Pattern Recognition Receptors and Inflammation - TLRs and Their Ligands

TLRs and Their Ligands

The TLR family is one of the best-characterized PRR families and is responsible for sensing invading pathogens outside of the cell and in intracellular endosomes and lysosomes (Akira et al., 2006). TLRs are characterized by N-terminal leucine-rich repeats (LRRs) and a transmembrane region followed by a cytoplasmic Toll/IL-1R homology (TIR) domain. Ten TLRs have been identified in humans and 12 in mice. Different TLRs recognize the different molecular patterns of microorganisms and self-components (Table 1).

 

 

TLR2 senses various components from bacteria, mycoplasma, fungi, and viruses. These components include the lipoproteins of bacteria and mycoplasma. TLR2 recognizes its ligands by forming a heterodimer with either TLR1 or TLR6 (Figure 1).

 

Figure 1. TLR2, TLR3, and TLR4 Signaling Pathways Lipoproteins and LPS are recognized on the cell surface by a heterodimer of TLR1/6 and TLR2, and by 2 sets of TLR4/MD2 complexes, respectively. Ligand stimulation recruits MyD88 and TIRAP to the TLR, and a complex of IRAKs and TRAF6 is subsequently formed. TRAF6 acts as an E3 ubiquitin ligase and catalyzes formation of a K63-linked polyubiquitin chain on TRAF6 itself and generation of an unconjugated polyubiquitin chain with an E2 ubiquitin ligase complex of Ubc13 and Uev1A. Ubiquitination activates a complex of TAK1, TAB1, and TAB2/3 resulting in the phosphorylation of NEMO and the activation of an IKK complex. Phosphorylated IkB is degraded, and the freed NF-kB translocates to the nucleus where it drives expression of cytokine genes. Simultaneously, TAK1 activates MAP kinase cascades leading to the activation of AP-1, which is also critical for the induction of cytokine genes.
LPS induces translocation of TLR4 to the endosome together with TRAM. TLR3 is present in the endosome and recognizes dsRNA. TLR3 and TLR4 activate TRIF-dependent signaling, which activates NF-kB and IRF3 resulting in the induction of proinflammatory cytokine genes and type I IFNs. TRAF6 and RIP1 activate NF-kB, whereas TRAF3 is responsible for phosphorylation of IRF3 by TBK1/IKK-i. NAP1 and SINTBAD are required for the activation of TBK1/IKK-i. Phosphorylated IRF3 translocates into the nucleus to induce expression of type I IFN genes.

 

Translocation : 염색체 전좌는 한 염색체의 일부가 다른 염색체로 옮겨지는 현상

 

 The resulting TLR1/TLR2 and TLR6/TLR2 complexes recognize distinct ligands (triacyl and diacyl lipoproteins, respectively). The crystal structures of the extracellular domains of TLR2, TLR1, and TLR6 revealed that they form M-shaped structures and that their cognate ligands interact with internal pockets formed by the TLR1/TLR2 or TLR6/TLR2 heterodimers (Jin et al., 2007).

 

Stimulation with TLR2 ligands, such as triacyl and diacyl lipoproteins, induces the production of various proinflammatory cytokines (but not type I IFNs) in macrophages and DCs.

 

However, another report showed that TLR2 in inflammatory monocytes induced type I IFNs in response to viral infection, suggesting that the cellular responses to TLR2 ligands differ depending on the cell types involved (Barbalat et al., 2009). TLR10 is related to TLR1 and TLR6 based on sequence similarity.

 

TLR10 seems to be functional in humans, although mouse TLR10 is disrupted by insertion of an endogenous retrovirus. The ligand for TLR10 has not been identified. TLR4 recognizes lipopolysaccharide (LPS) together with myeloid differentiation factor 2 (MD2) on the cell surface. LPS is a component derived from the outer membrane of Gramnegative bacteria and is known to be a cause of septic shock.

 

The crystal structure of a complex comprising TLR4, MD2, and LPS revealed that two complexes of TLR4-MD2-LPS interact symmetrically to form a TLR4 homodimer (Park et al., 2009). TLR4 is also involved in the recognition of viruses by binding to viral envelope proteins. In addition, TLR4 modulates the pathogenesis of H5N1 avian influenza virus infection by recognizing a DAMP rather than the virus itself (Imai et al., 2008). Acute lung injury caused by avian influenza virus infection produces endogenous oxidized phospholipids, which stimulate TLR4.

 

Mice lacking TLR4 were found to be resistant to avian fluinduced lethality TLR5 is highly expressed by DCs of the lamina propria (LPDCs) in the small intestine, where it recognizes flagellin from flagellated bacteria. In response to flagellin, LPDCs induce B cells to differentiate into IgA-producing plasma cells and trigger the differentiation of naive T cells into antigen-specific Th17 and Th1 cells (Uematsu et al., 2008).

 

TLR11, which is present in mice but not in humans, shows close homology to TLR5. TLR11 recognizes uropathogenic bacteria and a profilin-like molecule derived from the intracellular protozoan Toxoplasma gondii (Yarovinsky et al., 2005).

 

A set of TLRs, comprising TLR3, TLR7, TLR8, and TLR9, recognize nucleic acids derived from viruses and bacteria, as well as endogenous nucleic acids in pathogenic contexts (Akira et al., 2006).

 

Activation of these TLRs leads to the production of type I IFNs in addition to proinflammatory cytokines. TLR3 detects viral double-stranded (ds) RNA in the endolysosome. TLR3 is involved in the recognition of polyinosinic polycytidylic acid (poly I:C), a synthetic dsRNA analog. Although inoculation of mice with poly I:C induces the production of cytokines as well as type I IFNs in mice, TLR3 is essential for the production of cytokines such as IL-12p40, but not type I IFNs in sera (Kato et al., 2006).

