Takuma Shibata

Structural Reorganization of the Toll-Like Receptor 8 Dimer Induced by Agonistic Ligands

Dissecting TLR8 Interactions Toll-like receptors (TLRs) activate the innate immune system in response to invading pathogens. TLR7 and TLR8 recognize single-stranded RNA from viruses and also contribute to the pathogenesis of autoimmune diseases. Tanji et al. (p. 1426) now report the crystal structure of the unliganded TLR8 ectodomain and the TLR8 ectodomain bound to three different small-molecule agonists. Ligand binding to preformed TLR8 dimers induced conformational changes that brought the C-terminal domains closer together, presumably initiating downstream signaling. The crystal structure of unbound and ligand-bound Toll-like receptor 8 reveals ligand-induced conformational changes. Toll-like receptor 7 (TLR7) and TLR8 recognize single-stranded RNA and initiate innate immune responses. Several synthetic agonists of TLR7-TLR8 display novel therapeutic potential; however, the molecular basis for ligand recognition and activation of signaling by TLR7 or TLR8 is largely unknown. In this study, the crystal structures of unliganded and ligand-induced activated human TLR8 dimers were elucidated. Ligand recognition was mediated by a dimerization interface formed by two protomers. Upon ligand stimulation, the TLR8 dimer was reorganized such that the two C termini were brought into proximity. The loop between leucine-rich repeat 14 (LRR14) and LRR15 was cleaved; however, the N- and C-terminal halves remained associated and contributed to ligand recognition and dimerization. Thus, ligand binding induces reorganization of the TLR8 dimer, which enables downstream signaling processes.

A protein associated with Toll-like receptor (TLR) 4 (PRAT4A) is required for TLR-dependent immune responses.

Immune cells express multiple Toll-like receptors (TLRs) that are concomitantly activated by a variety of pathogen products. Although there is presumably a need to coordinate the expression and function of TLRs in individual cells, little is known about the mechanisms governing this process. We show that a protein associated with TLR4 (PRAT4A) is required for multiple TLR responses. PRAT4A resides in the endoplasmic reticulum, and PRAT4A knockdown inhibited trafficking of TLR1 and TLR4 to the cell surface and ligand-induced trafficking of TLR9 to lysosomes. Other cell-surface molecules were expressed normally on immunocytes from PRAT4A−/− mice. There was impaired cytokine production to TLR ligands, except to the TLR3 ligand poly(I:C), and to whole bacteria. Activation of antigen-specific T helper type 1 responses were also defective. Moreover, PRAT4A−/− bone marrow chimeric mice were resistant to lipopolysaccharide-induced sepsis. These results suggest that PRAT4A regulates the subcellular distribution and response of multiple TLRs and is required for both innate and adaptive immune responses.

Structural basis of CpG and inhibitory DNA recognition by Toll-like receptor 9

Innate immunity serves as the first line of defence against invading pathogens such as bacteria and viruses. Toll-like receptors (TLRs) are examples of innate immune receptors, which sense specific molecular patterns from pathogens and activate immune responses. TLR9 recognizes bacterial and viral DNA containing the cytosine–phosphate–guanine (CpG) dideoxynucleotide motif. The molecular basis by which CpG-containing DNA (CpG-DNA) elicits immunostimulatory activity via TLR9 remains to be elucidated. Here we show the crystal structures of three forms of TLR9: unliganded, bound to agonistic CpG-DNA, and bound to inhibitory DNA (iDNA). Agonistic-CpG-DNA-bound TLR9 formed a symmetric TLR9–CpG-DNA complex with 2:2 stoichiometry, whereas iDNA-bound TLR9 was a monomer. CpG-DNA was recognized by both protomers in the dimer, in particular by the amino-terminal fragment (LRRNT–LRR10) from one protomer and the carboxy-terminal fragment (LRR20–LRR22) from the other. The iDNA, which formed a stem-loop structure suitable for binding by intramolecular base pairing, bound to the concave surface from LRR2–LRR10. This structure serves as an important basis for improving our understanding of the functional mechanisms of TLR9.

