARD tetramers and then bind and activate the adaptor molecule MAVS. MAVS recruits TBK-1, which phosphorylates IRF3 to induce transcription of type I IFN genes [13,14,15].
PRR pathways are integral for mounting an effective defense against microbial infections. Several studies have investigated the relationship between microbial pathogens and their effects on the innate immune system [17,18].
Qureshi et al. investigated the response to bacterial lipopolysaccharide (LPS) in mice [17,18]. Their data showed that regulation of the response to LPS was localized to mouse chromosome 4 at the Lps locus, which encodes TLR4. This study identified TLR4 as a critical regulator of the host response to the pathogenic LPS [17,18].
Oosting et al. looked at the relationship between TLR1/TLR2 heterodimers and their response to the Borrelia species . One important characteristic of TLR2 is that it can form heterodimers with TLR1 and TRL6 . Even though TLR2 has been recognized as the main receptor for Borrelia spp, there is evidence for roles of TLR1 and TLR6 . TLR1- and TLR6-knockout mice were each exposed to Borrelia. The TLR1 knockout mice showed increased expression of IFNγ, in contrast to the TLR6 knockout mice, which were not able to induce a significant IFNγ response. There were no significant changes in the proinflammatory cytokine production between the two knockout lines .
TLRs are vital sensors in pathogen detection, and identifying and characterizing TLR expression on the surface of immune cells is important for studying the cellular mechanism for viral clearance. Flow cytometry is typically the method of choice for analyzing the TLR profile on immune cells.
Generally, the entire cell population is stained with a viability stain, followed by immunolabeling with TLR-specific fluorescent antibody conjugates. To stain all live cells in a single-cell suspension, the cells are washed once with phosphate-buffered saline (PBS), and resuspended in a staining buffer such as Invitrogen Bioscience Flow Cytometry Staining Buffer at 1 x106 cells per mL. Next, 1 µl of a viability dye such as a Invitrogen Fixable Viability Stain is added to the cells. Cells are then incubated at room temperature or on ice for 30 minutes, protected from light, washed once with 1 mL of PBS, and resuspend in 100 μL of staining buffer.
For cell-surface TLRs with extracellular domains, such as TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10, cells are directly stained with a fluorescent antibody conjugate that recognizes the TLR of interest (or a cocktail of spectrally compatible fluorescent antibody conjugates) (Figure 3, Table 2). Cells are incubated for 30 minutes on ice, washed once with staining buffer, and then resuspended in 300 µL of staining buffer. Cells are ready to be analyzed with a flow cytometer such as the Invitrogen Attune NxT Flow Cytometer. If fixation of cells is required before acquisition, cells can be fixed using a fixation buffer such as the Invitrogen eBioscience IC Fixation Buffer. After a short incubation, cells are washed and resuspended in staining buffer before flow cytometry analysis.
For antibody staining of intracellular TLRs, such as TLR3, TLR7, TLR8, TLR9, TLR11, TLR12, and TLR13, cells must be fixed and permeabilized to allow antibody access to the antigen. Cells, which may have already been immunolabeled for cell-surface antigens, are fixed as described above and permeabilized using a permeabilization buffer such as the Invitrogen eBioscience Permeabilization Buffer. Once fixed and permeabilized, the cells are incubated with a fluorescent antibody conjugate that recognizes the TLR of interest (or a cocktail of spectrally compatible fluorescent antibody conjugates) (Table 2). Cells are incubated with the antibody cocktail for 30 minutes on ice, washed once with permeabilization buffer and once with staining buffer, and then resuspended in 300 µl of staining buffer. After resuspension, cells are ready to be analyzed by flow cytometry.
Background fluorescence may be a problem due to the high density of Fc receptors on immune cells . We recommend using an unlabeled blocking antibody such as the Invitrogen Fc Receptor Binding Inhibitor Polyclonal Antibody for human cells or the Invitrogen CD16/CD32 Monoclonal Antibody (clone 93) and Invitrogen Normal Rat Serum for mouse cells.
For intranuclear detection, e.g., antibody labeling of NF-κB and other transcription factors (Table 2), we recommend using the Invitrogen eBioscience FoxP3 / Transcription Factor Staining Buffer Set to fix and permeabilize the cells (which may have previously been immunolabeled for cell-surface antigens). After fixation and permeabilization, cells are incubated with one or a cocktail of spectrally compatible fluorescent antibody conjugates that recognize the transcription factors of interest. Cells are incubated 30 minutes on ice and washed once with the permeabilization buffer from FoxP3 / Transcription Factor Staining Buffer Set and once with staining buffer, and then resuspended in 300 µL of staining buffer. After resuspension, cells are ready to be analyzed by flow cytometry.
|TLR markers||Method of detection|
|TLR1||WB, IHC, ICC, IF, Flow|
|TLR2||WB, IHC, ICC, IF, Flow|
|TLR4||WB, IHC, ICC, IF, Flow|
|TLR5||WB, IHC, ICC, IF, Flow|
|TLR10||WB, IHC, ICC, IF, Flow|
|TLR6||WB, IHC, ICC, IF, Flow|
|TLR7||WB, IHC, ICC, IF, Flow|
|TLR8||WB, IHC, ICC, IF, Flow|
|TLR9||WB, IHC, ICC, IF, Flow|
|TLR11||WB, IHC, ICC, IF, Flow|
|TLR12||WB, IHC, ICC, IF, Flow|
|TLR13||WB, IHC, ICC, IF, Flow|
|NF-κB||WB, IHC, ICC, IF, Flow, ChIP|
|IFNγ||WB, IHC, ICC, IF, ELISA|
|TNFα||WB, IHC, ICC, IF, ELISA|
|IL-6||WB, IHC, ICC, IF, ELISA|
|IL-8||WB, IHC, ICC, IF, ELISA|
|IL-12||WB, IHC, ICC, IF, ELISA|
Figure 3. Intracellular flow cytometric analysis of TLR7 in 1 x 106 human BDCM (B cell with DC morphology) cells using 10 µL (0.5 µg) of Invitrogen TLR7 Monoclonal Antibody, PE conjugate (clone 4G6, Cat. No. MA5-16249) (red) and 0.5 µg of mouse IgG1 isotype control (green).
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