 

The crystal structure of TLR3 bound to dsRNA revealed that dsRNA binds to the N-terminal and C-terminal portions of TLR3 LRRs, and this ligand binding dimerizes two TLR3 molecules (Choe et al., 2005; Liu et al., 2008). Mouse TLR7 and human TLR7/8 recognize single-stranded (ss) RNAs from RNA viruses, as well as small purine analog compounds (imidazoquinolines). TLR7 also detects RNAs from bacteria such as Group B Streptococcus in endolysosomes in conventional DCs (cDCs) (Mancuso et al., 2009).

 

TLR9 senses unmethylated DNA with CpG motifs derived from bacteria and viruses. Although the CpG motif was thought to be essential for TLR9 stimulation, the DNA sugar backbone of 20 deoxyribose also mediates TLR9 recognition (Haas et al., 2008). In addition to DNA, TLR9 also recognizes hemozoin, a crystalline metabolite of hemoglobin produced by the malaria parasite (Coban et al., 2005). TLR9 directly binds to hemozoin, and a crude extract of the malaria parasite elicits parasite-antigen-specific immune responses via TLR9 (Coban et al., 2010).

 

However, another report shows that TLR9 recognizes malaria parasite DNA contained in purified hemozoin, and that hemozoin only transports malaria parasite DNA to the endosome, where TLR9 is present (Parroche et al., 2007). Further studies will clarify the role of TLR9 in the recognition of malaria parasite components.

 

TLR7 and TLR9, but not TLR3, are highly expressed in plasmacytoid DCs (pDCs), a cell type that produces large amounts of type I IFNs in response to virus infection. Accumulating evidence underscores the importance of the localization of TLRs in the cell for their recognition by ligand (Barton and Kagan, 2009).

 

Given that self-nucleotides are potent TLR ligands and may facilitate autoimmunity, TLRs that recognize self-nucleotides are compartmentalized to avoid unwanted activation. Although TLR1, TLR2, TLR4, TLR5, and TLR6 are present on the plasma membrane, TLR3, TLR7, and TLR9 are mainly present on the endoplasmic reticulum (ER) membrane. It has been proposed that self-nucleic acids are degraded by extracellular or endosomal DNases prior to recognition by TLRs. Nucleic acid-sensing TLRs are recruited from the ER to endolysosomes following stimulation by their ligands (Figure 2).

 

Figure 2. Nucleic Acid Sensing by TLR7 and TLR9 TLR7 and TLR9 recognize viral ssRNA and CpG DNA, respectively. Stimulation with ligands or infection by viruses induces trafficking of TLR7 and TLR9 from the ER to the endolysosome via UNC93B1. TLR9 undergoes cleavage by proteases present in the endolysosome. A complex of MyD88, IRAK-4, TRAF6, TRAF3, IRAK-1, IKK-a, and IRF7 is recruited to the TLR. Phosphorylated IRF7 translocates into the nucleus and upregulates the expression of type I IFN genes. Viruses that have entered the cytoplasm are engulfed by autophagosomes and deliver viral nucleic acids to the endolysosome. An HMGB1-DNA complex released from damaged cells is captured by RAGE. Autoantibodies recognizing self-DNA or -RNA bind to FcgRIIa. LL37, an antimicrobial peptide, associates with endogenous DNA. These proteins are responsible for the delivery of endogenous nucleic acids to endolyosomes where they are recognized by TLR7 or TLR9.

 

 

The mechanism by which nucleotide-recognizing TLRs are recruited from the ER to the endolysosome compartment remains to be clarified. However, a forward genetics screen in mice revealed that UNC93B1 (an ER protein with 12 membrane-spanning domains) is responsible for TLR3, TLR7, and TLR9 signaling by governing the translocation of these TLRs from the ER to the endolysosome (Kim et al., 2008; Tabeta et al., 2006). When TLR9 is recruited from the ER to the endolysosome, it undergoes processing by proteases, such as cathepsins, in the endolysosome (Ewald et al., 2008; Park et al., 2008).

 

The processed form of TLR9 is responsible for CpG-DNA recognition. It has been shown that cathepsins B, K, and L and asparagine endopeptidase are required for TLR9 responses (Asagiri et al., 2008; Matsumoto et al., 2008; Sepulveda et al., 2009). Currently, it remains unclear whether TLR7 is also cleaved in the endolysosome, although endosomal acidification is required for the sensing of TLR7 ligands. TLR7 and TLR9 are essential for virus-induced type I IFN production by pDCs (Kato et al., 2005). Viral nucleotides can interact with TLR7 and TLR9 in pDCs after they have been endocytosed. Alternatively, once the viruses invade pDCs and virions are present in the cytoplasm, they can be delivered to the endolysosome where TLR7 and TLR9 are recruited for viral sensing. pDCs take advantage of a cellular process called autophagy in which self-proteins and damaged organelles are degraded in double-membraned vesicles called autophagosomes (Lee et al., 2007). In the absence of ATG5, a protein essential for autophagosome formation, pDCs fail to produce type I IFNs in response to virus infection, suggesting that the cytoplasmic virions are engulfed by autophagosomes and then fuse with lysosomes. However, ATG5 is also required for responses to CpGDNA. Therefore, autophagy may control either the endosomal maturation required for CpG-DNA sensing or the TLR9 signaling pathways in pDCs, or both. TLR-mediated microbial recognition is very important for host defense against pathogens. On the other hand, excess responses to TLR ligands induce lethal septic shock syndrome. These observations indicate that appropriate activation of TLRs is vital for eradicating invading pathogens without causing harmful damage to the host.