Toll-like receptor 8 senses degradation products of single-stranded RNA

Toll-like receptor 8 (TLR8) recognizes viral or bacterial single-stranded RNA (ssRNA) and activates innate immune systems. TLR8 is activated by uridine- and guanosine-rich ssRNA as well as by certain synthetic chemicals; however, the molecular basis for ssRNA recognition has remained unknown. In this study, to elucidate the recognition mechanism of ssRNA, we determined the crystal structures of human TLR8 in complex with ssRNA. TLR8 recognized two degradation products of ssRNA—uridine and a short oligonucleotide—at two distinct sites: uridine bound the site on the dimerization interface where small chemical ligands are recognized, whereas short oligonucleotides bound a newly identified site on the concave surface of the TLR8 horseshoe structure. Site-directed mutagenesis revealed that both binding sites were essential for activation of TLR8 by ssRNA. These results demonstrate that TLR8 is a sensor for both uridine and a short oligonucleotide derived from RNA.

An essential role for the N-terminal fragment of Toll-like receptor 9 in DNA sensing

Toll-like receptor 9 (TLR9) is an innate immune sensor for microbial DNA that erroneously responds to self DNA in autoimmune disease. To prevent autoimmune responses, Toll-like receptor 9 is excluded from the cell surface and silenced until the N-terminal half of the ectodomain (TLR9N) is cleaved off in the endolysosome. Truncated Toll-like receptor 9 (TLR9C) senses ingested microbial DNA, although the precise role of the truncation remains controversial. Here we show that TLR9 is expressed on the surface of splenic dendritic cells. Following the cleavage of TLR9 in the endolysosome, N-terminal half of the ectodomain remains associated with truncated TLR9, forming the complex TLR9N+C. The TLR9-dependent cytokine production by Tlr9(-/-) dendritic cells is rescued by a combination of TLR9N and TLR9C, but not by TLR9C alone. These results demonstrate that the TLR9N+C complex is a bona fide DNA sensor.

Structural Analysis Reveals that Toll-like Receptor 7 Is a Dual Receptor for Guanosine and Single-Stranded RNA

Toll-like receptor 7 (TLR7) is a single-stranded RNA (ssRNA) sensor in innate immunity and also responds to guanosine and chemical ligands, such as imidazoquinoline compounds. However, TLR7 activation mechanism by these ligands remain largely unknown. Here, we generated crystal structures of three TLR7 complexes, and found that all formed an activated m-shaped dimer with two ligand-binding sites. The first site conserved in TLR7 and TLR8 was used for small ligand-binding essential for its activation. The second site spatially distinct from that of TLR8 was used for a ssRNA-binding that enhanced the affinity of the first-site ligands. The first site preferentially recognized guanosine and the second site specifically bound to uridine moieties in ssRNA. Our structural, biochemical, and mutagenesis studies indicated that TLR7 is a dual receptor for guanosine and uridine-containing ssRNA. Our findings have important implications for understanding of TLR7 function, as well as for therapeutic manipulation of TLR7 activation.

Essential role for Toll-like receptor 7 (TLR7)-unique cysteines in an intramolecular disulfide bond, proteolytic cleavage and RNA sensing

Toll-like receptor 7 (TLR7) an innate immune sensor for microbial RNA, erroneously responds to self-derived RNA. To avoid autoimmune responses, TLR7 is suggested to be silenced until the N-terminal half of the TLR7 ectodomain (TLR7N) is cleaved off. Resultant truncated TLR7 (TLR7C) is thought to signal microbial RNA. We here show that TLR7N remains associated with TLR7C through a disulfide bond. By N-terminal amino acid sequencing, TLR7C was found to start at 461E or 462A. The newly established monoclonal anti-TLR7N showed that endogenous TLR7 in bone marrow-derived dendritic cells was almost all cleaved and cleaved TLR7N remained in endolysosomes. TLR7N in endolysosomes was linked with TLR7C by a disulfide bond. In contrast, TLR9 did not have a disulfide bond between TLR9N and TLR9C fragments. Among the cysteines unique to the ectodomain of TLR7 but not TLR9 (Cys98, Cys445, Cys475 and Cys722), Cys98 in TLR7N and Cys475 in TLR7C were required for an intramolecular disulfide bond. These cysteines were also needed for proteolytic cleavage of and RNA sensing by TLR7, but not for TLR7 trafficking from endoplasmic reticulum to endosomes. No response was seen in TLR7 mutants lacking the proteolytic cleavage site or TLR7C alone. These results demonstrate requirement for proteolytic cleavage and TLR7N in TLR7 responses and indicate RNA sensing by TLR7N + TLR7C.

Nucleic acid-sensing TLRs and autoimmunity: novel insights from structural and cell biology

Invasion of pathogenic microorganisms or tissue damage activates innate immune signaling receptors that sample subcellular locations for foreign molecular structures, altered host molecules, or signs of compartment breaches. Upon engagement of innate immune receptors an acute but transient inflammatory response is initiated, aimed at the clearance of pathogens and cellular debris. Among the molecules that are sensed are nucleic acids, which activate several members of the transmembrane Toll‐like receptor (TLR) family. Inappropriate recognition of nucleic acids by TLRs can cause inflammatory pathologies and autoimmunity. Here, we review the mechanisms involved in triggering nucleic acid‐sensing TLRs and indicate checkpoints that restrict their activation to endolysosomal compartments. These mechanisms are crucial to sample the content of endosomes for nucleic acids in the context of infection or tissue damage, yet prevent accidental activation by host nucleic acids under physiological conditions. Decoding the molecular mechanisms that regulate nucleic acid recognition by TLRs is central to understand pathologies linked to unrestricted nucleic acid sensing and to develop novel therapeutic strategies.

Targeting cell surface TLR7 for therapeutic intervention in autoimmune diseases

Toll-like receptor 7 (TLR7) senses microbial-derived RNA but can also potentially respond to self-derived RNA. To prevent autoimmune responses, TLR7 is thought to localize in endolysosomes. Contrary to this view, we show here that TLR7 is present on the cell surface of immune cells and that TLR7 responses can be inhibited by an anti-TLR7 antibody. The anti-TLR7 antibody is internalized with TLR7 and accumulates in endolysosomes as an immune complex. TLR7 responses in dendritic cells, macrophages and B cells are all inhibited by the anti-TLR7 antibody. Furthermore, the anti-TLR7 antibody inhibits in vivo cytokine production induced by a TLR7 ligand. Spontaneous TLR7 activation in Unc93b1(D34A/D34A) mice causes lethal inflammation. Progressive inflammation such as splenomegaly, thrombocytopenia and chronic active hepatitis are ameliorated by anti-TLR7 antibody treatment. These results demonstrate that cell surface TLR7 is a promising target for therapeutic intervention in autoimmune diseases.

Intracellular TLR4/MD-2 in macrophages senses Gram-negative bacteria and induces a unique set of LPS-dependent genes

Toll-like receptor (TLR)4/MD-2, a sensor for LPS, delivers the MyD88-dependent signal from the cell surface, then traffics to endolysosomes and delivers the TRIF/TICAM-1-dependent signal. Both signals are thought to be dependent on cell surface TLR4/MD-2. Although TLR4/MD-2 is located also in recycling endosomes, the Golgi apparatus or the endoplasmic reticulum, little is known about a role for intracellular TLR4/MD-2 in LPS responses. We here studied intracellular LPS sensing in macrophages. PRAT4A (protein associated with TLR4 A) is a cochaperone for a general chaperone gp96 and required for cell surface expression of TLR4/MD-2. Cell surface TLR4/MD-2 was undetectable on PRAT4A(-/-) thioglycollate-elicited peritoneal macrophages (P-Macs) and bone marrow-derived macrophages (BM-Macs). LPS responses were all abolished in PRAT4A(-/-) P-Macs, whereas a part of LPS responses remained detectable in PRAT4A(-/-) BM-Macs. Of note, LPS responses in PRAT4A(-/-) BM-Macs were not necessarily dependent on TRIF/TICAM-1 signaling. PRAT4A(-/-) BM-Macs showed unimpaired production of both TRIF/TICAM-1-dependent chemokine RANTES (CCL5) and MyD88-dependent chemokine MCP-1 (CCL2). Moreover, up-regulation of co-stimulatory molecules, CD40 and CD86 was not altered. In contrast, TRIF/TICAM-1-dependent production of type I IFN was profoundly impaired. In response to heat-killed bacteria Escherichia coli, BM-Macs also required PRAT4A-independent TLR4/MD-2 for production of MCP-1 (CCL2) and RANTES (CCL5) and for up-regulation of CD40 and CD86, indicating that intracellular TLR4/MD-2 is able to sense phagocytosed bacteria and activate immune responses. These results demonstrate that intracellular TLR4/MD-2 is responsible for unique set of LPS responses.